Note: Descriptions are shown in the official language in which they were submitted.
CA 02588369 2007-05-11
METHODS OF TREATMENT AND PREVENTION OF METABOLIC BONE
DISEASES AND DISORDERS
FIELD OF THE INVENTION
[1] The present invention relates generally to methods for the treatment
and/or prevention
of metabolic bone disease or disorders, and more particularly to treatment
and/or prevention
of metabolic bone diseases or disorders associated with loss of bone mass and
density, such
as osteoporosis and osteopenic diseases using agents that inhibit the
expression and/or
activity of Lp-PLA2 protein.
BACKGROUND
[2] Lipoprotein-Associated Phospholipase A2 (Lp-PLA2), also previously
known in the
art as Platelet Activating Factor Acetly Hydrolase (PAF acetyl hydrolase) is a
member of the
super family of phospholipase A2 enzymes that are involved in hydrolysis of
lipoprotein
lipids or phospholipids. It is secreted by several cells that play a major
role in the systemic
inflammatory response to injury, including lymphocytes, monocytes, macrophage,
T
Lymphocytes and mast cells.
131 During the conversion of LDL to its oxidised form, Lp-PLA:, is
responsible for
hydrolysing the sn-2 ester of oxidatively modified phosphatidylcholine to give
lyso-
phosphatidylcholine and an oxidatively modified fatty acid. Lp-PLA2 hydrolyzes
the sn2
position of a truncated phospholipid associated with oxidized LDL. As a
result, there is a
generation of 2 inflammatory cell homing mediators (non-esterfied fatty acids
(NEFA) and
LYSO PC) Both NEFA and LYSO PCs are chematractants for circulating monocytes,
play a
role in the activation of macrophages and increase oxidative stress as well as
affecting the
functional and the immediate responses of T lymphocytes. Lp-PLA2 is bound in
humans and
pigs to the LDL molecule via lipoprotein B, and once in the arterial wall the
oxidized LDL is
susceptible to hydrolysis by Lp-PLA2.
[4] Both of these products of Lp-PLA2 action are potent chemoattractants
for circulating
monocytes. As such, this enzyme is thought to be responsible for the
accumulation of cells
loaded with cholesterol ester in the arteries, causing the characteristic
'fatty streak' associated
with the early stages of atherosclerosis, and inhibition of the Lp-PLA2 enzyme
may be useful
in preventing the build up of this fatty streak (by inhibition of the
formation of
lysophosphatidylcholine), and useful in the treatment of atherosclerosis.
10566615.1
CA 02588369 2007-05-11
- 2 -
[5] In addition, it is proposed that Lp-PLA2 plays a direct role in LDL
oxidation. This is
due to the poly unsaturated fatty acid-derived lipid peroxide products of Lp-
PLA2 action
contributing to and enhancing the overall oxidative process. In keeping with
this idea, Lp-
PLA2 inhibitors inhibit LDL oxidation. Lp-PLA2 inhibitors may therefore have a
general
application in any disorder that involves lipid peroxidation in conjunction
with the enzyme
activity, for example in addition to conditions such as atherosclerosis and
diabetes other
conditions such as rheumatoid arthritis, myocardial infarction and reperfusion
injury.
[6] Lp-PLA2 is responsible for hydrolysing the sn-2 ester of oxidatively
modified
phosphatidylcholine to give lyso-phosphatidylcholine (lysoPC) and an
oxidatively modified
fatty acid. Both of these products of Lp-PLA2 action are potent
chemoattractants for
circulating monocytes. Therefore, Lp-PLA2 is thought to be responsible for the
accumulation
of cells loaded with cholesterol ester in the arteries, characteristic of
atherosclerosis.
[71 Osteopenia and osteoporosis are characterized by low bone mass and
structural
deterioration of bone tissue, leading to bone fragility and an increased
susceptibility to
fractures. Osteoporosis affects 44 million Americans, or 55 percent of the
people 50 years of
age and older. One in two women and one in four men over age 50 will have an
osteoporosis-
related fracture in her/his remaining lifetime. Osteopenia and osteoporosis
are responsible for
more than 1.5 million fractures annually. The estimated national direct
expenditures
(hospitals and nursing homes) for osteoporotic hip fractures were $18 billion
dollars in 2002,
and the cost is rapidly rising.
[8] One approach, for example, for treating bone disorders is inhibition
of the osteoclast
proton pump. See e.g., Blair et al., Science 1989, 245, 855-857; Finbow et
al., Biochem. J.
1997, 324, 697-712; Forgac, M. Soc. Gen. Physiol. Ser. 1996, 51, 121-132;
Baron et al., J.
Cell. Biol. 1985, 101, 2210-2222; Farina et al., Exp. Opin. Ther. Patents
1999, 9, 157-168;
and David, P. and Baron, R. "The Vacuolar II+TPase: A Potential Target for
Drug
Development in Bone Diseases" Exp. Opin. Invest. Drugs 1995, 4, 725-740.
191 Another approach to drug discovery for treating bone-related (and
other) diseases
involves the control of cellular signal transduction. See, for example,
Missbach et al., "A
Novel Inhibitor of the Tyrosine Kinase Src Suppresses Phosphorylation of Its
Major Cellular
Substrates and Reduces Bone Resorption in Vitro and in Rodent Models In Vivo."
Bone
1999, 24, 437-449; Connolly et al., Bioorg. & Med. Chem. Lett. 1997, 7, 2415-
2420; Trump-
Kallmeyer et al., J. Med. Chem. 1998, 41, 1752-1763; Klutchko et al., J. Med.
Chem. 1998,
41, 3276-3292; Legraverend et al., Bioorg. & Med. Chem. 1999, 7, 1281-1293;
Chang et al.,
0566615.1
CA 02588369 2007-05-11
- 3 -
Chem. & Biol. 1999, 6, 361-375; Lev et al. Nature 1995, 376, 737-784; Palmer
et al., J. Med.
Chem. 1997, 40, 1519-1529.
[10] Some approaches for the treatment of bone disorders such as osteoporosis
include, for
example, estrogens, bisphosphonates, calcitonin, flavonoids, and selective
estrogen receptor
modulators. Other approaches include peptides from the parathyroid hormone
family,
strontium ranelate, and growth hormone and insulin-like growth response (see,
for example,
Reginster et al. "Promising New Agents in Osteoporosis," Drugs R & D 1999, 3,
195-201).
[11] The variety of different approaches represented by the therapeutic agents
currently
available or under study evidence the variety of biological factors
influencing the competing
processes of bone production and resorption. Although progress has been made
towards
developing therapeutic agents for osteoporosis and other bone disorders, to
date, there is no
cure for osteopenia and osteoporosis. Current medication for osteopenia and
osteoporosis is
aimed at reducing fracture risk and alleviating symptoms related to fracture.
SUMMARY
[121 The present invention relates to methods for treating and preventing
metabolic bone
diseases and disoders by inhibiting Lp-PLA2, including inhibiting the
expression and/or
activity of Lp-PLA2. Metabolic bone diseases and disorders amenable to
treatment and/or
prevention by the methods of the present invention are diseases and disoders
associated with
loss of bone mass and density and include but are not limited osteoporosis and
osteopenic
related diseases. Osteoporosis and osteopenic related diseases include but are
not limited to
Paget's diseases, hyperparathyroidism and related diseases.
1131 In one embodiment, the methods as disclosed herein comprise administering
to a
patient in need thereof for treating or preventing a metabolic bone disease, a
pharmaceutical
composition comprising an effective amount of an agent which inhibits Lp-PLA2,
for
example an agent which inhibits the expression of Lp-PLA2 and/or the activity
of Lp-PLA2
protein. It is not intended that the present invention to be limited to any
particular stage of the
disease (e.g. early or advanced).
[14] In some embodiments as disclosed herein methods to prevent osteoporosis
and/or
osteopenic diseases are effected by inhibiting the expression of Lp-PLA2
and/or inhibiting the
protein activity of Lp-PLA2. Accordingly some embodiments provide methods for
inhibiting
Lp-PLA2 by blocking enzyme activity and some embodiments provide methods for
inhibiting
10566615.1
CA 02588369 2007-05-11
- 4 -
Lp-PLA2 by reducing and/or downregulating the expression of Lp-PLA2 RNA. In
some
embodiments preventing and/or reducing loss of bone mass and/or loss of bone
density leads
to preventing or reducing symptoms associated with metabolic bone diseases or
disorders
such as osteoporosis and/or osteopenic diseases.
[15] In one aspect the methods as disclosed herein provide methods of treating
and/or
preventing a metabolic bone disorder or disease in a patient, such as a human
patient, wherein
the methods comprise administering to the patient in need thereof a
pharmaceutical
composition comprising an effective amount of an agent that inhibits the
activity and/or
expression of the Lp-PLA2 protein. Such a metabolic bone disease or disorder
includes
metabolic bone diseases and disorders associated with loss of bone mass and/or
loss of bone
density. Such metabolic bone diseases include but are not limited to
osteoporosis and
osteopenic related diseases such as diseases with bone marrow abnormalities..
These include,
including dyslipidemia, type II diseases, metabolic syndrome, insulin
resistance, Paget's
disease, hyperparathyroidism and related diseases. In a further embodiment,
the patient
administered an effective amount of an agent that inhibits the activity or
expression of the
Lp-PLA2 protein is a human.
[16] The method of this invention can be effected by administering an
effective amount of
a reversible or irreversible Lp-PLA2 inhibitor. Examples of reversible
inhibitors are small
molecules and compounds such as 1-(N-(2-(diethylamino)ethyl)-N-(4-(4-
trifluoromethylphenypbenzy1)-aminocarbonylmethyl)-2-(4-fluorobenzypthio-5,6-
trimethylenepyrimidin-4-one (which is also known as SB480848). N-(2-
diethylaminoethyl)-
2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4,5,6,7-tetrahydro-
cyclopentapyrimidin-1-y11-N-(4'-
trifluoromethyl-biphenyl-4-ylmethypacetamide, N-(1-(2-Methoxyethyl)piperidin-4-
y1)-2-[2-
(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-l-y1]-N-(4'-trifluoromethylbipheny1-
4-ylmethyl)-
acetamide, methyl 2-[4-( {[2-[2-(2,3-difluorophenypethy1]-4-oxopyrido[2,3-
d]pyrimidin-
1(4H)-yl] acetyl } {[4'-(trifluoromethyl)-4-biphenylyl]methyllamino)-1-
piperidiny1]-2-
methylpropanoate, or a salt thereof.
[17] Also, the method of this inventon comprises identifying a patient at risk
for
developing a clinical degree of a metabolic bone disease such as osteopenia or
having a
clinical degree of osteopenia and, for those at risk or having such a
condition, administering
an effective amount of an Lp-PLA2 inhibitor to those in need thereof,
and,optionally,
monitoring the effectiveness of the treatment. Bone density and biomarkers can
be used to
identify patients who could benefit from the therapy as disclosed herein.
10566615.1
CA 02588369 2007-05-11
-5-
1181 The methods can further comprise administering additional therapeutic
agents used in
the treatment of metabolic bone diseases. For example, where the metabolic
bone disorder is
osteoporosis one can treat the patient with the likes of bisphosphates such as
alendronate,
ibandronate, risedronate, calcitonin, raloxifene, a selective estrogen
modulator (SERM),
estrogen therapy, hormone replacement therapy (ET/HRT) and teriparatide.
BRIEF DESCRIPTION OF FIGURES
[19] Figure 1 shows the region of medial femoral condyle scanned and analyzed
by micro
CT. Bold dashed line represents cut plane. Area to the right indicates wedge
of medial
femoral condyle scanned. Dotted line box indicates approximate area of
analysis.
[20] Figure 2 shows examples of diabetes mellitus/hypercholesteremia (DM/HC)-
induced
osteoporotic changes and the effects of the treatment in trabecular bone.
Panel 2A shown
bone matrix density on non-treated non-DM/HC control animals, and panel Fig 2B
shows an
example from a DM/HC non-treated animals as compared to panel Fig 2C showing
DM/HC
animals treated with an inhibitor of Lp-PLA2.
1211 Figure 3 shows examples of DM/HC-induced changes in the trabecular bone
environment, as shown in Fig 3B as compared to control non-DM/HC animals,
shown in Fig
3A.
[22] Figure 4 shows the Lp-PLA2 inhibitor restored normal bone marrow and
abnormalities in bone marrow such abnormal bone marrow homeostasis. Examples
of
DM/HC-induced changes in the trabecular bone environment are shown in panels
Fig4A-D as
compared to DM/HC animals administered an Lp-PLA2 inhibitor, which are shown
in panels
Fig4 E-G. Figure 4 shows representative images of examples from n = 4 animals
per group.
[23] Figure 5 shows the Lp-PLA2 inhibitor preserved normal bone marrow and
prevented
abnormal bone marrow, such as as abnormal bone marrow homeostasis in remote
trabacular
bone. Examples of low magnification images of DM/HC-induced changes in the
trabecular
bone environment are shown in panels Fig5A and B as compared to DM/HC animals
administered a Lp-PLA2 inhibitor, which are shown in panels Fig5 C and D.
[24] Figure 6 shows the Lp-PLA2 inhibitor preserved normal bone marrow and
prevented
abnormal bone marrow, such as as abnormal bone marrow homeostasis in remote
trabacular
bone. Examples of high magnification images of DM/HC-induced changes in the
trabecular
bone environment are shown in panels Fig6 A-C as compared to DM/HC animals
administered a Lp-PLA2 inhibitor, which are shown in panels Fig 6 D-E.
10566615.1
CA 02588369 2007-05-11
- 6 -
[25] Figure 7 shows examples of DM/HC-induced TUNEL positive cells within the
trabecular bone environment as shown in panels Fig 7A-D, as compared to the
effects of
animals treated with the Lp-PLA2 inhibitor SB480848, as shown in Fig 7 E-H.
Figure 7
shows representative images of examples from n = 4 animals per group.
[26] Figure 8 shows the results of MLO-A5 cells treated with 5 tM LysoPC after
7 days,
showing in panel B reduced mineralization in LysoPC treated cells as compared
to control
treatment in Panel 8A which has increased intensity of Alizarin red staining.
[27] Figure 9 shows MLO-A5 cells treated with 5 tM LysoPC after 7 days show
reduced
alkaline phosphatase staining. Figure 9A and 9B show examples of alkaline
phosphatase
staining in control treated cells, and Figure 9C and 9D show examples of
alkaline
phosphatase staining in cells treated with 5 M LysoPC, and Figure 9E and 9F
show
examples of alkaline phosphatase staining in cells treated with 20 M LysoPC,
showing
reduced alkaline phosphatase staining of MLO-A5 cells treated with at least 5
M LysoPC at
7 days as compared to control cells at 7 days.
[28] Figure 10 shows the results from the proliferation assay, showing MLO-A5
cells
treated with 5 uM LysoPC for 18 hours show reduced proliferation.
DETAILED DESCRIPTION
[29] The inventors have discovered that animals prone to pathological features
of
metabolic bone diseases exhibit reduced loss of bone density and bone mass
when treated
with an Lp-PLA2 inhibitor. Animals treated with an Lp-PLA2 inhibitor showed
increased
bone density and reduced death of osteocytes and osteoblasts as compared to
animals not
treated with the Lp-PLA2 inhibitor. The animals treated with an Lp-PLA2
inhibitor also had
normal trabecular bone marrow as compared to animals not treated with the Lp-
PLA2
inhibitor. The latter showed trabecular bone marrow abnormalities such as
increased
extracellular material, reduced cellularity and shrinkage of adipocytes,
demonstrating
pathological signs of abnormal bone marrow homeostasis. Therefore, the
inventors have
discovered that Lp-PLA2 inhibitors can be used in treating or preventing
metabolic bone
diseases and disorders, particularly metabolic bone diseases and disorders
associated with
loss of bone mass and/or loss of bone density. Such metabolic bone diseases
include
osteoporosis and osteopenic related diseases caused by or associated with
dyslipidemia, type
10566615.1
CA 02588369 2007-05-11
- 7 -
II diseases, metabolic syndrome, insulin resistance, Paget's disease, and
hyperparathyroidism
and related diseases.
Definitions
1301 For convenience, certain terms employed in the entire application
(including the
specification, examples, and appended claims) are collected here. Unless
defined otherwise,
all technical and scientific terms used herein have the same meaning as
commonly
understood by one of ordinary skill in the art to which this invention
belongs.
1311 The term "metabolic bone disease" as used herein refers to a varied
assortment of
bone diseases and disorders characterized by gradual and progressive loss of
bone tissue.
Metabolic bone diseases amenable to prevention and/or treatment using the
methods as
described herein are metabolic bone diseases whereby there is a condition of
diffusely
decreased bone density and/or diminished bone strength. Such diseases are
characterized by
histological appearance. Two examples are osteoporosis which is a common
metabolic bone
disorder characterized by decreased mineral and bone matrix, and osteomalacia
which is
characterized by decreased mineral but intact bone matrix.
1321 The term "osteopenic diseases" or "osteopenia" are used interchangeably
herein, and
refer to conditions with decreased calcification and/or bone density, and is a
descriptive term
used to refer to all skeletal systems in which the condition is noted.
Osteopenia also refers to
a reduced bone mass due to inadequate osteiod synthesis.
1331 The term "osteoporosis" refers to conditions in which decreased mineral
or bone
matrix and reduced bone mass occurs.
[34] The term "disease" or "disorder" is used interchangeably herein, and
refers to any
alteration in state of the body or of some of the organs, interrupting or
disturbing the
performance of the functions and/or causing symptoms such as discomfort,
dysfunction,
distress, or even death to the person afflicted or those in contact with a
person. A disease or
disorder can also relate to a distemper, ailing, ailment, malady, disorder,
sickness, illness,
complaint, inderdisposion or affectation.
1351 The term "abnormalities in bone marrow" or "abnormal bone marrow" is used
to refer
to a dysfunctional or abnormal morphological characteristic of bone marrow,
for example
where the bone marrow comprises loss or death of osteocytes and/or
osteoblasts, resulting in,
for example fast bone turn over or reduced bone formation. Abnormal bone
marrow can also
be used to refer to presence of cells or structures not typically present in
normal bone
10566615.1
CA 02588369 2007-05-11
- 8 -
marrow, and/or altered morphology of cells present in the bone marrow, for
example
presence of increased extracellular material, or altered morphology of
adipocytes or reduced
numbers of cells present in the bone marrow. Bone marrow abnormalities
referred to herein
are defined as those where general abnormalities of the biological balance in
the bone
marrow is indicated, for example, including but not limited to viral or
bacterial infections in
the bone marrow, cellular infiltration of the bone marrow, abnormalities of
the bone marrow
haematopoiesis, proliferation of malignant neoplasms in the bone marrow and
concentration
changes in cell growth differentiaion factors.
[36] The term "agent" refers to any entity which is normally not present or
not present at
the levels being administered in the cell. Agent can be selected from a group
comprising:
chemicals; small molecules; nucleic acid sequences; nucleic acid analogues;
proteins;
peptides; aptamers; antibodies; or fragments thereof. A nucleic acid sequence
can be RNA or
DNA, and can be single or double stranded, and can be selected from a group
comprising;
nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid
analogues, for
example peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked
nucleic
acid (LNA) etc. Such nucleic acid sequences include, for example, but are not
limited to,
nucleic acid sequence encoding proteins, for example that act as
transcriptional repressors,
antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for
example but are
not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense
oligonucleotides etc.
A protein and/or peptide or fragment thereof can be any protein of interest,
for example, but
are not limited to: mutated proteins; therapeutic proteins and truncated
proteins, wherein the
protein is normally absent or expressed at lower levels in the cell. Proteins
can also be
selected from a group comprising; mutated proteins, genetically engineered
proteins,
peptides, synthetic peptides, recombinant proteins, chimeric proteins,
antibodies, midibodies,
minibodies, triabodies, humanized proteins, humanized antibodies, chimeric
antibodies,
modified proteins and fragments thereof. Alternatively, the agent can be
intracellular within
the cell as a result of introduction of a nucleic acid sequence into the cell
and its transcription
resulting in the production of the nucleic acid and/or protein inhibitor of Lp-
PLA2 within the
cell. In some embodiments, the agent is any chemical, entity or moiety,
including without
limitation synthetic and naturally-occurring non-proteinaceous entities. The
agents of
particular interest are small molecules that reversibly or irreversibly
inhibit Lp-PLA2. These
include unsubstituted or substituted alkyl, aromatic, or heterocyclyl
compounds as further
illustrated below.
10566615.1
CA 02588369 2013-10-22
- 9 -
[37] The term "inhibiting" as used herein means that the expression or
activity of Lp-PLA2
or variants or homologues thereof is reduced to an extent, and/or for a time,
sufficient to
produce the desired effect. The reduction in activity can be due to affecting
one or more
characteristics of Lp-PLA2 including decreasing its catalytic activity or by
inhibiting a co-
factor of Lp-PLA2 or by binding to Lp-PLA2 with a degree of avidity that is
such that the
outcome is that of treating or preventing a metabolic bond disorder. In
particular, inhibition
of Lp-PLA2 can be determined using an assay for Lp-PLA2 inhibition by using
the bioassay
for Lp-PLA2 protein as disclosed herein.
[38] As used herein, the term "Lp-PLA2" refers to the protein target to be
inhibited by the
methods as disclosed herein. Lp-PLA2 is used interchangeably with lipoprotein
associated
phospholipase A2, also previously known in the art as Platelet Activating
Factor Acetyl
Hydrolase (PAF acetyl hydrolase). Human Lp-PLA2 is encoded by nucleic acid
corresponding to accession No: U20157 (SEQ ID NO:!) or Ref Seq ID: N1\4_005084
(SEQ
ID NO:2) or and the human Lp-PLA2 corresponds to protein sequence
corresponding to
accession No: NP 005075 (SEQ ID NO:3), which are disclosed in U.S. Patent
5,981,252.
[39] As used herein, "gene silencing" or "gene silenced" in reference to an
activity of n
RNAi molecule, for example a siRNA or miRNA refers to a decrease in the mRNA
level in a
cell for a target gene by at least about 5%, about 10%, about 20%, about 30%,
about 40%,
about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%,
about
100% of the mRNA level found in the cell without the presence of the miRNA or
RNA
interference molecule. In one preferred embodiment, the mRNA levels are
decreased by at
least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.
[40] As used herein, the term "RNAi" refers to any type of interfering RNA,
including but
are not limited to, siRNAi, shRNAi, endogenous microRNA and artificial
microRNA. For
instance, it includes sequences previously identified as siRNA, regardless of
the mechanism
of down-stream processing of the RNA (i.e. although siRNAs are believed to
have a specific
method of in vivo processing resulting in the cleavage of mRNA, such sequences
can be
incorporated into the vectors in the context of the flanking sequences
described herein).
[41] As used herein an "siRNA" refers to a nucleic acid that forms a double
stranded RNA,
which double stranded RNA has the ability to reduce or inhibit expression of a
gene or target
gene when the siRNA is present or expressed in the same cell as the target
gene, for example
Lp-PLA2. The double stranded RNA siRNA can be formed by the complementary
strands. In
CA 02588369 2013-10-22
- 10 -
one embodiment, a siRNA refers to a nucleic acid that can form a double
stranded siRNA.
The sequence of the siRNA can correspond to the full length target gene, or a
subsequence
thereof. Typically, the siRNA is at least about 15-50 nucleotides in length
(e.g., each
complementary sequence of the double stranded siRNA is about 15-50 nucleotides
in length,
and the double stranded siRNA is about 15-50 base pairs in length, preferably
about 19-30
base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21,
22, 23, 24, 25, 26,
27, 28, 29, or 30 nucleotides in length).
[42] As used herein "shRNA" or "small hairpin RNA" (also called stem loop) is
a type of
siRNA. In one embodiment, these shRNAs are composed of a short, e.g. about 19
to about 25
nucleotide, antisense strand, followed by a nucleotide loop of about 5 to
about 9 nucleotides,
and the analogous sense strand. Alternatively, the sense strand can precede
the nucleotide
loop structure and the antisense strand can follow.
[43] The terms "microRNA" or "miRNA" are used interchangeably herein are
endogenous
RNAs, some of which are known to regulate the expression of protein-coding
genes at the
posttranscriptional level. Endogenous microRNA are small RNAs naturally
present in the
genome which are capable of modulating the productive utilization of mRNA. The
term
artificial microRNA includes any type of RNA sequence, other than endogenous
microRNA,
which is capable of modulating the productive utilization of mRNA. MicroRNA
sequences
have been described in publications such as Lim, et al., Genes & Development,
17, p. 991-
1008 (2003), Lim et al Science 299, 1540 (2003), Lee and Ambros Science, 294,
862 (2001),
Lau et al., Science 294, 858-861 (2001), Lagos-Quintana et al, Current
Biology, 12, 735-739
(2002), Lagos Quintana et al, Science 294, 853-857 (2001), and Lagos-Quintana
et al, RNA,
9, 175-179 (2003). Multiple microRNAs can also be incorporated into a
precursor molecule.
Furthermore, miRNA-like stem-loops can be expressed in cells as a vehicle to
deliver
artificial miRNAs and short interfering RNAs (siRNAs) for the purpose of
modulating the
expression of endogenous genes through the miRNA and or RNAi pathways.
[44] As used herein, "double stranded RNA" or "dsRNA" refers to RNA molecules
that
are comprised of two strands. Double-stranded molecules include those
comprised of a
single RNA molecule that doubles back on itself to form a two-stranded
structure. For
example, the stem loop structure of the progenitor molecules from which the
single-stranded
miRNA is derived, called the pre-miRNA (Bartel et al. 2004. Cell 116:281-297),
comprises a
dsRNA molecule.
CA 02588369 2013-10-22
- 11 -
[45] The terms "patient", "subject" and "individual" are used
interchangeably herein and
refer to an animal, particularly a human, to whom treatment is provided.
[46] The term "gene" used herein can be a genomic gene comprising
transcriptional and/or
translational regulatory sequences and/or a coding region and/or non-
translated sequences
(e.g., introns, 5'- and 3'- untranslated sequences and regulatory sequences).
The coding region
of a gene can be a nucleotide sequence coding for an amino acid sequence or a
functional
RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA and antisense RNA. A gene
can
also be an mRNA or cDNA corresponding to the coding regions (e.g. exons and
miRNA)
optionally comprising 5'- or 3' untranslated sequences linked thereto. A gene
can also be an
amplified nucleic acid molecule produced in vitro comprising all or a part of
the coding
region and/or 5'- or 3'- untranslated sequences linked thereto.
[47] The term "nucleic acid" or "oligonucleotide" or "polynucleotide" used
herein can
mean at least two nucleotides covalently linked together. As will be
appreciated by those in
the art, the depiction of a single strand also defines the sequence of the
complementary
strand. Thus, a nucleic acid also encompasses the complementary strand of a
depicted single
strand. As will also be appreciated by those in the art, many variants of a
nucleic acid can be
used for the same purpose as a given nucleic acid. Thus, a nucleic acid also
encompasses
substantially identical nucleic acids and complements thereof. As will also be
appreciated by
those in the art, a single strand provides a probe for a probe that can
hybridize to the target
sequence under stringent hybridization conditions. Thus, a nucleic acid also
encompasses a
probe that hybridizes under stringent hybridization conditions.
[48] Nucleic acids can be single stranded or double stranded, or can contain
portions of
both double stranded and single stranded sequence. The nucleic acid can be
DNA, both
genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain
combinations of
deoxyribo- and ribo- nucleotides, and combinations of bases including uracil,
adenine,
thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and
isoguanine.
Nucleic acids can be obtained by chemical synthesis methods or by recombinant
methods.
[49] A nucleic acid will generally contain phosphodiester bonds, although
nucleic acid
analogs can be included that can have at least one different linkage, e.g.,
phosphoramidate,
phosphorothioate, phosphorodithioate, or 0-methylphosphoroamidite linkages and
peptide
nucleic acid backbones and linkages. Other analog nucleic acids include those
with positive
backbones; non-ionic backbones, and non-ribose backbones, including those
described in
U.S. Pat. Nos. 5, 235,033 and 5, 034,506. Nucleic acids
CA 02588369 2007-05-11
- 12 -
containing one or more non-naturally occurring or modified nucleotides are
also included
within one definition of nucleic acids. The modified nucleotide analog can be
located for
example at the 5'-end and/or the 3'-end of the nucleic acid molecule.
Representative examples
of nucleotide analogs can be selected from sugar- or backbone-modified
ribonucleotides. It
should be noted, however, that also nucleobase- modified ribonucleotides, i.e.
ribonucleotides, containing a non naturally occurring nucleobase instead of a
naturally
occurring nucleobase such as uridines or cytidines modified at the 5-position,
e.g. 5-(2-
amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at
the 8-
position, e.g. 8- bromo guanosine; deaza nucleotides, e. g. 7 deaza-adenosine;
0- and N-
alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The 2' OH- group
can be
replaced by a group selected from H. OR, R. halo, SH, SR, NH2, NHR, NR2 or CN,
wherein
R is C- C6 alkyl, alkenyl or alkynyl and halo is F. Cl, Br or I. Modifications
of the ribose-
phosphate backbone can be done for a variety of reasons, e.g., to increase the
stability and
half- life of such molecules in physiological environments or as probes on a
biochip.
Mixtures of naturally occurring nucleic acids and analogs can be made;
alternatively,
mixtures of different nucleic acid analogs, and mixtures of naturally
occurring nucleic acids
and analogs can be made.
1501 The term "vector" used herein refers to a nucleic acid sequence
containing an origin
of replication. A vector can be a plasmid, bacteriophage, bacterial artificial
chromosome or
yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can
be either a
self replicating extrachromosomal vector or a vector which integrate into a
host genome.
[51] As used herein, the term "treating" includes reducing or alleviating
at least one
adverse effect or symptom of a condition, disease or disorder associated with
a metabolic
bone disease or disorder such as osteoporosis and osteopenic related
disorders. The term
treating is used to refer to the reduction of a symptom and/or a biochemical
marker of a
metabolic bone disease or disorder by some useful amount, an amount that can
be determined
by one skilled in the art. "Treating" with regards to osteoporosis refers to a
measurable
reduction in a biochemical marker of osteoporosis, or a reduction in the death
or loss of
osteocytes and/or osteoblasts, i.e. such results would be considered effective
treatments by
the methods as disclosed herein. Alternatively, treating with regards to
osteoporisis refers to a
measurable increase in bone or bone matrix mineralization, such as a
measurable increase in
re-mineralization. As alternative examples, a reduction in a symptom such as a
measurable
slowing of the rate of decline of bone density or a measurable cessation of
the rate bone
10566615.1
CA 02588369 2007-05-11
- 13 -
density loss, or a measurable increase in bone density would also be
considered as affective
treatments by the methods as disclosed herein.
[52] The term "effective amount" as used herein refers to the amount of agent
that reduces
or stops at least one symptom of the metabolic bone disease or disorder. An
example of an
effective amount would be considered as the amount sufficient to reduce a
symptom of the
disease or disorder by some measurable amount. One possible measure for
treating or
preventing osteoporosis or a metabolic bone disorder is a reduction in the
measured
parameter of at least about 10%. An effective amount as used herein would also
include an
amount sufficient to prevent or delay the development of a symptom of the
disease, alter the
course of a symptom of the disease or reverse a symptom of the disease.
[53] The term "vectors" is used interchangeably with "plasmid" to refer to a
nucleic acid
molecule capable of transporting another nucleic acid to which it has been
linked. Vectors
capable of directing the expression of genes and/or nucleic acid sequence to
which they are
operatively linked are referred to herein as "expression vectors". In general,
expression
vectors of utility in recombinant DNA techniques are often in the form of
"plasmids" which
refer to circular double stranded DNA loops which, in their vector form are
not bound to the
chromosome. Other expression vectors can be used in different embodiments of
the
invention, for example, but are not limited to, plasmids, episomes,
bacteriophages or viral
vectors, and such vectors can integrate into the host's genome or replicate
autonomously in
the particular cell. Other forms of expression vectors known by those skilled
in the art which
serve the equivalent functions can also be used. Expression vectors comprise
expression
vectors for stable or transient expression encoding the DNA.
[54] The articles "a" and "an" are used herein to refer to one or to more than
one (i.e., to at
least one) of the grammatical object of the article. By way of example, "an
element" means
one element or more than one element.
Lp-PLA7: General Information
[55] Lp-PLA2 is also referred to in the art as aliases Lp-PLA2, LDL-PLA2,
lipoprotein
associated phospholipase A2, PLA2G7, phospholipase A, (group VII), or Platelet
Activating
Factor Acetyl Hydrolase (PAF acetyl hydrolase or PAFAH). Human Lp-PLA2 is
encoded by
nucleic acid corresponding to GenBank Accession No: U20157 (SEQ ID NO:1) or
Ref Seq
ID: NM _005084 (SEQ ID NO:2) and the human Lp-PLA2 corresponds to protein
sequence
10566615.1
CA 02588369 2013-10-22
- 14 -
corresponding to GenBank Accession No: NP 005075 (SEQ ID NO:3), which are
disclosed
in U.S. Patent 5,981,252.
[56] Phospholipase A2 enzyme Lipoprotein Associated Phospholipase A2 (Lp-
PLA2), the
sequence, isolation and purification thereof, isolated nucleic acids encoding
the enzyme, and
recombinant host cells transformed with DNA encoding the enzyme are disclosed
in WO
95/00649 (SmithKline Beecham plc). A subsequent publication from the same
group further
describes this enzyme (Tew D et al, Arterioscler Thromb Vas Biol 1996:16; 591-
9) wherein it
is referred to as LDL-PLA2 and later patent application (WO 95/09921, Icos
Corporation)
and a related publication in Nature (Tjoelker et al, vol 374, 6 April 1995,
549) describe the
enzyme PAF-AH which has essentially the same sequence as Lp-PLA2.
[57] It has been shown that Lp-PLA2 is responsible for the conversion of
phosphatidylcholine to lysophosphatidylcholine, during the conversion of low
density
lipoprotein (LDL) to its oxidised form. The enzyme is known to hydrolyse the
sn-2 ester of
the oxidised phosphatidylcholine to give lysophosphatidylcholine and an
oxidatively
modified fatty acid. Both products of Lp-PLA2 action are biologically active
with
lysophosphatidylcholine, in particular having several pro-atherogenic
activities ascribed to it
including monocyte chemotaxis and induction of endothelial dysfunction, both
of which
facilitate monocyte-derived macrophage accumulation within the artery wall.
[58] The inventors have discovered that animals prone to disorders
characterized by
metabolic bone disorders have been found to exhibit normal or close to normal
bone matrix
normal bone density and normal bone marrow characteristics when treated with
an Lp-PLA2
inhibitor. Such animals treated with an inhibitor to Lp-PLA2 also showed
reduced signs of
loss of osteoblasts and osteocytes, and reduced accumulation of bone marrow
abnormalities
as compared to animals not treated with the inhibitor. Therefore, Lp-PLA2
inhibitors can be
used to treat and/or prevent metabolic bone diseases and disorders such as
osteoporosis and
osteopenic related diseases bone marrow abnormalities and Paget's disease.
Agents that inhibit Lp-PLA2
[59] The present invention relates to the inhibition of Lp-PLA2. In some
embodiments
inhibition is inhibition of nucleic acid transcripts encoding Lp-PLA2 such as
by inhibition of
messenger RNA (mRNA). In alternative embodiments, inhibition of Lp-PLA2 is
effected by
the inhibition of the expression and/or inhibition of activity of the gene
product of Lp-PLA2,
CA 02588369 2013-10-22
- 15 -
including the polypeptide or protein of Lp-PLA20r its isoforms. As used
herein, the term
"gene product" refers to RNA transcribed from a gene, or a polypeptide encoded
by a gene or
translated from RNA.
[60] In some embodiments, inhibition of Lp-PLA2 is by an agent such as nucleic
acids,
nucleic acid analogues, peptides, phage, phagemids, polypeptides,
peptidomimetics,
ribosomes, aptamers, antibodies, small or large organic or inorganic
molecules, or any
combination thereof. These agents include agents that function as inhibitors
of Lp-PLA2
expression, inhibitors of mRNA encoding Lp-PLA2 being one example.
[61] Agents useful in the methods as disclosed herein can also inhibit gene
expression (i.e.
suppress and/or repress the expression of the gene). Such agents are referred
to in the art as
"gene silencers" and are commonly known to those of ordinary skill in the art.
Examples
include a nucleic acid sequence, for an RNA, DNA or nucleic acid analogue, and
can be
single or double stranded, and can be selected from a group comprising nucleic
acid encoding
a protein of interest, oligonucleotides, nucleic acids, nucleic acid analogues
such as peptide
nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acids
(LNA) and
derivatives thereof etc. Nucleic acid agents also include, but are not limited
to nucleic acid
sequences encoding proteins that act as transcriptional repressors, antisense
molecules,
ribozymes, small inhibitory nucleic acid sequences, for example but are not
limited to RNAi,
shRNAi, siRNA, micro RNAi (miRNA), antisense oligonucleotides,etc.
[62] As used herein, agents useful in the method as inhibitors of Lp-PLA2
expression
and/or inhibition of Lp-PLA2 function can be any type of entity. These include
small
molecules nucleic acid sequences, nucleic acid analogues, proteins, peptides
or fragments
thereof.
Small molecules
[63] Particularly useful agents are small molecules such as small synthetic
compounds that
inhibit Lp-PLA2. Irreversible or reversible inhibitors of Lp-PLA2 can be used
in the methods
of the present invention.
[64] Irreversible inhibitors of Lp-PLA2 are disclosed in patent
applications WO 96/13484,
W096/19451, WO 97/02242, W097/217675, W097/217676, WO 97/41098, and
W097/41099 (SmithKline Beecham plc) disclose inter alia various series of
4-thionyl/sulfinyl/sulfonyl azetidinone compounds which are inhibitors of the
enzyme Lp-
PLA2. These are irreversible, acylating inhibitors (Tew eta!, Biochemistry,
37, 10087,
1998).
CA 02588369 2014-06-10
- 16 -
[65] Synthetic small molecule Lp-PLA2 inhibitors effective in humans are
commonly
known by persons of ordinary skill and include those undergoing pre-clinical
and clinical.
A number of applications have been filed and published by SmithKline Beecham
and its
successor GlaxoSmithKline. A list of relevant published applications assigned
to same is:
W001/60805, W002/30904, W003/016287, W000/66567, W003/042218, W003/042206,
W003/042179, W003/041712, W003/086400, W003/087088, W002/30911,
W099/24420, W000/66566, W000/68208, W000/10980, and W02005/021002. In
addition, reference is made to U.S. provisional application 60/829,327.
[66] Other Lp-PLA2 inhibitors useful in the methods as disclosed herein are
described in
published patent applications, for example W02006063791-A1, W02006063811-Al,
W02006063812-A1, W02006063813-A1, all in the name of Bayer Healthcare; and
US2006106017-A1 assigned to Korea Res. Inst. Bioscience & Biotechnology.
[67] Lp-PLA2 inhibitors encompassed for use in the methods as disclosed
herein also
include other compounds, such as statins and/or Niacin and fenofibrate. Such
Lp-PLA2
agents can also be admininstered with the Lp-PLA2 inhibitor agents as
disclosed herein.
[68] It is believed that any or all of the compounds disclosed in these
documents are
useful for prophylaxis or treatment of metabolic bone disorders including
preventing or
treating osteoporosis or osteopenic diseases.
[69] The porcine model of decreased bone density as described herein below
and
exemplified in the Methods can be used by one of ordinary skill in the art to
determine
which of the disclosed compounds or other inhibitors of Lp-PLA2, for example
antibodies,
or RNAi are effective for the treatment and/or prevention of metabolic bone
diseases or
disorders as claimed herein. In some embodiments, agents inhibiting Lp-PLA2
can be
assessed in animal models for effect on increasing bone density and/or bone
mass. For
example, one can use the porcine model of hyperglycemia and
hypercholesterolemia as
disclosed in the Examples herein, where the bone marrow is abnormal and having
decreased bone density, for example the decreased bone matrix, in which the
bone
matrix can be assessed in the present and
CA 02588369 2013-10-22
- 17 -
absence of inhibitors for Lp-PLA2 by methods commonly known by persons in the
art. In
some embodiments, assessment of bone density and/or markers of metabolic bone
diseases
such as osteoporosis can be used, as disclosed herein.
[70] In a particular embodiment, Lp-PLA2 inhibitors as disclosed in U.S.
patent 6,649,619
and 7,153,861, (and International Application WO 01/60805) and U.S. patent
7,169,924 (and
International Patent Application WO 02/30911), are useful in the methods
disclosed herein
for the prophylaxis or for the treatment of metabolic bone diseases or
disorder such as
osteoporosis or osteopenia. In some embodiments, the Lp-PLA2 inhibitors as
disclosed in
U.S. publication No. 2005/0033052A1, and International Patent Applications WO
02/30904,
WO 03/042218, WO 03/042206, W003/042179, WO 03/041712, WO 03/086400, and WO
03/87088 are reversible Lp-PLA2 inhibitors.
[71] Formula (I) One can use a group of reversible Lp-PLA2 inhibitors that are
disclosed in international application WO 01/60805, from which arose U.S.
patents 6,649,619
and 7,153,861. A narrower group of compounds of interest are those of formula
(I) described
in WO 01/60805 and claimed in US patents 6,649,619 and 7,153,861, namely:
0
NRa
I
R2X NRb
4 5
N,
(I)
[72] wherein:
[73] Ra and Rb together with the pyrimidine ring carbon atoms to which they
are attached
form a fused 5-membered carbocyclic ring;
[74] R2 is phenyl, substituted by one to three fluorine atoms;
[75] R3 is methyl or Co _3)alkyl substituted by NR8R9; or
[76] R3 is Het-C(02)alkyl in which Het is a 5- to 7- membered heterocyclyl
ring having N
and in which N is unsubstituted or substituted by Co _oalkyl;
CA 02588369 2007-05-11
- 18 -
[77] R4 and R5 together form a 4-(4-trifluoromethylphenyl)phenyl moiety;
[78] R8 and R9 which can be the same or different are selected from the group
consisting
of hydrogen, or Co _6)alkyl);
[79] X is S, or a pharmaceutically acceptable salt thereof
[801 Of even more interest are the following compounds, all within the scope
of formula
(I) and disclosed in the application and patents noted above:
[81] 1-(N-(2-(diethylamino)ethyl)-N-(4-(4-trifluoromethylphenyl)benzyl)-
aminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5,6-trimethylenepyrimidin-4-one,
used in the
pig study described herein;
[82] 1 -(N-(2-(diethylamino)ethyl)-N-(4-(4-trifluoromethylphenypb enzy1)-
aminoc arbonylmethyl)-2-(2,3 -difluorob enzypthio -5 ,6-trimethyl enepyrimidin-
4-one ;
[83] 1-(N-(2-(diethylamino)ethyl)-N-(4-(4-trifluoromethylphenyl)benzyl)amino-
carbonylmethyl)-2-(3,4-difluorobenzyl)thio-5,6-trimethylenepyrimidin-4-one;
[84] 1-(N-(2-(diethylamino)ethyl)-N-(4-(4-trifluoromethylphenyl)benzypamino-
carbonylmethyl)-2-(2,3,4-trifluorobenzypthio-5,6-trimethylenepyrimidin-4-one;
[85] 1-(N-(2-(diethylamino)ethyl)-N-(4-(4-trifluoromethylphenyl)benzyl)amino-
carbonylmethyl)-2-(2-fluorobenzyl)thio-5,6-trimethylenepyrimidin-4-one;
[86] 1-(N-methyl-N-(4-(4-trifluoromethylphenyl)benzyl)aminocarbonylmethyl)-2-
(4-
fluorobenzyl)thio-5,6-trimethylenepyrimidin-4-one;
[87] 1-(N-(2-(1-piperidino)ethyl)-N-(4-(4-trifluoromethylphenyl)benzyl)amino-
carbonylmethyl)-2-(4-fluorobenzyl)thio-5,6-trimethylenepyrimidin-4-one;
[88] 1-(N-(1-ethylpiperidin-4-y1)-N-(4-(4-trifluoromethylphenyl)benzypamino-
carbonylmethyl)-2-(4-fluorobenzyl)thio-5,6-trimethylenepyrimidin-4-one;
[89] 1-(N-(2-ethylamino-2-methylpropy1)-N-(4-(4-trifluoromethylphenyl)benzyl)-
aminocarbonylmethyl)-2-(4-fluorobenzypthio-5,6-trimethylenepyrimidin-4-one;
[90] N-(2-tert-butylaminoethyl)-N-(4-(4-trifluoromethylphenyl)benzypamino-
carbonylmethyl)-2-(4-fluorobenzyl)thio-5,6-trimethylenepyrimidin-4-one;
[91] 1-(N-(1-methylpiperidin-4-y1)-N-(4-(4-trifluoromethylphenyl)benzy1)-
aminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5,6-trimethylenepyrimidin-4-one;
[92] 1-(N-(1-isopropylpiperidin-4-y1)-N-(4-(4-trifluoromethylphenyl)benzyp-
aminocarbonylmethyl)-2-(4-fluorobenzypthio-5,6-trimethylenepyrimidin-4-one;
10566615.1
CA 02588369 2014-06-10
- 19 -
[93] 1-(N-(1-(2-methoxyethyppiperidin-4-A-N-(4-(4-
trifluoromethylphenyl)benzy1)-
aminocarbonylmethyl)-2-(4-fluorobenzypthio-5,6-trimethylenepyrimidin-4-one;
[94] 1-(N-(2-(ethylamino)ethyl)-N-(4-(4-trifluoromethylphenyl)benzyl)-
aminocarbonylmethyl)-244-fluorobenzypthio-5,6-trimethylenepyrimidin-4-one; or
a
pharmaceutically acceptable salt of these compounds.
Methods for preparing these compounds are disclosed in the noted documents.
[95] A second process for making 1-(N-(2-(diethylamino)ethyl)-N-(4-(4-
trifluoromethylphenyl)benzy1)-aminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5,6-
trimethylenepyrimidin-4-one can be found in application WO 03/016287 (U.S.
publication
No 20050014793A1), which is incorporated herein by reference in its entirety.
[96] Formula (II)
[97] A further group of compounds which can be useful in practicing the
methods of this
invention are disclosed in WO 02/30911; US patent 7,169,924 corresponds to
this
international application. The generic formula in that case, represented here
as formula (II),
is as follows:
0
NI I
R13
X N R
6
R4
(II)
[98] in which:
[99] RI is an aryl group, optionally substituted by 1, 2, 3 or 4
substituents which can be
the same or different selected from C(16)alkyl, C(l.6)alkoxy, C(1_6)alkylthio,
hydroxy,
halogen, CN, and mono to perfluoro-C(l.4)alkyl;
1100] R2 is halogen, C(1_3)alkyl, C(1_3)alkoxy, hydroxyC(1.3)alkyl,
C(1_3)alkylthio, C(1_
3)alkylsulphinyl, aminoC(1_3)alkyl, mono- or di-C(l.3)alkylaminoC(1_3)alkyl,
C(1_
3)alkylcarbonylaminoC(1_3)alkyl,
C(1_3)alkoxyC(1_3)alkylcarbonylaminoC(1_3)alkyl, C(l_
3)alkylsulphonylam inoC(l_3)alkyl, C1_3)alkylcarboxy,
C(l_3)alkylcarboxyC(l.3)alkyl, and
[101] R3 is hydrogen, halogen, C(13)alkyl, or hydroxyC(l_3)alkyl; or
CA 02588369 2007-05-11
- 20 -
[102] R2 and R3 together with the pyrimidone ring carbon atoms to which they
are attached
form a fused 5-or 6-membered carbocyclic ring; or
[103] R2 and R3 together with the pyrimidone ring carbon atoms to which they
are attached
form a fused benzo or heteroaryl ring ring optionally substituted by 1, 2, 3
or 4 substituents
which can be the same or different selected from halogen, Co _4)alkyl, cyano,
Co _oalkoxy,
Co_oalkylthio or mono to perfluoro-Co_zoalkyl;
[104] R4 is hydrogen, Co _oalkyl which can be unsubstituted or substituted by
1, 2 or 3
substituents selected from hydroxy, halogen, OR7, COR7, carboxy, COOR7,
CONR9R10,
NR9R10, NR7COR8, mono- or di-(hydroxyC(i_oalkyl)amino and N-
hydroxyC(1_6)allcyl-
N-Co _oallcylamino; or
[105] R4 is Het-C(04)alkyl in which Het is a 5- to 7- membered heterocyclyl
ring
comprising N and optionally 0 or S, and in which N can be substituted by COR7,
COOR7,
CONR9R1O, or Co _oalkyl optionally substituted by 1, 2 or 3 substituents
selected from
hydroxy, halogen, OR7, COR7, carboxy, COOR7, CONR9R1 or NR9R10, for instance,
piperidin-4-yl, pyrrolidin-3-y1;
[106] R5 is an aryl or a heteroaryl ring optionally substituted by 1, 2, 3 or
4 substituents
which can be the same or different selected from Co _oalkyl, Co _oalkoxy, Co
_oalkylthio,
arylCo_oalkoxy, hydroxy, halogen, CN, COR7, carboxy, COOR7, NR7COR8, CONR9R10,
S02NR9R1 0,NR, 7
SO2R8, NR9R10, mono to perfluoro-Co_zoalkyl and mono to perfluoro-
Co _Loalkoxy;
[107] R6 is an aryl or a heteroaryl ring which is further optionally
substituted by 1, 2, 3 or 4
substituents which can be the same or different selected from Co -18)alkyl, Co
-18)alkoxy,
Co _oalkylthio, C(i_oallcylsulfonyl, ary1C(1_6)alkoxy, hydroxy, halogen, CN,
COR7,
carboxy, COOR7, CONR9R10, NR7COR8, S02NR9R10, NR7S02R8, NR9R10, mono to
perfluoro-C(1_4)alkyl and mono to perfluoro-C(1_4)alkoxy, or C(5_10)alkyl;
[108] R7 is hydrogen or Co _12)alkyl, for instance Co _4)alkyl (e.g. methyl or
ethyl);
[109] R8 is hydrogen, 0Co_6)alkyl, or Co _12)alkyl, for instance C(l4)alkyl
(e.g. methyl
or ethyl);
10566615.1
CA 02588369 2007-05-11
- 21 -11101 R9 and R10 which can be the same or different is each selected
from hydrogen, or
C(112)alkyl, or R9 and Rl together with the nitrogen to which they are
attached form a 5-
to 7 membered ring optionally containing one or more further heteroatoms
selected from
oxygen, nitrogen and sulphur, and optionally substituted by one or two
substituents selected
from hydroxy, oxo, C(14)alkyl, Co _zoalkylcarboxy, aryl, e.g. phenyl, or
aralkyl, e.g benzyl,
for instance morpholine or piperazine; and
[111] X is C(2_4)alkylene, optionally substituted by 1,2 or 3 substituents
selected from
methyl and ethyl, or CH=CH.
1112] All salts of formula (II), as well, can be used in the instant method of
treatment.
1113] Of particular interest are the compounds of formula (II) here, where, as
noted in WO
02/30911 for formula (I) there, R1 can be a phenyl group optionally
substituted by 1, 2, 3 or 4
substituents which can be the same or different selected from halo, C1_C6
alkyl,
trifluoromethyl or C1_C6alkoxy. More specifically, phenyl is unsubstituted or
substituted by
1, 2, 3 or 4 halogen substituents, particularly, from 1 to 3 fluoro groups,
and most
particularly, 2,3-difluoro, 2,4-difluoro or 4-fluoro.
[114] A further embodiment of formula (II) here, is where Y is ¨CH?Cl-b-.
1115] In addition, of interest are compounds of formula (II) where R2 is
hydrogen, by
default, or is halo, CI_C6alkyl, mono to perfluoro- C1_C4 alkyl, mono to
perfluoro C1_C46
alkoxy, or Ci_C6alkoxy; particularly mono to perfluoro- Ci_C4alkyl, mono to
perfluoro-
C1_C4alkoxy, or C1_C6alkoxy. Of particular interest are the compounds of
formula (II) where
R2 is other than hydrogen, n in (R2)õ is 1, 2, or 3, and the substitution
pattern is meta and/or
para, particularly para, i.e. a 4-position substituent. See also those
compounds where R2 is 4-
trifluoromethyl or 4-trifluoromethoxy.
[116] R3 and R4 can be the same or different and are methyl, ethyl, n-propyl,
or n-butyl. Of
particular interest are those compounds of formula (II) herein where R3 and R4
are the same
and are methyl, or ethyl; methyl is of particular interest.
[117] R5 can be hydrogen, C(1_6) alkyl which is a straight chain, or
branched. Of particular
interest is methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, t-
butyl, n-pentyl or n-
hexyl.
[118] It will be appreciated that within the compounds of formula (II) herein
there is a
further sub-group of compounds in which:
[119] R1 is phenyl substituted by 2,3 difluoro;
10566615.1
CA 02588369 2007-05-11
- 22 -
[120] R2 and R3, together with the pyrimidine ring carbon atoms to which they
are
attached, form a fused 5-membered cyclopentenyl ring;
[121] R4 is 2-(diethylamino)ethyl;
[122] R5 is phenyl;
[123] R6 is phenyl substituted by trifluoromethyl at the 4-position, or thien-
2-y1 substituted
by trifluoromethyl in the 5-position; and
1124] X is (CH2)2.
[125] Particular compounds of formula (II) herein of interest are:
1126] N-(2-diethylaminoethyl)-212-(2-(2,3-difluorophenypethyl)-4-oxo-4,5,6,7-
tetrahydro-cyclopentapyrimidin-l-yll-N-(4'-trifluoromethyl-biphenyl-4-
ylmethypacetamide;
[127] N-(2-diethylaminoethyl)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4,5,6,7-
tetrahydro-cyclopentapyrimidin-1-y1]-N-(4'-trifluoromethyl-bipheny1-4-
ylmethypacetamide
bitartrate;
[128] N-(2-ethylamino-2-methyl-propy1)-2-(2-(2-(2,3-difluoropheny1)-ethyl)-4-
oxo-
4,5,6,7-tetrahydro-cyclopentapyrimidin-1-y1)-N-(4'-trifluoromethyl-bipheny1-4-
ylmethyl)-
acetamide bitartrate;
[129] N-(2-t-butylaminoethyl)-2-(2-(2-(2,3-difluoropheny1)-ethyl)-4-oxo-
4,5,6,7-
tetrahydro-cyclopentapyrimidin-1-y1)-N-(4'-trifluoromethyl-biphenyl-4-
ylmethyl)acetamide
bitartrate;
[130] N-(1-ethyl-piperidin-4-y1)-2-(2-(2-(2,3-difluoropheny1)-ethyl)-4-oxo-
4,5,6,7-
tetrahydro-cyclopentapyrimidin-1-y1)-N-(4'-trifluoromethyl-biphenyl-4-
ylmethyDacetamide
bitartrate;
[131] N-(2-diethylaminoethyl)-2-(2-(2-(4-fluoro-2-(trifluoromethyl)pheny1)-
ethyl)-4-oxo-
4,5,6,7-tetrahydro-cyclopentapyrimidin-l-y1)-N-(4'-trifluoromethyl-bipheny1-4-
ylmethyl)-
acetamide bitartrate;
[132] N-(2-diethylaminoethyl)-2-(2-(2-(4-fluoro-3-(trifluoromethyl)phenyl)-
ethyl)-4-oxo-
4,5,6,7-tetrahydro-cyclopentapyrimidin-1-y1)-N-(4'-trifluoromethyl-biphenyl-4-
ylmethyl)-
acetamide bitartrate;
[133] N-(2-diethylaminoethyl)-2-(2-(2-(3-chloro-4-fluoropheny1)-ethyl)-4-oxo-
4,5,6,7-
tetrahydro-cyclopentapyrimidin-l-y1)-N-(4'-trifluoromethyl-biphenyl-4-
ylmethypacetamide
bitartrate;
10566615.1
CA 02588369 2007-05-11
- 23 -
[134] (+/-)-N-(2-diethylaminoethyl)-2-(2phenyl-propy1)-4-oxo-4,5,6,7-
tetrahydro-
cyclopentapyrimidin-1-y1)-N-(4'-trifluoromethyl-biphenyl-4-ylmethypacetamide
bitartrate;
[135] N-(2-diethylamino ethyl)-2-(2-(2-(2 ,4-difluoropheny1)-ethyl)-4-o xo -4
,5 ,6,7-
tetrahydro-cyc lopentapyrimidin-1 -y1)-N-(4'-trifluoromethyl-biphenyl-4-
ylmethypacetamide
bitartrate;
[136] N-(2 -diethyl aminoethyl)-2-(2-(2-(2 ,5-difluoropheny1)-ethyl)-4-oxo-
4,5 ,6,7-
tetrahydro-cyc lop entapyrimidin-1 -y1)-N-(4' -tri fluorom ethyl-bipheny1-4-
ylm ethyl)acetamide
bitartrate;
[137] N-(2 -diethyl amino ethyl)-2-(2-(2-(3 ,4-difluoropheny1)- ethyl)-4-o
xo -4,5 ,6,7-
tetrahydro-cyclopentapyrimidin-l-y1)-N-(4'-trifluoromethyl-bipheny1-4-
ylmethyDacetamide;
[138] N-(2-diethylamino ethyl)-2-(2-(2-(2-fluoropheny1)- ethyl)-4-oxo-4,5
,6,7-tetrahydro-
cyclopentapyrimidin- 1-y1)-N- (4'-trifluoromethyl-bipheny1-4-
ylmethyl)acetamide ;
[139] N-(2 -diethyl amino ethyl)-2-(2-(2 -(3 -fluoropheny1)- ethyl)-4-oxo-
4,5 ,6,7- tetrahydro-
cyclopentapyrimidin-1 -y1)-N-(4'-tri fluoromethyl-bipheny1-4-ylmethyl)ac
etamide bitartrate;
[140] N-(2-diethylaminoethyl)-2-(2-(2-(3-chloropheny1)-ethyl)-4-oxo-4,5,6,7-
tetrahydro-
cyclopentapyrimidin-l-y1)-N-(4'-trifluoromethyl-biphenyl-4-ylmethypacetamide;
[141] N-(2-diethylamino ethyl)-2-(2-(2-(4-chloropheny1)-ethyl)-4-oxo-4,5
,6,7-tetrahydro -
cyc lopentapyrimi din- 1-y1)-N-(4'-trifluoromethyl-bipheny1-4-
ylmethyl)acetamide ;
[142] N-(2-diethylaminoethyl)-2-(2-(2-(4-methylpheny1)-ethyl)-4-oxo-4,5,6,7-
tetrahydro-
cyclopentapyrimidin-l-y1)-N-(4'-trifluoromethyl-bipheny1-4-ylmethyl)acetamide;
[143] N-(2-diethylaminoethyl)-2-(2-(2-(4-(trifluoromethyl)pheny1)-ethyl)-4-oxo-
4,5,6,7-
tetrahydro-cyc lop entapyrimidin-1 -y1)-N-(4' -tri fluoromethyl-bipheny1-4-ylm
ethyl)acetamide ;
[144] N-(2 -diethylamino ethyl)-2 -(2-(2-(4-methoxypheny1)- ethyl)-4-oxo-4,5
,6,7-
tetrahydro-cyc lop entapyrimidin-l-y1)-N- (4'-trifluoromethyl-bipheny1-4-ylm
ethypacetamide
bitartrate;
[145] N-(2-diethyl amino ethyl)-2-(2-(2 -(4-(trifluoromethoxy)pheny1)-ethyl)-4-
o xo-4,5 ,6,7 -
tetrahydro-cyc lopentapyrimidin-l-y1)-N-(4'-tri fluorom ethyl-bipheny1-4-ylm
ethypacetamide
bitartrate;
[146] or the free base of any of the bitartrate salts , or another
pharmaceutically acceptable
salt.
[147] Further, of interest are compounds of formula (III), disclosed in WO
02/30904:
10566615.!
CA 02588369 2007-05-11
- 24 -
0
R2
R3
R4 N RL R6 (III)
[148] in which:
[149] R1 is an aryl group, optionally substituted by 1, 2, 3 or 4 substituents
which can be the
same or different selected from C(i_oalkyl, C(i_oalkoxy, C(i_oalkylthio,
hydroxy,
halogen, CN, mono to perfluoro-C(1_4)allcyl, mono to perfluoro-
C(1_4)alkoxyaryl, and
ary1C(1_4)alkyl;
[150] R2 is halogen, C(13)alkyl, C(1_3)alkoxy, hydroxyC(1_3)alkyl,
C(1_3)alkylthio,
C(1_3)alkylsulphinyl, aminoC(1_3)alkyl, mono- or di-
C(1_3)alkylaminoC(1_3)alkyl,
C(1_3)alkylcarbonylaminoC(1_3)alkyl,
C(1_3)alkoxyC(1_3)alkylcarbonylaminoC(1_3)alkyl,
Co _3)alkylsulphonylaminoC(1_3)alkyl, C(1_3)alkylcarboxy,
C(1_3)alkylcarboxyC(1-3)alkyl,
and
[151] R3 is hydrogen, halogen, C(13)alkyl, or hydroxyC(1_3)alkyl; or
[152] R2 and R3 together with the pyridone ring carbon atoms to which they are
attached
form a fused 5-or 6-membered carbocyclic ring; or
[153] R2 and R3 together with the pyridone ring carbon atoms to which they are
attached
form a fused benzo or heteroaryl ring optionally substituted by 1, 2, 3 or 4
substituents which
can be the same or different selected from halogen, Co cyano, Co
_3)alkoxyC(1 _
3)alkyl, Co _zoalkoxy or Co _Loalkylthio, or mono to perfluoro-Co_zoalkyl;
[154] R4 is hydrogen, C(l6)alkyl which can be unsubstituted or substituted by
1, 2 or 3
substituents selected from hydroxy, halogen, OR7, COR7, carboxy, COOR7,
CONR9R10,
NR9R10, NR7COR8, mono- or di-(hydroxyC(i_oalkyl)amino and N-hydroxyC(i_oalkyl-
N-C(i_oalkylamino; or
[155] R4 is Het-C(04)alkyl in which Het is a 5- to 7- membered heterocyclyl
ring
comprising N and optionally 0 or S, and in which N can be substituted by COR7,
COOR7,
10566615.1
CA 02588369 2007-05-11
- 25 -
CONR9R10, or Co _oallcyl optionally substituted by 1, 2 or 3 substituents
selected from
hydroxy, halogen, OR7, COR7, carboxy, COOR7, CONR9R10 or NR9R10, for instance,
piperidin-4-yl, pyrrolidin-3-y1;
[156] R5 is an aryl or a heteroaryl ring optionally substituted by 1, 2, 3 or
4 substituents
which can be the same or different selected from C(16)alkyl, Co_oalkoxy,
Co_oalkylthio,
arylCo_oalkoxy, hydroxy, halogen, CN, COR7, carboxy, COOR7, NR7COR8, CONR9R10,
SO2NR9R1 ,NR, 7
SO2R8, NR9R10, mono to perfluoro-Co_Loalkyl and mono to perfluoro-
Co_zoalkoxy;
[157] R6 is an aryl or a heteroaryl ring which is further optionally
substituted by 1, 2, 3 or 4
substituents which can be the same or different selected from Co_oalkyl,
Co_oalkoxy,
Co_oalkylthio, Co_oalkylsulfonyl, arylCo_oalkoxy, hydroxy, halogen, CN, COR7,
carboxy, COOR7, CONR9R10, NR7COR8, S02NR9R10, NR7S02R8, NR9R10, mono to
perfluoro-Co_zoallcyl and mono to perfluoro-Co_zoalkoxy, or C(5_10)alkyl;
[158] R7 and R8 are independently hydrogen or Co _12)alkyl, for instance Co
_4)alkyl (e.g.
methyl or ethyl);
[159] R9 and R1 which can be the same or different is each selected from
hydrogen, or
C(l_12)alkyl, or R9 and R1 together with the nitrogen to which they are
attached form a 5-
to 7 membered ring optionally containing one or more further heteroatoms
selected from
oxygen, nitrogen and sulphur, and optionally substituted by one or two
substituents selected
from hydroxy, oxo, Co _Loalkyl, Co _coalkylearboxy, aryl, e.g. phenyl, or
aralkyl, e.g benzyl,
for instance morpholine or piperazine; and
[160] X is a C(2_4)alkylene group (optionally substituted by 1, 2 or 3
substituents selected
from methyl and ethyl), CH=CH, (CH2)nS or (CH2)n0 where n is 1, 2 or 3;
[161] or a pharmaceutically acceptable salt thereof.
[162] Of particular interest are those compounds of formula (III) where R2 and
R3 together
with the pyridone ring carbon atoms to which they are attached form a fused
benzo or
heteroaryl ring optionally substituted by 1, 2, 3 or 4 substituents which can
be the same or
different selected from halogen, Co _4)alkyl, cyano, Co_zoalkoxy or Co
_4)alkylthio, or
mono to perfluoro-C(1_4)alkyl. Preferably, R1 is phenyl optionally substituted
by halogen,
10566615.1
CA 02588369 2007-05-11
- 26 -
Co oalkyl, trifluoromethyl, Co _oalkoxy, preferably, from 1 to 3 fluoro, more
preferably,
2,3-difluoro. Representative examples of R4 include piperidin-4-y1 substituted
at the 1-
position by methyl, isopropyl, 1-(2-methoxyethyl), 1-(2-hydroxyethyl), t-
butoxycarbonyl or
ethoxycarbonylmethyl; ethyl substituted at the 2-position by amino ethyl; 1-
ethylpiperidinylmethyl; piperidin-4-y1; 3-diethylaminopropyl; 4-pyrrolidin-1-
ylbutyl and 1-
ethylpyrrolidin-3-yl. Preferably R4 is 1-(2-methoxyethyl)piperidin-4-yl, 1-
methylpiperidin-
4-y1 or 1-ethylpyn-olidin-3-yl. Representative examples of R5 include phenyl
and pyridyl.
Preferably, R5 is phenyl. Representative examples of R6 include phenyl
optionally
substituted by halogen, or trifluoromethyl, preferably at the 4-position and
hexyl. Preferably,
R6 is phenyl substituted by trifluoromethyl at the 4-position. Further
representative examples
of R6 include phenyl substituted by 1 or more Co _3)alkyl. Preferably, R6 is
phenyl
substituted by ethyl in the 4-position. Preferably, R5 and R6 together form a
4-
(phenyl)phenyl or a 2-(phenyl)pyridinyl substituent in which the remote phenyl
ring can be
optionally substituted by halogen or trifluoromethyl, preferably at the 4-
position. Preferably
X is C(2_4)allcylene, more preferably C(2_3)alkylene, most preferably, (CH2)2,
or CH2S.
[163] It will be appreciated that within the group of compounds comprising
formula (III)
there is sub-group of compounds in which:
[164] R1 is phenyl substituted by 2,3-difluoro;
[1651 R2 and R3, together with the pyridone ring carbon atoms to which they
are attached,
form a fused benzo or pyrido ring;
[166] R4 is 1-(2-methoxyethyl)piperidin-4-y1;
[167] R5 and R6 together form a 4-(phenyl)phenyl substituent in which the
remote phenyl
ring is substituted by trifluoromethyl, preferably at the 4-position; and
[168] X is CH2S or (CH2)2.
[169] The following compounds of formula (III) are of interest:
[170] N-(2-diethylaminoethyl)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-
1-y1]-N-
(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[171] N-(2-diethylaminoethyl)-2-[2-(2-(2,3-difluorophenypethyl)-4-oxo-4H-
quinolin-1-
y11-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide;
[172] N-(2-diethylaminoethyl)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4H-
quinolin-1-
ylkN-(4'-trifluoromethylbiphenyl-4-ylmethyDacetamide bitartrate;
10566615.1
CA 02588369 2007-05-11
- 27 -
[173] N-(2-diethylaminoethyl)-2-[2-(2,3-difluorobenzylthio)-4-oxo-5,6-
trimethylene-
pyridin-1-y1]-N-(4T-trifluoromethylbiphenyl-4-ylmethyl)acetamide bitartrate;
[174] N-(1-(2-methoxyethyl)piperidin-4-y1)-242-(2,3-difluorobenzylthio)-4-oxo-
4H-
quinolin-l-yll-N-(4'-trifluoromethylbiphenyl-4-ylmethypacetamide;
[175] N-(1-methylpiperidin-4-y1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-
quinolin-l-yl]-
N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[176] N-(1-methylpiperidin-4-y1)-2-[2-(2-(2,3-difluorophenypethyl)-4-oxo-4H-
[1,8]naphthyridin-1-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide
bitartrate;
[177] N-(1-(2-methoxyethyl)piperidin-4-y1)-212-(2-(2,3-difluorophenyeethyl)-4-
oxo-4H-
[1,8]naphthyridin -1-yll-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide;
[178] N-(1-(2-methoxyethyl)piperidin-4-y1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-
4H-
[1,8]naphthyridin -1-y1]-N-(41-trifluoromethylbipheny1-4-ylmethyl)acetamide
bitartrate;
[179] N-(1-ethylpiperidin-4-y1)-242-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-
1-yll-N-
(4'-ethylbiphenyl-4-ylmethypacetamide bitartrate;
[180] N-(1-ethylpiperidin-4-y1)-245-(2-(2,3-difluorophenypethyl)-2-methy1-7-
oxo-7H-
thiazolo[4,5-b]pyridin-4-y11-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide
bitartrate;
[181] ( )N - (1-ethylpyrrolidin-3-y1)-242-(2-(2,3-difluorophenypethyl)-4-oxo-
4H-quinolin-
l-yli-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
[182] (+)N - ( 1-ethylpyrrolidin-3-y1)-242-(2,3-difluorobenzylthio)-4-oxo-
4H-quinolin-1-
y11-N-(4'-trifluoromethy1bipheny1-4-y1methy1)acetamide bitartrate;
[183] N-(1-(2-methoxyethyppiperidin-4-y1)-242-(2,3-difluorobenzylthio)-4-oxo-
4H-
quinolin-1-yll-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[184] N-(1-(2-methoxyethyl)piperidin-4-y1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-
4H-
quinolin-1-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide
dihydrochloride;
[185] N-(1-(2-methoxyethyl)piperidin-4-y1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-
4H-
quinolin-l-A-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide mono
paratoluenesulphonate;
[186] N-(1-(2-methoxyethyppiperidin-4-y1)-242-(2-(2,3-difluorophenyl)ethyl)-4-
oxo-4H-
[1,81naphthyridin -1-y11-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide
bitartrate;
[187] N-(1-(2-methoxyethyppiperidin-4-y1)-242-(2-(2,3-difluorophenypethyl)-4-
oxo-4H-
[1,8]naphthyridin -1-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide
monohydrochloride;
10566615.1
CA 02588369 2007-05-11
- 28 -
[188] N-(1-(2-methoxyethyl)piperidin-4-y1)-242-(2-(2,3-difluorophenypethyl)-4-
oxo-4H-
[ 1,8] naphthyridin -1 -y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide
dihydrochloride;
[189] N-(2-diethylaminoethyl)-2-[2-(4-fluorobenzylthio)-4-oxo-4H-quinolin-1 -
y11-N-(4'-
trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
[190] N-(2-diethylaminoethyl)-242-(4-fluorobenzylthio)-4-oxo-5,6-
trimethylenepyridin-1-
y1]-N-(4'-trifluoromethyl-bipheny1-4-ylmethypacetamide bitartrate;
1191] N-(2-diethylaminoethyl)-2-[2-(2,3-difluorobenzylthio)-4-oxo-5,6-
trimethylene-
pyridin-l-yl] -N-(4'-trifluoromethyl-biphenyl-4-ylmethyDacetamide bitartrate;
[192] N-(2-diethylaminoethyl)-2-[2-(4-fluorophenypethyl)-4-oxo-4H-quinolin-1-
3/1]-N-(4'-
trifluoromethylbiphenyl-4-ylmethypacetamide;
[193] N-(2-diethylaminoethyl)-2-[2-(2-(3,4-difluorophenypethyl)-4-oxo-4H-
quinolin-1-
y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[194] N-(2-diethylaminoethyl)-242-(2-(2-fluorophenypethyl)-4-oxo-4H-quinolin-1-
y1]-N-
(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
[195] N-(2-diethylaminoethyl)-2-[2-(2-(3-chlorophenyl)ethyl)-4-oxo-4H-quinolin-
1-y11-N-
(4'-trifluoromethylbiphenyl-4-ylmethypacetamide bitartrate;
[196] N-(2-diethylaminoethyl)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4H-
[1,8]naphthyridin-1-y1)1-N-(4'-trifluorornethylbipheny1-4-ylmethypacetarnide
bitartrate;
[197] N-(1-ethylpiperidin-4-y1)-2-[2-(2-(2,3-difluorophenypethyl)-4-oxo-4H-
[1,8]naphthyridin-l-y1)]-N-(4'-trifluoromethylbiphenyl-4-ylmethypacetamide;
[198] N-(1-ethylpiperidin-4-y1)-242-(2-(2,3-difluorophenypethyl)-4-oxo-4H-
quinolin-1-
y11-N-(4'-trifluoromethylbiphenyl-4-ylmethyl)acetamide bitartrate;
[199] N-(2-pyn-olidin- 1-ylethyl)-2-[2-(2-(2,3-difluorophenypethyl)-4-oxo-4H-
quinolin-1-
yfl-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[200] N-(1-isopropylpiperidin-4-y1)-242-(2-(2,3-difluorophenypethyl)-4-oxo-4H-
quinolin-
1-y1)-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[201] N-(2-piperidin-1-ylethyl)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4H-
quinolin- 1-
y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
[202] N-(2-diethylaminoethyl)-2-[2-(2,3-difluorobenzylthio)7-fluoro-4-oxo-4H-
quinolin-
1 -y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[203] N-(2-diethylaminoethyl)-5-[2-(2-(2,3-difluorophenyl)ethyl)-2-methyl-7-
oxo-7H-
thieno[3,2-b]pyridin-4-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide
bitartrate;
10566615.1
CA 02588369 2007-05-11
- 29 -
[2041 N-(2-diethylaminoethyl)-2-[2-(2-(2,3-difluorophenyl)ethyl)-5,6-dimethyl-
4-oxo-4H-
pyridin-1-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
[205] N-(2-diethylaminoethyl)-242-(2-(2,3-difluorophenyl)ethyl)-5-ethyl-4-oxo-
4H-
pyridin-1-y11-N-(4t-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[206] N-(1-(2-methoxyethyl)piperidin-4-y1)-2-[2-(2-(2,3-difluorophenyl)ethyl)-
4-oxo-4H-
quinolin-l-y11-N-(4'-trifluoromethylbiphenyl-4-ylmethypacetamide bitartrate;
12071 N-(1-methylpiperidin-4-y1)-242-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4H-
quinolin-l-
y11-N-(4'-trifluoromethylbiphenyl-4-ylmethypacetamide bitartrate;
1208] N-(2-diethylaminoethyl)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4H-
thieno[3,4-
b]pyridin-1-y11-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
[209] N-(1-ethylpiperidin-4-y1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-
quinolin-1-y1]-N-
(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[210] N-(2-pyrrolidin-1-ylethyl)-212-(2,3-difluorobenzylthio)-4-oxo-4H-
quinolin-1-y1]-N-
(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
[211] N-(1-ethylpiperidin-4-y1)-246-(2-(2,3-difluorophenypethyl)-2-methy1-4-
oxo-4H-
pyrazolo[3,4-b]pyridin-7-y11-N-(4'-trifluoromethylbipheny1-4-
ylmethyl)acetamide bitartrate;
[212] N-(1-isopropylpiperidin-4-y1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-
quinolin-l-
y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[213] N-(1-ethylpiperidin-4-ylmethyl)-2-[2-(2-(2,3-difluorophenypethyl)-4-oxo-
4H-
quinolin-l-y11-N-(4?-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[214] N-(3-diethylaminopropy1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-
1-y1]-N-
(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
12151 N-(4-pyrrolidin-1-ylbuty1)-242-(2-(2,3-difluorophenypethyl)-4-oxo-4H-
quinolin-1-
y11-N-(4'-trifluoromethylbiphenyl-4-ylmethypacetamide bitartrate;
[216] N-(3-diethylaminopropy1)-2-[2-(2-(2,3-difluorophenypethyl)-4-oxo-4H-
quinolin-1-
y11-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
1217] N-(4-pyrrolidin-1-ylbuty1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-
quinolin-1-y1]-N-
(4'-trifluoromethylbiphenyl-4-ylmethyl)acetamide bitartrate;
[218] N-(1-ethylpiperidin-4-y1)-245-(2,3-difluorobenzylthio)-7-oxo-7H-
thieno[3,2-
b]pyridin-4-yli-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[219] N-(2-diethylaminoethyl)-245-(2-(2,3-difluorophenyl)ethyl)-2-methyl-7-oxo-
7H-
thiazolo[4,5-b]pyridin-4-y11-N-(41-trifluoromethylbipheny1-4-ylmethypacetamide
bitartrate;
10566615.1
CA 02588369 2007-05-11
- 30 -
[220] N-(2-diethylaminoethyl)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-
1-y1]-N-
(4'-ethylbipheny1-4-ylmethypacetamide bitartrate;
[221] N-(2-diethylaminoethyl)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4H-
quinolin-1-
y1]-N-(4'-ethylbipheny1-4-ylmethyl)acetamide bitartrate;
[222] N-(2-diethylaminoethyl)-242-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-1-
y11-N-
(4'-isopropylbiphenyl-4-ylmethypacetamide bitartrate;
[223] N-(2-diethylaminoethyl)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4H-
quinolin-1-
y1]-N-(4'-isopropylbipheny1-4-ylmethypacetamide bitartrate;
[224] N-(2-diethylaminoethyl)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-
[1,8]naphthyridin-
1-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyDacetamide bitartrate;
[225] N-(1-ethylpiperidin-4-y1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-
[1,8]naphthyridin-
1-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
[226] N-(2-diethylaminoethyl)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4H-
quinolin-1-
y11-N-(4'-methylbipheny1-4-ylmethypacetamide bitartrate;
[227] N-(2-diethylaminoethyl)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-
1-y1]-N-
(4'-methylbipheny1-4-ylmethypacetamide bitartrate;
[228] N-(1-ethoxycarbonylmethylpiperidin-4-y1)-2-[2-(2-(2,3-
difluorophenypethyl)-4-oxo-
4H-quinolin-l-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide
bitartrate;
[229] N-(1-isopropylpiperidin-4-y1)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-
4H-
[1,8]naphthyridin-1-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide
bitartrate;
[230] N-(2-diethylaminoethyl)-242-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-l-
yll-N-
(3',4'-dimethylbiphenyl-4-ylmethypacetamide bitartrate;
[231] N-(1-(t-butoxycarbonyl)piperidin-4-y1)-212-(2-(2,3-difluorophenyDethyl)-
4-oxo-
4H41,8]naphthyridin-1 -y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide
bitartrate;
[232] N-(2-diethylaminoethyl)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-
l-y1]-N-
(31,4'-difluorobipheny1-4-ylmethypacetamide bitartrate;
[233] N-(2-diethylaminoethyl)-246-(2,3-difluorobenzylthio)-4-oxo-4H-thieno-
[2,3-b]pyridin-7-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide
bitartrate;
[234] N-(1-methylpiperidin-4-y1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-
[1,8]naphthyridin-1-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide
bitartrate;
[235] N-(1-ethylpiperidin-4-y1)-2-[2-(2-(2,3,4-trifluorophenylethyl)-4-oxo-4H-
quinolin-l-
y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
10566615.1
CA 02588369 2007-05-11
-31 -
[236] N-(2-diethylaminoethyl)-246-(2,3-difluorobenzylthio)-2-methy1-4-oxo-2,4-
dihydro-
pyrazolo[3,4-b]pyridin-7-y1]-N-(4'-trifluoromethylbipheny1-4-
ylmethyl)acetamide bitartrate;
[237] N-(1-ethylpiperidin-4-y1)-2-[6-(2-(2,3-difluorophenypethyl)-2-ethyl-4-
oxo-2,4-
dihydropyrazolo[3,4-b]pyridin-7-y1]-N-(4'-trifluoromethylbipheny1-4-
ylmethypacetamide
bitartrate;
[238] N-(1-ethylpiperidin-4-y1)-246-(2-(2,3-difluorophenypethyl)-2-isopropy1-4-
oxo-2,4-
dihydropyrazolo[3,4-b]pyridin-7-y1]-N-(4'-trifluoromethylbipheny1-4-
ylmethyDacetamide
bitartrate;
[239] N-(1-ethylpiperidin-4-y1)-242-(2-(2,3-difluorophenypethyl)-4-oxo-4H-
quinolin-1-
y1]-N-(4'-ethylbipheny1-4-ylmethypacetamide bitartrate;
[240] N-(1-isopropylpiperidin-4-y1)-2-[5-(2-(2,3-difluorophenypethyl)-2-methy1-
7-oxo-
7H-thiazolo[4,5-b]pyridin-4-y1]-N-(4'-trifluoromethylbipheny1-4-
ylmethypacetamide
bitartrate;
1241] N-(1-(2-methoxyethyppiperidin-4-y1)-245-(2-(2,3-difluorophenypethyl)-2-
methyl-
7-oxo-7H-thiazolo[4,5-b]pyridin-4-y1]-N-(4'-trifluoromethylbipheny1-4-
ylmethypacetamide
bitartrate;
[242] N-(1-ethylpiperidin-4-y1)-2-[2-(2-(2,3-difluorophenypethyl)-4-oxo-5,6-
trimethylene-
pyridin-l-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[243] N-(1-methylpiperidin-4-y1)-242-(2-(2,3-difluorophenypethyl)-4-oxo-5,6-
trimethylenepyridin-l-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide
bitartrate;
[244] N-(1-(2-methoxyethyl)piperidin-4-y1)-242-(2-(2,3-difluorophenyl)ethyl)-4-
oxo-5,6-
trimethylenepyridin-1-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide
bitartrate;
[245] N-(1-isopropylpiperidin-4-y1)-242-(2-(2,3-difluorophenyDethyl)-4-oxo-5,6-
trimethylenepyridin-l-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide
bitartrate;
1246] N-(1-ethylpiperidin-4-y1)-2-[5-(2-(2,3-difluorophenypethyl)-2-methyl-7-
oxo-2,7-
dihydropyrazolo[4,3-b]pyridin-4-y1]-N-(4'-trifluoromethylbipheny1-4-
ylmethypacetamide
bitartrate;
[247] N-(1-ethylpiperidin-4-y1)-245-(2-(2,3-difluorophenypethyl)-1-methy1-7-
oxo-1,7-
dihydropyrazolo[4,3-b]pyridin-4-y1]-N-(4'-trifluoromethylbipheny1-4-
ylmethypacetamide
bitartrate;
1248] N-(1-ethylpiperidin-4-y1)-242-(2-(2,3-difluorophenypethyl)-4-oxo-5,6-
trimethylene-
pyridin-l-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
10566615.1
CA 02588369 2007-05-11
- 32 -
[249] N-(1-ethylpiperidin-4-y1)-2-[2-(2-(2,3-difluorophenypethyl)-7-methy1-4-
oxo-4H-
[1,8]naphthyridin-1-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyDacetamide
bitartrate;
1250] N-(2-diethylaminoethyl)-242-(2-(2,3-difluorophenypethyl)-7-methyl-4-oxo-
4H-
[1,8]naphthyridin-1-y11-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide
bitartrate;
[251] N-(1-methylpiperidin-4-y1)-2-[2-(2-(2,3-difluorophenyl)ethyl)-7-methyl-4-
oxo-4H-
[1,8]naphthyridin-1-y11-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide
bitartrate;
[252] N-(1-isopropylpiperidin-4-y1)-242-(2-(2,3-difluorophenypethyl)-7-methy1-
4-oxo-
4H41,81naphthyridin-1-y1]-N-(41-trifluoromethylbipheny1-4-ylmethyl)acetamide
bitartrate;
[253] N-(1-(2-methoxyethyl)piperidin-4-y1)-242-(2-(2,3-difluorophenypethyl)-7-
methyl-
4-oxo-4H-[1,8]naphthyridin-1-y1]-N-(41-trifluoromethylbipheny1-4-
ylmethypacetamide
bitartrate;
[254] N-(1-methylpiperidin-4-y1)-212-(2,3-difluorobenzylthio)-4-oxo-5,6-
trimethylene-
pyridin-1-yll-N-(4'-trifluoromethylbipheny1-4-ylmethyDacetamide bitartrate;
[255] N-(1-ethylpiperidin-4-y1)-242-(2,3-difluorobenzylthio)-4-oxo-5,6-
trimethylene-
pyridin-l-y11-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
1256] N-(1-isopropylpiperidin-4-y1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-5,6-
trimethylene-
pyridin-l-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[257] N-(1-(2-methoxyethyl)piperidin-4-y1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-
5,6-
trimethylenepyridin-l-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide
bitartrate;
[258] N-(2-diethylaminoethyl)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-5,6-
tetramethylenepyridin-1-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide
bitartrate;
[259] N-(1-methylpiperidin-4-y1)-242-(2,3-difluorobenzylthio)-4-oxo-4H-
quinolin-l-y1]-
N-(4'-chlorobipheny1-4-ylmethypacetamide bitartrate;
[260] N-(1-methylpiperidin-4-y1)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4H-
quinolin-1-
yll-N-(4'-chlorobipheny1-4-ylmethypacetamide bitartrate;
[261] N-(1-ethylpiperidin-4-y1)-242-(2-(2,3-difluorophenypethyl)-4-oxo-4H-
quinolin-1-
yll-N-(4'-chlorobiphenyl-4-ylmethyDacetamide bitartrate;
[262] N-(1-(2-methoxyethyl)piperidin-4-y1)-242-(2-(2,3-difluorophenypethyl)-4-
oxo-4H-
quinolin-1-y1]-N-(4'-chlorobipheny1-4-ylmethypacetamide bitartrate;
[263] N-(1-isopropylpiperidin-4-y1)-242-(2-(2,3-difluorophenypethyl)-4-oxo-4H-
quinolin-
1-y1]-N-(4'-chlorobipheny1-4-ylmethyl)acetamide bitartrate;
[264] N-(2-diethylaminoethyl)-2-[6-(2-(2,3-difluorophenyl)ethyl)-2-methyl-4-
oxo-4H-
pyrazolo[3,4-b]pyridin-7-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyDacetamide
bitartrate;
10566615.1
CA 02588369 2007-05-11
- 33 -
[265] N-(1-(t-butoxycarbonyl)piperidin-4-y1)-2-[2-(2,3-difluorobenzylthio)-4-
oxo-4H-
[1,8]naphthyridin-l-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide;
[266] N-(1-ethylpiperidin-4-y1)-2-[6-(2-(2,3-difluorophenypethyl)-2-(2-
methoxyethyl)-4-
oxo-4H-pyrazolo[3,4-13]pyridin-7-y1]-N-(4'-trifluoromethylbipheny1-4-
ylmethypacetamide
bitartrate;
[267] N-(2-diethylaminoethyl)-2-[4-oxo-2-(2-(2,3,4-trifluorophenypethyl)-4H-
quinolin-1-
y1]-N -(4'-trifluoromethyl-biphenyl-4-ylmethypacetamide bitartrate;
12681 N-(2-diethylaminoethyl)-242-(2-(2,4-difluorophenypethyl)-4-oxo-4H-
quinolin-1-
y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
[269] N-(2-diethylamino ethyl)-2-[2-(2-(3-fluorophenyl)ethyl)-4-oxo-4H-
quinolin-1-yl] -N-
(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
1270] N-(piperidin-4-y1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-l-yli-
N-(4'-
trifluoromethylbiphenyl-4-ylmethyl)acetamide bitartrate;
[271] N-(piperidin-4-y1)-2- [24242,3 -difluorophenypethyl)-4-o xo-4H-quino lin-
1-y11-N-
(4 '-tiifluoromethylbipheny1-4-ylmethypacetamide bitartrate;
[272] N-(piperidin-4-y1)-242-(2-(2,3-difluorophenyl)ethyl)-4-oxo-5,6-
trimethylenepyridin-
l-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
[273] N-(piperidin-4-y1)-242-(2-(2,3-difluorophenypethyl)-4-oxo-4H-
[1,8]naphthyridin-l-
y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
[274] N-(piperidin-4-y1)-2- [2-(2,3-difluo rob enzylthio)-4-oxo-4H-[1,
8]naphthyridin-l-y11-
N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide trifluoroacetate;
[275] N-(2-ethylaminoethyl)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-1-
y1]-N-(4'-
trifluoromethylbipheny1-4-ylmethypacetamide;
[276] N-(2-ethylamino ethyl)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-o xo-4H-
quino lin-l-y1J-
N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide;
[277] N-(1-(2-hydroxyethyl)piperidin-4-y1)-2-[2-(2-(2,3-difluorophenypethyl)-4-
oxo-4H-
quinolin-l-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethyl)acetamide bitartrate;
or the free base
thereof, or another pharmaceutically acceptable salt.
[278] Formula (IV)
[279] Also of interest are compounds of formula (IV)
10566615.1
CA 02588369 2007-05-11
-34-
0
w
1 1
R \ N/\ X!!
(R2)n
0 N efa
R5',0 N7-
R4 R3 (IV)
[280] wherein:
[281] RI is an aryl group, unsubstituted or substituted by 1, 2, 3 or 4
substituents which can
be the same or different selected from the group consisting of Ci-C6 alkyl, CI
_C6 alkoxy,
C1_C6 alkylthio, aryl C1_C6 alkoxy, hydroxy, halo, CN, COR6, COOR6, NR6COR7,
CONR8R9, SO2NR8R9, NR6S02R7, NR8R9, halo C1_C4 alkyl, and halo C1_C4 alkoxy;
[282] W is CH and X is N, or W is N and X is CH, W and X are both CH, or W and
X are
N;
[283] Y is C2-C4allcyl,
[284] R2 is hydrogen, CI_C6 alkyl, C1_C6 alkoxy, C1_C6 alkylthio, aryl C1_C6
alkoxy,
hydroxy, halo, CN, COR6, carboxy, COOR6, NR6COR7, CONR8R9, SO2NR8R9,
NR6S02R7, NR8R9, mono to perfluoro- CI_C6 alkyl, or mono to perfluoro- C1E6
alkoxy;
[285] n is 0-5;
[286] R3 is CI-CI alkyl;
[287] R4 is Ci-C4 alkyl;
[288] R5 is hydrogen, Ci_Cio alkyl, C2_C10 alkenyl, C2_C10 alkynyl, halo
C1_C4 alkyl, C3-C8
cycloalkyl, C3-C8cycloalkyl, C3-C8cycloalkyl C1_C4 alkyl, C5-C8cycloalkenyl,
C5-C8cycloalkenyl C1_C4 alkyl, 3-8-membered heterocycloalkyl, 3-8-membered
heterocycloalkyl CI.C4 alkyl, C6-C14 aryl, C6-C14 aryl Ci_Cio alkyl,
heteroaryl, or heteroaryl
Ci_Cioalkyl; wherein each group is optionally one or more times by the same
and/or a
different group which is C1_C6 alkoxy, C1.C6 alkylthio, aryl C1_C6 alkoxy,
hydroxy, halo, CN,
NR8R9, or halo C1_C4 alkoxy
[289] R6 and R7 are independently hydrogen or Ci_Cioalkyl;
10566615.1
CA 02588369 2007-05-11
- 35 -
[290] R8 and R9 are the same or different and are hydrogen or Ci_Cio alkyl, or
R9 and R1
together with the nitrogen to which they are attached form a 5- to 7 membered
ring optionally
containing one or more further heteroatoms selected from oxygen, nitrogen and
sulphur, and
optionally substituted by one or two substituents selected from the group
consisting of
hydroxy, oxo, C1_C4 alkyl, Ci.C4 alkylcarboxy, aryl, and aryl CI_C4 alkyl;
[291] or a pharmaceutically acceptable salt thereof.
[292] Without intending to exclude any defined substituents and/or their
recited radicals
from the scope of formula (IV), the following R groups and the associated
radicals are of
particular interest:
[293] As regards R1, it can be an phenyl group optionally substituted by 1, 2,
3 or 4
substituents which can be the same or different selected from halo, C1_C6
alkyl,
trifluoromethyl or C1.C6 alkoxy. More specifically, phenyl is unsubstituted or
substituted by
1, 2, 3 or 4 halogen substituents, particularly, from 1 to 3 fluoro groups,
and most
particularly, 2,3-difluoro, 2,4-difluoro or 4-fluoro.
[294] A further embodiment of formula (I) is where Y is ¨CH2CH2-=
[295] The invention also provides a compound of formula (I) in which R2 is
hydrogen, by
default, or is halo, C1_C6 alkyl, mono to perfluoro- C1_C4 alkyl, mono to
perfluoro C1_C46
alkoxy, or CI_C6alkoxy; particularly mono to perfluoro- C1_C4 alkyl, mono to
perfluoro-
CI_C4alkoxy, or C1_C6alkoxy. Of particular interest are the compounds where R2
is other
than hydrogen, n in (R2)õ is 1, 2, or 3, and the substitution pattern is meta
and/or para,
particularly para, i.e. a 4-position substituent. Exemplified compounds
include those where
R2 is 4-trifluoromethyl or 4-trifluoromethoxy.
[296] R3 and R4 can be the same or different and are methyl, ethyl, n-propyl,
or n-butyl.
Of particular interest are those compounds of formula (I) where R3 and R4 are
the same and
are methyl, or ethyl; methyl is of particular interest.
[297] R5 can be hydrogen, C(1_6) alkyl which is a straight chain, or branched.
Of particular
interest is methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, t-
butyl, n-pentyl or n-
hexyl.
[298] Any of the compounds described herein above can be prepared in
crystalline or
non-crystalline form, and, if crystalline, can be solvated, e.g. as the
hydrate. This invention
includes within its scope stoichiometric solvates (e.g. hydrates).
[299] Certain of the compounds described herein can contain one or more chiral
atoms, or
can otherwise be capable of existing as two enantiomers. The compounds useful
in the
10566615 1
CA 02588369 2013-10-22
- 36 -
methods as described herein include mixtures of enantiomers as well as
purified enantiomers
or enantiomerically enriched mixtures. Also included within the scope of the
invention are
the individual isomers of the compounds represented by formulas (I) - (IV), as
well as any
wholly or partially equilibrated mixtures thereof. The present invention also
covers the
individual isomers of the claimed compounds as mixtures with isomers thereof
in which one
or more chiral centers are inverted. Also, it is understood that any tautomers
and mixtures of
tautomers of the claimed compounds are included within the scope of the
compounds of
formulas (I) ¨ (IV). The different isomeric forms can be separated or resolved
one from the
other by conventional methods, or any given isomer can be obtained by
conventional
synthetic methods or by stereospecific or asymmetric syntheses.
[300] Syntheses of the Compounds of Formula (I), (II), (III) and (IV)
[301] Methods for preparing compounds of formula (I), (II) and (III) have been
published
in the patent literature. For example, methods for making formula (I) can be
found in WO
01/60805 and W003/016287. Methods for making compounds of formula (II) have
been set
out in WO 02/30911. And methods for making compounds of formula (III) can be
found in
WO 02/30904. This document provides methods for making compounds of formula
(IV),
methods copied from US provisional application 60/829,327.
[302] Some examples of syntheses are provided below. To differentiate between
the
several generic groups of compounds in the examples herein, materials relating
to formula (I)
will be labeled as " Example of Synthesis Approach (1)-1" et seq., for formula
(II) "Example
of Synthesis Approach (II)-1" et seq., for formula (III), "Example of
Synthesis Approach
(III)-1 et seq., and for formula (I), "Example of Synthesis Approach (IV)-1,
et seq.
[303] Synthesis of Formula (I)
[304] Compounds of formulae (I) can be prepared by processes scheme I, as
disclosed in
WO 01/60805:
[305] Scheme I
CA 02588369 2007-05-11
- 37 -
o
o 0
A
Ra R2 X Rb
Ra I
N
0 A (c) Nlij: .11E------
(CH2)n
R2 X N Rb -all¨ R2 XN Rb
(rn) LX) COOR _
( I
,s
I
AR'
4......(22....,,--- (CH )
(II) 1 2 n I
(CH2),,
R2X N Fe COOH (XIX) I
i 12,-Z-R4-YR'NHR3 0001215
0.,(CH2),, (III) R2-01-
12-L1 (VII)
1 4 (d)
,NõR¨ZR5
R" Y (b)
i R5-Z-R4-YR.NFP-00-(CH2),-L' 0
R` (I) (V) L1-(CH2)n-COOR15 (X)
.).,)tx Fe
(9) 0 HN 1
A
(k)
Ra S N
12b
ijaC 1
(CH2)n
(d) R2 X N Rb
(XI) I ,,
R2-CH,-OH (IX) R2-CH2-1_, (VII) H (IV)
COOR "
0
R2-CH2-1_, (VII)
0HNAx1R
(d) R150-00-(CH2)n-NH2 (0
al2 (XV)
11): S N Rb 0
L2 N Rb I
1 0._ ,(CH2)õ Alia
HN 1
SCN¨ CO¨ C¨Ra
0(CH2)n Fis 1 4 5
1 4 5 N, ,R ¨Z12"
r y S N Rb
R3
_N( , , Fe (VI)
R ¨Z12" H CI-130
r,
R' (VIII) (XII) I (XIII)
(h) 125-Z-R4-YR0NR3-00-(CH2L-NH,
(XIV)
\ )
SCN¨ CO¨ C¨Ra
CH,0 7\ Rb
.41f-,...... CICO¨ C ¨Ra 1202C, ,.-12a (1)
C -A
CO2CCH2¨R'
_I
(XIII) 0) CH20 ''.....Rb
HO Rb
(XVIII)
(XVI)
(XVII)
in which:
[306] L3 is a C(1-6)alkyl group, for instance methyl;
1307] R15 is a C(l6)alkyl group, for instance ethyl or t-butyl and
[308] Ll, L2, Ra, Rb, Rc, R2, R3, R4, R5, n, X, Y and Z are as defined in WO
01/60805.
1309] An exemplary reaction for making a compound of formula (I) of interest
is as
follows:
Example of Synthesis Approach (I)-1(a)
[310] 1-(N-(2-(Diethylamino)ethyl)-N-(4-(4-
trifluoromethylphenyl)benzyDaminocarbonyl-
methyl)-2-(4-fluorobenzyl)thio-5,6-trimethylenepyrimidin-4-one
0
1 110
S N
40 40) CF,
F yo drat,
SOõ.....,,N III*
10566615.1
CA 02588369 2007-05-11
- 38 -
[311] Intermediate B69 of WO 01/60805 (87.1g, 0.26 mol.) was suspended in
dichloromethane (2.9 litre). 1-Hydroxybenzotriazole hydrate (35.2g, 0.26 mol.)
and 1-(3-
dimethylaminopropy1)-3-ethylcarbodiimide hydrochloride (99.7g, 0.52 mol.) were
added and
the suspension stirred for 45 minutes by which time complete solution had been
obtained.
Intermediate A30 of WO 01/60805 (91.2g, 0.26 mol.) was added as a solution in
dichloromethane (100m1) over 5 minutes and the solution stirred for 4 hours.
Saturated
ammonium chloride solution:water mixture (1:1, 1 litre) was added and the
solution stirred
for 10 minutes. The organic phase was separated and extracted with saturated
ammonium
chloride:water mixture (1:1, 1 litre), extracts were pH 6. The organic phase
was separated
and extracted with water (1 litre) containing acetic acid (10m1), extract pH
5. The
dichloromethane layer was separated and extracted with saturated sodium
carbonate
solution:water:saturated brine mixture (1:3:0.2, 1 litre), pH 10.5, then with
saturated
brine:water mixture (1:1, 1 litre). The brown solution was dried over
anhydrous sodium
sulfate in the presence of decolourising charcoal (35g), filtered and the
solvent removed in
vacuo to give a dark brown foam. The foam was dissolved in iso-propyl acetate
(100m1) and
the solvent removed in vacuo. The dark brown gummy residue was dissolved in
boiling iso-
propyl acetate (500m1), cooled to room temperature, seeded and stirred
overnight. The pale
cream solid produced was filtered off and washed with iso-propyl acetate
(100m1). The solid
was sucked dry in the sinter for 1 hour then recrystallized from iso-propyl
acetate (400m1).
After stirring overnight the solid formed was filtered off, washed with iso-
propyl acetate
(80m1) and dried in vacuo to give the title compound, 110g, 63.5% yield. 1H
NMR (CDC13,
ca 1.9:1 rotamer mixture) 8 0.99 (6H, t), 2.10 (2H, m), 2.50 (4H, q),
2.58/2.62 (2H, 2 x t),
2.70/2.82 (2H, 2 x t), 2.86 (2H, t), 3.28/3.58 (2H, 2 x t), 4.45/4.52 (2H, 2 x
s), 4.68/4.70 (2H,
2 x s), 4.93 (2H, s), 6.95 (2H, m), 7.31 (2H, d), 7.31/7.37 (2H, 2 x m),
7.48/7.52 (2H, d), 7.65
(2H, m), 7.72 (2H, m); MS (APCI) (M+H)+ 667; mp 125 C (by DSC - assymetric
endotherm).
Example of Synthesis Approach (I)-1(b)
[312] 1-(N-(2-(Diethylamino)ethyl)-N-(4-(4-
trifluoromethylphenyl)benzypaminocarbonyl-
methyl)-2-(4-fluorobenzypthio-5,6-trimethylenepyrimidin-4-one bitartrate
[313] Prepared from intermediates A30 and B69 in WO 01/60805 by the method of
Example 1 in WO 01/60805. 1H-NMR (d6-DMSO, ca 1:1 rotamer mixture) E 0.92/0.99
(6H,2x t), 1.99 (2H,m), 2.54 (6H,m), 2.68/2.74 (4H,m), 3.36 (2H,m), 4.21
(2H,$), 4.37/4.44
10566615.1
CA 02588369 2013-10-22
- 39 -
(2H,2x s), 4,63/4.74 (2H,2x s), 4,89/5.13 (2H,2x s), 7.08/7.14 (2H,2x m), 7.36-
7.50 (4H, m),
7.64/7.70 (2H,2x d), 7.83 (4H,m); MS (APCI+) found (M+1) = 667; C36H38F4N402S
requires 666.
Example of Synthesis Approach (1)4 (c)
[314] 1-(N-(2-(Diethylamino)ethyl)-N-(4-(4-
trifluoromethylphenyl)benzyl)aminocarbonyl-
methyl)-2-(4-fluorobenzypthio-5,6-trimethylenepyrimidin-4-one hydrochloride
[315] The free base from Example (I)-1(a) (3.00g, 0.0045 mol) was suspended
with stirring
in isopropanol (30 ml) and warmed to 450C to give a clear solution. The
solution was then
cooled to ambient temperature and conc. hydrochloric acid (0.40 ml, 0.045 mol)
was added.
The resultant slurry was then stirred at ambient temperature for 35 minutes,
before being
cooled to 00C for 35 minutes. The slurry was then filtered and washed with
isopropanol (10
ml), followed by heptane (30 ml), before being dried under vacuum to give the
title
compound as a white solid (3.00 g, 95%). 1H NMR (CDC13) 6 1.38 (6H, t), 2.08
(2H, m),
2.82 (2H, t), 2.99 (2H, t), 3.19 (4H, m), 3.35 (2H, m), 3.97 (2H, s), 4.42
(2H, s), 4.81 (2H, s),
4.99 (2H, s), 6.87 (2H, t), 7.26 (2H, t), 7.33 (2H, d), 7.41 (2H, d), 7.53
(2H, d), 7.71 (2H, d),
11.91 (1H, s).
[316] Synthesis of Formula (II)
[317] A description of how to make the compounds of formula (II) and examples
of
intermediates and final products for the compounds named above can be found in
published
international application WO 02/30911. A last-step method for making a
compound useful in
this invention is Example (II)-1.
Example of Synthesis Approach (II)-1
[318] N-(2-Diethylaminoethyl)-242-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4,5,6,7-
tetrahydro-
cyclopentapyrimidin-1-y1]-N-(4'-trifluoromethyl-bipheny1-4-ylmethypacetamide
bitartrate
0
N11)10FF
F 401
yo 011
EtN
,
[319] A solution of /V,N-diethyl-N-(41-trifluoromethyl-bipheny1-4-ylmethyl)-
ethane-1,2-
diamine (Int D4 in WO 02/30911) (0.50g, 1.44mmol), 1-(3-dimethylaminopropy1)-3-
ethylcarbodiimide (0.56g, 1.45mmol), 1-hydroxybenzotriazole hydrate (0.12g)
and 2-(2-[2-
CA 02588369 2007-05-11
- 40 -
(2,3-difluoropheny1)-ethy1]-4-oxo-4,5,6,7-tetrahydro-cyclopentapyrimidin-1-y1)-
acetic acid
(Int Cl in WO 02/30911) (0.48g, 1.44mmol) in dichloromethane (10m1) was
stirred at
ambient temperature overnight then diluted with dichloromethane (30m1), washed
with
aqueous sodium bicarbonate and evaporated. The residue was purified by
chromatography
(10g silica cartridge, ethyl acetate-acetone) to give the title compound as a
yellow foam (free
base) (0.50g, 52%). 1H-NMR (DMSO, rotamer mixture) 8 0.83-0.89 (6H, m), 1.98
(2H, m),
2.40 (4H, m), 2.45-2.82 (10H, m), 3.02 (2H, m), 4.64/4.75 (2H,2x s), 4.96/5.19
(2H,2x s),
7.11-7.40 (5H, m), 7.65 (2H, m), 7.84 (4H, m); MS (APCI+) found (M+1) = 667;
[320] 37H39F5N402 requires 666.
[321] d-Tartaric acid (0.09g, 0.60mmol) was added to a solution of the free
base (0.40g,
0.60mmol) in methanol (10m1) with stirring. The resulting solution was
evaporated to yield
the salt (0.49g). 1H-NMR (DMSO, rotamer mixture) 8 0.85-0.97 (611, m), 1.91-
2.00 (2H, m),
2.40-2.49 (4H, m), 2.54-2.82 (10H, m), 3.02-3.46 (2H, m), 4.20 (211, s),
4.64/4.75 (2H, 2x s),
4.97/5.18 (2H, 2x s), 7.11-7.40 (511, m), 7.65 (2H, m), 7.84 (4H, m); MS
(APCI+) found
(M+1) = 667; C37H39F5N402 requires 666.
[322] Following this process, or alternatively other processes described in WO
02/30911,
one can prepare the other compounds named above that have the structure of
formula (II).
[323] Synthesis of Formula (III)
[324] The overall synthesis of compounds of formula (III) is illustrated in
the following
scheme III, as presented in W002/30904:
[325] Scheme III
10566615.1
CA 02588369 2007-05-11
- 41 -
Ho 0
...A.xR2 0
V2
(s)
0 1
131-CH,-L. + j.......,_1+ =N =___ s ___,... 0 0
123
(XXIV) 0 0
H214 R3
0
(XXVI)
X NH (XXVII)
(XXIII) (vv)
0 /0
R2
P o
-CH,-COOR"
(XXVIII)
(VI) (c) 0 N R3
0 0 H
-%-S
I I. L R2
2
1_,-CH2-COOR"
(c)
HN R3 (VI)
(XXII) L.,
COO R11
0 0 \1) 0
(....1.XLI (XXIV) (r) j,,,Jtx R2 R1502C.,,1
õ i e IV-CH2-L
XXV) R1 4 +
R1.., ,..-
X N
0 N R3 Xk 0
L'COOR" L
(XX) COOR" (XXI)
w 4( (0)
(v)
0 0 0 0
R2 (, (b) R1502C1.kx R2
R1 X N R" A R2
(a) i A AR2 )
., ....___ 13 1.,. X I I
X N Fe R.."` Rt,
N R3
X N R3
Oyi (I) R6-R5-CH2NHR4 L. L.
(III) (n) COON COOR" (XIX) L.
COOR"
R4.... N ' (IV)"=""- R5- RG (c)
R12,. o R2 1_,-CH,-COOR"
R12,. o
(e) R2 (d) (VI)
/.. 1
R' ¨XH R'.... ...-C11 0
L2 N R3 (XIX) X N R3
(VII)
R'2-OH(VIII) R,..õ,õ A
R2
X N R3
(X) (1) \ 0)
H
(V) \L.1 OH 41() (h)
R12 o R2 R14
R12 ,, o I., ',.
L2 R2 R
N R3 R1¨ XH X N R3 R2
0 I
_ . I (XIX) , ...ril R2 (d) ...= ,
1 - R'Y N R"
(IX)
L2 ...N R3 R.._ ,. . , (XVI)
I _ (e) X N R-
(XVII)
0 I _
(XIV) 0
1 (9) (XV) 12,-Y-CH2-L (m)
(XVI)
OH 1:114
OH
R2
I (to R1302c ., R2 0) R'302CX R2 LL x R2
I I
HO N R3HO N R3 H2N R.3 Me
N R3
(XI) (XII)
(XIII) (XVIII)
[326] Referring to this scheme, the ester (IV) is usually prepared by N-1
alkylation of (V)
using (VI), in which R11 is as hereinbefore defined e.g. (VI) is t-butyl
bromoacetate or ethyl
bromoacetate, in the presence of a base e.g. BuLi in THF or sodium hydride in
N-methyl
pyrrolidinone (NMP) (step c).
10566615.1
CA 02588369 2007-05-11
- 42 -
[327] When X is CH2S, the key intermediate (IV) can be synthesised by reacting
(XX) with
dimethyloxosulfonium methylide, generated via the treatment of
trimethylsulfoxonium iodide
with sodium hydride at low temperature, to yield a sulfur ylid (XXII) (step
q). Subsequent
treatment of (XXII) with carbon disulfide in the presence of diisopropylamine,
followed by
R1CH2-L4, where L4 is a leaving group, yields intermediate (IV) (step r).
[328] Alternatively, when X is CH2S, the R1X substituent can be introduced by
displacement of a leaving group L2 (e.g. Cl) (step e) either on a pyridine
(VIII) or pyridine
N-oxide (XIV), to give 2-substituted pyridines (VII) and (XV). Transformation
of (VII) or
(XV) to the 4-pyridone (V) is accomplished by deprotection of the 4-oxygen
(e.g. using
(Ph3P)3RhC1 when in aq. ethanol when R12 = ally1) (step d), followed, for
(XVI), by removal
of the N-oxide substituent, using hydrogen in the presence of Pd/C in acetic
acid (step k).
The pyridine (VIII) or pyridine N-oxide (XIV) can be prepared by steps (i),
(h), (g), (f), and
(j), in which:
[329] (j) treatment of (VIII) with m-chloroperbenzoic acid in dichloromethane;
[330] (f) treatment of (IX) with R120H (X), in which R12 is allyl, and sodium
hydride in
DMF;
[331] (g) treatment of (XI) with phosphorus oxychloride;
[332] (h) treatment of (XII) with aq HC1 with heating;
[333] (i) treatment of (XIII) with di-lower alkyl malonate and sodium alkoxide
in alcohol
(in which R13 is C(16)alkyl, typically R13 = Et); and
[334] R1- CH2SH (XIX) is typically prepared from the thioacetate, which is
formed from
the con-esponding alkyl bromide R1-CH2Br.
[335] Alternatively, when X is CH2S and R2 and R3, together with the pyridone
ring
carbon atoms to which they are attached, form a fused benzo ring, intermediate
(IV) can be
synthesised from known starting materials by steps (s), (c) and (v) in which:
[336] (s) treatment of Meldrum's acid (XXIII) with sodium hydride at low
temperature,
followed by reaction with phenylisothiocyanate and subsequent treatment with
R1CH2-L4;
[337] (c) as hereinbefore discussed;
[338] (v) treatment of (XXV) with trifluoroacetic acid.
10566615.1
CA 02588369 2013-10-22
- 43 -
[339] When X is alkylene, it is preferable to use steps (m) and (h)
(intermediates (XVII),
(XVIII)) or steps (n) and (p) (intermediates (XIX), (XX), (XXI)) in which:
[340] (h) transformation of a 4-substituted pyridine into a 4-pyridone e.g. by
treatment of
(XVII) R14 = Cl with aq HCI and dioxan, or deprotection of R14 = OR12, e.g.
using
conditions of step (d).
[341] (m) chain extension of a 2-alkyl pyridine, e.g. where X = YCH2CH2 by
treatment of
a 2-methylpyridine (XVIII) with R1-Y-CH2-L4 (XVI) in which L4 is a leaving
group and a
strong base, such as BuLi, in THF.
[342] In the alternative route, the 3-ester group is removed from intermediate
(XIX) R15 =
Co _6)alkyl by heating in diphenyl ether where R15 = tBu (step n);
Intermediate (XIX) is
formed from the 2,6-dioxo-1,3-oxazine (XX) and ester (XXI) by treatment with a
base such
as NaH in DMF or 1,8-diazabicyclo[5.4.0]undec-7-ene in dichloromethane.
[343] Synthesis of (XX) from known starting materials can be achieved via
steps (w) and
(c) or steps (y) and (c) in which:
[344] (w) treatment of (XXVII) with azidotrimethylsilane in THF;
[345] (y) treatment of (XXVI) with phosgene;
[346] (c) as hereinbefore described.
[347] See W002/30904 for additional details and exposition of how to make
compounds of
formula (III).
Example of Synthesis Approach (III)-1
[348] N-(1-(2-Methoxyethyl)piperidin-4-y1)-2-[2-(2,3-difluorobenzylthio)-4-oxo-
4H-
quinolin-l-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide
0
I 1.1F
S N
L.r0
[349] The free base was prepared from Int. El and Int. A42 by the method of
Example 1 in
WO 02/30904, except using DMF as solvent in place of dichloromethane. 1.97g of
this
material was crystallised from n.butyl acetate (10m1) to give the title
compound (1.35g). 'H-
NMR (CD30D) 6 1.7-2.05 (4H, m), 2.05-2.3 (2H, 2xt), 2.5-2.65 (2H, m), 2.95-3.1
(2H, m),
CA 02588369 2013-10-22
=
-44-
3.3 (3H, s), 3.45-3.55 (2H, m), 3.9-4.05 + 4.4-4.5 (1H, 2xm), 4.37 + 4.48 (2H,
2xs), 4.71 +
4.87 (2H, 2xbr s), 5.31 + 5.68 (2H, 2xs), 6.44 + 6.52 (1H, 2xs), 6.95-7.3 (3H,
m), 7.35-7.85
(11H, m), 8.2-8.35 (1H, m); MS (APCI+) found (M+1) 736; C44138F5N303S requires
735.
[350] Synthesis of Formula (IV)
[351] The following flow chart illustrates a process for making the compounds
of this
invention.
0 0
Me0)Br + I-K)
K2CO3, MeCN, reflux
0 si CF3
0
Me0-5c ri`-') +
H2N 1101
1 ________________________________________________ 1
1) NaBH(OAc),, DCE, AcOH
F WIY) 2) aq. Na2CO3
1 I
F 0iii
IW N X
0 Me01_ NDA
OH 11 * CF3
I I
HATU, DIPEA, DMF
1
0
F õ,õ,Jn
, ,
F
W- N X 0 CF3
y, 0N
0
MeOictV,.)
[352] In addition, the reader is referred to published PCT application WO
03/016287 for
chemistries that can be useful in preparing some of the intermediates set out
in this flow
chart. Those chemistries, to the extent they are useful in this case, are
incorporated herein by
reference as though it was fully set out herein. In addition, reference is
made to the syntheses
set out in published PCT applications WO 01/60805, WO 02/30911, WO 02/30904,
WO
03/042218, WO 03/042206, WO 03/041712, WO 03/086400, and WO 03/87088, and co-
pending US provisional application 60/829,327. To the extent the reader wishes
to prepare
the compounds of formula (IV) by using intermediates, reagents, solvents,
times,
temperatures, etc., other than those in the route on the foregoing page, these
published PCT
applications and co-pending US applications can
CA 02588369 2013-10-22
- 45 -
provide useful guidance. To the extent the chemistries in these applications
are pertinent to
making the instant compounds.
[353] Intermediate (IV)-A1{[41-(Trifluoromethyl)-4-biphenylyl]methyll amine
H2N = it
[354] The preparation of this compound was described in WO 02/30911 as
Intermediate D7.
[355] Intermediate (IV)-A2 ({4'-{(Trifluoromethypoxy]-4-biphenyly1}methypamine
hydrochloride
F F
F1,11 0Y-F
[356] A solution of 41-[(trifluoromethypoxy]-4-biphenylcarbonitrile (prepared
from {4-
[(trifluoromethypoxy]phenyllboronic acid by a method analogous to that
described for the
4'-trifluoromethyl analogue, Intermediate D6 of WO 02/30911) (66.6g) in
ethanol (2000m1)
and concentrated hydrochloric acid (100m1) was hydrogenated over Pearlman's
catalyst
(10g) at 25psi until reduction was complete. The catalyst was removed by
filtration through
celite, then the solvent was removed in vacuo to obtain the desired product.
[357] LCMS Rt = 2.212 minutes; m/z [M+H] = 251.0
[358] Intermediates for making formula (IV)
[359] Intermediate (IV)-A3
[360] Methyl 2-methyl-2-(4-oxo-1-piperidinyl)propanoate
oo
[361] A mixture of methyl 2-bromo-2-methylpropanoate (80.87m1, 5 equiv), 4-
piperidone
hydrochloride monohydrate (19.6g, 1 equiv), acetonitrile (200m1) and potassium
carbonate
(69.1g, 4 equiv) was heated at reflux under nitrogen with mechanical stirring
for 17.5h then
cooled in an ice bath before adding diethyl ether (100m1). Filtration through
celite followed
by flash chromatography (silica, 10-50% ethyl acetate in hexane) and
evaporation of the
product fractions gave the desired product as a yellow oil (14.28g).
[362] 1H NMR (CDC13) 6 1.41 (6H,$), 2.47 (4H,m), 2.88 (4H,m), 3.73 (3H,$).
[363] Intermediate (IV)-A4
[364] Ethyl 2-methy1-2-(4-oxo-1-piperidinyl)propanoate
CA 02588369 2007-05-11
- 46 -
0
,----..0-1-7c tsi,õõ--
1365] A mixture of ethyl 2-bromo-2-methylpropanoate (48.3m1, 5 equiv), 4-
piperidone
hydrochloride monohydrate (100g, 1 equiv), acetonitrile (1216m1) and potassium
carbonate
(353g, 4 equiv) was heated at reflux under nitrogen with mechanical stirring
for 20h then
cooled in an ice bath before adding diethyl ether (approx. 1400m1). The
mixture was filtered
through celite, evaporated in vacuo, then excess bromoester distilled off (50
C still head
temperature/10 Ton). Flash chromatography (silica, 5-30% ethyl acetate in
hexane) and
evaporation of the product fractions gave the crude product as a yellow oil.
To remove some
remaining bromo ester contaminant this was partitioned between ethyl acetate
and 2M
aqueous hydrochloric acid. The organic layer was discarded and the aqueous
layer was
basified with sodium carbonate, saturated with sodium chloride and extracted
with ethyl
acetate. Drying and evaporation of the organic extracts gave the desired
product as a yellow
oil (54.7g).
[366] 1H NMR (CDC13) (5 1.27 (3H,t), 1.40 (6H,$), 2.47 (4H,m), 2.90 (4H,m),
4.20 (2H,q).
[367] Intermediate (IV)-A5
[368] 1,1-Dimethylethyl 2-methy1-2-(4-oxo-1-piperidinyl)propanoate
o ro
>(0-)1XN''''.--
[369] A mixture of 1,1-dimethylethyl 2-bromo-2-methylpropanoate (8.0g, 1.1
equiv), 4-
piperidone hydrochloride (5.0g, 1 equiv), acetone (50m1) and potassium
carbonate (13.0g, 3
equiv) was heated at reflux with stifling for 24h, then filtered and the
filtrate evaporated. The
crude residue was used in the next step without purification.
[370] ES+MS m/z [M+H-tBu] =186.1
[371] Intermediate (IV)-B1
[372] Methyl 2-methyl-2-[4-(([4'-(trifluoromethyl)-4-biphenylyl]methyl} amino)-
1-
piperidinyl]propanoate
o F
.F F
[373] A mixture of methyl 2-methy1-2-(4-oxo-1-piperidinyl)propanoate (Int. A3)
(14.28g, 1
equiv), {[4'-(trifluoromethyl)-4-biphenylyl]methyl}amine (Int. Al) (19.6g,
0.85 equiv), DCE
(300m1), acetic acid (3.8m1, 0.90 equiv) and sodium triacetoxyborohydride
(20.7g, 1.25
10566615.1
CA 02588369 2007-05-11
- 47 -
equiv) was stirred at room temperature under nitrogen for 17.5h. Aqueous
sodium carbonate
(2M solution, excess) was added and stirred for 4h, then the mixture was
extracted with a
mixture of diethyl ether and THF. The organic extracts were backwashed with
water and
brine, dried over sodium sulfate and filterered through a pad of silica gel
which was rinsed
with 2.5% methanol in DCM. After evaporation in vacuo, the crude product was
crystallised
from ether/hexane, finally at ice bath temperature, which after drying yielded
a white solid
(20.9g).
[374] LCMS Rt = 2.070 minutes; m/z [M+H]+ = 435.2
[375] 1H NMR (d6-DMS0) (5 1.15-1.32 (8H, m), 1.75-187(2H,m), 1.97-2.12 (2H,m),
2.27-
2.40 (1H, m), 2.77-2.90(2H,m), 3.60 (3H,$), 3.76 (2H,$), 7.46 (2H, d,
J=8.03Hz), 7.67 (2H, d,
J=8.28Hz), 7.80 (2H, d, J=8.53Hz), 7.88 (2H, d, 8.03Hz)
[376] Intermediate (IV)-B2
[377] Ethyl 2-methyl-2[4-( 44'-(trifluoromethyl)-4-biphenylyl]methyll amino)-1-
piperidinyl]propanoate
0
_______________ N 410 F
rH
[378] A mixture of ethyl 2-methy1-2-(4-oxo-1-piperidinyl)propanoate (Int. A4)
(25.6g, 1.2
equiv), {[4'-(trifluoromethyl)-4-biphenylyl]methyl}amine (Int. Al) (31.1g, 1.0
equiv), DCE
(400m1) and acetic acid (6.3m1, 1.1 equiv) was stirred at room temperature
under nitrogen.
Sodium triacetoxyborohydride (33.5g, 1.5 equiv) was added and stirring
contined for 19
hours. Aqueous sodium carbonate (2M solution, excess) was added and stirred
for 1.5h, then
the mixture was extracted with a mixture of diethyl ether and THF. The organic
extracts
were backwashed with water and brine, filterered through a pad of silica gel,
dried over
sodium sulfate and evaporated in vacuo. The desired product was obtained as a
white solid
(44.2g) which was used without further purification.
[379] LCMS Rt = 2.194 minutes; rn/z [M+H] = 449.3
[380] IH NMR (d6-DMS0) 6 1.06-1.32 (11H,m), 1.74-1.89 (2H,m), 1.99-2.14 (2H,
m),
2.25-2.39 (1H. m), 2.69-2.89 (2H, m), 3.75 (2H, s), 4.01-4.12 (2H, m), 7.45
(2H, d, J=7.55
Hz), 7.67 (2H, d, J=7.81 Hz), 7.79 (2H, d, J=8.06 Hz), 7.88 (2H. d, J=8.06Hz)
[381] Intermediate (IV)-B3
[382] Ethyl 2-methy1-2-{41({4'4(trifluoromethypoxy]-4-biphenyly1}methyl)amino]-
1-
piperidinyl}propanoate
10566615.1
CA 02588369 2007-05-11
- 48 -
11¨>¨N
\--F
\ ______________ H
F F
[383] A mixture of ethyl 2-methy1-2-(4-oxo-1-piperidinyl)propanoate (Int. A4)
(1.09g, 1.2
equiv), ({41-[(trifluoromethyl)oxy]-4-biphenylyl}methyl)amine hydrochloride
(Int. A2)
(1.28g, 1.0 equiv), DCE (21m1) and acetic acid (0.27m1, 1.1 equiv) was stirred
at room
temperature under nitrogen. Sodium triacetoxyborohydride (1.42g, 1.5 equiv)
was added and
stirring contined for 3 hours. Aqueous sodium carbonate (2M solution, excess)
was added
and stirred for 45min, then the mixture was partitioned with a mixture of
diethyl ether/THF
and water. The organic extracts were backwashed with water and brine, and
dried over
sodium sulfate and evaporated in vacuo. The desired product was obtained as a
light yellow
solid (2.14g) which was used without further purification.
[384] LCMS Rt = 2.244 minutes; m/z [M+H]+ = 465.3
[385] Intermediate (IV)-B4
[386] 1,1-Dimethylethyl 2-methy1-2-[4-(44'-(trifluoromethyl)-4-
biphenylylimethyllamino)-1-piperidinyl]propanoate
0
04 _______________ 410
ts1
¨/\ )-11
[387] A mixture of 1,1-dimethylethyl 2-methy1-2-(4-oxo-1-
piperidinyl)propanoate (Int. A6)
(370mg, 1.2 equiv), {[4'-(trifluoromethyl)-4-biphenylyl]methyll amine (Int.
Al) (397mg, 1
equiv), sodium triacetoxyborohydride (400mg, 1.5 equiv), DCM (10m1) and acetic
acid
(0.076m1, 1 equiv) was combined and stirred at room temperature until LCMS
confirmed
disappearance of the amine starting material (approx. 18 hours). Aqueous
sodium carbonate
was added and then extracted with DCM. The organics were dried over sodium
sulfate and
concentrated to give a solid (420mg) that was used without further
purification.
[388] LCMS Rt = 2.24 minutes; m/z [M+Hr = 477.3
[389] Intermediate (IV)-C1
[390] [242-(2,3-Difluorophenypethy1]-4-oxo-1(41/)-quinazolinyliacetic acid
40
F arL
141
OH
10566615.1
CA 02588369 2007-05-11
- 49 -
[391] The preparation of this compound was described in WO 02/30911 as
Intermediate
C43.
[392] Intermediate (IV)-C2
[393] [242-(2,3-Difluorophenypethy11-4-oxo-1,8-naphthyridin-1(4H)-yl]acetic
acid
i
F \
I I
F 40
N N
Y
OH
[394] The preparation of this compound was described in WO 02/30904 as
Intermediate
E21.
[395] Intermediate (IV)-C3
[396] [242-(2,4-Difluorophenypethy1]-4-oxo-1(411)-quinazolinyl]acetic acid
=
F NI 0
40 N
yo
F OH
[397] The preparation of this compound was described in WO 02/30911 as
Intermediate
C45.
[398] Intermediate (IV)-C4
[399] Ethyl [242-(2,4-difluorophenypethyl]-4-oxopyrido[2,3-d]pyrimidin-1 (411)-
yl] acetate
F N'Ill
F 140 N N
o
[400] A mixture of ethyl (2,4-dioxo-3,4-dihydropyrido[2,3-d]pyrimidin-1(211)-
ypacetate
(WO 02/30911, Intermediate B52) (40.8g, 1.2 equiv) and 3-(2,4-difluoropheny1)-
propanimidamide (made by methods analogous to those described for the 2,3-
difluoro
isomer, Intermediates Al to A3 of WO 02/30911) (30.0g, 1 equiv) was fused in a
150 C oil
bath for 25 min, then cooled quickly to room temperature in a water bath.
Chromatography
(silica, crude product loaded in DCM and eluted with 50-100% ethyl acetate in
hexane) gave
the desired product (43.56g).
[401] LCMS Rt = 2.521 minutes; m/z [M+H] = 374.1
10566615.1
CA 02588369 2007-05-11
- 50 -
[402] 1HNMR (CDC13) 5 1.31 (3H, t), 3.13 (2H, m), 3.26 (2H, m), 4.28 (2H, q),
5.27 (2H,
s), 6.82 (2H, m), 7.34 (1H, m), 7.50 (1H, m), 8.65 (1H, m), 8.74 (1H, m).
[403] Intermediate (IV)-05
1404] [2-[2-(2,3-Difluorophenyl)ethyl]-4-oxopyrido[2,3-d]pyrimidin-1 (4 11)-
yl] acetic acid
NIX)I I
F *
N N
[r0
OH
[405] The preparation of this compound was described in WO 02/30911 as
Intermediate
C35.
[406] Intermediate (IV)-05
[407] [242-(2,4-Difluorophenyl)ethy1]-4-oxopyrido[2,3-d]pyrimidin-1(411)-
yl]acetic acid
NIX)I I
N N
F 1101
OH
[408] Ethyl [2-[2-(2,4-difluorophenyl)ethy1]-4-oxopyrido[2,3-cflpyrimidin-
1(4H)-yliacetate
(Int. Cl) (32.76g, 1 equiv) was dissolved in ethanol (350m1) and water (70m1),
cooled in ice,
then aqueous lithium hydroxide (2M solution, 43.42m1, 0.99 equiv) was added.
Stirring was
continued for 2h at room temperature. The solution was concentrated in vacuo
and the
residue was redissolved in water (700m1) and saturated aqueous sodium
bicarbonate (50m1),
then washed with ethyl acetate (200m1). The aqueous layer was acidified to pH
2 with 2M
hydrochloric acid, and the precipitate was filtered off, washed with ice water
(50m1) and
dried in vacuo (50 C, 16h) to obtain the desired product (23.2g).
[409] IHNMR (d6-DMS0) 5 2.4-2.6 (4H, m), 5.24 (2H, s), 7.04 (1H, m), 7.22
(1H, m),
7.48 (1H, m), 7.60 (1H, m), 8.47 (1H, m), 8.84 (1H, m).
Example (IV)-1
[410] Methyl 2-[4-( { [242-(2,3-difluorophenypethy1]-4-oxo-1(41/)-
quinazolinyl] acetyl} {[4'-
(trifluoromethyl)-4-biphenylyl]methyl} amino)-1-piperidiny1]-2-
methylpropanoate 2,3-
dihydroxybutanedioate (salt)
10566615.1
CA 02588369 2007-05-11
- 51 -
=
F ea
111,1 INS
FFF
y 411
OH 0
0
HO
0
OH
[411] A mixture of [2-[2-(2,3-difluorophenypethy1]-4-oxo-1(411)-
quinazolinyl]acetic acid
(Int. Cl) (100mg, 1 equiv), methyl 2-methy1-244-(114'-(trifluoromethyl)-4-
biphenylyl]methyllamino)-1-piperidinyl]propanoate (Int. B1) (130mg, 1.03
equiv), DIPEA
(0.1m1, 3.6 equiv), acetonitrile (2m1) and HATU (130mg, 1.4 equiv) was stirred
at room
temperature for lh, then evaporated and redissolved in acetonitrile.
Purification by reverse
phase HPLC (Preparative Method A) gave methyl 244-({[242-(2,3-
difluorophenypethyl]-4-
oxo-1(41/)-quinazolinyl] acetyl } { [4'-(trifluoromethyl)-4-biphenylyl]methyl}
amino)-1-
piperidiny1]-2-methylpropanoate (128mg).
[412] LCMS Rt = 2.686 minutes; m/z [M+H] = 761.3
[413] 11-1 NMR (CDC13) 5 1.33 (3H, s), 1.36 (3H, s), 1.83-2.02 (4H, m), 2.36-
2.48 (2H, m),
2.87-2.91 (1H, m), 3.06-3.09 (2H, m), 3.16-3.20 (2H, m), 3.26-3.29 (1H, m),
3.71-3.73 (3H,
m), 4.02/4.51 (1H, 2x br m), 4.74 (1H, s), 4.92 (1H, s), 5.12 (1H, s), 5.56
(1H, s), 7.00-7.19
(3H, m), 7.32-7.37 (1H, m), 7.48-7.62 (5H, m), 7.72-7.81 (5H, m), 8.22-8.28
(1H, m).
1414] The free base was converted to the bitartrate salt by adding L-tartaric
acid (1.675g,
1.0 equiv) in one portion and stirred for 30 minutes at room temperature. The
solution was
concentrated in vacuo to an off-white powder that was dried in a vacuum oven
at room
temperature.
Example of Synthesis Approach (IV)-2
14151 Methyl 2-[4-({[242-(2,3-difluorophenypethy1]-4-oxo-1,8-naphthyridin-
1(411)-
yl]acetyl} 1[4'-(trifluoromethyl)-4-biphenylyl]methyl}amino)-1-piperidiny1]-2-
methylpropanoate 2,3-dihydroxybutanedioate (salt)
I N I
F
yo 40
0 9H 0
0 =
OH
105666151
CA 02588369 2007-05-11
- 52 -
[416] A mixture of [242-(2,3-difluorophenypethy1]-4-oxo-1,8-naphthyridin-1(4H)-
yl]acetic
acid (Int. C2) (100mg, 1 equiv), carbonyldiimidazole (50mg, 1.05 equiv) and
dimethyl-
acetamide (4m1) was stirred at 60 C for 30 min then methyl 2-methy1-214-({[4'-
(trifluoro-
methyl)-4-biphenylyl]methyl}amino)-1-piperidinyl]propanoate (Int. B1) (132mg,
1.05 equiv)
was added and the temperature raised to 80 C for 2h. A further portion of
carbonyldiimidazole (0.5 equiv) was added and stirring continued at 80 C for
15h. After
cooling the crude mixture was applied to reverse phase HPLC (Preparative
Method A) to
obtain methyl 2-[4-( {[2-[2-(2,3-difluorophenyl)ethyl]-4-oxo-1,8-naphthyridin-
1(4H)-y1]-
acetyl} 1[4'-(trifluoromethyl)-4-biphenylyllmethyll amino)-1-piperidiny1]-2-
methylpropanoate
(99mg).
[417] LCMS Rt = 2.845 minutes; m/z [M+H]+ = 761.3
[418] 1H NMR (CDC13) (5 1.28 (3H, s), 1.31 (3H, s), 1.73-2.05 (4H, m), 2.25
(1H, t), 2.39-
2.46 (1H, m), 2.96-2.99 (1H, m), 3.00-3.12 (4H, m), 3.19 (1H, s), 3.68-3.73
(3H, m),
4.11/4.41 (1H, 2x br m), 4.73 (1H, s), 4.97 (1H, s), 5.51 (1H, s), 6.29-6.34
(1H, m), 7.06-7.20
(2H, m), 7.35-7.41 (1H, m), 7.48-7.58 (2H, m), 7.68-7.84 (6H, m), 8.60-8.68
(1H, m), 8.87-
8.91 (1H, m).
[419] This was converted to the bitartrate salt by a method analogous to that
described for
Example of Synthesis Approach (I).
Example of Synthesis Approach (I7)-3
[420] Ethyl 2-[4-({[2-[2-(2,3-difluorophenyl)ethy1]-4-oxo-1(411)-
quinazolinyllacety1}{[4'-
(trifluoromethyl)-4-biphenylyl]methyl}amino)-1-piperidiny1]-2-methylpropanoate
2,3-
dihydroxybutanedioate (salt)
Nil
F 14,1
F
S F
Lf0 qp)
OH
0
OH
[421] A mixture of [2-[2-(2,3-difluorophenypethyl]-4-oxo-1(411)-
quinazolinyl]acetic acid
(Int. Cl) (115mg, 1 equiv), ethyl 2-methy1-244-({[4'-(trifluoromethyl)-4-
biphenylyl]methyl}amino)-1-piperidinyl]propanoate (Int. B2) (150mg, 1 equiv),
HATU
(151mg, 1.2 equiv), DMF (2.7m1) and DIPEA (0.17m1, 3 equiv) was shaken at room
temperature for 5h. The reaction mixture was partitioned between ethyl
acetate/methanol and
10566615.1
CA 02588369 2007-05-11
- 53 -
aqueous sodium bicarbonate, then the organic layer was brine-washed and dried.
Flash
chromatography (silica, 3-4% methanol in DCM) gave ethyl 244-U[24242,3-
difluorophenypethy1]-4-oxopyrido[2,3-d]pyrimidin-1(41/)-yl]acetyl} {[4'-
(trifluoromethyl)-4-
biphenylyl]methyl}amino)-1-piperidiny1]-2-methylpropanoate as a white solid
(190mg).
[422] LCMS Rt = 2.55 minutes; m/z [M+H] = 775.3
[423] 'H NMR (CDC13) ö 1.18-1.40 (9H, m), 1.61-2.09 (4H, m), 2.22-2.45 (2H,
m), 2.75-
2,85 (1H, m), 2.90-3.34 (5H, m), 3.71/4.66 (1H, 2x m), 4.12-4.26 (2H, m), 4.70-
4.85 (3H, m),
5.08 (1H, s), 6.80-6.88 (1H,m), 6.95-7.13 (3H, m), 7.27-7.33 (1H, m), 7.34-
7.52 (3H,m),
7.56-7.62 (1H, m), 7.63-7.77 (4H, m), 8.29-8.44 (2H, m).
[424] This was converted to the bitartrate salt by a method analogous to that
described in
Example of Synthesis Approach (I).
Example of Synthesis Approach (IV)-4
[425] Ethyl 2-{4-[([242-(2,3-difluorophenyl)ethyl]-4-oxo-1(41/)-
quinazolinyllacetyl}({4'-
[(trifluoromethypoxy]-4-biphenyly1}methypamino]-1-piperidinyll-2-
methylpropanoate 2,3-
dihydroxybutanedioate (salt)
0
F I 0F
IIIP N Ail y 0,4 o 40 w F
ThY)cN N OH 0
H0A0H
0 =
OH
[426] A mixture of [242-(2,3-difluorophenypethy11-4-oxo-1(411)-
quinazolinyl]acetic acid
(Int. Cl) (124mg, 1.2 equiv), ethyl 2-methy1-2-{44({4'-[(trifluoromethypoxy]-4-
biphenyly1}methyl)amino]-1-piperidinyllpropanoate (Int. B3) (139mg, 1 equiv),
DMF
(1.2m1) and DIPEA (0.16m1, 3 equiv) was shaken at room temperature for 30 min,
then
HATU (176mg, 1.5 equiv) was added and shaking continued for 4h. Reverse phase
HPLC
(Preparative Method B) gave ethyl 2- {4-[ {[242-(2,3-difluorophenypethy1]-4-
oxo-1(411)-
quinazolinyl]acetyl}({4'-[(trifluoromethypoxy]-4-biphenyly1}methypamino]-1-
piperidinyl} -
2-methylpropanoate as a white solid (174mg).
[427] LCMS Rt = 2.77 minutes; m/z [M+H] = 791.3
[428] 'H NMR (CDCI3) Characteristic peaks: 15 1.21-1.42 (9H, m), 1.58-2.08
(4H, m), 2.20-
2,48 (2H, m), 2.71-5.1 (13H, br m), 6.79-6.87 (1H, d), 6.92-7.11 (3H, m), 7.30-
7.46 (5H, m),
7.48-7.63 (5H, m), 8.26-8.40 (1H, m)
10566615.1
CA 02588369 2007-05-11
- 54 -
[429] This was converted to the bitartrate salt by a method analogous to that
described in
Example of Synthesis Approach (I).
[430] Example of Synthesis Approach (IV)-5
[431] Methyl 2-[4-({[242-(2,4-difluorophenypethy1]-4-oxo-1(411)-
quinazolinyl]acetyl} {[4'-
(trifluoromethyl)-4-biphenylyl]methyl}amino)-1-piperidiny1]-2-methylpropanoate
2,3-
dihydroxybutanedioate (salt)
=
40 FF
F Y
0 r.-N OH
''.0)L7cN HOOH
0 =
OH
1432] mixture of [242-(2,4-difluorophenypethy1]-4-oxo-1(4H)-
quinazolinyl]acetic acid
(Int. C3) (100mg, 1 equiv), methyl 2-methy1-244-({[4'-(trifluoromethyl)-4-
biphenylyl]methyl}amino)-1-piperidinyl]propanoate (Int. B1) (130mg, 1.03
equiv), DIPEA
(0.1m1, 2 equiv), acetonitrile (2m1) and HATU (130mg, 1.4 equiv) was stirred
at room
temperature for lh, then evaporated and redissolved in acetonitrile.
Purification by reverse
phase HPLC (Preparative Method B) gave methyl 244-({[242-(2,4-
difluorophenypethy1]-4-
oxo-1(411)-quinazolinyllacetyl} {[4'-(trifluoromethyl)-4-biphenylyl]methyll
amino)-1-
piperidiny1]-2-methylpropanoate (126mg).
[433] LCMS Rt = 2.698 minutes; m/z [M+H] = 761.3
[434] 'H NMR (CDC13) .5 1.30 (3H, s), 1.34 (3H s), 1.81-2.03 (4H, m), 2.29-
2.35 (1H, m),
2.39-2.45 (1H, m), 2.82-2.87 (1H, m), 3.00-3.14 (4H, m), 3.19-3.24 (1H, m),
3.70-3.73 (3H,
m), 4.00/4.51 (1H, 2x br m), 4.74 (1H, s), 4.91 (1H, s), 5.10 (1H, s), 5.54
(1H, s), 6.77-6.84
(1H, m), 6.87-6.98 (1H, m), 7.28-7.43 (2H, m), 7.48-7.61 (5H, m), 7.73-7.81
(5H, m), 8.23-
8.29 (1H, m).
[435] This was converted to the bitartrate salt by a method analogous to that
described in
Example of Synthesis Approach (I).
Example Synthesis Approach (IV)-6
[436] Ethyl 244-({[212-(2,4-difluorophenypethy1]-4-oxo-1(4H)-
quinazolinyl]acetyll {[4'-
(trifluoromethyl)-4-biphenylyl]methyll amino)-1-piperidiny1]-2-
methylpropanoate 2,3-
dihydroxybutanedioate (salt)
10566615.1
CA 02588369 2007-05-11
- 55 -
=
I
F
O Lro
0 s HoOH
0H
0 =
OH
[437] A mixture of [242-(2,4-difluorophenyl)ethy1]-4-oxo-1(411)-
quinazolinyliacetic acid
(Int. C3) (120mg, 1 equiv), ethyl 2-methy1-244-({[41-(trifluoromethyl)-4-
biphenylyl]methyll-
amino)-1-piperidinyl]propanoate (Int. B2) (204mg, 1.3 equiv), DMF (1.4m1) and
DIPEA
(0.183m1, 3 equiv) was shaken at room temperature, then HATU (206mg, 1.5
equiv) was
added with vigorous agitation and shaking continued for 1.5h. A further
portion of
Intermediate D5 (12mg, 0.1 equiv) was added then shaking was continued for 2
days.
Reverse phase HPLC (Preparative Method B) gave ethyl 244-(1[242-(2,4-
difluorophenyl)ethy11-4-oxo-1(4H)-quinazolinyllacetyll [4'-(trifluoromethyl)-4-
biphenyly11-
methyllamino)-1-piperidiny1]-2-methylpropanoate as a white solid (173mg).
[438] LCMS Rt = 2.751 minutes; miz [M+H]+ = 775.3
[439] 1H NMR (CDC13) (5 (mixture of rotomers) Characteristic peaks:1.22-1.47
(9H, m),
1.63-2.10 (4H, m), 2.16-5.11 (15H, br m), 6.75-6.88 (2H, m), 7.14-7.80 (12H,
m), 8.26-8.40
(1H, m).
[440] This was converted to the bitartrate salt by a method analogous to that
described in
Example of Synthesis Approach (I).
Example Synthesis Approach (IV)-7
[441] Ethyl 2- {4-{ {[242-(2,4-difluorophenypethy1]-4-oxo-1(411)-
quinazolinyl]acetyll({4t-
[(trifluoromethypoxy]-4-biphenylyl}methyl)amino]-1-piperidinyll-2-
methylpropanoate 2,3-
dihydroxybutanedioate (salt)
0
I
0)<F
F kip FF
0
r,1\1 OH
HOyz.j. 0H
0
OH
[442] A mixture of [242-(2,4-difluorophenypethy1]-4-oxo-1(411)-
quinazolinyl]acetic acid
(Int. C3) (114mg, 1.1 equiv), ethyl 2-methy1-2-{4-[({4'-[(trifluoromethypoxy]-
4-
biphenylyllmethypamino]-1-piperidinyl}propanoate (Int. B3) (139mg, 1 equiv),
DMF
10566615.1
CA 02588369 2007-05-11
- 56 -
(1.2m1) and DIPEA (0.16m1, 3 equiv) was shaken at room temperature, then HATU
(176mg,
1.5 equiv) was added with vigorous agitation and shaking continued for 30 mm.
A further
portion of Intermediate D5 (2 lmg, 0.2 equiv) was added, followed lh later by
further HATU
(23mg, 0.2 equiv), then shaking was continued for 18h. Reverse phase HPLC
(Preparative
Method B) gave ethyl 2- {4-[1[2-[2-(2,4-difluorophenypethyl]-4-oxo-1(41/)-
quinazolinyl]-
acetyl)( {4'-[(trifluoromethypoxy]-4-biphenylyl}methyeamino]-1-piperidinyl} -2-
methylpropanoate as a white solid (149mg).
[443] LCMS Rt = 2.793 minutes; m/z [M+11]+ = 791.3
[444] 'H NMR (CDC13) Characteristic peaks: (5 1.20-1.45 (9H, m), 1.58-2.12
(4H, m), 2.14-
2.48 (2H,m), 2.620-5.11 (11H, m), 6.59-6.72 (1H, m), 6.73-6.90 (2H, m), 7.16-
7.64 (11H.
m), 8.25-8.40 (1H, m).
1445] This was converted to the bitartrate salt by a method analogous to that
described in
Example of Synthesis Approach (II).
Example Synthesis Approach (IV)-8
[446] 2-[4-( {[242-(2,3-Difluorophenyflethyl]-4-oxo-1,8-naphthyridin-1(411)-
yl]acetyll {[4'-
(trifluoromethyl)-4-biphenylyl]methyllamino)-1-piperidiny1]-2-methylpropanoic
acid
trifluoroacetate
o
F --.. F
I I F
FO
yo 411 . F
0
r,N
F>1)-L.
HO OH
[447]
F
F
[447] A mixture of 1,1-dimethylethyl 2-methy1-244-( {[4'-(trifluoromethyl)-4-
biphenylyl]methyl}amino)-1-piperidinyl]propanoate (Int. B4) (1 equiv),
[24242,3-
difluorophenypethy1]-4-oxo-1,8-naphthyridin-1(411)-yl]acetic acid (Int. C2)
(1.2 equiv),
DIPEA (3 equiv) and DMF (1.0m1) is stirred at room temperature for 5min. HATU
(1.5
equiv) is added in 1 portion and stirred an additional 5 mm. The crude
reaction mixture is
concentrated, filtered through a plug of silica eluted with acetone and
evaporated to obtain
crude 1,1-dimethylethyl 2-[4-({[2-[2-(2,3-difluorophenyl)ethy1]-4-oxo-1,8-
naphthyridin-
1(41/)-yl]acetyl} { [4'-(trifluoromethyl)-4-biphenylyl]methyl } amino)-1-
piperidiny1]-2-methyl
propanoate.
10566615.1
CA 02588369 2007-05-11
- 57 -
[448] The proponate, without isolation, is dissolved in a 1:1 mixture of TFA
and DCM and
stirred at RT for 4h. Evaporation and prepative HPLC (Method A) gives the
captioned
compound.
[449] Other salts can be prepared by conventional means. The free base can
also be
prepared by conventional means.
Example Synthesis Approach (IV)-9
[450] 2- [4-( [242-(2,3-Difluorophenypethyl] -4-oxo-1(411)-quinazolinyl]
acetyl} {[4'-
(trifluoromethyl)-4-biphenylyl]methyl}amino)-1-piperidiny1]-2-methylpropanoic
acid
trifluoroacetate
F, NI!
,F; el
0
F>1)t,
OH
[451] A mixture of 1,1-dimethylethyl 2-methy1-2-[4-({[4'-(trifluoromethyl)-4-
biphenylAmethyllamino)-1-piperidinyl]propanoate (Int. B4) (1 equiv), [21242,3-
difluorophenypethy1]-4-oxo-1(4H)-quinazolinyl]acetic acid (Int. Cl) (1.2
equiv), DIPEA (3
equiv) and DMF (1.0m1) is stirred at room temperature for 5min. HATU (1.5
equiv) is added
in 1 portion and stirred an additional 5 min. The crude reaction mixture is
concentrated,
filtered through a plug of silica eluted with acetone and evaporated to obtain
crude 1,1-
dimethylethyl 2-[4-( {[2-[2-(2,3-difluorophenyl)ethy1]-4-oxo-1(411)-
quinazolinyl]acetyll {[4'-
(trifluoromethyl)-4-biphenylyl]methyl}amino)-1-piperidiny1]-2-
methylpropanoate.
[452] The proponate, without isolation, is dissolved in a 1:1 mixture of TFA
and DCM and
stirred at RT for 4h. Evaporation and prepative HPLC (Method A) gives the
captioned
compound.
Example Synthesis Approach (IV)-1O
[453] Methyl 2-[4-({[2-[2-(2,3-difluorophenypethy1]-4-oxopyrido[2,3-
d]pyrimidin-1(411)-
yl]acetyl} {[4'-(trifluoromethyl)-4-biphenylyl]methyl} amino)-1-piperidiny1]-2-
methyl-
propanoate
10566615.!
CA 02588369 2007-05-11
-58-
F
F
IljY)
Sy
F
0
14541 A mixture of [242-(2,3-difluorophenypethy1]-4-oxopyrido[2,3-d]pyrimidin-
1(411)-
yliacetic acid (Int. D1) (20.7g, 1.3 equiv), methyl 2-methy1-2-[4-({[4'-
(trifluoromethyl)-4-
biphenylyl]methyl} amino)-1-piperidinyl]propanoate (Int. B1) (20.0g, 1.3
equiv), DIPEA
(24.0m1, 3 equiv) and DMF (184m1) was mechanically stirred, then HATU (27.1g,
1.5 equiv)
was added in one portion and stirring continued for 2h. The reaction mixture
was partioned
between diethyl ether/THF (1:1) and sodium carbonate (1M, excess). The organic
layer was
washed with water and brine, dried and evaporated. Chromatography was run
sequentially
on three silica columns (firstly 3:1 Et0Ac/hexanes; secondly 2% Me0H in DCM;
thirdly 1:1
Et0Ac/hexanes to 100% Et0Ac). Product fractions were evaporated to obtain the
desired
product as an amorphous pink solid (27.5g).
[455] LCMS Rt = 2.702 minutes; m/z [M+H]+ = 762.3
[456] Crystallisation: A mixture of methyl 2-[4-({[2-[2-(2,3-
difluorophenypethy1]-4-
oxopyrido[2,3-d]pyrimidin-1(411)-yl]acetyl) {[4'-(trifluoromethyI)-4-
biphenylyl]methyll-
amino)-1-piperidiny1]-2-methylpropanoate (8.0g) and ethanol (200m1) was warmed
until
fully dissolved. The solution was stirred magnetically for 24h at room
temperature, then
filtered and 7.5g of solid collected. These solvated crystals were placed into
a 60 C vacuum
oven with a nitrogen bleed to hold the vacuum at approximately 630 Ton for 24h
to provide
the unsolvated, crystalline title compound (7.15g), m.p. 150 C.
[457] IHNMR (CD30D) 6 1.25 (3H, s), 1.30 (3H, s), 1.63-1.99 (4H, m), 2.16-2.28
(1H,
m), 2.3-2.43 (1H, m), 2.89-2.98 (1H, m), 2.98-3.08 (2H, m), 3.16-3.30 (3H, m),
3.66-3.69
(3H, m), 4.02/4.38 (1H, 2x br m), 4.69 (1H, s), 4.87 (1H, s), 5.4/5.73 (2H, 2x
s), 6.99-7.19
(3H, m), 7.29-7.35 (1H, m), 7.50-7.61 (3H, m), 7.64-7.82 (5H, m), 8.48-8.57
(1H, m), 8.80-
8.89 (1H, m) See Figure 1 below.
Example Synthesis Approach (IV)-11
[458] Methyl 2444 {[242-(2,3-difluorophenypethyl]-4-oxopyrido[2,3-d]pyrimidin-
1(411)-
yl]acetyl} {[4'-(trifluoromethyl)-4-biphenylyl]methyl} amino)-1-piperidiny1]-2-
methyl-
propanoate 2,3-dihydroxybutanedioate (salt)
10566615.1
CA 02588369 2007-05-11
- 59 -
F FE
I I
F NN F
0
H0,1 C7c1,..),.OH
0 =
OH
[459] Methyl 2-[4-({[2-[2-(2,3-difluorophenyl)ethy1]-4-oxopyrido[2,3-
cflpyrimidin-1(411)-
yl]acetyl} {[4'-(trifluoromethyl)-4-biphenylyl]methyl}amino)-1-piperidiny1]-2-
methylpropanoate (8.5g, 1 equiv) was suspended in methanol (100m1) and warmed
to 50 C
until the solid dissolved. L-Tartaric acid (1.675g, 1.0 equiv) was added in
one portion and
stirred for 30 minutes at room temperature. The solution was concentrated in
vacuo to an off-
white powder that was dried in a vacuum oven at room temperature.
[460] LCMS Rt = 2.697 minutes; m/z [M+H] = 762.3
[461] NMR (d6-DMS0) E 1.17 (3H, s), 1.23 (3H, s), 1.47-1.91 (4H, m), 1.98-
2.41 (1H,
m), 2.16-2.33 (1H, m), 2.80-3.26 (6H, m), 3.50-3.67 (3H, m), 3.95/4.17 (1H, 2x
hr m), 4.61
(1H, s), 4.85 (1H, s), 5.39/5.69 (2H, 2x s), 7.08-7.39 (4H, m), 7.53-7.70 (3H,
m), 7.72-7.97
(5H, m), 8.42-8.54 (1H, m), 8.85-8.95 (1H, m)
Example Synthesis Approach (IV)-12
[462] Ethyl 2-[4-( {[242-(2,3-difluorophenyl)ethy1]-4-oxopyrido[2,3-
cflpyrimidin-1(4H)-y11-
acetyl} { [4'-(trifluoromethyl)-4-biphenylyl]methyl} amino)-1-pip eridinyl] -2-
methylpropanoate
2,3-dihydroxybutanedioate (salt)
Njtr) FE
I I
F tiL
N N
yo el
0 OH
1-1(30H
0 =
OH
[463] A mixture of [2-[2-(2,3-difluorophenypethy1]-4-oxopyrido[2,3-c]pyrimidin-
1(41-1)-
yl]acetic acid (Int. D1) (116mg, 1 equiv), ethyl 2-methy1-244-({[4'-
(trifluoromethyl)-4-
biphenylyl]methyl}amino)-1-piperidinyl]propanoate (Int. B2) (150mg, 1 equiv),
HATU
(151mg, 1.2 equiv), DMF (2.72m1) and DIPEA (0.17m1, 3 equiv) was shaken at
room
temperature for 3.25h. The reaction mixture was partitioned between ethyl
acetate/ methanol
and aqueous sodium bicarbonate, the organic layer was brine-washed, dried and
treated with
activated charcoal (250mg). Flash chromatography (silica, 3-4% methanol in
DCM) gave
10566615.1
CA 02588369 2007-05-11
- 60 -
ethyl 2-[4-(1[212-(2,3-difluorophenypethy1]-4-oxopyrido[2,3-d]pyrimidin-1(411)-
y11-
acetyl} {{4'-(trifluoromethyl)-4-biphenylyl]methyllamino)-1-piperidiny1]-2-
methylpropanoate
as a white solid (178mg).
[464] LCMS Rt = 2.58 minutes; ni/z [M+Hr = 776.3
[465] 11-1 NMR (CDC13) 5 1.20-1.40 (9H, m), 1.56-2.02 (4H, m), 2.19-2.44 (2H,
m), 2.88-
3.20, (4H, m), 3.22-3.40 (2H, m), 3.81/4.58 (1H, 2x m), 4.11-4.27 (2H, m),
4.69/4.84 (2H, 2x
s), 5.17/5.49 (2H, 2x s), 6.95-7.14 (3H, m), 7.25-7.31 (1H, m), 7.38-7.54 (3H,
m), 7.54, 7.61
(1H, m), 7.62-7.79 (4H, m), 8.57-8.75 (2H, m)
1466] This was converted to the bitartrate salt by a method analogous to that
described for
Example of Synthesis Approach (II).
Example Synthesis Approach (IV)-13
[467] Ethyl 2- {4-[1[242-(2,3-difluorophenypethy1]-4-oxopyrido[2,3-d]pyrimidin-
1(411)-
yliacetyl} ({4'4(trifluoromethypoxy]-4-biphenylyl}methypamino]-1-piperidinyl} -
2-
methylpropanoate 2,3-dihydroxybutanedioate (salt)
1 I
F
N N
,N OH 0
0OH
[468] A mixture of [212-(2,3-difluorophenyl)ethy1]-4-oxopyrido[2,3-c]pyrimidin-
1(411)-
yl]acetic acid (Int. D1) (114mg, 1.1 equiv), ethyl 2-methy1-2-
{44({4'4(trifluoromethyDoxy]-
4-biphenylyl}methypamino]-1-piperidinyllpropanoate (Int. B4) (139mg, 1 equiv),
DMF
(1.2m1) and DIPEA (0.16m1, 3 equiv) was shaken at room temperature for 30 min,
then
HATU (176mg, 1.5 equiv) was added and shaking continued for 3h. Reverse phase
HPLC
(Preparative Method B) gave ethyl 2- {44 {[242-(2,3-difluorophenypethy11-4-oxo-
1(411)-
quinazolinyflacetyl}({4'1(trifluoromethypoxy]-4-biphenylyllmethyl)amino]-1-
piperidiny11-2-methylpropanoate as a white solid (166mg).
[469] LCMS Rt = 2.87 minutes; m/z [M+H]+ = 792.3
[470] 'H NMR (CDC13) 5 1.18-1.42 (9H, m), 1.54-2.04 (4H, m), 2.12-2.46 (2H,
m), 2.86-
3,21 (4H, m), 3.21-3.41 (2H, m), 3.79/4.57 (1H, 2x m), 4.10-4.27 (2H, m), 4.68
(1H, s), 4.82
(1H, s), 5.17 (1H, s), 5.47 (1H, s), 6.94-7.16 (3H, m), 7.20-7.36 (3H, m),
7.37-7.48 (3H, m),
7.48-7.61 (3H, m), 8.56-8.76 (2H, m).
10566615.1
CA 02588369 2007-05-11
- 61 -
[471] This was converted to the bitartrate salt by a method analogous to that
described for
Example of Synthesis Approach (II).
Example Synthesis Approach (IV)-14
[472] 1-Methylethyl 2-[4-( [242-(2,3-difluorophenypethyl] -4-oxopyrido [2,3 -
d]pyrimidin-
1(4H)-yljacetyll {[4'-(trifluoromethyl)-4-biphenylyl]methyl}amino)-1-
piperidiny1]-2-
methylpropanoate 2,3-dihydroxybutanedioate (salt)
N)L
F
I I
N N
Lo el
0 rN OH 0
HO
0 =
OH
1473] A mixture of 1-methylethyl 2-methy1-244-({[4'-(trifluoromethyl)-4-
biphenyly1]-
methyl} amino)-1-piperidinyl]propanoate (Int. B3) (420 mg, 1 equiv), [2-[2-
(2,3-
difluorophenypethy1]-4-oxopyrido[2,3-d]pyrimidin-1(411)-yl]acetic acid (Int.
D1) (300mg, 1
equiv), HATU (396mg, 1.2 equiv), DIPEA (0.22m1, 1.5 equiv) and DMF (3.0m1) was
stirred
at room temperature for 30 min. The crude reaction mixture was applied
directly to reverse-
phase HPLC (Preparative Method A) to obtain 1-methylethyl 244-(112-[2-(2,3-
difluorophenypethy1]-4-oxopyrido[2,3-d]pyrimidin-1(4H)-yl]acetyl} {[4'-
(trifluoromethyl)-4-
biphenylyl]methyl} amino)-1-piperidiny1]-2-methylpropanoate (171mg).
[474] LCMS Rt = 2.837 minutes; m/z [M+11]+ = 790.3
[475] 1HNMR (CD30D) (5 1.16-1.37 (12H, m), 1.62-2.01 (4H, m), 2.27-2.55 (2H,
m), 2.95-
3.12 (3H, m), 3.12-3.29 (3H, m), 4.06/4.40 (1H, 2x br m), 4.71 (1H, s), 4.89
(1H, s), 4.92-
5.07 (1H, m), 5.43/5.76 (2H, 2x s), 7.00-7.21 (3H, m), 7.29-7.38 (1H, m), 7.49-
7.65 (3H, m),
7.65-7.87 (5H, m), 8.48-8.58 (1H, m), 8.81-8.90 (1H, m).
[476] This was converted to the bitartrate salt by a method analogous to that
described for
Example of Synthesis Approach (II).
Example Synthesis Approach (IV)-15
[477] 1-Methylethyl 2- {4-[ {[2-[2-(2,3-difluorophenypethyl]-4-oxopyrido[2,3-
d]pyrimidin-
1 (411)-yl] acetyl}(14'-[(trifluoromethypoxy]-4-biphenyly1}methyl)amino]-1-
piperidinyl} -2-
methylpropanoate 2,3-dihydroxybutanedioate (salt)
10566615.1
CA 02588369 2007-05-11
- 62 -
F 11 I
FsN N
yo 41 (D)<FF
OH
N H0,1(1.A0H
0
OH
14781 A mixture of 1-methylethyl 2-methy1-2-{44({4'-[(trifluommethypoxy]-4-
biphenylyl}methypamino]-1-piperidinyllpropanoate (Int. B5) (80 mg, 1 equiv),
[2-[2-(2,3-
difluorophenypethyl]-4-oxopyrido[2,3-cflpyrimidin-1(411)-yl]acetic acid (Int.
D1) (67mg, 1
equiv), HATU (400mg, 5 equiv), DIPEA (0.22m1, 1.5 equiv) and DMF (2.0m1) was
stirred at
room temperature for 30 mm. The crude reaction mixture was applied directly to
reverse-
phase HPLC (Preparative Method A) to obtain 1-methylethyl 2-{44112-[2-(2,3-
difluorophenypethyl]-4-oxopyrido[2,3-c]pyrimidin-1 (4 11)-yl] acetyl}({4'-
[(trifluoromethypoxy]-4-biphenyly1}methyDamino]-1-piperidiny1}-2-
methylpropanoate
(25mg).
[479] LCMS Rt = 2.952 minutes; rn/z [M+H] = 806.4
[480] NMR (DMSO-d6) (5 1.09-1.25 (12H, m), 1.47-1.91 (4H, m), 2.05-2.20
(1H, m),
2.21-2.38 (1H, m), 2.87-3.07 (3H, m), 3.08-3.22 (3H, m), 3.95/4.17 (1H, 2x br
m), 4.59 (1H,
s), 4.75-4.97 (2H, m), 5.38/5.68 (2H, 2x s), 7.90-7.21 (1H, m), 7.21-7.36 (3H,
m), 7.42-7.55
(3H, m), 7.55-7-64 (2H, m), 7.66-7.77 (2H, m), 7.77-7.85 (1H, m), 8.43-8.52
(1H, m), 8.86-
8.95 (1H, m)
14811 This was converted to the bitartrate salt by a method analogous to that
described for
Example of Synthesis Approach (II).
Example Synthesis Approach (IV)-16
14821 Methyl 2-[4-({[242-(2,4-difluorophenypethy1]-4-oxopyrido[2,3-d]pyrimidin-
1 (4 I-1)-
yllacetyll {[4'-(trifluoromethyl)-4-biphenylyl]methyl} amino)-1-pip eridinyl] -
2-
methylpropanoate 2,3-dihydroxybutanedioate (salt)
WinFF
I
N N F
OH
HO
N OH
0 =
OH
10566615.1
CA 02588369 2007-05-11
- 63 -
14831 A mixture of [2-[2-(2,4-difluorophenypethy1]-4-oxopyrido[2,4-d]pyrimidin-
1(411)-
yl]acetic acid (Int. D2) (100mg, 1 equiv), methyl 2-methy1-244-({[4'-
(trifluoromethyl)-4-
biphenylyl]methyl}amino)-1-piperidinyl]propanoate (Int. B1) (130mg, 1.03
equiv), DIPEA
(0.16m1, 3 equiv), acetonitrile (2m1) and HATU (130mg, 1.2 equiv) was stirred
at room
temperature for lh, then evaporated and redissolved in acetonitrile.
Purification by reverse
phase HPLC (Preparative Method B) gave methyl 2-[4-({[242-(2,4-
difluorophenypethy11-4-
oxopyrido[2,3-cflpyrimidin-1(4H)-yl]acetyl) {[4'-(trifluoromethyl)-4-
biphenylyl]methyl} amino)-1-piperidiny1]-2-methylpropano ate (145mg).
[484] LCMS Rt = 2.716 minutes; m/z [M+H]+ = 762.3
[485] 1H NMR (CDC13) .5 1.27 (3H, s), 1.33 (3H, s), 1.69-1.98 (4H, m), 2.22-
2.29 (1H, m),
2.36-2.43 (1H, m), 2.96-3.08 (3H, m), 3.13-3.24 (3H, m), 3.69-3.72 (3H, m),
4.04/4.41 (1H,
2x br m), 4.72 (1H, s), 4.91 (1H, s), 5.41/5.73 (2H, 2x s), 6.84-6.97 (2H, m),
7.34-7.44 (2H,
m), 7.54-7.63 (3H, m), 7.69-7.83 (5H, m), 8.55-8.60 (111, m), 8.86-8.91 (1H,
m).
[486] This was converted to the bitartrate salt by a method analogous to that
described for
Example of Synthesis Approach (II).
Example Synthesis Approach (IV)-17
[487] Ethyl 2-[4-( {[2-[2-(2,4-difluorophenyl)ethyl]-4-oxopyrido[2,3-
d]pyrimidin-1(411)-
yl] acetyl} {[4'-(trifluoromethyl)-4-biphenylyl]methyllamino)-1-piperidiny1]-2-
methylpropanoate 2,3-dihydroxybutanedioate (salt)
I I
40 NN
N 9H 0
H0,11,011
=
0 OH
[488] A mixture of [2-[2-(2,4-difluorophenypethy1]-4-oxopyrido[2,4-d]pyrimidin-
1(411)-
yllacetic acid (Int. D2) (120mg, 1 equiv), ethyl 2-methy1-244-({[4'-
(trifluoromethyl)-4-
biphenylyl]methyl}amino)-1-piperidinyl]propanoate (Int. B2) (198mg, 1.3
equiv), DMF
(1.4m1) and DIPEA (0.178m1, 3 equiv) was shaken at room temperature for 1.5h,
then HATU
(200mg, 1.5 equiv) was added with vigorous agitation and shaking continued for
1.5h. A
further portion of Intermediate D2 (12mg, 0.1 equiv) was added then shaking
was continued
for 2 days. Reverse phase HPLC (Preparative Method B) gave ethyl 2-[4-( {[2-{2-
(2,4-
10566615.1
CA 02588369 2007-05-11
- 64 -
difluorophenypethy1]-4-oxopyrido[2,3-cflpyrimidin-1(41/)-yllacetyll {[4'-
(trifluoromethyl)-4-
biphenylyl]methyl}amino)-1-piperidiny1]-2-methylpropanoate as a white solid
(170mg).
[489] LCMS Rt = 2.827 minutes; m/z [M+H] = 776.3
[490] 'H NMR (CDC13) Characteristic peaks: (3 1.14-1.43 (9H, m), 1.57-2.05
(4H, m), 2.10-
2.46 (2H, m), 2.84-3.11 (3H, m), 3.12-3.34 (3H, m), 3.65/3.85 (1H, m), 4.06-
4.27 (2H, m),
4.65/4.85 (2H, s), 5.15/5.45 (2H, s), 6.62-6.89 (2H, m), 7.18-7.34 (1H, m),
7.37-7.82 (9H, m),
8.59-8.77 (2H, m).
[491] This was converted to the bitartrate salt by a method analogous to that
described for
Example of Synthesis Approach (II).
Example Synthesis Approach (IV)-18
[492] Ethyl 2- {4-[ {[212-(2,4-difluorophenypethy1]-4-oxopyrido[2,3-
cflpyrimidin-1(411)-
yllacetyl}({4'4(trifluoromethypoxy]-4-biphenyly1}methypamino]-1-piperidiny11-2-
methylpropanoate 2,3-dihydroxybutanedioate (salt)
Njr)I I
40 N N y 0 F
)<F o F
OH
0
HOy),J
. OH
0
OH
[493] A mixture of [242-(2,4-difluorophenyl)ethy1]-4-oxopyrido[2,4-d]pyrimidin-
1(411)-
yl]acetic acid (Int. D2) (114mg, 1.1 equiv), ethyl 2-methy1-2-
{44({4'4(trifluoromethypoxy]-
4-biphenylyllmethypamino]-1-piperidinyl}propanoate (Int. B4) (139mg, 1 equiv),
DMF
(1.2m1) and DIPEA (0.16m1, 3 equiv) was shaken at room temperature, then HATU
(176mg,
1.5 equiv) was added with vigorous agitation and shaking continued for 2h.
Reverse phase
HPLC (Preparative Method B) gave ethyl 2-{44{[242-(2,4-difluorophenypethyl]-4-
oxopyrido[2,3-d]pyrimidin-1(411)-yl]acetyl}({4'4(trifluoromethyl)oxy]-4-
biphenyly1}-
methyl)amino]-1-piperidiny11-2-methylpropanoate as a white solid (149mg).
[494] LCMS Rt = 2.801 minutes; miz [M+H] = 792.3
[495] 'H NMR (CDC13)45 1.18-1.40 (9H, m), 1.61-2.02 (4H, m), 2.20-2.44 (2H,
m), 2.83-
3.35 (6H, br m), 3.79/4.57 (1H, 2x br m), 4.07-4.27 (2H, m), 4.68/4.81 (2H, 2x
s), 5.14/5.46
(2H, 2x br m), 6.62-6.90 (2H, 2x m), 7.18-7.63 (10H, m), 8.59-8.75 (2H, m).
[496] This was converted to the bitartrate salt by a method analogous to that
described for
Example of Synthesis Approach (II).
10566615.1
CA 02588369 2007-05-11
- 65 -
Example Synthesis Approach (IV)-I9
[497] 1-Methylethyl 2-[4-({[242-(2,4-difluorophenyl)ethy11-4-oxopyrido[2,3-
d]pyrimidin-
1(411)-yl] acetyl} {[4'-(trifluoromethyl)-4-biphenylyl]methyl} amino)-1-
piperidiny1]-2-
methylpropanoate 2,3-dihydroxybutanedioate (salt)
NJ'
ii F
N N
yo
F
0
_ OH
0 =
OH
[498] A mixture of 1-methylethyl 2-methyl-244-({[4'-(trifluoromethyl)-4-
biphenylyl]methyl}amino)-1-piperidinyl]propanoate (Int. B3) (70 mg, 1 equiv),
[24242,4-
difluorophenypethy1]-4-oxopyrido[2,4-d]pyrimidin-1(411)-yl] acetic acid (Int.
D2) (52.2mg, 1
equiv), HATU (69mg, 1.2 equiv), DIPEA (0.04m1, 1.5 equiv) and DMF (1.0m1) was
stirred at
room temperature for 10min. The crude reaction mixture was applied directly to
reverse-
phase HPLC (Preparative Method A) to obtain 1-methylethyl 244-W24242,4-
difluorophenypethy1]-4-oxopyrido [2,3-c]pyrimidin-1(411)-yl]acetyl} ([4'-
(trifluoromethyl)-4-
biphenylyl]methyl}amino)-1-piperidiny11-2-methylpropanoate (20mg).
[499] LCMS Rt = 2.910 minutes; m/z [M+H] = 790.4
[500] 1H NMR (d6-DMS0) 6 1.08-1.27 (12H, m), 1.40-1.90 (4H, m), 2.03-2.35 (2H,
m),
2.85-3.24 (6H, m), 3.95/4.17 (1H, 2x br m), 4.61 (1H, s), 4.80-4.97 (2H, m),
5.36/5.67 (2H,
2x s), 6.96-7.10 (1H, m), 7.13-7.28 (1H, m), 7.28-7.38 (1H, m), 7.39-7.54 (1H,
m), 7.54-7.68
(3H, m), 7.72-7.98 (5H, m), 8.43-8.52 (1H, m), 8.86-8.95 (1H, m)
[501] This was converted to the bitartrate salt by a method analogous to that
described for
Example of Synthesis Approach (II).
[502] Example Synthesis Approach (IV)-20
[503] 1-Methylethyl 2- {44 {[242-(2,4-difluorophenypethyl]-4-oxopyrido [2,3-
d]pyrimidin-
1(411)-yll acetyl} ( {4'-[(trifluoromethyl)oxy]-4-biphenyly1} methypamino]-1-
piperidinyll -2-
methylpropanoate 2,3-dihydroxybutanedioate (salt)
10566615.1
CA 02588369 2007-05-11
- 66 -
N)
I I
1101 N./^rey 0FF o up
0 ='
OH
[504] A mixture of 1-methylethyl 2-methy1-2-{4-[({4'4(trifluoromethypoxy]-4-
biphenylyllmethypamino]-1-piperidinyl}propanoate (Int. B5) (80 mg, 1 equiv),
[24242,3-
difluorophenypethy1]-4-oxopyrido[2,4-d]pyrimidin-1(41i)-yl]acetic acid (Int.
D2) (57mg, 1
equiv), HATU (76mg, 1.2 equiv), DIPEA (0.04m1, 1.5 equiv) and DMF (1.0m1) was
stirred at
room temperature for 10min. The crude reaction mixture was applied directly to
reverse-
phase HPLC (Preparative Method A) to obtain 1-methylethyl 2- {44 {[242-(2,4-
difluorophenypethy1]-4-oxopyrido[2,3-d]pyrimidin-1(4H)-yl] acetyl } (
{4'4(trifluoromethyl)-
oxy]-4-biphenylyl}methypamino]-1-piperidinyll-2-methylpropanoate (47mg).
1505] LCMS Rt = 2.909 minutes; m/z [M+H] = 806.4
[506] 1H NMR (d6-DMS0) 6 1.09-1.27 (12H, m), 1.50-1.90 (4H, m), 2.03-2.17 (1H,
m),
2.20-2.37 (1H, m), 2.88-3.18 (6H, m), 3.94/4.17 (1H, 2x m), 4.60 (1H, s), 4.74-
4.96 (2H, m),
5.36/5.66 (2H, 2x br s), 6.96-7.09 (1H, m), 7.14-7.32 (2H, m), 7.39-7.55 (4H,
m), 7.55-7.66
(2H, m), 7.66-7.87 (3H, m), 8.43-8.54 (1H, m), 8.85-8.96 (1H, m).
[507] This was converted to the bitartrate salt by a method analogous to that
described for
Example of Synthesis Approach (II).
Example Synthesis Approach (IV)-21
[508] Methyl 2- {41 4212-(2,3-difluorophenypethy11-4-oxopyrido[2,3-c]pyrimidin-
1(411)-
yl] acetyl } ( {4'4(trifluoromethypoxy]-4-biphenylyll methyl)amino]-1-pip
eridinyl} -2-
methylpropanoate dihydroxybutanedioate (salt)
NIX)I I
F,
w F
HO C.'?F
. OH
0 =
OH
[509] A mixture of methyl 2-methy1-2-{4-[({4'1(trifluoromethyDoxy]-4-
biphenylyllmethypamino]-1 piperidinyllpropanoate (Int. B6) (145 mg, 1 equiv),
[242-(2,3-
difluorophenypethy1]-4-oxopyrido[2,3-d]pyrimidin-1(4H)-yliacetic acid (Int.
D1) (122mg, 1
10566615.1
CA 02588369 2007-05-11
- 67 -
equiv), DIPEA (0.084m1, 1.5 equiv) and DMF (2.0m1) was stirred at room
temperature for
5min. The HATU (160mg, 1.3 equiv) was added in 1 portion and stirred an
additional 1 hour
under nitrogen. The crude reaction mixture was applied directly to reverse-
phase HPLC
(Preparative Method A) to obtain methyl 2- {4-[ {[242-(2,3-
difluorophenypethyl]-4-
oxopyrido[2,3-d]pyrimidin-1(4H)-yllacety1}({4'-[(trifluoromethyDoxy]-4-
biphenylyl}methyl)amino]-1-piperidinyl} -2-methylpropanoate (116mg).
[510] LCMS Rt = 2.721 minutes; m/z [M+H] = 778.3
[511] 11-1 NMR (CDC13) Characteristic peaks: 6 1.53-1.62 (6H, m), 3.46-5.99
(22H, m),
7.01-7.21 (3H, m), 7.30-7.43 (3H, m), 7.50-7.78 (6H, m), 8.54-8.60 (1H, m),
8.86-8.94 (1H,
m).
[512] This was converted to the bitartrate salt by a method analogous to that
described for
Example of Synthesis Approach (II).
Example Synthesis Approach (IV)-22
[513] 2-[4-( {[2-[2-(2,3-Difluorophenypethy1]-4-oxopyrido[2,3-d]pyrimidin-
1(41/)-
yl]acetyll {[4'-(trifluoromethyl)-4-biphenylyl]methyl}amino)-1-piperidiny1]-2-
methylpropanoic acid trifluoroacetate
0
N'ArI I
F. N N FF
ty0
0
F>rfi,
OH
[514] A mixture of 1,1-dimethylethyl 2-methy1-244-({[4'-(trifluoromethyl)-4-
biphenylyl]methyl}amino)-1-piperidinyl]propanoate (Int. B7) (150 mg, 1 equiv),
[2-[2-(2,3-
difluorophenyl)ethy1]-4-oxopyrido[2,3-d]pyrimidin-1(411)-yl] acetic acid (Int.
D1) (130mg,
1.2 equiv), DIPEA (0.164m1, 3 equiv) and DMF (1.0m1) was stirred at room
temperature for
5min. HATU (180mg, 1.5 equiv) was added in 1 portion and stirred an additional
5 min.
The crude reaction mixture was concentrated, filtered through a plug of silica
eluted with
acetone and evaporated to obtain crude 1,1-dimethylethyl 2-[4-({[2-[2-(2,3-
difluorophenypethy1]-4-oxopyrido[2,3-d]pyrimidin-1(414)-yl]acetyl} {[4'-
(trifluoromethyl)-4-
biphenylyl]methyl}amino)-1-piperidiny1]-2-methylpropanoate.
[515] LCMS Rt = 2.823 minutes; m/z [M+Hr = 804.4
10566615.1
CA 02588369 2007-05-11
- 68 -
[516] This intermediate, without isolation, was dissolved in a 1:1 mixture of
TFA and DCM
and stirred at RT for 4h. Evaporation and prepative HPLC (Method A) gave the
desired 2-[4-
( { [2-[2-(2,3-difluorophenypethy1]-4-oxopyrido [2,3 -d]pyrimidin-1(41/)-yl]
acetyl} { [4'-
(trifluoromethyl)-4-biphenylyl]methyl} amino)-1-piperidiny1]-2-methylpropanoic
acid
trifluoroacetate (70mg).
[517] LCMS Rt = 2.554 minutes; m/z [M-1-11]+ = 748.2
[518] 11-1 NMR (d6-DMS0) d 1.44 (3H, s), 1.51 (3H, s), 1.70-2.30 (4H, m), 2.41-
2.56 (2H,
m), 2.94-3.54 (6H, m), 4.44-4.95 (3H, m), 5.42/5.76 (2H, 2x hr s), 7.07-7.38
(4H, m), 7.54-
7.75 (3H, m), 7.76-7.99 (5H, m), 8.42-8.54 (1H, m), 8.85-8.98 (1H, m).
[519] Other salts can be prepared by conventional means. The free base can
also be
prepared by conventional means.
[520] In some embodiments, compounds useful as inhibitors of Lp-PLA2 useful in
the
methods as disclosed herein are:
[521] 1-(N-(2-(diethylamino)ethyl)-N-(4-(4-trifluoromethylphenyl)benzy1)-
aminocarbonylmethyl)-2-(4-fluorobenzypthio-5,6-trimethylenepyrimidin-4-one,
also referred
to as "SB480848" or the USAN name "darapladib" which is a pyrimidinone-based
compound
and a reversible inhibitor of Lp-PLA2 and is used in the Examples herein,
[522] N-(2-diethylaminoethyl)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4,5,6,7-
tetrahydro-
cyclopentapyrimidin-1-y1]-N-(4'-trifluoromethyl-bipheny1-4-ylmethypacetamide;
[523] N-(1-(2-Methoxyethyppiperidin-4-y1)-242-(2,3-difluorobenzylthio)-4-oxo-
4H-
quinolin-l-y1]-N-(4'-trifluoromethylbipheny1-4-ylmethypacetamide; and
[524] methyl 2-[4-({[242-(2,3-difluorophenyl)ethy1]-4-oxopyrido[2,3-
d]pyrimidin-1(41-1)-
yl] acetyl} {[4'-(trifluoromethyl)-4-biphenylyl]methyll amino)-1-piperidiny1]-
2-
methylpropanoate.
[525] Pharmaceutically acceptable salts of 1-(N-(2-(diethylamino)ethyl)-N-(4-
(4-
trifluoromethylphenyl)benzy1)-aminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5,6-
trimethylenepyrimidin-4-one, AKA SB480848, and used in the Examples herein; N-
(2-
diethylaminoethyl)-2-[2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-4,5,6,7-tetrahydro-
cyclopentapyrimidin-1-y1]-N-(4'-trifluoromethyl-bipheny1-4-ylmethypacetamide;
N-(1-(2-
Methoxyethyl)piperidin-4-y1)-242-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-l-
y1]-N-(4'-
trifluoromethylbipheny1-4-ylmethypacetamide; and methyl 2-[4-( {[242-(2,3-
difluorophenypethy1]-4-oxopyrido[2,3-d]pyrimidin-1(4H)-yl] acetyl } { [4'-
(trifluoromethyl)-4-
10566615.1
CA 02588369 2007-05-11
- 69 -
biphenylyl]methyl}amino)-1-piperidiny1]-2-methylpropanoate are also useful as
inhibitors of
Lp-PLA2 for use in the methods as disclosed herein..
[526] Nucleic acid inhibitors of Lp-PLA2
[527] In some embodiments, agents that inhibit Lp-PLA2 are nucleic acids.
Nucleic acid
inhibitors of Lp-PLA2 are, for example, but not are limited to, RNA
interference-inducing
molecules, for example but are not limited to siRNA, dsRNA, stRNA, shRNA and
modified
versions thereof; where the RNA interference molecule silences the gene
expression of Lp-
PLA2. In some embodiments, the nucleic acid inhibitor of Lp-PLA2 is an anti-
sense
oligonucleic acid, or a nucleic acid analogue, for example but are not limited
to DNA, RNA,
peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), or locked
nucleic acid
(LNA) and the like. In alternative embodiments, the nucleic acid is DNA or
RNA, and
nucleic acid analogues, for example PNA, pcPNA and LNA. A nucleic acid can be
single or
double stranded, and can be selected from a group comprising nucleic acid
encoding a protein
of interest, oligonucleotides, PNA, etc. Such nucleic acid sequences include,
for example, but
are not limited to, nucleic acid sequence encoding proteins that act as
transcriptional
repressors, antisense molecules, ribozymes, small inhibitory nucleic acid
sequences, for
example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi),
antisense
oligonucleotides etc.
[528] In some embodiments single-stranded RNA (ssRNA), a form of RNA
endogenously
found in eukaryotic cells can be used to form an RNAi molecule. Cellular ssRNA
molecules
include messenger RNAs (and the progenitor pre-messenger RNAs), small nuclear
RNAs,
small nucleolar RNAs, transfer RNAs and ribosomal RNAs. Double-stranded RNA
(dsRNA)
induces a size-dependent immune response such that dsRNA larger than 30bp
activates the
interferon response, while shorter dsRNAs feed into the cell's endogenous RNA
interference
machinery downstream of the Dicer enzyme.
[529] Lp-PLA2 can be reduced by inhibition of the expression of Lp-PLA2
polypeptide or
by "gene silencing" methods commonly known by persons of ordinary skill in the
art.
[530] RNA interference (RNAi) provides a powerful approach for inhibiting the
expression
of selected target polypeptides. RNAi uses small interfering RNA (siRNA)
duplexes that
target the messenger RNA encoding the target polypeptide for selective
degradation. siRNA-
dependent post-transcriptional silencing of gene expression involves cutting
the target
messenger RNA molecule at a site guided by the siRNA.
10566615.1
CA 02588369 2007-05-11
- 70 -
[531] RNA interference (RNAi) is an evolutionally conserved process whereby
the
expression or introduction of RNA of a sequence that is identical or highly
similar to a target
gene results in the sequence specific degradation or specific post-
transcriptional gene
silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene
(see
Coburn, G. and Cullen, B. (2002)J. of Virology 76(18):9225), thereby
inhibiting expression
of the target gene. In one embodiment, the RNA is double stranded RNA (dsRNA).
This
process has been described in plants, invertebrates, and mammalian cells. In
nature, RNAi is
initiated by the dsRNA-specific endonuclease Dicer, which promotes processive
cleavage of
long dsRNA into double-stranded fragments termed siRNAs. siRNAs are
incorporated into a
protein complex (termed "RNA induced silencing complex," or "RISC") that
recognizes and
cleaves target mRNAs. RNAi can also be initiated by introducing nucleic acid
molecules,
e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the
expression of target
genes. As used herein, "inhibition of target gene expression" includes any
decrease in
expression or protein activity or level of the target gene or protein encoded
by the target gene
as compared to a situation wherein no RNA interference has been induced. The
decrease can
be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as
compared to
the expression of a target gene or the activity or level of the protein
encoded by a target gene
which has not been targeted by an RNA interfering agent.
15321 "Short interfering RNA" (siRNA), also referred to herein as "small
interfering RNA"
is defined as an agent which functions to inhibit expression of a target gene,
e.g., by RNAi.
An siRNA can be chemically synthesized, can be produced by in vitro
transcription, or can be
produced within a host cell. In one embodiment, siRNA is a double stranded RNA
(dsRNA)
molecule of about 15 to about 40 nucleotides in length, preferably about 15 to
about 28
nucleotides, more preferably about 19 to about 25 nucleotides in length, and
more preferably
about 19, 20, 21, 22, or 23 nucleotides in length, and can contain a 3' and/or
5' overhang on
each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The
length of the
overhang is independent between the two strands, i.e., the length of the
overhang on one
strand is not dependent on the length of the overhang on the second strand.
Preferably the
siRNA is capable of promoting RNA interference through degradation or specific
post-
transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).
[533] siRNAs also include small hairpin (also called stem loop) RNAs (shRNAs).
In one
embodiment, these shRNAs are composed of a short (e.g., about 19 to about 25
nucleotide)
antisense strand, followed by a nucleotide loop of about 5 to about 9
nucleotides, and the
10566615.1
CA 02588369 2013-10-22
- 71 -
analogous sense strand. Alternatively, the sense strand can precede the
nucleotide loop
structure and the antisense strand can follow. These shRNAs can be contained
in plasmids,
retroviruses, and lentiviruses and expressed from, for example, the pol III U6
promoter, or
another promoter (see, e.g., Stewart, et al. (2003) RNA Apr;9(4):493-501.
[534] The target gene or sequence of the RNA interfering agent can be a
cellular gene or
genomic sequence, e.g. the Lp-PLA2 sequence. An siRNA can be substantially
homologous
to the target gene or genomic sequence, or a fragment thereof As used in this
context, the
term "homologous" is defined as being substantially identical, sufficiently
complementary, or
similar to the target mRNA, or a fragment thereof, to effect RNA interference
of the target.
In addition to native RNA molecules, RNA suitable for inhibiting or
interfering with the
expression of a target sequence include RNA derivatives and analogs.
Preferably, the siRNA
is identical to its target.
[535] The siRNA preferably targets only one sequence. Each of the RNA
interfering
agents, such as siRNAs, can be screened for potential off-target effects by,
for example,
expression profiling. Such methods are known to one skilled in the art and are
described, for
example, in Jackson et al, Nature Biotechnology 6:635-637, 2003. In addition
to expression
profiling, one can also screen the potential target sequences for similar
sequences in the
sequence databases to identify potential sequences which can have off-target
effects. For
example, according to Jackson et al. (Id.) 15, or perhaps as few as 11
contiguous nucleotides
of sequence identity are sufficient to direct silencing of non-targeted
transcripts. Therefore,
one can initially screen the proposed siRNAs to avoid potential off-target
silencing using the
sequence identity analysis by any known sequence comparison methods, such as
BLAST.
[536] siRNA molecules need not be limited to those molecules containing only
RNA, but,
for example, further encompasses chemically modified nucleotides and non-
nucleotides, and
also include molecules wherein a ribose sugar molecule is substituted for
another sugar
molecule or a molecule which performs a similar function. Moreover, a non-
natural linkage
between nucleotide residues can be used, such as a phosphorothioate linkage.
For example,
siRNA containing D-arabinofuranosyl structures in place of the naturally-
occurring D-
ribonucleosides found in RNA can be used in RNAi molecules according to the
present
invention (U.S. Pat. No. 5,177,196). Other examples include RNA molecules
containing the
o-linkage between the sugar and the heterocyclic base of the nucleoside, which
confers
nuclease resistance and tight complementary strand binding to the
oligonucleotidesmolecules
CA 02588369 2007-05-11
- 72 -
similar to the oligonucleotides containing 2'-0-methyl ribose, arabinose and
particularly D-
arabinose (U.S. Pat. No. 5,177,196).
[537] The RNA strand can be derivatized with a reactive functional group of a
reporter
group, such as a fluorophore. Particularly useful derivatives are modified at
a terminus or
termini of an RNA strand, typically the 3' terminus of the sense strand. For
example, the 2'-
hydroxyl at the 3' terminus can be readily and selectively derivatized with a
variety of
groups.
1538] Other useful RNA derivatives incorporate nucleotides having modified
carbohydrate
moieties, such as 2'0-allcylated residues or 2'-0-methyl ribosyl derivatives
and 2'-0-fluoro
ribosyl derivatives. The RNA bases can also be modified. Any modified base
useful for
inhibiting or interfering with the expression of a target sequence can be
used. For example,
halogenated bases, such as 5-bromouracil and 5-iodouracil can be incorporated.
The bases
can also be alkylated, for example, 7-methylguanosine can be incorporated in
place of a
guanosine residue. Non-natural bases that yield successful inhibition can also
be
incorporated.
[539] The most preferred siRNA modifications include 2'-deoxy-2'-fluorouridine
or locked
nucleic acid (LNA) nucleotides and RNA duplexes containing either
phosphodiester or
varying numbers of phosphorothioate linkages. Such modifications are known to
one skilled
in the art and are described, for example, in Braasch et al., Biochemistry,
42: 7967-7975,
2003. Most of the useful modifications to the siRNA molecules can be
introduced using
chemistries established for antisense oligonucleotide technology. Preferably,
the
modifications involve minimal 2'-0-methyl modification, preferably excluding
such
modification. Modifications also preferably exclude modifications of the free
5'-hydroxyl
groups of the siRNA.
[540] siRNA and miRNA molecules having various "tails" covalently attached to
either
their 3'- or to their 5'-ends, or to both, are also known in the art and can
be used to stabilize
the siRNA and miRNA molecules delivered using the methods of the present
invention.
Generally speaking, intercalating groups, various kinds of reporter groups and
lipophilic
groups attached to the 3' or 5' ends of the RNA molecules are well known to
one skilled in the
art and are useful according to the methods of the present invention.
Descriptions of
syntheses of 3'-cholesterol or 3'-acridine modified oligonucleotides
applicable to preparation
of modified RNA molecules useful according to the present invention can be
found, for
example, in the articles: Gamper, H. B., Reed, M. W., Cox, T., Virosco, J. S.,
Adams, A. D.,
10566615.1
CA 02588369 2007-05-11
- 73 -
Gall, A., Scholler, J. K., and Meyer, R. B. (1993) Facile Preparation and
Exonuclease
Stability of 3'-Modified Oligodeoxynucleotides. Nucleic Acids Res. 21145-150;
and Reed,
M. W., Adams, A. D., Nelson, J. S., and Meyer, R. B.,Jr. (1991) Acridine and
Cholesterol-
Derivatized Solid Supports for Improved Synthesis of 3'-Modified
Oligonucleotides.
Bioconjugate Chem. 2 217-225 (1993).
[541] Other siRNAs useful for targeting Lp-PLA2 expression can be readily
designed and
tested. Accordingly, siRNAs useful for the methods described herein include
siRNA
molecules of about 15 to about 40 or about 15 to about 28 nucleotides in
length, which are
homologous to an Lp-PLA2 gene. Preferably, the Lp-PLA2 targeting siRNA
molecules have
a length of about 19 to about 25 nucleotides. More preferably, the Lp-PLA2
targeting siRNA
molecules have a length of about 19, 20, 21, or 22 nucleotides. The Lp-PLA2
targeting
siRNA molecules can also comprise a 3' hydroxyl group. The Lp-PLA2 targeting
siRNA
molecules can be single-stranded or double stranded; such molecules can be
blunt ended or
comprise overhanging ends (e.g., 5', 3'). In specific embodiments, the RNA
molecule is
double stranded and either blunt ended or comprises overhanging ends.
1542] In one embodiment, at least one strand of the Lp-PLA2 targeting RNA
molecule has a
3' overhang from about 0 to about 6 nucleotides (e.g., pyrimidine nucleotides,
purine
nucleotides) in length. In other embodiments, the 3' overhang is from about 1
to about 5
nucleotides, from about 1 to about 3 nucleotides and from about 2 to about 4
nucleotides in
length. In one embodiment the Lp-PLA2 targeting RNA molecule is double
stranded - one
strand has a 3' overhang and the other strand can be blunt-ended or have an
overhang. In the
embodiment in which the Lp-PLA2 targeting RNA molecule is double stranded and
both
strands comprise an overhang, the length of the overhangs can be the same or
different for
each strand. In a particular embodiment, the RNA of the present invention
comprises about
19, 20, 21, or 22 nucleotides which are paired and which have overhangs of
from about 1 to
about 3, particularly about 2, nucleotides on both 3' ends of the RNA. In one
embodiment,
the 3' overhangs can be stabilized against degradation. In a preferred
embodiment, the RNA
is stabilized by including purine nucleotides, such as adenosine or guanosine
nucleotides.
Alternatively, substitution of pyrimidine nucleotides by modified analogues,
e.g., substitution
of uridine 2 nucleotide 3' overhangs by 2'-deoxythymidine is tolerated and
does not affect
the efficiency of RNAi. The absence of a 2' hydroxyl significantly enhances
the nuclease
resistance of the overhang in tissue culture medium.
10566615.1
CA 02588369 2007-05-11
- 74 -
15431 Lp-PLA2 mRNA has been successfully targeted using siRNAs and such siRNA
or
vectors for preparing them are commercially available, for example from
Invitrogen. In some
embodiments, assesment of the expression and/or knock down of Lp-PLA2 protein
using such
Lp-PLA2 siRNAs can be determined using commercially available kits, for
example but are
not limited to PLAC assay from diaDexus. Others can be readily prepared by
those of skill in
the art based on the known sequence of the target mRNA. To avoid doubt, the
sequence of a
human Lp-PLA2 cDNA is provided at, for example, GenBank Accession Nos. :
U20157
(SEQ ID NO:1) or NM 005084 (SEQ ID NO:2). The sequence at U20157 is the
following
(SEQ ID NO:1):
[544] 1 gctggtcgga ggctcgcagt gctgtcggcg agaagcagtc gggtttggag cgcttgggtc
61 gcgttggtgc gcggtggaac gcgcccaggg accccagttc ccgcgagcag ctccgcgccg
121 cgcCtgagag actaagctga aactgctgct cagctcccaa gatggtgcca cccaaattgc
181 atgtgctttt ctgcctctgc ggctgcctgg ctgtggttta tccttttgac tggcaataca
241 taaatcctgt tgcccatatg aaatcatcag catgggtcaa caaaatacaa gtactgatgg
301 ctgctgcaag ctttggccaa actaaaatcc cccggggaaa tgggccttat tccgttggtt
361 gtacagactt aatgtttgat cacactaata agggcacctt cttgcgttta tattatccat
421 cccaagataa tgatcgcctt gacacccttt ggatcccaaa taaagaatat ttttggggtc
481 ttagcaaatt tcttggaaca cactggctta tgggcaacat tttgaggtta ctctttggtt
541 caatgacaac tcctgcaaac tggaattccc ctctgaggcc tggtgaaaaa tatccacttg
601 ttgttttttc tcatggtctt ggggcattca ggacacttta ttctgctatt ggcattgacc
661 tggcatctca tgggtttata gttgctgctg tagaacacag agatagatct gcatctgcaa
721 cttactattt caaggaccaa tctgctgcag aaatagggga caagtcttgg ctctacctta
781 gaaccctgaa acaagaggag gagacacata tacgaaatga gcaggtacgg caaagagcaa
841 aagaatgttc ccaagctctc agtctgattc ttgacattga tcatggaaag ccagtgaaga
901 atgcattaga tttaaagttt gatatggaac aactgaagga ctctattgat agggaaaaaa
961 tagcagtaat tggacattct tttggtggag caacggttat tcagactctt agtgaagatc
1021 agagattcag atgtggtatt gccctggatg catggatgtt tccactgggt gatgaagtat
1081 attccagaat tcctcagccc ctctttttta tcaactctga atatttccaa tatcctgcta
1141 atatcataaa aatgaaaaaa tgctactcac ctgataaaga aagaaagatg attacaatca
1201 ggggttcagt ccaccagaat tttgctgact tcacttttgc aactggcaaa ataattggac
1261 acatgctcaa attaaaggga gacatagatt caaatgtagc tattgatctt agcaacaaag
1321 cttcattagc attcttacaa aagcatttag gacttcataa agattttgat cagtgggact
1381 gcttgattga aggagatgat gagaatctta ttccagggac caacattaac acaaccaatc
1441 aacacatcat gttacagaac tcttcaggaa tagagaaata caattaggat taaaataggt
1501 ttttt
[545] siRNA sequences are chosen to maximize the uptake of the antisense
(guide) strand of
the siRNA into RISC and thereby maximize the ability of RISC to target human
Lp-PLA2
mRNA for degradation. This can be accomplished by scanning for sequences that
have the
lowest free energy of binding at the 5'-terminus of the antisense strand. The
lower free
energy leads to an enhancement of the unwinding of the 5'- end of the
antisense strand of the
siRNA duplex, thereby ensuring that the antisense strand will be taken up by
RISC and direct
the sequence-specific cleavage of the human Lp-PLA2 mRNA.
1546] In a preferred embodiment, the siRNA or modified siRNA is delivered in a
pharmaceutically acceptable carrier. Additional carrier agents, such as
liposomes, can be
added to the pharmaceutically acceptable carrier.
10566615.1
CA 02588369 2007-05-11
- 75 -
[547] In another embodiment, the siRNA is delivered by delivering a vector
encoding small
hairpin RNA (shRNA) in a pharmaceutically acceptable carrier to the cells in
an organ of an
individual. The shRNA is converted by the cells after transcription into siRNA
capable of
targeting, for example, Lp-PLA2. In one embodiment, the vector can be a
regulatable vector,
such as tetracycline inducible vector.
[548] In one embodiment, the RNA interfering agents used in the methods
described herein
are taken up actively by cells in vivo following intravenous injection, e.g.,
hydrodynamic
injection, without the use of a vector, illustrating efficient in vivo
delivery of the RNA
interfering agents, e.g., the siRNAs used in the methods of the invention.
[549] Other strategies for delivery of the RNA interfering agents, e.g., the
siRNAs or
shRNAs used in the methods of the invention, can also be employed, such as,
for example,
delivery by a vector, e.g., a plasmid or viral vector, e.g., a lentiviral
vector. Such vectors can
be used as described, for example, in Xiao-Feng Qin et al. Proc. Natl. Acad.
Sci. U.S.A., 100:
183-188. Other delivery methods include delivery of the RNA interfering
agents, e.g., the
siRNAs or shRNAs of the invention, using a basic peptide by conjugating or
mixing the RNA
interfering agent with a basic peptide, e.g., a fragment of a TAT peptide,
mixing with cationic
lipids or formulating into particles.
[550] As noted, the dsRNA, such as siRNA or shRNA can be delivered using an
inducible
vector, such as a tetracycline inducible vector. Methods described, for
example, in Wang et
al. Proc. Natl. Acad. Sci. 100: 5103-5106, using pTet-On vectors (BD
Biosciences Clontech,
Palo Alto, CA) can be used. In some embodiments, a vector can be a plasmid
vector, a viral
vector, or any other suitable vehicle adapted for the insertion and foreign
sequence and for
the introduction into eukaryotic cells. The vector can be an expression vector
capable of
directing the transcription of the DNA sequence of the agonist or antagonist
nucleic acid
molecules into RNA. Viral expression vectors can be selected from a group
comprising, for
example, reteroviruses, lentiviruses, Epstein Barr virus-, bovine papilloma
virus, adenovirus-
and adeno-associated-based vectors or hybrid virus of any of the above. In one
embodiment,
the vector is episomal. The use of a suitable episomal vector provides a means
of maintaining
the antagonist nucleic acid molecule in the subject in high copy number extra
chromosomal
DNA thereby eliminating potential effects of chromosomal integration.
[551] RNA interference molecules and nucleic acid inhibitors useful in the
methods as
disclosed herein can be produced using any known techniques such as direct
chemical
synthesis, through processing of longer double stranded RNAs by exposure to
recombinant
10566615.!
CA 02588369 2013-10-22
- 76 -
Dicer protein or Drosophila embryo lysates, through an in vitro system derived
from S2 cells,
using phage RNA polymerase, RNA-dependant RNA polymerase, and DNA based
vectors.
Use of cell lysates or in vitro processing can further involve the subsequent
isolation of the
short, for example, about 21-23 nucleotide, siRNAs from the lysate, etc.
Chemical synthesis
usually proceeds by making two single stranded RNA-oligomers followed by the
annealing
of the two single stranded oligomers into a double stranded RNA. Other
examples include
methods disclosed in WO 99/32619 and WO 01/68836 that teach chemical and
enzymatic
synthesis of siRNA. Moreover, numerous commercial services are available for
designing
and manufacturing specific siRNAs (see, e.g., QIAGEN Inc., Valencia, CA and
AMBION
Inc., Austin, TX)
[552] In some embodiments, an agent is protein or polypeptide or RNAi agent
that inhibits
expression of Lp-PLA and/or activity of the Lp-PLA2 protein. In such
embodiments cells can
be modified (e.g., by homologous recombination) to provide increased
expression of such an
agent, for example by replacing, in whole or in part, the naturally occurring
promoter with all
or part of a heterologous promoter so that the cells express the natural
inhibitor agent of Lp-
PLA2, for example protein or miRNA inhibitor of Lp-PLA2 at higher levels. The
heterologous promoter is inserted in such a manner that it is operatively
linked to the desired
nucleic acid encoding the agent. See, for example, PCT International
Publication No. WO
94/12650 by Transkaryotic Therapies, Inc., PCT International Publication No.
WO 92/20808
by Cell Genesys, Inc., and PCT International Publication No. WO 91/09955 by
Applied
Research Systems. Cells also can be engineered to express an endogenous gene
comprising
the agent under the control of inducible regulatory elements, in which case
the regulatory
sequences of the endogenous gene can be replaced by homologous recombination.
Gene
activation techniques are described in U.S. Patent No. 5,272,071 to Chappel;
U.S. Patent No.
5,578,461 to Sherwin et al.; PCT/US92/09627 (W093/09222) by Selden et al.; and
PCT/US90/06436 (W091/06667) by Skoultchi et al. The agent can be prepared by
culturing
transformed host cells under culture conditions suitable to express the miRNA.
The resulting
expressed agent can then be purified from such culture (i.e., from culture
medium or cell
extracts) using known purification processes, such as gel filtration and ion
exchange
chromatography. The purification of the peptide or nucleic acid agent
inhibitor of Lp-PLA2
can also include an affinity column containing agents which will bind to the
protein; one or
more column steps over such affinity resins as concanavalin A-agarose, heparin-
toyopearlTM
or Cibacrom blue 3GA SepharoseTM; one or more steps involving hydrophobic
interaction
CA 02588369 2007-05-11
- 77 -
chromatography using such resins as phenyl ether, butyl ether, or propyl
ether;
immunoaffnity chromatography, or complementary cDNA affinity chromatography.
15531 In one embodiment, the nucleic acid inhibitors of Lp-PLA2 can be
obtained
synthetically, for example, by chemically synthesizing a nucleic acid by any
method of
synthesis known to the skilled artisan. The synthesized nucleic acid
inhibitors of Lp-PLA,
can then be purified by any method known in the art. Methods for chemical
synthesis of
nucleic acids include, but are not limited to, in vitro chemical synthesis
using phosphotriester,
phosphate or phosphoramidite chemistry and solid phase techniques, or via
deoxynucleoside
H-phosphonate intermediates (see U.S. Patent No. 5,705,629 to Bhongle).
15541 In some circumstances, for example, where increased nuclease stability
is desired,
nucleic acids having nucleic acid analogs and/or modified internucleoside
linkages can be
preferred. Nucleic acids containing modified internucleoside linkages can also
be synthesized
using reagents and methods that are well known in the art. For example,
methods of
synthesizing nucleic acids containing phosphonate phosphorothioate,
phosphorodithioate,
phosphoramidate methoxyethyl phosphoramidate, formacetal, thioformacetal,
diisopropylsilyl, acetamidate, carbamate, dimethylene-sulfide (-CH2-S-CH2),
diinethylene-
sulfoxide (-CH2-SO-CH2), dimethylene-sulfone (-CH2-S02-CH2), 2'-0-alkyl, and
2'-deoxy-2'-
fluor ' phosphorothioate internucleoside linkages are well known in the art
(see Uhlmann et
al., 1990, Chem. Rev. 90:543-584; Schneider et al., 1990, Tetrahedron Lett.
31:335 and
references cited therein). U.S. Patent Nos. 5,614,617 and 5,223,618 to Cook,
et al., 5,714,
606 to Acevedo, et al, 5,378,825 to Cook, et al., 5,672,697 and 5,466, 786 to
Buhr, et al., 5,
777,092 to Cook, et al., 5,602,240 to De Mesmacker, et al., 5,610,289 to Cook,
et al. and
5,858,988 to Wang, also describe nucleic acid analogs for enhanced nuclease
stability and
cellular uptake.
15551 Synthetic siRNA molecules, including shRNA molecules, can be obtained
using a
number of techniques known to those of skill in the art. For example, the
siRNA molecule
can be chemically synthesized or recombinantly produced using methods known in
the art,
such as using appropriately protected ribonucleoside phosphoramidites and a
conventional
DNA/RNA synthesizer (see, e.g., Elbashir, S.M. et al. (2001) Nature 411:494-
498; Elbashir,
S.M., W. Lendeckel and T. Tuschl (2001) Genes & Development 15:188-200;
Harborth, J. et
al. (2001) J. Cell Science 114:4557-4565; Masters, J.R. et al. (2001) Proc.
Natl. Acad. Sci.,
USA 98:8012-8017; and Tuschl, T. et al. (1999) Genes & Development 13:3191-
3197).
Alternatively, several commercial RNA synthesis suppliers are available
including, but are
10566615.1
CA 02588369 2007-05-11
- 78 -
not limited to, Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO,
USA),
Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research
(Sterling, VA,
USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK). As such, siRNA
molecules are not overly difficult to synthesize and are readily provided in a
quality suitable
for RNAi. In addition, dsRNAs can be expressed as stem loop structures encoded
by plasmid
vectors, retroviruses and lentiviruses (Paddison, P.J. et al. (2002) Genes
Dev. 16:948-958;
McManus, M.T. etal. (2002) RNA 8:842-850; Paul, C.P. etal. (2002) Nat.
Biotechnol.
20:505-508; Miyagishi, M. et al. (2002) Nat. Biotechnol. 20:497-500; Sui, G.
et al. (2002)
Proc. Natl. Acad. Sci., USA 99:5515-5520; Brummelkamp, T. etal. (2002) Cancer
Cell
2:243; Lee, N.S., etal. (2002) Nat. Biotechnol. 20:500-505; Yu, J.Y., etal.
(2002) Proc. Natl.
Acad. Sci., USA 99:6047-6052; Zeng, Y., etal. (2002) Mol. Cell 9:1327-1333;
Rubinson,
D.A., et al. (2003) Nat. Genet. 33:401-406; Stewart, S.A., et al. (2003) RNA
9:493-501).
These vectors generally have a polIII promoter upstream of the dsRNA and can
express sense
and antisense RNA strands separately and/or as a hairpin structures. Within
cells, Dicer
processes the short hairpin RNA (shRNA) into effective siRNA.
[556] The targeted region of the siRNA molecule of the present invention can
be selected
from a given target gene sequence, e.g., a Lp-PLA2 coding sequence, beginning
from about
to 50 nucleotides, from about 50 to 75 nucleotides, or from about 75 to 100
nucleotides
downstream of the start codon. Nucleotide sequences can contain 5' or 3' UTRs
and regions
20 nearby the start codon. One method of designing a siRNA molecule of the
present invention
involves identifying the 23 nucleotide sequence motif AA(N19)TT (where N can
be any
nucleotide), and selecting hits with at least 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%,
65%, 70% or 75% G/C content. The "TT" portion of the sequence is optional.
Alternatively,
if no such sequence is found, the search can be extended using the motif
NA(N21), where N
25 can be any nucleotide. In this situation, the 3' end of the sense siRNA
can be converted to
TT to allow for the generation of a symmetric duplex with respect to the
sequence
composition of the sense and antisense 3' overhangs. The antisense siRNA
molecule can
then be synthesized as the complement to nucleotide positions 1 to 21 of the
23 nucleotide
sequence motif. The use of symmetric 3' TT overhangs can be advantageous to
ensure that
the small interfering ribonucleoprotein particles (siRNPs) are formed with
approximately
equal ratios of sense and antisense target RNA-cleaving siRNPs (Elbashir et
al. (2001) supra
and Elbashir et al. 2001 supra). Analysis of sequence databases, including but
are not limited
to the NCBI, BLAST, Derwent and GenSeq as well as commercially available
oligosynthesis
10566615.1
CA 02588369 2007-05-11
- 79 -
software such as Oligoengine , can also be used to select siRNA sequences
against EST
libraries to ensure that only one gene is targeted.
[557] Delivery of RNA Interfering Agents: Methods of delivering RNA
interfering agents,
e.g., an siRNA, or vectors containing an RNA interfering agent, to the target
cells ( e.g., cells
of the brain or other desired target cells, for cells in the central and
peripheral nervous
systems), can include, for example (i) injection of a composition containing
the RNA
interfering agent, e.g., an siRNA, or (ii) directly contacting the cell, e.g.,
a cell of the brain,
with a composition comprising an RNA interfering agent, e.g., an siRNA. In
another
embodiment, RNA interfering agents, e.g., an siRNA can be injected directly
into any blood
vessel, such as vein, artery, venule or arteriole, via, e.g., hydrodynamic
injection or
catheterization. In some embodiments, the siRNA is delivered to the bone
marrow, where Lp-
PLA2 is secreted from bone marrow-derived cells such as leukocytes.
[558] Administration can be by a single injection or by two or more
injections. The RNA
interfering agent is delivered in a pharmaceutically acceptable carrier. One
or more RNA
interfering agents can be used simultaneously. The RNA interfering agents,
e.g., the siRNAs
targeting Lp-PLA2 mRNA, can be delivered singly, or in combination with other
RNA
interfering agents, e.g., siRNAs, such as, for example siRNAs directed to
other cellular genes.
Lp-PLA2 siRNAs can also be administered in combination with other
pharmaceutical agents
which are used to treat or prevent metabolic bone diseases or disorders.
[559] In one embodiment, specific cells are targeted with RNA interference,
limiting
potential side effects of RNA interference caused by non-specific targeting of
RNA
interference. The method can use, for example, a complex or a fusion molecule
comprising a
cell targeting moiety and an RNA interference binding moiety that is used to
deliver RNA
interference effectively into cells. For example, an antibody-protamine fusion
protein when
mixed with an siRNA, binds siRNA and selectively delivers the siRNA into cells
expressing
an antigen recognized by the antibody, resulting in silencing of gene
expression only in those
cells that express the antigen. The siRNA or RNA interference-inducing
molecule binding
moiety is a protein or a nucleic acid binding domain or fragment of a protein,
and the binding
moiety is fused to a portion of the targeting moiety. The location of the
targeting moiety can
be either in the carboxyl-terminal or amino-terminal end of the construct or
in the middle of
the fusion protein.
[560] A viral-mediated delivery mechanism can also be employed to deliver
siRNAs to cells
in vitro and in vivo as described in Xia, H. etal. (2002) Nat Biotechnol
20(10):1006).
10566615.1
CA 02588369 2007-05-11
- 80 -
Plasmid- or viral-mediated delivery mechanisms of shRNA can also be employed
to deliver
shRNAs to cells in vitro and in vivo as described in Rubinson, D.A., et al.
((2003) Nat. Genet.
33:401-406) and Stewart, S.A., et al. ((2003) RNA 9:493-501).
[561] RNA interfering agents, for e.g., an siRNA, can also be introduced into
cells via the
vascular or extravascular circulation, the blood or lymph system, and the
cerebrospinal fluid.
[562] The dose of the particular RNA interfering agent will be in an amount
necessary to
effect RNA interference, e.g., post translational gene silencing (PTGS), of
the particular
target gene, thereby leading to inhibition of target gene expression or
inhibition of activity or
level of the protein encoded by the target gene.
[563] It is also known that RNAi molecules do not have to match perfectly to
their target
sequence. Preferably, however, the 5' and middle part of the antisense (guide)
strand of the
siRNA is perfectly complementary to the target nucleic acid sequence.
1564] Accordingly, the RNAi molecules functioning as nucleic acid inhibitors
of Lp-PLA2
in the present invention are for example, but are not limited to, unmodified
and modified
double stranded (ds) RNA molecules including short-temporal RNA (stRNA), small
interfering RNA (siRNA), short-hairpin RNA (shRNA), microRNA (miRNA), double-
stranded RNA (dsRNA), (see, e.g. Baulcombe, Science 297:2002-2003, 2002). The
dsRNA
molecules, e.g. siRNA, also can contain 3' overhangs, preferably 3'UU or 3'TT
overhangs. In =
one embodiment, the siRNA molecules of the present invention do not include
RNA
molecules that comprise ssRNA greater than about 30-40 bases, about 40-50
bases, about 50
bases or more. In one embodiment, the siRNA molecules of the present invention
are double
stranded for more than about 25%, more than about 50%, more than about 60%,
more than
about 70%, more than about 80%, more than about 90% of their length. In some
embodiments, a nucleic acid inhibitor of Lp-PLA2 is any agent which binds to
and inhibits
the expression of Lp-PLA2 mRNA, where the expression of Lp-PLA2 mRNA or a
product of
transcription of nucleic acid encoded by SEQ ID NO:1 or 2 is inhibited.
[565] In another embodiment of the invention, agents inhibiting Lp-PLA2 are
catalytic
nucleic acid constructs, such as, for example ribozymes, which are capable of
cleaving RNA
transcripts and thereby preventing the production of wildtype protein.
Ribozymes are targeted
to and anneal with a particular sequence by virtue of two regions of sequence
complementary
to the target flanking the ribozyme catalytic site. After binding, the
ribozyme cleaves the
target in a site specific manner. The design and testing of ribozymes which
specifically
recognize and cleave sequences of the gene products described herein, for
example for
10566615.1
CA 02588369 2013-10-22
- 81 -
cleavage of Lp-PLA2 or homologues or variants thereof can be achieved by
techniques well
known to those skilled in the art (for example Lieber and Strauss, (1995) Mol
Cell Biol
15:540.551.
[566] Proteins and peptide inhibitors of Lp-PLA2
[567] In some embodiments, agent that inhibit Lp-PLA2 are proteins and/or
peptide
inhibitors or fragments of inhibitors of Lp-PLA2, for example, but are not
limited to mutated
proteins; therapeutic proteins and recombinant proteins. Proteins and peptides
inhibitors can
also include for example mutated proteins, genetically modified proteins,
peptides, synthetic
peptides, recombinant proteins, chimeric proteins, antibodies, humanized
proteins,
humanized antibodies, chimeric antibodies, modified proteins and fragments
thereof.
[568] In some embodiments, the agents that inhibit Lp-PLA2 are dominant
negative variants
of Lp-PLA2, for example a non-functional variant of Lp-PLA2.
[569] Antibodies
[570] In some embodiments, inhibitors of genes and/or gene products useful in
the methods
of the present invention include, for example, antibodies, including
monoclonal, chimeric
humanized, and recombinant antibodies and antigen-binding fragments thereof.
In some
embodiments, neutralizing antibodies can be used as inhibitors of the Lp-PLA2
enzyme.
Antibodies are readily raised in animals such as rabbits or mice by
immunization with the
antigen. Immunized mice are particularly useful for providing sources of B
cells for the
manufacture of hybridomas, which in turn are cultured to produce large
quantities of
monoclonal antibodies.
[571] In one embodiment of this invention, the inhibitor to the gene products
identified
herein can be an antibody molecule or the epitope-binding moiety of an
antibody molecule
and the like. Antibodies provide high binding avidity and unique specificity
to a wide range
of target antigens and haptens. Monoclonal antibodies useful in the practice
of the present
invention include whole antibody and fragments thereof and are generated in
accordance with
conventional techniques, such as hybridoma synthesis, recombinant DNA
techniques and
protein synthesis.
[572] Useful monoclonal antibodies and fragments can be derived from any
species
(including humans) or can be formed as chimeric proteins which employ
sequences from
more than one species. Human monoclonal antibodies or "humanized" murine
antibody are
also used in accordance with the present invention. For example, murine
monoclonal
CA 02588369 2007-05-11
- 82 -
antibody can be "humanized" by genetically recombining the nucleotide sequence
encoding
the murine Fv region (i.e., containing the antigen binding sites) or the
complementarily
determining regions thereof with the nucleotide sequence encoding a human
constant domain
region and an Fc region. Humanized targeting moieties are recognized to
decrease the
immunoreactivity of the antibody or polypeptide in the host recipient,
permitting an increase
in the half-life and a reduction the possibly of adverse immune reactions in a
manner similar
to that disclosed in European Patent Application No. 0,411,893 A2. The murine
monoclonal
antibodies should preferably be employed in humanized form. Antigen binding
activity is
determined by the sequences and conformation of the amino acids of the six
complementarily
determining regions (CDRs) that are located (three each) on the light and
heavy chains of the
variable portion (Fv) of the antibody. The 25-1(Da single-chain Fv (scFv)
molecule,
composed of a variable region (VL) of the light chain and a variable region
(VH) of the
heavy chain joined via a short peptide spacer sequence, is the smallest
antibody fragment
developed to date. Techniques have been developed to display scFv molecules on
the surface
of filamentous phage that contain the gene for the scFv. scFv molecules with a
broad range of
antigenic-specificities can be present in a single large pool of scFv-phage
library. Some
examples of high affinity monoclonal antibodies and chimeric derivatives
thereof, useful in
the methods of the present invention, are described in the European Patent
Application EP
186,833; PCT Patent Application WO 92/16553; and US Patent No. 6,090,923.
15731 Chimeric antibodies are immunoglobin molecules characterized by two or
more
segments or portions derived from different animal species. Generally, the
variable region of
the chimeric antibody is derived from a non-human mammalian antibody, such as
murine
monoclonal antibody, and the immunoglobin constant region is derived from a
human
immunoglobin molecule. Preferably, both regions and the combination have low
immunogenicity as routinely determined.
15741 One limitation of scFv molecules is their monovalent interaction with
target antigen.
One of the easiest methods of improving the binding of a scFv to its target
antigen is to
increase its functional affinity through the creation of a multimer.
Association of identical
scFv molecules to form diabodies, triabodies and tetrabodies can comprise a
number of
identical Fv modules. These reagents are therefore multivalent, but
monospecific. The
association of two different scFv molecules, each comprising a VH and VL
domain derived
from different parent Ig will form a fully functional bispecific diabody. A
unique application
of bispecific scFvs is to bind two sites simultaneously on the same target
molecule via two
10566615.1
CA 02588369 2007-05-11
- 83 -
(adjacent) surface epitopes. These reagents gain a significant avidity
advantage over a single
scFv or Fab fragments. A number of multivalent scFv-based structures has been
engineered,
including for example, miniantibodies, dimeric miniantibodies, minibodies,
(scFv)2,
diabodies and triabodies. These molecules span a range of valence (two to four
binding sites),
size (50 to 120 lcDa), flexibility and ease of production. Single chain Fv
antibody fragments
(scFvs) are predominantly monomeric when the VH and VL domains are joined by,
polypeptide linkers of at least 12 residues. The monomer scFv is
thermodynamically stable
with linkers of 12 and 25 amino acids length under all conditions. The
noncovalent diabody
and triabody molecules are easy to engineer and are produced by shortening the
peptide
linker that connects the variable heavy and variable light chains of a single
scFv molecule.
The scFv dimers are joined by amphipathic helices that offer a high degree of
flexibility and
the miniantibody structure can be modified to create a dimeric bispecific
(DiBi) miniantibody
that contains two miniantibodies (four scFv molecules) connected via a double
helix. Gene-
fused or disulfide bonded scFv dimers provide an intermediate degree of
flexibility and are
generated by straightforward cloning techniques adding a C-terminal Gly4Cys
sequence.
scFv-CH3 minibodies are comprised of two scFv molecules joined to an IgG CH3
domain
either directly (LD minibody) or via a very flexible hinge region (Flex
minibody). With a
molecular weight of approximately 80 kDa, these divalent constructs are
capable of
significant binding to antigens. The Flex minibody exhibits impressive tumor
localization in
mice. Bi- and tri-specific multimers can be formed by association of different
scFv
molecules. Increase in functional affinity can be reached when Fab or single
chain Fv
antibody fragments (scFv) fragments are complexed into dimers, trimers or
larger aggregates.
The most important advantage of multivalent scFvs over monovalent scFv and Fab
fragments
is the gain in functional binding affinity (avidity) to target antigens. High
avidity requires that
scFv multimers are capable of binding simultaneously to separate target
antigens. The gain in
functional affinity for scFv diabodies compared to scFv monomers is
significant and is seen
primarily in reduced off-rates, which result from multiple binding to two or
more target
antigens and to rebinding when one Fv dissociates. When such scFv molecules
associate into
multimers, they can be designed with either high avidity to a single target
antigen or with
multiple specificities to different target antigens. Multiple binding to
antigens is dependent on
correct alignment and orientation in the Fv modules. For full avidity in
multivalent scFvs
target, the antigen binding sites must point towards the same direction. If
multiple binding is
not sterically possible then apparent gains in functional affinity are likely
to be due the effect
10566615.1
CA 02588369 2007-05-11
- 84 -
of increased rebinding, which is dependent on diffusion rates and antigen
concentration.
Antibodies conjugated with moieties that improve their properties are also
contemplated for
the instant invention. For example, antibody conjugates with PEG that
increases their half-life
in vivo can be used for the present invention. Immune libraries are prepared
by subjecting the
genes encoding variable antibody fragments from the B lymphocytes of naive or
immunized
animals or patients to PCR amplification. Combinations of oligonucleotides
which are
specific for immunoglobulin genes or for the immunoglobulin gene families are
used.
Immunoglobulin germ line genes can be used to prepare semisynthetic antibody
repertoires,
with the complementarity-determining region of the variable fragments being
amplified by
PCR using degenerate primers. These single-pot libraries have the advantage
that antibody
fragments against a large number of antigens can be isolated from one single
library. The
phage-display technique can be used to increase the affinity of antibody
fragments, with new
libraries being prepared from already existing antibody fragments by random,
codon-based or
site-directed mutagenesis, by shuffling the chains of individual domains with
those of
fragments from naive repertoires or by using bacterial mutator strains.
[575] Alternatively, a SCID-hu mouse, for example the model developed by
Genpharm, can
be used to produce antibodies, or fragments thereof. In one embodiment, a new
type of high
avidity binding molecule, termed peptabody, created by harnessing the effect
of multivalent
interaction is contemplated. A short peptide ligand was fused via a semirigid
hinge region
with the coiled-coil assembly domain of the cartilage oligomeric matrix
protein, resulting in a
pentameric multivalent binding molecule. In prefered embodiment of this
invention, ligands
and/or chimeric inhibitors can be targeted to tissue- or tumor-specific
targets by using
bispecific antibodies, for example produced by chemical linkage of an anti-
ligand antibody
(Ab) and an Ab directed toward a specific target. To avoid the limitations of
chemical
conjugates, molecular conjugates of antibodies can be used for production of
recombinant
bispecific single-chain Abs directing ligands and/or chimeric inhibitors at
cell surface
molecules. Alternatively, two or more active agents and or inhibitors attached
to targeting
moieties can be administered, wherein each conjugate includes a targeting
moiety, for
example, a different antibody. Each antibody is reactive with a different
target site epitope
(associated with the same or a different target site antigen). The different
antibodies with the
agents attached accumulate additively at the desired target site. Antibody-
based or non-
antibody-based targeting moieties can be employed to deliver a ligand or the
inhibitor to a
10566615.1
CA 02588369 2013-10-22
- 85 -
target site. Preferably, a natural binding agent for an unregulated or disease
associated
antigen is used for this purpose.
Bioassay for Identifying Lp-PLA/ Inhibitors:
[576] Screen for inhibition of Lp-PLA2 protein
[577] In some embodiments, the methods of the present invention relate to use
of inhibitors
of Lp-PLA2 for the prevention and/or treatment of metabolic bone diseases or
disorders, for
example osteoporosis and osteopenic related diseases. Where necessary, agents
that inhibit
Lp-PLA2 protein are assessed using a bioassay, as disclosed in U.S. Patent
5,981,252. One
such assay is testing the effect of the agent on the recombinant Lp-PLA2
protein. In one
assay, for example, recombinant Lp-PLA2 is purified to homogeneity from
baculovirus
infected Sf9 cells, using a zinc chelating column, blue sepharose affinity
chromatography and
an anion exchange column. Following purification and ultrafiltration, the
enzyme can be
stored at 6mg/m1 at 4 C. Assay buffer comprises Tris-HC1 (50 mM), NaC1 (150
mM) and
1mM CHAPS, pH 7.4 at room temperature. Activity is measured by an increase in
emission
at 535 nm on hydrolysis of N-((6-(2,4-dinitrophenyl) amino)hexanoy1)-2-(4,4-
difluoro-5,7-
dimethy1-4-bora-3a,4a-diaza-s-indacene-3-pentanoy1)-1-hexadecanoyl-sn-glycero-
3-
phosphoethanolamine, triethylammonium salt (PED6, Molecular Probes catalogue
reference
D-23739) as substrate, using a fluorometric plate reader with 384 well
microtitre plates.
Reaction is initiated by the addition of enzyme (approx 400 pM final by
weight) and substrate
(5 1.1M final) to inhibitor in a total volume of 10 microlitres.
0
CH3(CH2)14- 0-0CH
2 02N
H3C F, ,F (012)4-F-0?Fl 0 01
N N 0 CH2 0 -P- OCH2 CH2 NH - C - (CH2)5NH NO2
0
HC
(CH3CH2)3NH
(PED6)
[578] The compounds as disclosed herein, for example as disclosed in the
sections entitled
of Examples of synethesis were tested and were found to have IC50 values in
the range 0.1 to
10 nM.
CA 02588369 2007-05-11
- 86 -
Uses of agents that inhibit Lp-PLA2 for the prevention and/or treatment of
metabolic
bone diseases or disorders.
[579] One aspect of the present invention relates to methods for the treatment
and/or
prevention of metabolic bone diseases or disorders. These include metabolic
bone diseases
and/or disorders are characterized by abnormalities in the bone marrow. The
method uses
agents that inhibit the expression and/or function of Lp-PLA/. In some
embodiments, the
metabolic bone disease or disorder is osteoporosis and osteopenia, and in
other embodiments
the metabolic bone disease or disorders can be caused by metabolic diseases
such as
dyslipidemia, type II diabetes, metabolic syndrome or insulin resistance and
the like.
[580] Bone marrow homeostasis is critical for tissue repair and regeneration.
Disturbed
bone marrow homeostasis may lead to aging and tissue degeneration. As
disclosed herein,
inhibition of Lp-PLA2 can also be used for the prevention and/or treatment of
metabolic bone
marrow diseases or disorders and bone marrow abnormalities.
[581] In other embodiments, agents inhibiting Lp-PLA2 as disclosed herein are
useful in the
treatment and/or prophylaxis of diseases where metabolic bone disease occurs
or disorders
where loss of bone mass, or bone density has been determined to play a role,
such as
osteoporosis and related osteopenic diseases, Paget's disease,
hyperparathyroidism and
related diseases, such as for example dyslipidemia, type II diabetes,
metabolic syndrome or
insulin resistance and the like.
[582] In some embodiments, the osteopenic related disease or osteoporosis is
associated
with the pen i and post menopausal conditions. Also encompassed are the
treatment and
prophylaxis of Paget's disease, hyperealcernia associated with bone neoplasms
and all the
types of osteoporotic diseases as classified below according to their
etiology: Primary
osteoporosis, hypercalcemia, involutional osteoporosis, Type I or
postmenopausal
osteoporosis, Type II or senile osteoporosis, Juvenile osteoporosis,
Idiopathic in young adults
osteoporosis, Secondary osteoporosis, Endocrine abnormality, Hyperthyroidism,
Hypogonadism, Ovarian agenesis, or Turner's syndrome, Hyperadrenocorticism or
Cushing's
syndrome, Hyperparathyroidism, Bone marrow abnormalities, Multiple myeloma and
related
disorders, and Systemic mastocytosis, disseminated carcinoma osteoporosis,
Gaucher's
disease, Connective tissue abnormalities, Osteogenesis imperfecta,
Homocystinuria, Ehlers-
Danlos syndrome, Marfan's syndrome, Menke's syndrome, Miscellaneous causes
Immobilisation or weightlessness, Sudeck's atrophy, chronic obstructive
pulmonary disease,
10566615.1
CA 02588369 2007-05-11
- 87 -
chronic alcoholism, chronic heparin administration and chronic ingestion of
anticonvulsant
drugs
[583] Patients amenable to treatment by agents inhibiting Lp-PLA2 as disclosed
herein
include patients at risk of disease but not showing symptoms (for example
asymptomatic
patients), as well as patients presently showing symptoms. In the case of
osteoporosis,
virtually anyone, particularily women are at risk of suffering from
osteoporosis if he or she
lives long enough.
[584] The methods as disclosed herein are especially useful for patients who
do have a
known genetic risk of metabolic bone diseases and/or disorders, for example
osteoporosis.
Such patients include those as identified to have risk of having a metabolic
bone disease or
disorder, as disclosed herein in the section entitled "osteoporosis risk
factors" below, which
include analysis of risk factors and analysis of genetic or biochemical
markers. In some
embodiments, patients are women, for example post menopausal, or women at
least 65 years
of age, or patients who have had previous fractures or have relatives who have
had a
metabolic bone disease, for example osteoporosis. Patients can be identified
as having
increased risk of developing metabolic bone disease using methods commonly
known by
person of ordinary skill in the art, and are disclosed herein in the section
entitled "methods to
identify patients at risk of, or having a metabolic bone disease or
osteoporosis" below, which
include analysis of genetic and/or biochemical markers for metabolic bone
diseases as
disclosed herein.
[585] Osteoporosis
[586] Osteoporosis (gr: osteon bone; poros hole) is described in general terms
as a reduction
in bone density with retention of a normal chemical composition. More
specifically,
osteoporosis is a generalized, progressive diminution of bone density, i.e.
bone mass per unit
volume, causing skeletal weakness, although the ratio of mineral to organic
elements is
unchanged. 30 to 40% of the skeletal mass must be lost in order to reliably
diagnose
osteoporosis by radiology. Contemporary medicine distinguishes between primary
and
secondary osteoporosis (The Merck Manual of Diagnosis and Therapy, 17th ed.,
1999).
Primary osteoporosis includes idiopathic osteoporosis, rare but occurring in
children and
young adults; postmenopausal osteoporosis, occurring between the ages of 50
and 75; and
involutional or senile osteoporosis associated with the normal process of
aging. In some
embodiments, osteoporosis is characterized by increased osteoclast activity
and a disruption
of the feedback mechanism between the serum calcium level and the parathyroid
hormone
10566615.1
CA 02588369 2007-05-11
- 88 -
(PTH) secretion. It occurs mainly uniformly throughout the whole skeleton.
Secondary
osteoporosis, accounting for less than 5% of all osteoporosis cases, includes
endocrine
dysfunctions. It starts mostly at the main skeleton and progresses
centrifugally. Osteoporosis
is characterized by pain in the respective bones, diffuse back pain, vertebral
body collapse,
pathological fractures, in particular, fracture of the neck of the femur. The
goal of the
management of all types of osteoporosis is therefore to decrease pain, to
prevent fractures and
to maintain the body functions.
15871 The cause of osteoporosis has not been fully clarified. According to one
theory,
osteoporosis is a calcium dysfunction and the use of calcium supplements has
been widely
suggested. However, so far, no reossification of the osteoporotic bone after
calcium therapy
could be demonstrated.
15881 Other metabolic bone disorders and osteopenic related diseases.
15891 In some embodiments, agents inhibiting Lp-PLA2 using the agents as
disclosed herein
are useful in preventing and treating metabolic bone diseases and disorders.
Additional
examples of metabolic bone diseases include osteoporosis. Osteoporosis is a
common clinical
feature and common complication in patients affected with chronic inflammatory
diseases
with joint manifestations. These include rheumatoid arthritis (RA), Juvenile
Rheumatoid
Arthritis (JRA), psoriatic arthritis, Reiter's syndrome (reactive arthritis),
Crohn's disease,
ulcerative colitis and sarcoidosis (Orcel, et al., Bone demineralization and
cytokines; Rev
Rhum Mal Osteoartic.1992; 59:16S-22S; Brown, et al., The radiology of
rheumatoid arthritis.
Am Fam Physician. 1995. 52:1372-80; De Vos, et al., Bone and joint diseases in
inflammatory bowel disease. Aliment Pharmacol Ther. 1998;12(5):397-404;
Falcini, et al.,
The primary role of steroids on the osteoporosis in juvenile rheumatoid
patients evaluated by
dual energy X-ray absorptiometry. J Endocrinol Invest. 1996;19(3):165-9;
Scutellari, et al.,
Rheumatoid arthritis: sequences. Eur J Radiol. 1998: Suppl 1:S31-8).
[590) Rheumatoid arthritis is associated with a decrease in bone mass (Cortet,
et al.,
Evaluation of bone mineral density in patients with rheumatoid arthritis.
Influence of disease
activity and glucocorticoid therapy. Rev Rhum Engl Ed. 1997 July-Sep. 30,
1997; 64(7-
9):451-8). Typical changes of an inflammatory arthritis include juxta-
articular osteoporosis,
cartilage loss, and cortical or marginal bone erosions (Lawson, et al., Lyme
arthritis:
radiologic findings. Radiology. 1985;154(1):37-43; Grassi, et al., The
clinical features of
rheumatoid arthritis. Eur J Radiol. 1998;1:S18-24).
10566615.1
CA 02588369 2007-05-11
- 89 -
[591] Three forms of bone disease (bone loss) have been described in
rheumatoid arthritis,
namely: focal bone loss affecting the immediate subchondral bone and bone at
the joint
margins; periarticular osteopenia adjacent to inflamed joints; and generalized
osteoporosis
involving the axial and appendicular skeleton (Goldring, et al., Mechanisms of
bone loss in
inflammatory arthritis: diagnosis and therapeutic implications. Arthritis Res.
2000; 2(1):33-
7).
[592] During chronic inflammatory joint diseases, such as rheumatoid
arthritis, synovial
cells produce large amounts of cytokines leading to increased local bone
resorption and juxta-
articular bone destructions (Orcel, P et al., Bone demineralization and
cytokines. Rev Rhum
Mal Osteoartic. 1992; 59(6 Pt 2):16S-22S).
[593] Homocysteinemia (the accumulation of homocysteine in plasma and tissue)
is the
result of deficiencies of certain enzymes and/or substrates involved in the
transmethylation
pathways. It is caused by the accumulation of homocysteine and its two
disulfides in plasma
and tissue (Mudd et al., The Metabolic Basis of Inherited Disease, New York,
McGraw-Hill,
1978, p. 458). Homocysteinemia is associated with juvenile arteriosclerosis,
recurrent arterial
and venous thromboembolic manifestations and osteoporosis. The latter may be
due to the
fact that homocysteine also interferes with collagen synthesis, and it is this
interaction that
may be significant in the development of defective bone matrix and
osteoporosis (Am J Med
Sci, 273, 1977, p. 120). Folic acid has been described as a successful tool
for the treatment of
hyperhomocysteinemia (Brattstrom et al., Metabolism, Vol. 34, No. 11, 1985, p.
1073). The
metabolite transforming homocysteine to methionine is the active form of folic
acid: 5-
methyl-tetrahydrafolic acid (5-MTHF). Depending on the degree of methylene
tetrahydrofolate reductase (MTHFR) dysfunction, the body can less or more
easily transform
the various forms of folates into 5-MTHF.
[594] In some embodiments, agents that inhibit Lp-PLA2 as disclosed herein are
also useful
in the treatment of other metabolic bone disorders, for example but not
limited to patients
with metabolic bone diseases due to chronic inflammatory diseases with joint
manifestations,
for example but not limited to rheumatoid arthritis (RA), Juvenile Rheumatoid
Arthritis
(JRA), psoriatic arthritis, Reiter's syndrome (reactive arthritis), Crohn's
disease, ulcerative
colitis, sarcoidosis. Other patients with metabolic bone diseases include, for
example but are
not limited to, related osteopenic diseases, Paget's disease,
hyperparathyroidism and related
diseases, such as for example dyslipidemia, type II diabetes, metabolic
syndrome or insulin
resistance and the like, primary osteoporosis, involutional osteoporosis, Type
I or
10566615.1
CA 02588369 2007-05-11
- 90 -
postmenopausal osteoporosis, Type II or senile osteoporosis, oral bone loss,
metabolic
peridontilis, Juvenile Idiopathic osteoporosis in young adults, Secondary
osteoporosis,
Endocrine abnormality, Hyperthyroidism, Hypogonadism, Ovarian agenesis or
Turner's
syndrome, Hyperadrenocorticism or Cushing's syndrome, Hyperparathyroidism,
Bone
marrow abnormalities, Multiple myeloma and related disorders, Systemic
mastocytosis,
Disseminated carcinoma, Gaucher's disease, Connective tissue abnormalities,
Osteogenesis
imperfecta, Homocystinuria, Ehlers-Danlos syndrome, Marfan's syndrome, Menke's
syndrome, Miscellaneous causes, Immobilisation or weightlessness, Sudeck's
atrophy,
Chronic obstructive pulmonary disease, Chronic alcoholism, Chronic heparin
administration
and Chronic ingestion of anticonvulsant drugs.
[595] In some embodiments, agents that inhibit Lp-PLA7 as disclosed herein are
also useful
in the treatment of other metabolic bone disorders, for example but not
limited to patients
with metabolic bone diseases where the metabolic bone disease or disorder
involves bone
reabsorbtion, for example Paget's Disease, primary and secondary
hyperparathyroidism,
humoral hypercalcameia of malignancy and various cancers, where bone
resorbtion is
increased.
[596] Metabolic bone disorders with abnormal bone marrow.
[597] The bone marrow is an organ responsible for part of haematopoiesis, and
since it is in
contact with the medullary cavity and cancellus bone it is possible that, as a
result of bone
marrow abnormalities, abnormalities in the bone and other surrounding tissues
can occur and
result in serious illness in the patient. Bone marrow abnormalities referred
to here are defined
as those where general abnormality of the biological balance in the bone
marrow is indicated,
such as viral and bacterial infections in the bone marrow, cellular
infiltration of the bone
marrow, abnormalities of the bone marrow haematopoiesis, proliferation of
malignant
neoplasms in the bone marrow and concentration changes in cell growth-
differentiation
factors.
[598] Bone marrow abnormalites include, but are not limited to, anemia,
immunodeficiency,
bone marrow, depression, bone marrow fibrosis, bone marrow necrosis and
degeneration.
[599] Metabolic bone diseases or disorders and abnormal bone marrow can be
brought
about by many pathogenic factors such as pyogenic bacteria, tuberculosis,
syphilis, fungi and
specified viruses or exogenous matter is termed osteomyelitis and, when there
is
osteomyelitis, as a result of the impeded blood circulation and infiltration
of neutrophils into
the bone marrow region, there occur surrounding bone decalcification and
tissue breakdown,
10566615.1
CA 02588369 2007-05-11
- 91 -
with resulting pain. While the occurrence of acute osteomyelitis is declining
due to the
widespread use of antibiotics, as a result of the appearance of resistant
microorganisms,
osteomyelitis which from the outset follows a subacute or chronic course
remains a problem
[Green, etal., J. Bone Joint Serg., 63-A, p107-114 (1981)].
[600] In rheumatoid arthritis, it has been reported that there is an increase
in the
concentration of components which induce a proliferation of synoviocytes in
the bone
marrow and, moreover, that abnormal myelocytes are found within the bone
marrow and
changes in cell ratios such as an increase in the T cell ratio are shown
[Ochi, T., Igaku no
Ayumi, 161, p609-613 (1992)]. Since myelocytes differentiate into neutrophils,
it can be
expected that the number of neutrophils showing abnormal activity will
increase in the bone
marrow and contribute to an aggravation of the condition. Moreover, since the
progress of a
condition where marked changes in the bone marrow are exhibited is rapid and
the outlook
for the patient is severe [Ochi, T. et al., Arthritis Rheum., 31, p37 (1988)],
there is the
possibility that early stage improvement in the pathological changes in the
bone marrow
could be linked to the cure of the disease. In addition, in the treatment of
rheumatoid arthritis
there is also the problem that there is a considerable likelihood of multiple
agents which show
serious side effects being used concomitantly, such as steroids which display
a variety of
adverse-side effects and gold preparations compounds which exhibit
hematopoiesis decrease.
[601] In leukaemia, irrespective of cell type and whether it is acute or
chronic, or whether it
is myelogenicor lymphocytic leukaemia, the bone marrow is the location of a
markedly
increased production of leukaemia cells, and normal blood components decline.
Again, in
multiple myeloma, a principal feature is the proliferation of tumours of
plasma cells, which
are cells at the end of the B cell lineage, and a multiplicity of these is
produced in the bone
marrow at sites of active haematopoiesis. In leukaemia, multiple myeloma and
the like, an
increase in cell growth-differentiation factor activity in the bone marrow and
an abnormal
proliferation of cells are found, and the abnormalities in the biological
balance in the bone
marrow are believed to be closely connected with the presentation and
continuance of the
diseased state.
[602] Thus, inflammatory cell infiltration into the bone marrow, abnormal cell
proliferation
or an abnormal increase in cell growth-differentiation factor activity in the
bone marrow are
closely related with many bone marrow abnormalities, and the agents that
inhibit Lp-PLA2 as
disclosed herein can prevent and/or correct the biological balance in the bone
marrow and
used for the prevention and/or treatment of bone marrow abnormalities. The
aforementioned
10566615.1
CA 02588369 2013-10-22
- 92 -
disorders as disclosed herein to which bone marrow abnormalities are disclosed
are given as
examples, and there are no restrictions thereto.
[603] In yet another aspect, as disclosed herein methods for the treatment
and/or
prophylaxis of a metabolic bone disease or disorder, including secondary
conditions of a
metabolic bone disease are provided. A metabolic bone disease or disorder also
encompasses
disease and disorders with overactivity of osteocasts. In some embodiments,
disease or
disorders associated with osteocast activity includes for example but are not
limited to
osteoporosis, Paget's disease, hypercalcemeia, rheumatoid arthritis, cancer,
metastatic bone
destruction, oral bone loss, metabolic peridontitis and immune disorders.
[604] Thus, treatment can be directed to a patient who is affected with
asymptomatic
metabolic bone diseases; it can prevent bone density loss and/or improve bone
matrix density.
The efficacy of treatment can be determined by monitoring bone mineral density
(BMD) or
biochemical markers in biological samples. Biochemical markers include, for
example but
not limited to, measuring the presence of estrogen in the blood of women, and
calcium in the
urine, hypercalciuria, or increased excretion of calcium. Other markers also
include for
example, but are not limited to serum markers of bone turnover, for example
bone-specific
alkaline phosphatase (BSAP), osteocalcin (BGP), tartrate-resistant acid
phosphatase (TRAP)
and urinary collagen C-terminal extension peptides (CrossLaps) (Hotchkiss et
al, 2001; 29;7-
15). In some embodiments, diagnostic test for metabolic bone diseases and/or
osteoporosis
can be used, for example, as disclosed in European Patents EP1666883,
EP1680513 and
EP1639946.
[605] Some methods entail determining a baseline value of the bone density of
a patient
before administering a dosage of agent, and comparing this with a value for
bone density of
the patient after treatment. The same level of bone density or an increase in
bone density
indicates a positive treatment outcome (i.e., that administration of the agent
has achieved or
prevented a decrease in bone density loss). Alternatively, a decreased in the
rate of bone
density loss or of the rate of bone density loss also indicates a positive
outcome, for example
that administration has prevented or reduced the rate of the bone density
loss. If the value for
level of bone density decreases, a negative treatment outcome is indicated. In
general,
patients undergoing an initial course of treatment with an agent are expected
to show the
same level or an increase in bone density with successive dosages of an agent
as described
herein.
CA 02588369 2007-05-11
- 93 -
[606] In other methods to determine efficacy of treatment, a control value
(i.e., a mean and
standard deviation) of bone density is determined for a control population.
Typically the
individuals in the control population have not received prior treatment and do
not suffer from
a metabolic bone disease or disorder, for example osteoporosis. Measured
values of bone
density in a patient after administering an inhibitor agent of Lp-PLA2 as
disclosed herein are
then compared with the control value. An increase in the bone density in the
patient relative
to the control value (e.g.. an increase of at least 10% of bone density and/or
bone mass in a
patient approaching control values signals a positive treatment outcome. A
decrease in bone
density decrease signals a negative treatment outcome.
[607] In other methods, a control value of bone density is determined from a
control
population of patients who have undergone treatment with a therapeutic agent
that is effective
at halting the loss of bone density or increasing bone density. Measured
values of bone
density in the patient are compared with the control value.
[608] In other methods, a patient who is not presently receiving treatment by
agents that
inhibit Lp-PLA2 as disclosed herein, but has undergone a previous course of
treatment is
monitored for bone density to determine whether a resumption of treatment is
required. The
measured value of bone density in the test patient can be compared with a
level of bone
density previously achieved in the patient after a previous course of
treatment. An increase in
the rate of bone density loss and/or bone mass loss, or a decrease in bone
density and/or bone
mass relative to the previous measurement (e.g., a 10% increase of the rate of
bone density
loss and/or a decrease in bone density of about 10%) is an indication that
treatment with an
agent inhibitor of Lp-PLA2 can be resumed. Alternatively, the level of bone
density or bone
mass in the patient can be compared with a control bone densitity level of
determined in a
population of patients after undergoing a course of treatment. Alternatively,
the bone density
in a patient can be compared with a control value in populations of
prophylactically treated
patients who remain free of symptoms of disease, in particular free of
symptoms of metabolic
bone diseases, for example osteoporosis or osteopenic related diseases, or
populations of
therapeutically treated patients who show amelioration of a disease symptom.
[609] Methods to identify patients for risk of, or having a metabolic bone
disease or
osteoporosis.
[610] Patients amenable to treatment using the methods as disclosed herein
include patients
at risk of developing a metabolic bone disease or disorder such as
osteoporosis but not
10566615.1
CA 02588369 2013-10-22
- 94 -
showing symptoms, as well as patients showing symptoms of the metabolic bone
disease
patients with symptoms of osteoporosis.
[611] Patients can be screened for their likelihood of having or developing
osteoporosis
based on a number of bone density tests, imaging tests, biochemical and
genetic markers.
[612] A number of diagnostic tests are available for identifying patients who
have
osteoporosis or osteopenic related diseases, or patients at risk of developing
ostepoperosis or
osteopenic diseases. These include, but are not limited to a bone density
test, for example a
dual energy x-ray absorption scan (also known as DEXA or DXA scan by persons
of
ordinary skill in the art). A DEXA scan can identify a patient with a normal
bone density, low
bone mass or osteoporosis. In some embodiments, bone density testing involves
a low level
of radiation exposure. For example, in some embodiments an non-isotopic
detection method
for osteoblastic acitivity can be used, as disclosed in U.S. Patent 6,869,593.
[613] Other diagnostic tests to identify patients at risk of developing
osteoporosis or
osteopenic diseases include for example without limitation, X ray test to
identify the presence
of fractures or a history of fractures, bone mineral density (BMD) tests, for
example BMD
which can be used to determine bone health, bone scans, which identify a
patient with
metabolic bone disorders, for example cancer, bone lesions, abnormal bone
marrow and new
fractures. BMD tests provide a measurement known by persons in the art called
a T-score, a
number value that results from comparing the bone density to optimal bone
density. When a
T-score appears as a negative number such as -1, -2 or -2.5, the patient is
identified as having
low bone mass. The more negative the T-score, the greater risk the patient has
of developing
or having osteoporosis.
[614] In some embodiments, diagnostic tests to identify patients at risk of
developing
osteoporosis or osteopenic diseases include for example without limitation, a
three componet
X-ray bone densitometry scan, as disclosed in U.S. Patent 6,909,771. One can
also identify a
patient at risk or having a metabolic bone disorder, for example osteoporosis,
for example
using a quantitative ultrasound (QUS) measurements, for example ultrasound
bone analysis
measurement as disclosed in U.S. Patent 6,899,680.
[615] Other diagnostic methods can be used to identify a patient at increased
risk of
developing osteoporosis or with osteoporosis, for example diagnositic methods
as disclosed
in European patents 1666883, 1680513 and 1639946.
CA 02588369 2013-10-22
- 95 -
Other methods to diagnose a patient at risk of or having a metabolic bone
disease such as
osteoporosis and/or abnormal bone marrow includes measurement of Lp-PLA2
activity and/or
expression using the methods as disclosed herein, for example using the PLAC
test
commercially available from diaDexus.
[616] A number of biomarkers can identify a patient at risk of osteoporosis.
For example,
laboratory test are also useful in the methods of the present invention to
identify a patient at
risk of developing and/or having osteoporosis, for example measuring the
levels of a number
of biochemical markers in a biological sample including blood and/or urine.
Such
biochemical markers that can be measured include blood calcium levels, blood
vitamin D
levels, thyroid function, parathyroid hormone levels, estradiol levels to
measure estrogen
(useful in assessing the risk in women), follicle stimulating hormone (FSH) to
establish
menopause status, testosterone levels (in men) and osteocalcin levels to
measure bone
formation. One can perform clinincal tests such as levels of calcium and
phosphorus in blood
for diagnosis of metabolic bone diseases or disorders, as about 20% of
postmenopausal
women with osteoporosis exhibit hypercalciuria, or increased excretion of
calcium in urine.
Other biochemical markers include for example, but are not limited to serum
markers of bone
turnover such as bone-specific alkaline phosphatase (B SAP), osteocalcin
(BGP), tartrate-
resistant acid phosphatase (TRAP) and urinary collagen C-terminal extension
peptides
(CrossLaps) (Hotchkiss et al, 2001; 29;7-15).
[617] Such diagnostic tests are also useful to monitor response of the patient
to
administration of the Lp-PLA2 inhibitor agents as disclosed herein.
Accordingly, the dose and
therapeutic regime of administration of the Lp-PLA2 can be altered according
for maximal
benefit on improvement of a symptom of osteoporosis.
[618] In some embodiments, a patient at risk of osteoporosis include women
over 65 years
of age, post-menoplausal women under age 65 who have multiple risk factors,
women at
menopause, patients with abnormal spine x-rays, patients with long term oral
steroid use and
patients with hyperparathyroidism (an over active parathyroid gland).
[619] Osteoporosis risk factors
[620] One can also diagnose a patient with increased risk of developing a
metabolic bone
disease or disorder such as osteoporosis on the basis of risk factors. In some
embodiments,
patients identified to be at risk of metabolic bone diseases or disorders are
admininstered
agents that inhibit Lp-PLA2 as disclosed herein. Risk factors for osteoporosis
are known by
CA 02588369 2007-05-11
- 96 -
persons of ordinary skill in the art, and include advanced age: (i) patients
over the age of 65
years; and (ii) gender. Women are at greater risk than men as women lose bone
more rapidly
than men due to menopause - however, men are also at risk and constitute 20%
of the patient
population with osteoporosis; (iii) family and personal history, including a
family history of
osteoporosis, history of fracture on the mother's side of the family, and a
personal history of
any kind of bone fracture as an adult (after age 50); (iv) Race, with
caucasian and Asian
women at increased risk; (v) body type, with patients who are small-boned, for
example small
boned women who weigh less than 127 pounds are at increased risk; (vi)
menstrual history
and menopause, with a normal menopause alone increasing a woman's risk of
osteoporosis,
and patients having an early menopause or cessation of menstruation before
menopause
increasing the risk of developing ostoporosis significantly; (vii)
Hypogonadism (small
gonads, e.g., testosterone deficiency) in males; (viii) lifestyle behaviors
that increase
osteoporosis risk include for example but are not limited to: calcium and/or
vitamin D
deficiency; little or no exercise, especially weight-bearing exercise; alcohol
abuse; cigarette
smoking; (ix) patients with chronic diseases and medications, for example
certain types of
medications can damage bone and lead to what is termed "secondary
osteoporosis." This type
of osteoporosis is estimated to occur in almost 50% of pre-menopausal women
with
osteoporosis and from 30% to 60% of men with osteoporosis. Also, secondary
osteoporosis
can cause further bone loss in postmenopausal women and older men with primary
osteoporosis. Included in this category are certain medications to treat
endocrine disorders
such as hyperthyroidism, marrow disorders, collagen disorders,
gastrointestinal problems and
seizure disorders. Use of glucocorticoids (steroids) to treat diseases such as
asthma,
rheumatoid arthritis and inflammatory bowel disease, especially the oral form
of these
medications (at higher doses and over longer periods of time e.g., more than 2
months), can
be particularly damaging to bone.
16211 Other risk factors for osteopenic diseases and osteoporosis include, for
example,
patients with: a history of fractures as an adult, history of fractures in the
first degree relative,
advanced age, female patients, dementia, patients with poor health, fragility
or both, current
cigarette smoking, low body weight, anorexia nervosa, estrogen deficiency
(past menopause,
menopause before the age of 45, having both ovaries removed, or absence of
menstral periods
for a year or more prior to menopause, low testosterone levels in men, use of
certain
medications such as cortiosteriods and anticonvulsants, lifelong low calcium
intake,
10566615.1
CA 02588369 2013-10-22
- 97 -
excessive alcohol intake, inpaired eyesight despite adequate correction,
recurrent falls and
inadequate physical activity.
[622] The cause of osteoporosis are include the hormone deficiency (estrogen
or androgen),
hormone excess (cushing's syndrome or glucocorticoid administration,
thyrotoxicosis,
hyperparathyroidism, excessive vitamin D administration), immobilization,
tabacco,
malignancy, idiopathic or geriatric, and genetic disorders (Type I collagen
mutations, Ehlers-
danlos syndrome, Marfan's syndrome, Homocystinuria).
[623] In some embodiments, inhibitors of Lp-PLA2 can be administered to
patients
considered at especially high risk for developing osteoporosis, such patients
include for
example but without limitation: all women over age 65, women less than age 65
who are
postmenopausal and have one or more of the above described risk factors for
osteoporosis,
postmenopausal women who experience any type of bone fracture, and men who
have a
testosterone deficiency.
[624] In addition, one can also diagnose a patient with increased risk of
developing a
metabolic bone disease using genetic markers for the disease. Genetic markers
to identify a
patient at risk of developing the likes of osteoporosis are known to person of
ordinary skill in
the art. For example, polymorphisms or single nucleotide polymorphisms (SNPs)
present in
patients at risk for osteopoerisis are disclosed in U.S. Patent 6,825,336.
Patients at risk of
developing a metabolic bone disease or disorder such as osteoporosis have
variances in the
PPAR gamma gene, as disclosed in European Patent 1612279.
[625] Among the genetic disorders, osteogenesis imperfecta is caused by a
major mutation
in the gene encoding for type I collagen, the major collagen constituent of
bone. This causes
severe osteoporosis. Marfan's syndrome is caused by mutations in fibrillin
gene on
chromosome 15. Homocytinuria is caused by cystathionine beta-synthase
deficiency and
exhibits an autosomal recessive pattern of inheritance.
[626] Researchers believe that genetic factors play a dominant role in the
etiology of this
disease among the ethnic or gender difference. Several genes have been shown
to be
associated with low bone density and research has focused on identifying those
genes that
may act as markers of disease. Common allelic variations of the vitamin D
receptor gene
have been found to be associated with decreased bone density in certain
populations,
including premenopausal women and young girls (Wood, R. J. and Fleet, J. C.
Ann. Rev.
CA 02588369 2007-05-11
- 98 -
Nutrit. 1998 18:233-258). Bone mineral density has also been associated with
genetic
variation in the estrogen receptor gene, both by itself and in conjunction
with variations in the
vitamin D receptor gene (Willing et al. J. Bone Min. Res. 1998 13:695-705). In
Japanese
women, the HLA-A*24-B*07-DRB*01 halotype has been linked to low peak bone mass
(Tsuji et al. Hum. Immuno1.1998 59:243-249). A variant of the gene encoding
transforming
growth factor-beta 1 has also been associated with low bone mass in
osteoporotic women and
with low bone mass and increased bone turnover in normal women (Langdahl et
al. Bone
1997 20:289-294). A polymorphism of the COLIAI gene has been identified as a
potential
marker for low bone mass and vertebral fracture in women (Grant et al. Nat.
Genet. 1996
14:203-205). Devoto et al. (Eur. J. Hum. Genet. 1998 6:151-157) determined
that there was a
gene or genes on chromosome 1 of humans that was linked to low bone density.
Polymorphisms linked to osteoporosis have been described in the TGF-91 gene,
whose
protein product is abundant in bone and an important regulator of bone
resorption and
formation (Langdahl etal., 1997; Yamada etal., 1998; W097/28280). A
polymorphism in the
gene on chromosome 1 for tumor necrosis factor alpha receptor 2 has now been
shown to be
associated with low bone density. (Spotila et al. WO 0032826).
[627] Genes associated with osteoporosis include, but not limit to: alcitonin
receptor,
collagen subunit (alpha-1 (X)) 3, Kuestner et al Mol. Pharmacol. 46 (2), 246-
255 (1994);
insulin-like growth factor binding protein 1, Brewer et al., Biochem. Biophys.
Res. Commun.
152 (3), 1289-1297 (1988), Brinkman et al., EMBO J. 7 (8), 2417-2423 (1988),
Cubbage et
al., Mol. Endocrinol. 3 (5), 846-851 (1989), Alitalo et al., Hum. Genet. 83
(4), 335-338
(1989), Ekstand et al.,Genomics 6 (3), 413-418 (1990), Suwanichkul et al., J.
Biol. Chem.
265 (34), 21185-21193 (1990), Ehrenborg etal., Genomics 12(3), 497-502 (1992);
insulin-
like growth factor 1 receptor beta chain, Francke et al., Cold Spring Harb.
Symp. Quant. Biol.
51, 855-866 (1986), Ullrich et al., EMBO J. 5 (10), 2503-2512 (1986), Flier
etal., Proc. Natl.
Acad. Sci. U.S.A. 83 (3), 664-668 (1986), Abbott et al., J. Biol. Chem. 267
(15), 10759-
10763 (1992), Werner et al., Proc. Natl. Acad. Sci. U.S.A. 93 (16), 8318-8323
(1996), Grant
et al., J. Clin. Endocrinol. Metab. 83 (9), 3252-3257 (1998); interleukin 4
receptor, Idzerda et
al., J. Exp. Med. 171 (3), 861-873 (1990), Pritchard et al., Genomics 10 (3),
801-806 (1991);
Werner syndrome, Goto et al., Nature 355 (6362), 735-738 (1992).
[628] Diagnosis of osteoporosis is most often done in conjunction with a study
of bone
density by radiography.
10566615.1
CA 02588369 2007-05-11
- 99 -
[629] In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,
30). Usually,
however, it is not necessary to begin treatment until a patient reaches 40,
50, 60 or 70.
Treatment typically entails multiple dosages over a period of time. In some
embodiments,
treatment with an inhibitor agent of Lp-PLA2 can be monitored by assaying
presence of
biomarkers or density of bones as disclosed herein over time. If the bone
density indicates
low bone mass, additional treatment with agents inhibiting Lp-PLA2 as
disclosed herein are
recommended, and/or treatment of additional therapies for metabolic bone
disease, for
example osteoporosis. In the case of potential patients with an inherited risk
of developing
osteoporosis, treatment can begin antenatally by administering therapeutic
agent to the
mother or shortly after birth.
[630] Assessment of inhibitors of Lp-PLA2 in models of metabolic bone
diseases and
osteoporosis and/or osteopenia
[631] In some embodiments, agents inhibiting Lp-PLA2 can be assessed in animal
models
for effect reducing a symptom of a metabolic bone disease. For example, one
can use the
porcine model of hyperglycemia and hypercholesterolemia (DM/HC) as disclosed
herein,
where the animal exhibits symptoms of a metabolic bone disease, for example
reduced bone
matrix and abnormal bone marrow, in which bone matrix and/or abnormal bone
marrow can
be assessed in the presence and absence of inhibitors for Lp-PLA2 by methods
commonly
known by persons in the art. In some embodiments, bone density can be assessed
using
methods commonly known by persons of ordinary skill in the art, for example
BMD tests or
CT-micro assessment as disclosed in Example 1.
1632] In some embodiments, agents inhibiting Lp-PLA2 can be assessed in animal
models
for oesteoporosis, permitting analysis of the effects of Lp-PLA2 inhibitory
agents on bone
formation, bone repair and development and treatment, as well as assessment of
drug dosages
on the development, prognosis and recovery from metabolic bone diseases, for
example
osteoporosis. Animal models of osteoporosis are well known by person of
ordinary skill in
the art. One commonly well established model of postmenopausal bone loss are
ovariectomized (OVX) rodents.
[633] An effective dosage causes at least a measurable, statistically or
clinically significant
attenuation of at least one marker, symptom, or histological evidence
characteristic of
metabolic bone disorder and/or abnormal bone marrow. Markers, symptoms and
histological
evidence characteristic of osteoporosis include bone mass loss, decrease in
bone density, and
10566615.1
CA 02588369 2007-05-11
- 100 -
increase in blood calcium levels, and/or vitamin D levels, thyroid function,
parathyroid
hormone levels, estradiol levels to measure estrogen (useful in assessing the
risk in women),
follicle stimulating hormone (FSH) to establish menopause status, testosterone
levels (in
men) and osteocalcin levels to measure bone formation. Other biochemical
markers also
include calcium and phosphorus in blood, hypercalciuria, or increased
excretion of calcium in
urine, and presence of serum markers of bone turnover, for example bone-
specific alkaline
phosphatase (BSAP), osteocalcin (BGP), tartrate-resistant acid phosphatase
(TRAP) and
urinary collagen C-terminal extension peptides (CrossLaps) (Hotchkiss et al,
2001; 29;7-15).
1634] Assesment of inhibitors of Lp-PLA2 on models of metabolic bone diseases
or
disorders.
[635] The suitability of an inhibitor of Lp-PLA2 for the treatment of a
metabolic bone
disease can be assessed in any of a number of animal models for metabolic bone
diseases.
Animal models of osteoporisos are well known by person of ordinary skill in
the art. For
example, one commonly well established model of postmenopausal bone loss are
ovariectomized (OVX) rodents, for example rat and mouse models and non-rodent
animals,
as disclosed in Thompson et al, 1995; 17:125S-133S, Iwaniec et al, J. Bone
Miner Res, 2006,
21;1086-74; Wimalawansa et al, Calcif Tissue Int, 2000; 66: 56-60 which
demonstrate
postmenopausal cancellous bone loss. Examples of OVX non-rodent anomals,
include for
example but are not limited to monkeys (Hotchkiss et al, Bone, 2001; 29:7-15)
and sheep
(Sigrist et al, J Bone Miner Metab, 2007; 28: 28-35. Other models of
osteoporosis are known
in the art, for example OVX non-primate animals, such as for example mice,
rats, dogs,
rabbits, pigs and sheep, as disclosed in Bellino, Menopause, 2000: 7; 14-24
and Thorndike
and Turner, 1998, in search of an animal model for osteoporosis.
[636] One can also use the porcine model of hyperglycemia and
hypercholesterolemia
(DM/HC) as disclosed herein, where the animal exhibits symptoms of reduced
bone matrix
and abnormal bone marrow in which bone matrix and/or abnormal bone marrow can
be
assessed in the presence and absence of inhibitors for Lp-PLA2 by methods
commonly
known by persons in the art.
[637] One can also use animal models for oral bone loss, for example animal
models for
metabiolic peridontitis.
[638] Animals administered the compounds are evaluated for symptoms relative
to animals
not administered the compounds. A measurable change in the severity of a
symptom, (for
10566615.1
CA 02588369 2007-05-11
- 101 -
example a decrease in at least one symptom) or a delay in the onset of a
symptom in animals
treated with an Lp-PLA2 inhibitor versus untreated animals is indicative of
therapeutic
efficacy.
Formulations of compositions
[639] Compounds, for example agents inhibiting Lp-PLA2 as disclosed herein,
can be used
as a medicament or used to formulate a pharmaceutical composition with one or
more of the
utilities disclosed herein. They can be administered in vitro to cells in
culture, in vivo to cells
in the body, or ex vivo to cells outside of an individual that can later be
returned to the body
of the same individual or another. Such cells can be disaggregated or provided
as solid tissue.
[640] Compounds, for example agents inhibiting Lp-PLA2 as disclosed herein can
be used
to produce a medicament or other pharmaceutical compositions. Use of agents
inhibiting Lp-
PLA2 which further comprise a pharmaceutically acceptable carrier and
compositions which
further comprise components useful for delivering the composition to an
individual are
known in the art. Addition of such carriers and other components to the agents
as disclosed
herein is well within the level of skill in this art.
[641] Pharmaceutical compositions can be administered as a formulation adapted
for
delivery to the bone or direct contact with the bone marrow. In some
embodiments, the
compostions may be administered as a formulation adapted for systemic
delivery. In some
embodiments, the compostions may be administered as a formulation adapted for
delivery to
specific organs, for example but not limited to the liver, bone marrow or
systemic delivery.
[642] Alternatively, pharmaceutical compositions can be added to the culture
medium of
cells ex vivo. In addition to the active compound, such compositions can
contain
pharmaceutically-acceptable carriers and other ingredients known to facilitate
administration
and/or enhance uptake (e.g., saline, dimethyl sulfoxide, lipid, polymer,
affinity-based cell
specific-targeting systems). The composition can be incorporated in a gel,
sponge, or other
permeable matrix (e.g., formed as pellets or a disk) and placed in proximity
to the
endothelium for sustained, local release. The composition can be administered
in a single
dose or in multiple doses which are administered at different times.
[643] Pharmaceutical compositions can be administered by any known route. By
way of
example, the composition can be administered by a mucosal, pulmonary, topical,
or other
localized or systemic route (e.g., enteral and parenteral). The phrases
"parenteral
administration" and "administered parenterally" as used herein means modes of
10566615.1
CA 02588369 2007-05-11
- 102 -
administration other than enteral and topical administration, usually by
injection, and
includes, without limitation, intravenous, intramuscular, intraarterial,
intrathecal,
intraventricular, intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal,
transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular,
subarachnoid,
intraspinal, intracerebro spinal, and intrasternal injection, infusion and
other injection or
infusion techniques, without limitation.The phrases "systemic administration,"
"administered
systemically", "peripheral administration" and "administered peripherally" as
used herein
mean the administration of the agents as disclosed herein such that it enters
the animal's
system and, thus, is patient to metabolism and other like processes, for
example,
subcutaneous administration.
[644] The phrase "pharmaceutically acceptable" is employed herein to refer to
those
compounds, materials, compositions, and/or dosage forms which are, within the
scope of
sound medical judgment, suitable for use in contact with the tissues of human
beings and
animals without excessive toxicity, irritation, allergic response, or other
problem or
complication, commensurate with a reasonable benefit/risk ratio.
[645] The phrase "pharmaceutically acceptable carrier" as used herein means a
pharmaceutically acceptable material, composition or vehicle, such as a liquid
or solid filler,
diluent, excipient, solvent or encapsulating material, involved in carrying or
transporting the
subject agents from one organ, or portion of the body, to another organ, or
portion of the
body. Each carrier must be "acceptable" in the sense of being compatible with
the other
ingredients of the formulation, for example the carrier does not decrease the
impact of the
agent on the treatment. In other words, a carrier is pharmaceutically inert.
[646] Suitable choices in amounts and timing of doses, formulation, and routes
of
administration can be made with the goals of achieving a favorable response in
the subject
with a metabolic bone disease or disorder, such as osteoporosis or osteopenia
or a risk thereof
(i.e., efficacy), and avoiding undue toxicity or other harm thereto (i.e.,
safety). Therefore,
"effective" refers to such choices that involve routine manipulation of
conditions to achieve a
desired effect.
[647] A bolus of the formulation administered to an individual over a short
time once a day
is a convenient dosing schedule. Alternatively, the effective daily dose can
be divided into
multiple doses for purposes of administration, for example, two to twelve
doses per day.
Dosage levels of active ingredients in a pharmaceutical composition can also
be varied so as
to achieve a transient or sustained concentration of the compound or
derivative thereof in an
10566615.1
CA 02588369 2013-10-22
- 103 -
individual, especially in and around the bone or bone marrow, and to result in
the desired
therapeutic response or protection. But it is also within the skill of the art
to start doses at
levels lower than required to achieve the desired therapeutic effect and to
gradually increase
the dosage until the desired effect is achieved.
[648] The amount of agents inhibiting Lp-PLA2 administered is dependent upon
factors
known to a person skilled in the art such as bioactivity and bioavailability
of the compound
(e.g., half-life in the body, stability, and metabolism); chemical properties
of the compound
(e.g., molecular weight, hydrophobicity, and solubility); route and scheduling
of
administration, and the like. It will also be understood that the specific
dose level to be
achieved for any particular individual can depend on a variety of factors,
including age,
gender, health, medical history, weight, combination with one or more other
drugs, and
severity of disease.
[649] In some embodiments, treatment can also involve combination with other
existing
modes of treatment, for example existing agents for treatment for
osteoporosis, for example
but not limited to, estrogens, bisphosphonates, calcitonin, flavonoids, and
selective estrogen
receptor modulators. Other approaches include peptides from the parathyroid
hormone
family, strontium ranelate, and growth hormone and insulin-like growth
response.
[650] U.S. Patent. No. 5,478,579, describes a method for inducing and
enhancing the
absorption of calcium into mammalian bone tissue in order to treat metabolic
calcium
deficiencies in bone tissue, in particular osteoporosis. In that reference, it
was found that
ossification of mammalian bone tissue could be enhanced by orally
administering to a patient
an effective dose of calcium in combination with a flavonol aglycone
glycoside. It is believed
that the flavonol aglycone glycoside affords an advantageous function through
a chelation
delivery system. Flavonols possess a benzene ring structure having available
bonds to
function as a chelate. Therefore, flavonols, due to their particular molecular
structure, are
capable of holding and delivering certain minerals, including calcium, to
mammalian bone
tissue. Also bone tissue would naturally absorb flavonol glycosides from the
blood stream. It
is further disclosed that the combination of the flavonol aglycone glycoside
and calcium leads
to an increased bone mineral density which would not have been obtainable
through the use
of simple calcium supplements.
[651] Furthermore, quercetin, which is a related bioflavonoid and differs from
the
aforementioned flavonol compounds in that it does not contain the glycoside
residue, has
been shown to inhibit TNF-a induced expression of interleukin 8 (IL-8) and
monocyte
CA 02588369 2007-05-11
- 104 -
chemoattractant protein-1 (MCP-1) in cultured human synovial cells. It was
therefore
suggested that quercetin can be used in the treatment of rheumatoid arthritis
which is an
autoimmune disorder and involved synovial tissues of joints (Sato et al., The
Journal of
Rheumatology, 1997; 24:9, P. 1680). In addition, the relation between
interleukins and
cytokines and metabolic bone diseases was studied (Pumarino et al., Rev Med
Chile 1996;
124: p. 48). It could be shown that interleukin 1, 6 and 11, transforming
growth factor and
tumor necrosis factor stimulate osteoclast mediated bone resorption.
Interleukin 1 is the most
potent bone resorption agent. Although the role of interleukin 1, 6, 11 and
the tumor necrosis
factors is not quite clear, they appear to have a depressing effect on bone
formation.
[652] Cohen et al. (Israel Journal of Medical Sciences, 17, 1981, p. 1123)
investigated the
cause of an increased crystallinity index in bone tissue found in iliac crest
bone samples from
postmenopausal osteoporotic women by chemical analysis. The percentage of
crystallinity
should be regarded as an index that assumes that mature bone is only apatitic
and this
provides a measure of crystal size and perfection. It could be demonstrated by
Cohen et al.
that osteoporotic women have low total body magnesium stores. It could also be
shown that
magnesium exerts its action as a crystal poison in the nucleation and growth
of apatite and its
precrystalline intermediate. Therefore, osteoporotic bone, i.e. bone mineral
with a lower
magnesium content, has larger and more perfect crystals and bone mineral with
a higher
magnesium content has smaller and less perfect crystals than normal bone
mineral. It was
consequently suggested that the administering of magnesium supplements may be
used in
osteoporosis therapy.
[653] In some embodiments, agents that inhibit Lp-PLA2 as disclosed herein can
be
combined with other therapeutic agent to prevent and/or treat metabolic bone
disease or
disorders. Such agents can be any agent currently in use or being developed
for the treatment
and/or prevention of osteoporosis, where the agent can have a prophylactic
and/or a curative
effect and/or reduce a symptom of a metabolic bone disorder or disease.
[654] In embodiments where inhibitor agents of Lp-PLA2 as disclosed herein are
used for
the prevention and/or treatment of osteoporosis, the inhibitor agents of Lp-
PLA,) as disclosed
herein can be used in combination with medicaments commonly known by person of
ordinary skill in the art that are claimed to be useful as symptomatic
treatments of
osteoporosis. Examples of such medicaments include, but are not limited to,
agents known to
modify or inhibit osteoclast activity, promote osteoblast activity and/or
regulate cellular
events necessary for healthy bone marrow and healthy bone metabolism.
10566615.1
CA 02588369 2013-10-22
- 105 -
[655] In some embodiments, where the inhibitor agents of Lp-PLA2 as disclosed
herein are
used for the treatment of osteoporosis, the inhibitor agents of Lp-PLA2 as
disclosed herein
can be used in combination with those medicaments mentioned above that are
claimed to be
useful as symptomatic treatments of osteoporosis and/or disease-modifying
agents. Disease
modifying agents include, for example but are not limited to, estrogens,
bisphosphonates,
calcitonin, flavonoids, and selective estrogen receptor modulators. Other
approaches include
peptides from the parathyroid hormone family, strontium ranelate, and growth
hormone and
insulin-like growth response.
[656] Bisphosphates, for example bisphosphate acid compounds includes for
example,
Alendronate, for example Fosmax, Ibandronate, for example Boniva and
Risendronate, for
example Actonel. Bisphosphosphate acid compounds suppress excessive bone
resorption in
tumor-induced osteolysis, Paget's disease and osteoporosis, as disclosed in
U.S. Patent
6,555,529, and patents EP177433, EP337706, AU8551534, EP27982 and EP94714.
Bisphosphate acid compounds are alone disclosed in EP100718, U.S. Patent
4,234,645 and
4,067,971 EP84822, WO/9203451, WO/935052, W097/49711, W097/04785. In some
embodiments, a bisphosphate acid compounds for use with the methods as
disclosed herein
includes bisphosphoric acid compounds for the treatment of bone marrow
abnormalities, as
disclose in U.S. Patent 6,555,529. In some embodiments, a pyridopyrimidine is
useful with
the methods as disclosed herein, for example as disclosed in International
Application
WO/03000011.
[657] Calcitonin includes Miacalcin, Calcimar and Fortical. Selective estrogen
receptor
modulators (SERM) can also be used for the treatment of osteoporosis, i.e.
Roloxifene, sold
as Evista . Parathyroid hormone can also be used in combination with the
agents that inhibit
Lp-PLA2 as disclosed herein. Teriparatide is one such agents. It is sold as
Fortdoe. In some
embodiments, estrogen replacement and/or hormone replacement therapy can be
used in
combination with agents that inhibit Lp-PLA2 formation as disclosed herein. In
some
embodiments, an increase in calcium and/or vitamin D, and/or an increase in
physical activity
and/or increase in healthy lifestyle (for example stopping smoking, avoidance
of excessive
alcohol) can be combined with the methods as disclosed herein.
[658] One can also use indole derivatives with the agents as disclosed herein
for the
treatment and/or prevention of metabolic bone disorders as disclosed in U.S.
Patent
6,903,117.
CA 02588369 2013-10-22
- 106 -
One can further also use proton pump inhibitors with the agents as disclosed
herein for the
treatment and/or prevention of metabolic bone disorders, for example
osteoporosis, for
example as disclosed in International Patent Application W001/44257. One can
further also
use quinolones with the agents as disclosed herein for the treatment and/or
prevention of
metabolic bone disorders as disclosed in U.S. Patent Application 2003/0114486.
[659] In some embodiments, where the metabolic bone disorder or disease is a
bone marrow
neoplasm, agent useful for administration to the patient in combination with
the agents that
inhibit Lp-PLA2 as disclosed herein are cancer agents admininstered for
surgery,
chemotherapy, radiotherapy, hormone therapy and immunotherapy and the like.
Such agents
include chemotherapeutic agents covering three main categories of therapeutic
agent: (1)
other antiangiogenic agents that work by different mechanisms from those
defined
hereinbefore (linomide, angiostatin, razoxin, thalidomide), (ii) cytostatic
agents such as
antioestrogens ( tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene),
progestogens
(megestrol acetate), aromatase inhibitors (nastrozole, letrazole, vorazole,
exemestane),
antiprogestogens, antiandrogens (flutamide, nilutamide, bicalutamide,
cyproterone acetate),
LHRH agonists and antagonists (goserelin acetate, luprolide), inhibitors of
testosterone
5.alpha.-dihydroreductase (finasteride), anti- invasion agents
(metalloproteinase inhibitors
like marimastat and inhibitors of urokinase plasminogen activator receptor
function) and
inhibitors of growth factor function, (such growth factors include for example
EGF, FGFs,
platelet derived growth factor and hepatocyte growth factor such inhibitors
include growth
factor antibodies, growth factor receptor antibodies, tyrosine kinase
inhibitors and
serine/threonine kinase inhibitors); and (iii)
antiproliferative/antineoplastic drugs and
combinations thereof, as used in medical oncology, such as antimetabolites
(for example
antifolates like methotrexate, fluoropyrimidines like 5-fluorouracil, purine
and adenosine
analogues, cytosine arabinoside); antitumour antibiotics (for example
anthracyclines like
doxorubicin, daunomycin, epirubicin and idarubicin, mitomycin-C, dactinomycin,
mithramycin); platinum derivatives (cisplatin, carboplatin); alkylating agents
(nitrogen
mustard, melphalan, chlorambucil, busulphan, cyclophosphamide, ifosfamide,
nitrosoureas,
thiotepa); antimitotic agents (vinca alkaloids like vincristine and taxoids
like taxol, taxotere);
topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and
teniposide,
amsacrine, topotecan), which are useful in the methods as disclosed herein.
CA 02588369 2007-05-11
- 107 -
[660] Thus, combination treatment with one or more agents that inhibit Lp-PLA2
with one
or more other medical procedures can be practiced.
[661] In addition to administering an Lp-PLA2 inhibitor, one can also
administer one or
more other therapeutic agent which prevents or reduces bone demineralization,
or effects re-
mineralization. Examples are bisphosphonates, parathyroid hormone (PTH),
hormone
replacement therapy (HRT), selective estrogen receptor modulators (SERM),
calcitonin,
RANKL antibody, constitutively active androstane receptor (CaR) antagonist and
cathepsin
K inhibitors.
[662] Examples of bisphosphonates include, for example but are not limited to
Alendronate
(Fosamax - Merck), Risedronate (Actonel ¨ Proctor & Gamble), Ibandronate
(Boniva /Bonviva ¨ Roche/GSK/), Zoledronate (Reclaste/Aclasts - Novartis).
Examples of PTH drugs include teriparatide (Forteo /Forsteo ¨ Eli Lilly)
parathyroid
hormone. Hormone replacement therapy and/or selective estrogen receptor
modulators
include estrogen, Premarin being one example, raloxifene sold under the name
Evista by
Eli Lilly, and bazedoxifene under development by Wyeth. An example of a
calcitonin
therapy is miacacline sold by Novartis.
[663] One or more of these drugs, and drugs of these types, can be
administered at the same
time as the Lp-PLA2 inhibitor, or they may be administered at another time if
the affects of
the combination therapy is optimized by the latter regime. While it is
believed that the
dosage of these second therapeutics will most likely be those approved for use
as a stand-
alone therapy, it is possible that a particular combination may warrant
adjusting the dosage
up or down depending on factors such as drug/drug effects and/or the need or
response of a
particular patient.
1664] In addition, treatment can also comprise multiple agents to inhibit Lp-
PLA2
expression or activity. Other agents include the use of statins with Niacin
(see
http://www.genengnews.com/news/bnitem.aspx?name=6724568) and fenofibrate (see
http://www.genengnews.com/news/bnitem.aspx?name=14817756&taxid=19).
[665] Similarly, diagnosis according to the invention can be practiced with
other diagnostic
procedures. The bone marrow or blood or urine can be assayed for a change in
gene
expression profiles using disease-specific molecular diagnostics kits (e.g.,
custom made
arrays, multiplex QPCR, multiplex proteomic arrays). In addition, a non-
invasive diagnostic
procedure (e.g., CAT, MM, SPECT, or PET) can be used in combination to improve
the
10566615.1
CA 02588369 2007-05-11
- 108 -
accuracy and/or sensitivity of diagnosis. Early and reliable diagnosis is
especially useful to
for prevention and/or treatment for metabolic bone disorders as disclosed
herein.
[666] The amount which is administered to a patient is preferably an amount
that does not
induce toxic effects which outweigh the advantages which result from its
administration.
Further objectives are to reduce in number, diminish in severity, and/or
otherwise relieve
suffering from the symptoms of the disease in the individual in comparison to
recognized
standards of care.
[667] Dosages, formulations, dosage volumes, regimens, and methods for
analyzing results
aimed at inhibiting Lp-PLA2 expression and/or activity can vary. Thus, minimum
and
maximum effective dosages vary depending on the method of administration.
Suppression of
the clinical and histological changes associated with a metabolic bone disease
and/or disorder
can occur within a specific dosage range, which, however, varies depending on
the organism
receiving the dosage, the route of administration, whether agents that inhibit
Lp-PLA2 are
administered in conjunction with other co-stimulatory molecules, and the
specific regimen of
inhibitor of Lp-PLA2 administration.
[668] For oral or enteral formulations for use with the present invention,
tablets can be
formulated in accordance with conventional procedures employing solid carriers
well-known
in the art. Capsules employed for oral formulations to be used with the
methods of the present
invention can be made from any pharmaceutically acceptable material, such as
gelatin or
cellulose derivatives. Sustained release oral delivery systems and/or enteric
coatings for
orally administered dosage forms are also contemplated, such as those
described in U.S. Pat.
No. 4,704,295, "Enteric Film-Coating Compositions," issued Nov. 3, 1987; U.S.
Pat. No. 4,
556,552, "Enteric Film- Coating Compositions," issued Dec. 3, 1985; U.S. Pat.
No.
4,309,404, "Sustained Release Pharmaceutical Compositions," issued Jan. 5,
1982; and U.S.
Pat. No. 4,309,406, "Sustained Release Pharmaceutical Compositions," issued
Jan. 5, 1982.
[669] Examples of solid carriers include starch, sugar, bentonite, silica, and
other commonly
used carriers. Further non-limiting examples of carriers and diluents which
can be used in the
formulations of the present invention include saline, syrup, dextrose, and
water.
[670] Enteric Coated Formulation
[671] As regards formulations for administering the small chemical entities
for inhibitors of
Lp-PLA2 of the likes of formulas (I) ¨ (IV) as disclosed herein, one
particularly useful
embodiment is a tablet formulation comprising the Lp-PLA inhibitor with an
enteric polymer
casing. An example of such a preparation can be found in W02005/021002. The
active
10566615.1
CA 02588369 2007-05-11
- 109 -
material in the core can be present in a micronised or solubilised form. In
addition to active
materials the core can contain additives conventional to the art of compressed
tablets.
Appropriate additives in such a tablet can comprise diluents such as anhydrous
lactose,
lactose monohydrate, calcium carbonate, magnesium carbonate, dicalcium
phosphate or
mixtures thereof; binders such as microcrystalline cellulose,
hydroxypropylmethylcellulose,
hydroxypropyl-cellulose, polyvinylpyrrolidone, pre-gelatinised starch or gum
acacia or
mixtures thereof; disintegrants such as microcrystalline cellulose (fulfilling
both binder and
disintegrant functions) cross-linked polyvinylpyn-olidone, sodium starch
glycollate,
croscarmellose sodium or mixtures thereof; lubricants, such as magnesium
stearate or stearic
acid, glidants or flow aids, such as colloidal silica, talc or starch, and
stabilisers such as
desiccating amorphous silica, colouring agents, flavours etc. Preferably the
tablet comprises
lactose as diluent. When a binder is present, it is preferably
hydroxypropylmethyl cellulose.
Preferably, the tablet comprises magnesium stearate as lubricant. Preferably
the tablet
comprises croscarmellose sodium as disintegrant. Preferably, the tablet
comprises
microcrystalline cellulose.
[672] The diluent can be present in a range of 10¨ 80% by weight of the core.
The
lubricant can be present in a range of 0.25 ¨ 2% by weight of the core. The
disintegrant can
be present in a range of 1 ¨ 10% by weight of the core. Microcrystalline
cellulose, if present,
can be present in a range of 10 ¨ 80% by weight of the core.
[673] The active ingredient preferably comprises between 10 and 50% of the
weight of the
core, more preferably between 15 and 35% of the weight of the core.
(calculated as free base
equivalent). The core can contain any therapeutically suitable dosage level of
the active
ingredient, but preferably contains up to 150mg as free base of the active
ingredient.
Particularly preferably, the core contains 20, 30, 40, 50, 60, 80 or 100mg as
free base of the
active ingredient. The active ingredient can be present as the free base, or
as any
pharmaceutically acceptable salt. If the active ingredient is present as a
salt, the weight is
adjusted such that the tablet contains the desired amount of active
ingredient, calculated as
free base of the salt. Preferably, the active ingredient is present as a
hydrochloride salt.
[674] The core can be made from a compacted mixture of its components. The
components
can be directly compressed, or can be granulated before compression. Such
granules can be
formed by a conventional granulating process as known in the art. In an
alternative
embodiment, the granules can be individually coated with an enteric casing,
and then
enclosed in a standard capsule casing.
10566615.1
CA 02588369 2007-05-11
- 110 -
[675] The core is surrounded by a casing which comprises an enteric polymer.
Examples of
enteric polymers are cellulose acetate phthalate, cellulose acetate succinate,
methylcellulose
phthalate, ethylhydroxycellulose phthalate, polyvinylacetate pthalate,
polyvinylbutyrate
acetate, vinyl acetate-maleic anhydride copolymer, styrene-maleic mono-ester
copolymer,
methyl acrylate-methacrylic acid copolymer or methacrylate-methacrylic acid-
octyl acrylate
copolymer. These can be used either alone or in combination, or together with
other
polymers than those mentioned above. The casing can also include insoluble
substances
which are neither decomposed nor solubilised in living bodies, such as alkyl
cellulose
derivatives such as ethyl cellulose, crosslinked polymers such as styrene-
divinylbenzene
copolymer, polysaccharides having hydroxyl groups such as dextran, cellulose
derivatives
which are treated with bifunctional crosslinking agents such as
epichlorohydrin,
dichlorohydrin or 1, 2-, 3, 4-diepoxybutane. The casing can also include
starch and/or
dextrin.
1676] Preferred enteric coating materials are the commercially available
Eudragit0 enteric
polymers such as Eudragit0 L, Eudragit0 S and Eudragit NE used alone or with
a
plasticiser. Such coatings are normally applied using a liquid medium, and the
nature of the
plasticiser depends upon whether the medium is aqueous or non-aqueous.
Plasticisers for use
with aqueous medium include propylene glycol, triethyl citrate, acetyl
triethyl citrate or
Citroflex or Citroflex A2. Non-aqueous plasticisers include these, and also
diethyl and
dibutyl phthalate and dibutyl sebacate. A preferred plasticiser is Triethyl
citrate. The
quantity of plasticiser included will be apparent to those skilled in the art.
1677] The casing can also include an anti-tack agent such as talc, silica or
glyceryl
monostearate. Preferably the anti-tack agent is glyceryl monostearate.
Typically, the casing
can include around 5 ¨ 25 wt% Plasticiser and up to around 50 wt % of anti
tack agent,
preferably 1-10 wt.% of anti-tack agent.
[678] If desired, a surfactant can be included to aid with forming an aqueous
suspension of
the polymer. Many examples of possible surfactants are known to the person
skilled in the
art. Preferred examples of surfactants are polysorbate 80, polysorbate 20, or
sodium lauryl
sulphate. If present, a surfactant can form 0.1 ¨ 10% of the casing,
preferably 0.2¨ 5% and
particularly preferably 0.5 ¨ 2%
[679] In one embodiment, there is a seal coat included between the core and
the enteric
coating. A seal coat is a coating material which can be used to protect the
enteric casing from
possible chemical attack by any alkaline ingredients in the core. The seal
coat can also
10566615 1
CA 02588369 2014-06-10
- 111 -
provide a smoother surface, thereby allowing easier attachment of the enteric
casing. A
person skilled in the art would be aware of suitable coatings. Preferably the
seal coat is made
of an Opadry coating, and particularly preferably it is OpadryTM White OY-S-
28876.
[680] In one embodiment, the pharmaceutically active ingredient is 1-(N-(2-
(diethylamino)ethyl)-N-(4-(4-trifluoromethylphenyl)benzyl)aminocarbonylmethyl)-
2-(4-
fluorobenzypthio-5,6-trimethylenepyrimidin-4-one, or a salt thereof.
[681] One example of such an enteric-coated formulation, as described in
W02005/021002,
comprises varying amounts of 1-(N-(2-(diethylamino)ethyl)-N-(4-(4-
trifluoromethylphenyl)benzypaminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5,6-
trimethylenepyrimidin-4-one (called "active" in this example) as hydrochloride
salt.
[682] In that example, lactose monohydrate, microcrystalline cellulose, the
active
ingredient, the hydroxypropyl methyl cellulose and half of the croscarmellose
sodium were
screened into a 10 Litre Fielder high-shear blender (any suitable high shear
blender could be
used) and blended for 5 minutes at 300 rpm with the chopper off. The mixture
was then
granulated by the addition of about 750 ml water whilst continuing to blend.
The granules
were dried in a Glatt 3/5 fluid bed drier, screened by Comil into a Pharmatec
5 Litre bin
blender and then blended with any lactose anhydrous given in the formula plus
the remainder
of the croscarmellose sodium over 5 minutes at 20 rpm. Magnesium stearate was
screened
into the blender and the mixing process continued for a further 1 minute at 10
rpm. The
lubricated mix was compressed using a Riva Piccolla rotary tablet press fitted
with 9.5mm
round normal convex punches (any suitable tablet press could be used). The
sealcoat, and
subsequently the enteric coat, are applied by spraying of an aqueous
suspension of the coat
ingredients in a Manesty 10 coater using parameters for the coating process as
recommended
by the manufacturers of the coating polymers (again, any suitable coater could
be used).
[683] Other enteric-coated preparations of this sort can be prepared by one
skilled in the art,
using these materials or their equivalents.
EXAMPLES
[684] The examples presented herein relate to the methods and compositions for
the
prevention and/or treatment of metabolic diseases or disorders including but
not limited to
osteoporosis or osteopenia. Throughout this application, various publications
are referenced.
The following examples are
CA 02588369 2007-05-11
- 112 -
not intended to limit the scope of the claims to the invention, but are rather
intended to be
exemplary of certain embodiments. Any variations in the exemplified methods
which occur
to the skilled artisan are intended to fall within the scope of the present
invention.
METHODS
[685] Animal model: A diabetic/diabetic hypercholestrolemic pig model (DM/HC)
was
developed that mimics human-like atherosclerosis. Domestic Yorkshire boars
weighing 25-
30 kg and aged ¨4 months were castrated and made diabetic with a single
intravenous
injection of 125 mg/kg of streptozotocin (Sicor Pharmaceuticals, Irvine, CA).
After stabilized
for 1-2 weeks, the animals with elevated levels of plasma glucose (>150 mg/di)
were fed with
atherogenic (high-fat) diet as shown in Table 1 (Animal Specialties,
Quakertown, PA) to
achieve a cholesterol level of approximately 250-800 mg/d1. Maintainance of
the cholesterol
level was determined by method as shown in Table 2.
[686] Table 1. Diet for 2.0% cholesterol diet the components are:
Component Weight/Weight %
Purina* porcine grower meal 47.5 %
Lard 25.0%
Casein 11.1%
Dried whole milk 7.9 %
_
Peanut oil 2.37 %
Cholesterol 2.0 %
Wesson salt mix 2.37 %
Purina* vitamin mix 1.58 `)/0
Sodium cholate 1.58 %
Calcium carbonate 0.4 %
Choline chloride 0.2 %
*Purina Mills, LLC, Checkerboard Square, St. Louis, Missouri, 63164, USA.
These feeds
were prepared by Animal Specialties and Provisions, LLC, Quakertown, PA USA.
[687] For the 0.5% cholesterol diet the components are similar with the
exception of 0.5%
cholesterol and 20% lard. The animals were Yorkshire pigs that were castrated
males at the
age of 3-5 and were obtained from Archer Farms, Darlington, MD. These feeds
were
prepared by Animal Specialties and Provisions, LLC, Quakertown, PA USA.
[688] On days 1-2, animals were fed normal chow, followed by on days 3-14
animals were
fed a diet of 0.5% cholesterol, 2% lard and on day 14, cholesterol levels were
measured and
10566615.1
CA 02588369 2007-05-11
- 113 -
the diet adjusted accordingly to increase to 2% cholesterol, 10% lard if
cholesterol is <300
mg/di. Following induction of DM/HC, cholesterol was measured until
cholesterol levels are
stable between 300 and 800 mg/di, and following cholesterol stabilization,
cholesterol was
measured monthly. If cholesterol levels were unstable following initial
stabilization phase,
the diet of the animal was returned to the initial two-week measurement
schedule. Monthly
cholesterol levels were determined, including levels of total cholesterol,
LDL, HDL, VLDL
and triglycerides. Adjustment of the diet of the animal for a stable
cholersteol level was
determined according to the outlines shown in Table 2.
[689] Table 2. Cholesterol and Dietry adjustment.
Cho!ester Dietary Next Cholesterol Dietary
adjustment Next
ol level adjustment measure level choleste
rol
ment measure
ment
<250 Change to 2 weeks <300 mg/di Continue 25%
lard diet 2 weeks
mg/di 25% lard
diet.
300-800 mg/di Change to 75% lard (25% lard): 25% 2 weeks
normal diet
>800 mg/d1 Change to 100% 10% lard diet 2
weeks
>1000 mg/c11 Change to 50:50 mix of 10% lard diet
2 weeks
>1500 mg/di Change to normal diet 2 weeks*
300-800 No change. 2 weeks <300 mg/di Change to
25% lard diet 2 weeks
mg/di 10% lard
diet
300-800 mg/di No change Regular
schedule
>800 mg/di Change to 50:50 mixture with 10% lard
2 weeks
>1000 mg/di Change to 25:75 mixture with 10% lard
2 weeks
>1500 mg/c11 Normal chow 2 weeks
800-1000 Change to 2 weeks <300 mg/di Change to 25:75 mixture
with 10% lard 2 weeks
mg/di 50:50 mix of diet
10% lard
and normal
chow diet.
300-800 ring/d1 No change in diet (50:50) 10% lard 2 weeks
>800 mg/di Change to 25:75 mixture (10% lard)
2 weeks
>1000 mg/di Change to 25:75 mixture (10% lard)
2 weeks
>1500 mg/di Normal chow 2 weeks*
>1000 25:75 mix of 2 weeks <300 mg/di Change to
50:50 mix of 10% lard and 2 weeks
mg/di 10% lard normal chow diet.
and normal
chow diet.
300-800 mg/di No change in diet 25:75 mix of 10% 2 weeks
lard and normal chow diet
>800 mg/c11 No change in diet 2 weeks
>1000 mg/di Normal chow 2 weeks*
>1500 mg/di Normal chow 2 weeks*
>1500 Normal 2 weeks <300 mg/c11 Change to
100% of 10% lard diet. 2 weeks
mg/c11 chow diet.
300-800 mg/c11 Chge diet to 50:50 mix of 10% lard 2 weeks
10566615.1
CA 02588369 2007-05-11
- 114 -
and normal chow diet
>800 mg/di Normal chow 2
weeks*
>1000 mg/di Normal chow 2
weeks*
>1500 mg/d1 Normal chow 2
weeks*
[690] At one month after stabilization of choleresterol at 250-800mg/d1, the
animals were
randomized into two experimental groups, DM/HC (hyperglycemia and
hypercholesterolemia) group with no treatment and treatment group (10
mg/kg/day of SB-
480848, also referred to as 1-(N-(2-(diethylamino)ethyl)-N-(4-(4-
trifluoromethylphenyl)benzy1)-aminocarbonylmethyl)-2-(4-fluorobenzypthio-5,6-
trimethylenepyrimidin-4-one). The animals were sacrificed at 6-month after the
randomization. The animal protocol has been approved by the Institutional
Animal Care and
Use Committee of University of Pennsylvania.
[691] Two diabetic/hypereholesterolemic groups were evaluated: 1. DM/HC group
and 2.
DM/HC animals receiving Lp-PLA2 inhibitors. The experiments included: the
control group
(DM/HC group - 17 pigs) and the experimental group (DM/HC animals receiving Lp-
PLA2
inhibitors - 20 pigs). In addition blood cholesterol levels were maintained
between 300 and
800 mg/di in experimental animals, this range having been determined to
provide a better test
model. Blood cholesterol levels were monitored in all animals on a bimonthly
basis, as
shown in Table 4 and adjustments were made to the fat content of the feed
accordingly, as
shown in Table 2. The cholesterol and lard percent were in the range of 0.5-2%
and 10-25%,
respectively, and all animals received feed that contained cholesterol and
lard concentration
within that range. Blood samples were obtained at baseline, 1 month, 3 months,
and 6
months. Less than 1 ml/kg of blood were obtained each time. At the bimonthly
blood
cholesterol levels tests, levels of total cholesterol, LDL, HDL, VLDL and
triglycerides, blood
glucose, Lp-PLA2 and primary bone marrow cells (PBMCs) were tested (see Table
4).
[692] The animal number, selected to justify the minimum requirement for
statistical
validity were 2 groups of animals per experiment as follows: 1. Control group
(n-17);
Diabetic and hyperlipidemic; 2. Experimental group (n=20) Diabetic,
hyperlipidemie
receiving 10 mg/kg Lp-PLA2 inhibitor, as shown in Table 3.
[693] Domestic farm pigs, Yorkshire boars, ranging in weight between 25-35 kg
were
purchased from a local farm and placed in indoor housing under the care of a
veterinarian.
They were castrated 3-5 days in advance of the study start date. Test pigs
were made diabetic
by infusing one dose of streptozocin (125 mg/kg) IV in a period of 30 min. If
animals do not
become diabetic a second dose of (50 mg/kg) was administered. To avoid the
possible onset
10566615.1
CA 02588369 2007-05-11
- 115 -
of initial hypoglycemia, 20 g of glucose powder was added to the feed for the
first 2. The
blood glucose was measured using a glucometer every day before feeding for the
first 14
days and then once a week.
[694] Test animals were housed separately from control animals to avoid inter-
animal
transfer of drug due to colcophagia. All animals were fed an atherogenic diet
twice daily with
free access to water. The custom-made diet contained 0.5 and 2% cholesterol
and 10 and
25% lard, the components of which are shown in Table 1.
[695] Table 3. Schedule of animals and procedures (divided into 2 groups):
Animal number Timeline
Group 1: DM/HC N=17 7 months
Group 2: DM/HC receiving Lp- 7 months
N=20
PLA, inhibitors
Total N=37 7 months
[696] Table 4. Summary of Proceedures
Start 28 d 57 d 85 d - 113 d 141 d 168 d
196 d
Serum Glucose, LDL, HDL, X X X X X X X X
Triglycerides
Lp-PLA2 (frozen serum-EDTA) X X X X X X X X
PBMCs X X X X
Tissue harvest X
[697] Daily dosing began on Day 29, at which time each test animal was given a
daily dose
of 10 mg/kg SB-480848 (given as bolus equivalent in dog food). Animals were
NPO from
midnight the night before. Animals were euthanized on Day 196 and tissues
harvested
immediately.
[698] Histochemical Stains. Tissues were fixed in 4% Paraformaldehyde,
decalcified in
12.5% EDTA, dehydrated in a graded series of ethanol, followed by xylene,
embedded in
paraffin and sectioned (6 mm). Paraffin sections were dewaxed, rehydrated and
stained with
Harris Hematoxylin (Fisher Scientific, Kalamazoo, MI; 245-678) alcian blue
(Alcian Blue
8GX, Sigma, St. Louis, MO; A3157) (pH 1) and Eosin Y (Fisher Scientific
Kalamazoo, MI;
245-827) (H&E-AB) to determine cellularity, vascularization, tissue morphology
and
10566615.1
CA 02588369 2007-05-11
- 116 -
proteoglycan content. Inflammatory cells were identified via staining with
Protocol Wrights-
Giemsa Stain (Fisher Scientific, Kalamazoo, MI; 264-985).
[699] TUNEL Assay. Apoptosis was measured by the TUNEL assay. The TUNEL assay
takes advantage of the fact that during apoptosis nuclear endonucleases digest
genomic DNA
into fragments of multiples of approximately 200bp. To measure the fragmented
DNA, the
nucleotide ends were labeled using the FragEL DNA Fragmentation Kit
Colorimetric ¨
Klenow Enzyme (Calbiochem, EMD Biosciences, La Jolla, CA, Q1A21-1EA) according
to
manufacturer's instructions and positive DAB signal was visualized by
microscopy as
described below. DNase I recombinant, grade I (Roche Diagnostics,
Indianapolis, IN; 04 536
282 001) was used to generate TUNEL positive control sections according to
manufacturer's
instructions. Counterstained with Methyl Green. Analysis was performed on 4
animals from
each group, and representative images taken.
[700] Image Acquisition, Capture, and Analysis, Images were acquired with a
Retiga EXi
digital-cooled CCD camera with RGB electronic filter (QImaging, Canada) or
with an with
an RT Color Spot 1X HRD 100-NIK Model 2.2.1 camera (Diagnostic Instruments,
Sterling
Heights, MI) on either a Nikon Optiphot or on a Nikon E800 (Nikon, Melville,
NY) both
equipped with z-axis motors (Prior Scientific, Rockland, MA).
[701] Alkaline Phosphatase and Alizarin Red Staining. Alizarin Red and
Alkaline
Phosphatase Staining are bone marker proteins indicating the degree of
mineralization and
intensity of osteblast differentiation, respectively. MLO-A5 osteoblastic
cells (isolated from
14-day-old osteocalcin promoter-driven T-antigen transgenic mice, J Bone Miner
Res
12:2014-2023, 1997) were cultured on Collagenl coated plates at density
15000cells/ well in
12-well TC plates. After 24 hours, the cells were treated w/ 10mM 0- GP and
LysoPC at 5
[tM concentration for 3 and 7 days. Treatment was repeated every other day. At
each time
point alizarin red and alkaline phosphatase staining was performed. Alkaline
phosphatase
Detection Kit (Chemicon, Intl., Temecula, CA, SCR004) was used for detection
and
performed as per manufactures instructions. To visualize mineralization the
cells were
washed once with a 2% Alizarin Red S solution for 5 ¨ 10 min. at room
temperature followed
by one wash with water, two washes with PBS and fixation with
paraformaldehyde. The
results were then imaged with a digital cooled CCD camera.
[702] Cell Proliferation. MLO-A5 cells were plated in 48-well TC dishes at a
density of
5,000 cells/well and allowed to attach (5-6 hrs.). The cells were treated with
5 uM of Lyso
10566615 1
CA 02588369 2007-05-11
- 117 -
PC for 18 hrs followed by analysis with the CyQUANT Cell Proliferation Assay
Kit
(Molecular Probes) following manufacturers instructions to measure MLO-A5
proliferation.
EXAMPLE 1
[703] Bone density and micro-architecture is increased by inhibitors of Lp-
PLA,.
1704] Tissue sampling: The right knee joint was removed and dissected. The
femoral bone
was split in half in parallel to the long axis of the bone. A piece of bone
including both the
articular surface and growth plate region (-45-20 mm) was sliced off from the
front and
parallel to the femoral shaft (Figure. 1). The tissues were fixed in 4%
paraformaldehyde and
scanned within one week in a CT scanner (ACT-40, Scanco Corp.) with a voxel
resolution of
Am.
[705] MicroCT Analysis - Micro CT analysis generates 3 dimensional data
including bone
density and indices of micro-architecture. From the central region, i.e.,
between the articular
surface (top) and the growth plate (bottom), 250 slices (-5 mm2) were
analyzed. To avoid
15 artifact, the measurements were contoured away from the regions directly
below the articular
surface and the cutting edges. Paraformaldehyde fixed tissues (2x2 cm) were
analyzed using
microCT 40 (Scanco Medical, Bassersdorf, Switzerland) and a 3D reconstruction
of the
images slices was generated. The tissues were scanned at a medium resolution
with slice
thickness and slice increments of 20 p.m. A Sigma filter setting of 0.8, a
support value of 1,
20 and a threshold setting of 195. Scanning was performed at an energy
setting of 70000 (V) and
an intensity of 114 (AA) with an integration time of 200ms. All values for
tissue volume
(mm3), bone volume (mm3), bone volume/tissue volume, and mean/density (mg
HA/ccm)
were also calculated and recorded. The raw data were automatically segmented
and analyzed
with the CT Evaluation Program; the segmented data were imported and
displayed in CT
Ray V3Ø The image density was measured with the Distance3D tool of the CT
Evaluation
Program V5Ø
[706] The results of the MicroCT are summarized in Table 5 and examples of
DM/HC-
induced oestoporic changes are showin in Figure 2.
1707] Table 5. Results from MicroCT analysis. (P-value is t-test DM/HC vs.
SB480848)
10566615.1
CA 02588369 2007-05-11
- 118 -
Measurements Control DMIFIC 88480848 P value
SMI 0.68 0.10 0.997 0.11 0.65 0.10 0.028
BV/TV 0.25 0.01 0.21 0.01 0.25 0.01 0.016
BSIBV 23.18 1.55 27.16 0.81 24.40 0.51
0.012
Tb.Th 0.10 0.007 0.09 0.003 0.10 0.002
0.051
Tb.Sp 0.36 0.018 0.36 0.02 0.33 0.007
0.184
Tb.N 2.56 0.12 2.66 0.12 2.78 0.06 0.396
Conn-Dens. 31.38 3.46 34.97 2.74 35,69 1.22
0.828
[708] The structural model index (SMI) is an indicator of trabecular micro-
architecture with
0 indicating parallel plate-like formation (a stronger, more structurally
mature form) and 3
indicating cylindrical rod-like trabecular (more fragile, degenerating
structure). The higher
SMI in DM/HC animals shows degeneration of the bone micro-architecture, as
shown in
Figure 2B as compared with normal controls (Figure 2A). In Lp-PLA2 inhibitor
treated
animals, the DM/HC-induced degenerative changes were minimized, as shown in
figure 2C.
[709] The BV/TV (bone volume over total volume) is a volumetric indicator of
the bone
and the BS/BV (bone surface over bone volume) indicates bone structure. Strong
bones have
higher BV/TV and thin bones have higher BS/BV. DM/HC animals exhibited reduced
BV/TV and increased BS/BV, demononstrating DM/HC animals have osteoporotic
pathogenesis. On the contrary, DM/HC animals treated with the Lp-PLA2
inhibitor
SB480848 showed no osteoporotic changes.
[710] Tb.Th is a measurement of trabecular thickness. DM/HC animals showed
reduced
Tb.Th, whereas the reduced Tb.Th thichness was prevented in DM/HC animals
treated with
the Lp-PLA2 inhibitor.
[711] Other indicators used include: Trabecular separation (Tb.Sp), trabecular
number
(TbN), and connectivity density (ConriD), which showed no significant changes
between
control and DM/HC animals, or DM/HC animals treated with the Lp-PLA2 inhibitor
SB480848.
[712] During the study period, it was observed that animals administered the
Lp-PLA2
inhibitor were more responsive to external stimuli, demonstrated increased
activity in the
cage, and tended to respond more alertly to feeding and handing as compared to
the control
animals. Also, despite similar serum glucose and cholesterol levels, animals
treated with the
Lp-PLA2 inhibitor demonstrated an increase in weight as compared to control
animals (62.5
kg vs 50.9 kg for control animals) from a baseline of 26.9 kg and 30.3 kg
weight respectively.
Weight in animals administered the Lp-PLA2 inhibitor is a direct reflection of
their overall
10566615.1
CA 02588369 2007-05-11
- 119 -
well-being, insofar as that more sickly animals (i.e. the control animals) do
not eat. It was
observed that inhibition of inflammation by a LP-PLA, inhibitor results in
greater well-being
and health in the setting of systemic inflammation.
EXAMPLE 2
[7131 Lp-PLA, inhibtor restored bone marrow homeostasis.
[714] Histochemical staining with H&E-AB of trabecular bone areas showed
distinct
morphological changes between DM/HC animals and animals treated with the Lp-
PLA2
inhibitor SB480848. DM/HC animals showed marrow hypoplasia, fat atrophy, and
deposition of extracellular "gelatinous" material (gelatinous transformation)
as shown in
Figure 3B. Excess glycosaminoglycan, significantly alters the bone marrow
microenvironment, is detrimental to erythropoiesis, as well as osteogenesis.
In trabecular
bone marrow, striking abnormalities were observed in DM/HC animals, as shown
in Figure
3B and Figure 4A-D, with increased extracellular material, reduced
cellularility and
shrinkage of adipocyte as compared to control non-DM/HC animals as shown in
Figure 3A.
In contrast, the bone marrow of DM/HC animals treated with a Lp-PLA2 inhibtor
are shown
in Figure 4E-G, which was similar to normal control animals (for example non-
treated non-
DM/HC animals as shown in Figure 3A) and did not show abnormalities in bone
marrow that
occurred in non-treated DM/HC animals, demonstrating the Lp-PLA2 inhibotor
preserved
bone marrow homeostasis.
[715] Bone marrow homeostasis was also preserved in bone marrow located
remotely from
traebucular bone, as shown in a low magnification image of the bone marrow
remote from
the trabecular bone in Figure 5, animals treated with the Lp-PLA2 inhibitor
have normal bone
marrow as shown in Figures 5C and D, whereas abnormal homeostasis occurred in
non-
treated DM/HC animals, as shown in Figure 5A and B. Higher magnification
images of bone
marrow remote from the trabecular bone are shown in Figure 6D-F, where the Lp-
PLA2
inhibitor prevented DM/HC-induced bone marrow abnormalities that occurred in
non-treated
DM/HC animals (as shown in Figure 6A-C).
EXAMPLE 3
[716] Inhibition of loss of osteocytes and osteblasts by inhibitors of Lp-
PLA,,
[717] TUNEL staining of regions of trabecular bone were assessed in both DM/HC
animals
not treated and DM/HC animals treated with an Lp-PLA2 inhibitor. The animals
not treated
10566615.1
CA 02588369 2013-10-22
- 120 -
with the Lp-PLA2 inhibitor show dramatic increases in TUNEL positive cells
within the
trabecular bone, for example increased TUNEL positive osteocytes, and
increased TUNEL
positive osteoblasts on the surface the trabecular bone, as shown in Figure
7A, as compared
to animals treated with the Lp-PLA2 inhibtor, as shown in Figure 4B. Increased
apoptosis in
DM/HC animals demonstrates increase bone loss, fast bone turn over and reduced
bone
formation as compared to DM/HC animals treated with a Lp-PLA2 inhibitor, which
demonstrate less bone loss as demonstrated by micro CT analysis as shown in
Example 1.
EXAMPLE 4
[718] Activation of the Lp-PLA2 pathway by LypoPC prevents osteoblast
mineralization,
osteoblast differentiation and proliferation.
[719] Alizarin Red and Alkaline Phosphatase Staining are bone marker proteins
indicating
the degree of mineralization and intensity of osteblast differentiation,
respectively. MLO-A5
osteoblastic cells stained with alizarin Red following treatment with 5 M
LysoPC caused
decreased mineralization of MLO-A5 cells, compared to non-treated control
cells, after 7
days as shown by Alizarin Red Staining in Figure 8.
[720] Treatment with 51.IM LysoPC caused decreased alkaline phosphatase
staining of
MLO-A5 cells after 7 day treatment as compared to control, as shown in Figure
9.
[721] As shown, figure 10, MLO-A5 cells treated with 5 [tM LysoPC for 18 hours
show
reduced proliferation, as compared to non-treated cells, demonstrating that
activation of the
Lp-PLA2 pathway by LysoPC prevents proliferation of osteoblasts.
