U.S. patent application number 11/858827 was filed with the patent office on 2008-09-04 for compositions and methods for inducing osteogenesis.
This patent application is currently assigned to IRM LLC. Invention is credited to Sheng Ding, Nathanael S. Gray, Xu Wu.
Application Number | 20080214541 11/858827 |
Document ID | / |
Family ID | 32110195 |
Filed Date | 2008-09-04 |
United States Patent
Application |
20080214541 |
Kind Code |
A1 |
Wu; Xu ; et al. |
September 4, 2008 |
COMPOSITIONS AND METHODS FOR INDUCING OSTEOGENESIS
Abstract
The present invention provides compositions and methods for
differentiating and transdifferentiating mammalian cells into cells
of an osteoblast lineage.
Inventors: |
Wu; Xu; (San Diego, CA)
; Ding; Sheng; (San Diego, CA) ; Gray; Nathanael
S.; (San Diego, CA) |
Correspondence
Address: |
TOWMSEND & TOWNSED & CREW LLP
TWO EMBARCADERO CENTER, EIGHT FLOOR
SAN FRANCISCO
CA
94111
US
|
Assignee: |
IRM LLC
Hamilton HM LX
CA
Scripps Research Institute
La Jolla
|
Family ID: |
32110195 |
Appl. No.: |
11/858827 |
Filed: |
September 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10687220 |
Oct 15, 2003 |
7273864 |
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11858827 |
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60488699 |
Jul 18, 2003 |
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60418898 |
Oct 15, 2002 |
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Current U.S.
Class: |
514/234.2 ;
435/29; 435/352; 435/354; 435/363; 435/366; 435/377; 435/6.13;
514/263.1; 544/118; 544/277 |
Current CPC
Class: |
C07D 473/34 20130101;
C07D 473/18 20130101; C07D 473/16 20130101; A61P 7/00 20180101;
C07D 473/40 20130101; A61P 19/00 20180101; A61P 19/10 20180101 |
Class at
Publication: |
514/234.2 ;
544/277; 514/263.1; 544/118; 435/377; 435/354; 435/29; 435/6;
435/352; 435/363; 435/366 |
International
Class: |
A61K 31/5377 20060101
A61K031/5377; C07D 473/00 20060101 C07D473/00; A61K 31/52 20060101
A61K031/52; C12Q 1/02 20060101 C12Q001/02; C12Q 1/68 20060101
C12Q001/68; C12N 5/00 20060101 C12N005/00; C07D 295/00 20060101
C07D295/00 |
Claims
1. A compound of formula I: ##STR00025## wherein: R.sup.1 is a
member selected from the group consisting of hydrogen, halogen and
-L-R.sup.2; L is a member selected from the group consisting of
--O-- and --NR.sup.3--, wherein R.sup.3 is H, or R.sup.3 is
optionally taken together with R.sup.2 and the nitrogen to which
both are attached to form a heterocycle, optionally substituted
with C.sub.1-4alkyl; R.sup.2 is a member selected from the group
consisting of C.sub.1-4alkyl, C.sub.3-8cycloalkyl and
C.sub.0-2alkylaryl, substituted with 0-2 R.sup.2a groups that are
independently selected from the group consisting of halogen,
C.sub.1-4alkyl, C.sub.1-4alkoxy, --N(R.sup.2b, R.sup.2b),
--SO.sub.2N(R.sup.2b, R.sup.2b), --C(O)N(R.sup.2b, R.sup.2b) and
--O-aryl, or when said R.sup.2a groups are on adjacent ring atoms
they are optionally taken together to form a member selected from
the group consisting of --O--(CH.sub.2).sub.1-2--O--,
--O--C(CH.sub.3).sub.2CH.sub.2-- and --(CH.sub.2).sub.3-4--; each
R.sup.2b group is a member that is independently selected from the
group consisting of hydrogen and C.sub.1-4alkyl; R.sup.4 is a
member selected from the group consisting of C.sub.1-4alkyl,
C.sub.3-8cycloalkyl, C.sub.1-4alkylhydroxy, C.sub.0-2alkylaryl,
substituted with 0-2 R.sup.4a groups, and
C.sub.0-2alkylheterocycle, optionally substituted with
C.sub.1-4alkyl; each R.sup.4a group is a member independently
selected from the group consisting of hydrogen, halogen,
C.sub.1-4alkyl, C.sub.1-4alkoxy, and aryl, or when said R.sup.4a
groups are on adjacent ring atoms they are optionally taken
together to form --O--(CH.sub.2).sub.1-2--O--; R.sup.5 is hydrogen
and R.sup.6 is a member independently selected from the group
consisting of halogen, C.sub.1-4alkyl, --C(O)--C.sub.1-4alkyl,
--SO.sub.2--N(R.sup.2b, R.sup.2b), C.sub.1-4alkylhalo, --O-aryl and
--N(R.sup.7, R.sup.8), or when R.sup.5 and R.sup.6 are on adjacent
ring atoms they are optionally taken together to form
--O--(CH.sub.2).sub.1-2--O--; R.sup.7 is a member selected from the
group consisting of hydrogen, C.sub.1-4alkyl,
C.sub.1-4alkylhydroxy, aryl and --C(O)R.sup.7a; R.sup.7a is a
member selected from the group consisting of C.sub.1-4alkyl,
C.sub.1-4alkylhalo, C.sub.3-8cycloalkyl and aryl; R.sup.8 is a
member selected from the group consisting of H and C.sub.1-4alkyl,
or R.sup.7 and R.sup.8 are optionally taken together with the
nitrogen to which they are attached to form a heterocycle,
optionally substituted with C.sub.1-4alkyl; and all
pharmaceutically acceptable salts and hydrates thereof.
2. A compound of claim 1, wherein: R.sup.1 is a member selected
from the group consisting of: ##STR00026##
3. A compound of claim 1, wherein: R.sup.1 is ##STR00027##
4. A compound of claim 1, wherein: R.sup.4 is a member selected
from the group consisting of: ##STR00028##
5. A compound of claim 1, wherein: R.sup.4 is cyclohexyl.
6. A compound of claim 1, wherein: R.sup.5 is H and R.sup.6 is
morpholine.
7. A compound of claim 1, wherein: R.sup.1 is ##STR00029## R.sup.5
is H; and R.sup.6 is morpholine.
8. A compound of claim 1, wherein: R.sup.1 is ##STR00030## R.sup.5
is H; R.sup.6 is morpholine; and R.sup.4 is a member selected from
the group consisting of: ##STR00031##
9. A compound of claim 1, wherein the compound is a member selected
from the group consisting of: ##STR00032## ##STR00033##
##STR00034## ##STR00035## ##STR00036##
10. A compound of claim 1, wherein the compound is:
##STR00037##
11. A pharmaceutical composition comprising a compound of claim 1
and a pharmaceutically acceptable carrier.
12. A method of inducing osteogenesis, the method comprising:
contacting a mammalian cell with a compound of claim 1, whereby the
mammalian cell differentiates into a cell of an osteoblast
lineage.
13. The method of claim 12, wherein said compound of claim 1 is in
a pharmaceutically acceptable carrier.
14. The method of claim 12, wherein the mammalian cell is in a
mammal.
15. The method of claim 14, wherein the step of contacting is by
oral administration of the compound to the mammal.
16. The method of claim 14, wherein the step of contacting is by
intravenous administration of the compound to the mammal.
17. The method of claim 14, wherein the step of contacting is by
subcutaneous administration of the compound to the mammal.
18. The method of claim 14, wherein the step of contacting is by
intraperitoneal administration of the compound to the mammal.
19. The method of claim 12, further comprising detecting
differentiation of the mammalian cell into a cell of an osteoblast
lineage.
20. The method of claim 19, whereby differentiation of the
mammalian cell into a cell of an osteoblast lineage is detected by
detecting expression of an osteogenesis marker gene.
21. The method of claim 20, wherein the osteogenesis marker gene is
a gene selected from the group consisting of alkaline phosphatase,
collagen type I, osteocalcin, and osteoponin.
22. The method of claim 19, whereby differentiation of the
mammalian cell into a cell of an osteoblast lineage is detected by
detecting expression of a bone specific transcription factor.
23. The method of claim 22, wherein the bone specific transcription
factor is Cbfa1/Runx2.
24. The method of claim 12, wherein the mammalian cell is a stem
cell.
25. The method of claim 24, wherein the stem cell is a mesenchymal
stem cell.
26. The method of claim 25, wherein the mesenchymal stem cell is
isolated from a mouse.
27. The method of claim 26, wherein the mesenchymal stem cell is
murine embryonic mesoderm fibroblast cell.
28. The method of claim 25, wherein the mesenchymal stem cell is
isolated from a primate.
29. The method of claim 28, wherein the primate is a human.
30. The method of claim 12, wherein the mammalian cell is further
contacted with bone morphogenetic protein 4 (BMP-4).
31. The method of claim 30, wherein the mammalian cell is a
pre-adipocyte cell.
32. The method of claim 30, wherein the mammalian cell is a
myoblast cell.
33. The method of claim 12, wherein the mammalian cell is attached
to a solid support.
34. The method of claim 33, wherein the solid support is a three
dimensional matrix.
35. The method of claim 33, wherein the solid support is a planar
surface.
36. A method of inducing osteogenesis, the method comprising:
contacting a mammalian cell with a compound of claim 10, whereby
the mammalian cell differentiates into a cell of an osteoblast
lineage.
37. The method of claim 36, wherein the mammalian cell is in a
mammal.
38. The method of claim 36, wherein the step of contacting is by
oral administration of the compound to the mammal.
39. The method of claim 36, wherein the step of contacting is by
intravenous administration of the compound to the mammal.
40. The method of claim 36, wherein the step of contacting is by
subcutaneous administration of the compound to the mammal.
41. The method of claim 36, wherein the step of contacting is by
intraperitoneal administration of the compound to the mammal.
42. A method of treating a bone disorder, the method comprising:
contacting a mammalian cell with a compound of claim 1, whereby the
mammalian cell differentiates into a cell of an osteoblast
lineage.
43. The method of claim 42, wherein the bone disorder is associated
with defective osteoblasts.
44. The method of claim 43, wherein the bone disorder is
osteoporosis.
45. The method of claim 42, further comprising administering the
cell of an osteoblast lineage to an individual with the disorder,
thereby treating the disorder.
46. The method of claim 45, wherein the administration is by
surgical implantation.
Description
RELATED APPLICATIONS
[0001] This patent application claims the benefit of U.S.
Provisional Patent Application No. 60/418,898, filed Oct. 15, 2002,
and U.S. Provisional Patent Application No. 60/______, filed Jul.
18, 2003 (Attorney Docket No. 021288-001810), the teachings of both
of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Bone is a dynamic tissue, with a structure comprising a
mineral phase associated with an organic matrix. Homeostasis in the
adult skeleton requires a balance between bone resorption and bone
formation. During resorption, special cells on the bone's surface
dissolve bone tissue and create small cavities. During formation,
other cells fill the cavities with new bone tissue. An imbalance in
the bone remodeling cycle may cause bone loss that eventually leads
to bone-related diseases. Cells of an osteoblast lineage
(osteoblasts) play a key role in the balance of the bone remodeling
cycle by synthesizing and depositing new bone matrix
(osteogenesis). Cells of an osteoblast lineage differentiate from
mesenchymal stem cells (i.e., mesenchymal precursor cells).
Misregulation of the differentiation of mesenchymal precursor cells
into osteoblasts may account for several bone related diseases
associated with defective osteoblasts (see, e.g., Olsen et al, Ann.
Rev. Cell Dev. Biol. 16:191 (2000)), such as, for example,
osteoporosis, rickets, osteomalacia, McCune-Albright syndrome, and
Paget's disease.
[0003] Compositions that stimulating osteogenesis can conveniently
be used in therapeutic methods (e.g., oral administration) to treat
or prevent these and other disorders. In addition, cells of an
osteoblast lineage can conveniently be used in therapeutic methods
(e.g., transplantation) to treat or prevent these and other
disorders. Methods of stimulating osteogenesis have been described
in e.g., U.S. Pat. Nos. 6,369,029 and 5,942,225. It has been
difficult, however, to obtain osteoblasts in sufficient numbers to
enable effective therapy. For example, U.S. Pat. No. 5,942,225
describes in vitro culture conditions that induce differentiation
of human mesenchymal stem cells into cells of an osteogenic lineage
using a combination of a steroid (dexamethasone),
.beta.-glycerolphosphate and ascorbic acid. However, steroids such
as dexamethasone are potent anti-inflammatory drugs that have
multiple side-effects. Thus, compositions and methods for
appropriate regulation of the proliferation and differentiation of
mesenchymal stem cells into cells of an osteoblast lineage has
remained elusive.
[0004] Thus, there is a need in the art for compositions and
methods for inducing differentiation and transdifferentiation of
mammalian cells into cells of an osteoblast lineage in vivo and in
vitro. There is a particular need for small molecules that can
induce in vivo and in vitro differentiation and
transdifferentiation of mammalian cells into cells of an osteoblast
lineage. The present invention satisfies these and other needs.
SUMMARY OF THE INVENTION
[0005] The present invention provides novel compositions and
methods for inducing in vivo and in vitro differentiation and
transdifferentiation of mammalian cells into cells of an osteoblast
lineage.
[0006] One embodiment of the present invention provides compounds
of Formula I:
##STR00001##
wherein:
[0007] R.sup.1 is a functional group including, but not limited to,
hydrogen, halogen and -L-R.sup.2;
[0008] L is a functional group including, but not limited to, --O--
and --NR.sup.3--, wherein R.sup.3 is H, or R.sup.3 is optionally
taken together with R.sup.2 and the nitrogen to which both are
attached to form a heterocycle, optionally substituted with
C.sub.1-4alkyl;
[0009] R.sup.2 is a functional group including, but not limited to,
C.sub.1-4alkyl, C.sub.3-8cycloalkyl and C.sub.0-2alkylaryl,
substituted with 0-2 R.sup.2a groups that are independently
selected and that are functional groups including, but not limited
to, halogen, C.sub.1-4alkyl, C.sub.1-4alkoxy, --N(R.sup.2b,
R.sup.2b), --SO.sub.2N(R.sup.2b, R.sup.2b), --C(O)N(R.sup.2b,
R.sup.2b) and --O-aryl, or if R.sup.2a groups are present and if
the two R.sup.2a groups are on adjacent ring atoms, they are
optionally taken together to form a functional group including, but
not limited to, --O--(CH.sub.2).sub.1-2--O--,
--O--C(CH.sub.3).sub.2CH.sub.2-- and --(CH.sub.2).sub.3-4--;
[0010] each R.sup.2b group is independently selected and is a
functional group including, but not limited to, hydrogen and
C.sub.1-4alkyl;
[0011] R.sup.4 is a functional group including, but not limited to,
C.sub.1-4alkyl, C.sub.3-8cycloalkyl, C.sub.1-4alkylhydroxy,
C.sub.0-2alkylaryl, substituted with 0-2 R.sup.4a groups, and
C.sub.0-2alkylheterocycle, optionally substituted with
C.sub.1-4alkyl;
[0012] each R.sup.4a group is independently selected and is a
functional group including, but not limited to, hydrogen, halogen,
C.sub.1-4alkyl, C.sub.1-4alkoxy, and aryl, or if R.sup.4a groups
are present and if the two R.sup.4a groups are on adjacent ring
atoms, they are optionally taken together to form
--O--(CH.sub.2).sub.1-2--O--;
[0013] R.sup.5 is hydrogen, and R.sup.6 is a functional group
including, but not limited to, halogen, C.sub.1-4alkyl,
--C(O)--C.sub.1-4alkyl, --SO.sub.2--N(R.sup.2b, R.sup.2b),
C.sub.1-4alkylhalo, --O-aryl and --N(R.sup.7, R.sup.8), or when
R.sup.5 and R.sup.6 are on adjacent ring atoms they are optionally
taken together to form --O--(CH.sub.2).sub.1-2--O--;
[0014] R.sup.7 is a functional group including, but not limited to,
hydrogen, C.sub.1-4alkyl, C.sub.1-4alkylhydroxy, aryl and
--C(O)R.sup.7a;
[0015] R.sup.7a is a is a functional group including, but not
limited to, C.sub.1-4alkyl, C.sub.1-4alkylhalo, C.sub.3-8cycloalkyl
and aryl; and
[0016] R.sup.8 is a functional group including, but not limited to,
H and C.sub.1-4alkyl, or R.sup.7 and R.sup.8 are optionally taken
together with the nitrogen to which they are attached to form a
heterocycle, optionally substituted with C.sub.1-4alkyl.
[0017] The compounds of the present invention include all
pharmaceutically acceptable salts, isomers, solvates, hydrates and
prodrugs thereof.
[0018] In another embodiment, the present invention provides
methods of inducing osteogenesis. Mammalian cells are contacted
with a compound of Formula I, whereupon the mammalian cell
differentiates into a cell of an osteoblast lineage. The step of
contacting can be in vivo or in vitro. In view of their ability to
induce osteogenesis, the compounds of Formula I are useful for
treating bone disorders and diseases, such as osteoporosis,
rickets, osteomalacia, McCune-Albright syndrome, and Paget's
disease.
[0019] Another embodiment of the present invention provides methods
of treating bone disorders by contacting a mammalian cell with a
compound of Formula I, whereupon the mammalian cell differentiates
into a cell of an osteoblast lineage. The mammalian cell may be
further contacted with bone morphogenetic protein 4 (BMP-4). If the
mammalian cell is contacted with a compound of Formula I in vitro,
the differentiated cells are administered to an individual with the
disorder, thereby treating the disorder. In some embodiments, the
mammalian cell is attached to a solid support (e.g., a three
dimensional matrix or a planar surface).
[0020] In some embodiments, the mammalian cell is contacted with a
compound of Formula I in vivo. If the mammalian cell is contacted
with a compound of Formula I in vivo, the step of contacting may be
by oral, intravenous, subcutaneous, or intraperitoneal
administration of the compound to the mammal.
[0021] In some embodiments, the differentiation of the of the
mammalian cell into a cell of an osteoblast lineage is detected. In
some embodiments, the differentiation of the of the mammalian cell
into a cell of an osteoblast lineage is detected by detecting
expression of an osteogenesis marker gene (e.g., alkaline
phosphatase, collagen type I, osteocalcin, or osteoponin). In other
embodiments, the differentiation of the mammalian cell into a cell
of an osteoblast lineage is detected by detecting expression of a
bone specific transcription factor (e.g., Cbfa1/Runx2 or Osx).
[0022] In some embodiments, the mammalian cell is a stem cell,
(e.g., a mesenchymal stem cell, a pre-osteoblast cell, a
pre-adipocyte cell, or a myoblast cell). In some embodiments, the
mesenchymal stem cell is isolated from a mouse (e.g., a murine
embryonic mesoderm fibroblast cell) or from a primate (e.g., a
human).
[0023] Other embodiments and advantages of the present invention
will be apparent from the detailed description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 illustrates results demonstrating that Compound A is
a potent inducer of osteogenesis in multipotent C3H10T1/2 cells.
C3H10T1/2 cells were treated with DMSO alone (control); BMP-4 alone
(300 ng/mL); BMP-4 (100 ng/mL) and Compound A (1 .mu.M); or 0.5
.mu.M, 1 .mu.M, 1.2 .mu.M, 1.5 .mu.M, 1.8 .mu.M, 2 .mu.M, 5 .mu.M,
or 10 .mu.M Compound A alone. Alkaline phosphatase (ALP) activity
was measured after two, four, and six days of treatment.
[0025] FIG. 2 illustrates the results demonstrating that Compound A
is a potent inducer of osteogenesis in C3H10T1/2 cells, 3T3-L1,
MC-3T3E1 cells, and C2C12 cells. The cells were treated with DMSO
alone (control); BMP-4 alone (300 ng/mL); BMP-4 (100 ng/mL) and
Compound A (1 .mu.M); or 1 .mu.M, 2 .mu.M, or 10 .mu.M Compound A
alone. Cbfa1/Runx2 reporter gene activity was assayed after four
days of treatment.
[0026] FIG. 3 illustrates results demonstrating that Compound A is
a potent inducer of osteogenesis in multipotent 3T3-L1 cells, C2C12
cells, and MC-3T3E1 cells. The cells were treated with DMSO alone
(control); BMP-4 alone (300 ng/mL); BMP-4 (100 ng/mL) and Compound
A (1 .mu.M); or 0.1 .mu.M, 0.5 .mu.M, 1 .mu.M, 5 .mu.M, or 10 .mu.M
Compound A alone. ALP activity was measured after four days of
treatment.
[0027] FIG. 4 illustrates that Compound A (i.e., purmorphamine) is
a potent inducer of osteogenesis activity in mesenchymal (MSC)
progenitor cells. FIG. 4A illustrates results demonstrating that
Compound A is a potent inducer of osteogenesis in multipotent
C3H10T1/2 cells. C3H10T1/2 cells were treated with DMSO alone
(control); BMP-4 alone; and Compound A alone (2 .mu.M). Alkaline
phosphatase (ALP) activity was measured after two, four, and six
days of treatment. FIG. 4B illustrates Cbfa1 gene upregulation in
C3H10T1/2 cells in the presence of Compound A.
[0028] FIG. 5 illustrates morphological and transcriptional
analysis of osteogenesis induced by Compound A.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
I. Introduction
[0029] The present invention provides compounds, compositions and
methods for differentiating and transdifferentiating mammalian
cells into cells of an osteoblast lineage. More particularly, the
present invention provides compounds of Formula I that are useful
for differentiating and transdifferentiating mammalian cells into
cells of an osteoblast lineage. In some embodiments, a composition
comprising the compound of Formula I is provided. In other
embodiments, methods of inducing osteogenesis in mammalian cells
are provided. Osteogenesis can be induced in vivo or in vitro
according to the methods of the present invention.
II. Definitions
[0030] Unless defined otherwise, all technical and scientific terms
used herein generally have the same meaning as commonly understood
by one of ordinary skill in the art to which this invention
belongs. Generally, the nomenclature used herein and the laboratory
procedures for organic and analytical chemistry are those well
known and commonly employed in the art.
[0031] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon radical, or combination thereof, which
may be fully saturated, mono- or polyunsaturated and can include
di- and multivalent radicals, having the number of carbon atoms
designated (i.e. C.sub.1-C.sub.10 means one to ten carbons).
Examples of saturated hydrocarbon radicals include groups such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,
sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl,
homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl,
n-octyl, and the like. An unsaturated alkyl group is one having one
or more double bonds or triple bonds. Examples of unsaturated alkyl
groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl,
2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The
term "alkyl," unless otherwise noted, is also meant to include
those derivatives of alkyl defined in more detail below as
"heteroalkyl." Alkyl groups which are limited to hydrocarbon groups
are termed "homoalkyl".
[0032] The term "alkylene" by itself or as part of another
substituent means a divalent radical derived from an alkane, as
exemplified by --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and further
includes those groups described below as "heteroalkylene."
Typically, an alkyl (or alkylene) group will have from 1 to 24
carbon atoms, with those groups having 10 or fewer carbon atoms
being preferred in the present invention. A "lower alkyl" or "lower
alkylene" is a shorter chain alkyl or alkylene group, generally
having eight or fewer carbon atoms.
[0033] The terms "alkoxy," "alkylamino" and "alkylthio" (or
thioalkoxy) are used in their conventional sense, and refer to
those alkyl groups attached to the remainder of the molecule via an
oxygen atom, an amino group, or a sulfur atom, respectively.
[0034] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon radical, or combinations
thereof, consisting of the stated number of carbon atoms and from
one to three heteroatoms selected from the group consisting of O,
N, Si and S, and wherein the nitrogen and sulfur atoms may
optionally be oxidized and the nitrogen heteroatom may optionally
be quaternized. The heteroatom(s) O, N and S may be placed at any
interior position of the heteroalkyl group. The heteroatom Si may
be placed at any position of the heteroalkyl group, including the
position at which the alkyl group is attached to the remainder of
the molecule. Examples include --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--(CH.sub.3).sub.2,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.3. Similarly, the term
"heteroalkylene" by itself or as part of another substituent means
a divalent radical derived from heteroalkyl, as exemplified by
--CH.sub.2--CH.sub.2--S--CH.sub.2CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxy, alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied.
[0035] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include cyclopentyl,
cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the
like. Examples of heterocycloalkyl include
1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,
tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,
1-piperazinyl, 2-piperazinyl, and the like.
[0036] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "halo(C.sub.1-C.sub.4)alkyl" is mean to
include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,
3-bromopropyl, and the like.
[0037] The term "aryl" means, unless otherwise stated, a
polyunsaturated, typically aromatic, hydrocarbon substituent which
can be a single ring or multiple rings (up to three rings) which
are fused together or linked covalently. The term "heteroaryl"
refers to aryl groups (or rings) that contain from zero to four
heteroatoms selected from N, O, and S, wherein the nitrogen and
sulfur atoms are optionally oxidized, and the nitrogen atom(s) are
optionally quaternized. A heteroaryl group can be attached to the
remainder of the molecule through a heteroatom. Non-limiting
examples of aryl and heteroaryl groups include phenyl, 1-naphthyl,
2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,
3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,
4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,
4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,
2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl,
4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below.
[0038] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl and the like) including those alkyl groups in which a
carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0039] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl" and "heteroaryl") are meant to include both substituted and
unsubstituted forms of the indicated radical. Preferred
substituents for each type of radical are provided below.
[0040] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a
variety of groups selected from: --OR', .dbd.O, .dbd.NR',
.dbd.N--OR', --NR'R'', --SR', -halogen, --SiR'R''R''', --OC(O)R',
--C(O)R', --CO.sub.2R', --CONR'R'', --OC(O)NR'R'', --NR''C(O)R',
--NR''C(O)NR''R''', --NR''C(O).sub.2R', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --CN and --NO.sub.2 in a number
ranging from zero to (2m'+1), where m' is the total number of
carbon atoms in such radical. R', R'' and R''' each independently
refer to hydrogen, unsubstituted (C.sub.1-C.sub.8)alkyl and
heteroalkyl, unsubstituted aryl, aryl substituted with 1-3
halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or
aryl-(C.sub.1-C.sub.4)alkyl groups. When R' and R'' are attached to
the same nitrogen atom, they can be combined with the nitrogen atom
to form a 5-, 6-, or 7-membered ring. For example, --NR'R'' is
meant to include 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0041] Similarly, substituents for the aryl and heteroaryl groups
are varied and are selected from: -halogen, --OR', --OC(O)R',
--NR'R'', --SR', --R', --CN, --NO.sub.2, --CO.sub.2R', --CONR'R'',
--C(O)R', --OC(O)NR'R'', --NR''C(O)R', --NR''C(O).sub.2R',
--NR'--C(O)NR''R''', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --N.sub.3, --CH(Ph).sub.2,
perfluoro(C.sub.1-C.sub.4)alkoxy, and
perfluoro(C.sub.1-C.sub.4)alkyl, in a number ranging from zero to
the total number of open valences on the aromatic ring system; and
where R', R'' and R''' are independently selected from hydrogen,
(C.sub.1-C.sub.8)alkyl and heteroalkyl, unsubstituted aryl and
heteroaryl, (unsubstituted aryl)-(C.sub.1-C.sub.4)alkyl, and
(unsubstituted aryl)oxy-(C.sub.1-C.sub.4)alkyl.
[0042] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CH.sub.2).sub.q--U--, wherein T and U are
independently --NH--, --O--, --CH.sub.2-- or a single bond, and q
is an integer of from 0 to 2. Alternatively, two of the
substituents on adjacent atoms of the aryl or heteroaryl ring may
optionally be replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CH.sub.2--, --O--, --NH--, --S--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2NR'-- or a single bond, and r is an integer of from 1
to 3. One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of the aryl or heteroaryl ring
may optionally be replaced with a substituent of the formula
--(CH.sub.2).sub.n--X--(CH.sub.2).sub.t--, where s and t are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituent R' in --NR'-- and --S(O).sub.2NR'-- is selected from
hydrogen or unsubstituted (C.sub.1-C.sub.6)alkyl.
[0043] The terms "halo" or "halogen" as used herein refer to Cl,
Br, F or I substituents. The term "haloalkyl", and the like, refer
to an aliphatic carbon radicals having at least one hydrogen atom
replaced by a Cl, Br, F or I atom, including mixtures of different
halo atoms. Trihaloalkyl includes trifluoromethyl and the like as
preferred radicals, for example.
[0044] The terms "alkoxy," "alkylamino" and "alkylthio" (or
thioalkoxy) are used in their conventional sense, and refer to
those alkyl groups attached to the remainder of the molecule via an
oxygen atom, an amino group, or a sulfur atom, respectively.
[0045] As used herein, the term "heteroatom" is meant to include
oxygen (O), nitrogen (N) and sulfur (S).
[0046] The term "pharmaceutically acceptable salts" is meant to
include salts of the active compounds which are prepared with
relatively nontoxic acids or bases, depending on the particular
substituents found on the compounds described herein. When
compounds of the present invention contain relatively acidic
functionalities, base addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired base, either neat or in a suitable inert solvent. Examples
of pharmaceutically acceptable base addition salts include sodium,
potassium, calcium, ammonium, organic amino, or magnesium salt, or
a similar salt. When compounds of the present invention contain
relatively basic functionalities, acid addition salts can be
obtained by contacting the neutral form of such compounds with a
sufficient amount of the desired acid, either neat or in a suitable
inert solvent. Examples of pharmaceutically acceptable acid
addition salts include those derived from inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic,
p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
Also included are salts of amino acids such as arginate and the
like, and salts of organic acids like glucuronic or galactunoric
acids and the like (see, for example, Berge, S. M., et al,
"Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977,
66, 1-19). Certain specific compounds of the present invention
contain both basic and acidic functionalities that allow the
compounds to be converted into either base or acid addition
salts.
[0047] The neutral forms of the compounds may be regenerated by
contacting the salt with a base or acid and isolating the parent
compound in the conventional manner. The parent form of the
compound differs from the various salt forms in certain physical
properties, such as solubility in polar solvents, but otherwise the
salts are equivalent to the parent form of the compound for the
purposes of the present invention.
[0048] In addition to salt forms, the present invention provides
compounds which are in a prodrug form. Prodrugs of the compounds
described herein are those compounds that readily undergo chemical
changes under physiological conditions to provide the compounds of
the present invention. Additionally, prodrugs can be converted to
the compounds of the present invention by chemical or biochemical
methods in an ex vivo environment. For example, prodrugs can be
slowly converted to the compounds of the present invention when
placed in a transdermal patch reservoir with a suitable enzyme or
chemical reagent.
[0049] Certain compounds of the present invention can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. In general, the solvated forms are equivalent to unsolvated
forms and are intended to be encompassed within the scope of the
present invention. Certain compounds of the present invention may
exist in multiple crystalline or amorphous forms. In general, all
physical forms are equivalent for the uses contemplated by the
present invention and are intended to be within the scope of the
present invention.
[0050] Certain compounds of the present invention possess
asymmetric carbon atoms (optical centers) or double bonds; the
racemates, diastereomers, geometric isomers and individual isomers
are all intended to be encompassed within the scope of the present
invention.
[0051] The compounds of the present invention may also contain
unnatural proportions of atomic isotopes at one or more of the
atoms that constitute such compounds. For example, the compounds
may be radiolabeled with radioactive isotopes, such as for example
tritium (.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C).
All isotopic variations of the compounds of the present invention,
whether radioactive or not, are intended to be encompassed within
the scope of the present invention.
[0052] "Osteogenesis," as used herein, refers to proliferation of
bone cells and growth of bone tissue (i.e., synthesis and deposit
of new bone matrix). Osteogenesis also refers to differentiation or
transdifferentiation of progenitor or precursor cells into bone
cells (i.e., osteoblasts). Progenitor or precursor cells can be
pluripotent stem cells such as, e.g., mesenchymal stem cells.
Progenitor or precursor cells can be cells pre-committed to an
osteoblast lineage (e.g., pre-osteoblast cells) or cells that are
not pre-committed to an osteoblast lineage (e.g., pre-adipocytes or
myoblasts).
[0053] A "stem cell," as used herein, refers to any self-renewing
pluripotent cell or multipotent cell or progenitor cell or
precursor cell that is capable of differentiating into multiple
cell types. Stem cells suitable for use in the methods of the
present invention include those that are capable of differentiating
into cells of osteoblast lineage, e.g., osteoblasts. Suitable stem
cells for use in the methods of the present invention include, for
example, mesenchymal stem cells, pre-osteoblast cells,
pre-adipocyte cells, and myoblast cells. Mesenchymal stem cells
(MSC) are capable of differentiating into the mesenchymal cell
lineages, such as bone, cartilage, adipose, muscle, stroma,
including hematopoietic supportive stroma, and tendon, and play
important roles in repair and regeneration (see, e.g., Olsen, 2000,
supra). MSCs are identified by specific cell surface markers which
are identified with unique monoclonal antibodies as described in
e.g., U.S. Pat. No. 5,643,736.
[0054] "Differentiate" or "differentiation," as used herein, refers
to the process by which precursor or progenitor cells (i.e., stem
cells) differentiate into specific cell types, e.g., osteoblasts.
Differentiated cells can be identified by their patterns of gene
expression and cell surface protein expression. Typically, cells of
an osteoblast lineage express genes such as, for example, alkaline
phosphatase, collagen type I, osteocalcin, and osteoponin.
Typically, cells of an osteoblast lineage express bone specific
transcription factors such as, for example, Cbfa1/Runx2 and Osx
(see, e.g., Olsen et al, 2000 supra and Nakashima et al., Cell
108(1):17-29 (2002). Additional transcription factors that are
involved in osteoblast differentiation include, e.g., gsc, Dlx1,
Dlx5, Msx1, Cart1, Hoxa1, Hoxa2, Hoxa3, Hoxb1, rae28, Twist, AP-2,
Mf1, Pax1, Pax3, Pax9, TBX3, TBX4, TBX5, and Brachyury (see, e.g.,
Olsen et al, 2000 supra).
[0055] "Transdifferentiation" refers to the process refers to the
process by which precursor or progenitor cells (i.e., stem cells)
pre-committed to cell types of one lineage differentiate into
specific cell types of another lineage, e.g., pre-adipocytes
transdifferentiate into osteoblasts or myoblasts transdifferentiate
into osteoblasts. Transdifferentiated cells can be identified by
their patterns of gene expression and cell surface protein
expression. Typically, cells of an osteoblast lineage express genes
such as, for example, alkaline phosphatase, collagen type I,
osteocalcin, and osteoponin. Typically, cells of an osteoblast
lineage express bone specific transcription factors such as, for
example, Cbfa1/Runx2 and Osx (see, e.g., Olsen et al, 2000 supra
and Nakashima et al., Cell 108(1):17-29 (2002). Additional
transcription factors that are involved in osteoblast
differentiation include, e.g., gsc, Dlx1, Dlx5, Msx1, Cart1, Hoxa1,
Hoxa2, Hoxa3, Hoxb1, rae28, Twist, AP-2, Mf1, Pax1, Pax3, Pax9,
TBX3, TBX4, TBX5, and Brachyury (see, e.g., Olsen et al, 2000
supra).
[0056] A "solid support," as used herein in connection with
inducing osteogenesis, refers to a three-dimensional matrix or a
planar surface on which the stem cells can be cultured. The solid
support can be derived from naturally occurring substances (i.e.,
protein based) or synthetic substances. For example, matrices based
on naturally occurring substances may be composed of autologous
bone fragments or commercially available bone substitutes as
described in e.g., Clokie et al., J. Craniofac. Surg. 13(1): 111-21
(2002) and Isaksson, Swed. Dent. J. Suppl. 84:1-46 (1992). Suitable
synthetic matrices are described in, e.g. U.S. Pat. Nos. 5,041,138,
5,512,474, and 6,425,222. For example, biodegradable artificial
polymers, such as polyglycolic acid, polyorthoester, or
polyanhydride can be used for the solid support. Calcium carbonate,
aragonite, and porous ceramics (e.g., dense hydroxyapatite ceramic)
are also suitable for use in the solid support. Polymers such as
polypropylene, polyethylene glycol, and polystyrene can also be
used in the solid support. Cells cultured and differentiated on a
solid support that is a three-dimensional matrix typically grow on
all of the surfaces of the matrix, e.g., internal and external.
Cells cultured and differentiated on a solid support that is planar
typically grow in a monolayer. The term "solid-support" is also
used in the context of preparing the compounds of Formula I. In
this context, "solid-support" refers to a polymeric support, such
as a bead, that can be partially soluble in a suitable solvent or
completely insoluble, and is used to bind, for example, a reactant
or a reagent of the reaction. Suitable solid-supports include, but
are not limited to, PAL resin, Wang resin, and polystyrene
resin.
[0057] "Culturing," as used herein, refers to maintaining cells
under conditions in which they can proliferate, differentiate, and
avoid senescence. For example, in the present invention, cultured
mesenchymal stem cells proliferate and differentiate into cells of
an osteoblastic cell lineage. Cells can be cultured in growth media
containing appropriate growth factors, i.e., a growth factor
cocktail that contains, for example, bone morphogenetic protein-2
(BMP-2), bone morphogenetic protein-4 (BMP-4), bone morphogenetic
protein-7 (BMP-7), or another suitable member of the BMP family of
proteins.
III. Compounds of the Present Invention and Methods for their
Preparation
[0058] A. The Compounds of Formula I
[0059] In one aspect, the present invention provides compounds of
Formula I:
##STR00002##
[0060] In Formula I, R.sup.1 is a functional group including, but
not limited to, hydrogen, halogen and -L-R.sup.2. L, in connection
with R', is a functional group including, but not limited to, --O--
and --NR.sup.3--, wherein R.sup.3 is H, or R.sup.3 is optionally
taken together with R.sup.2 and the nitrogen to which both are
attached to form a heterocycle, optionally substituted with
C.sub.1-4alkyl. R.sup.2 is a functional group including, but not
limited to, C.sub.1-4alkyl, C.sub.3-8cycloalkyl and
C.sub.0-2alkylaryl, substituted with 0-2 R.sup.2a groups that are
independently selected and that are functional groups including,
but not limited to, halogen, C.sub.1-4alkyl, C.sub.1-4alkoxy,
--N(R.sup.2b, R.sup.2b), --SO.sub.2N(R.sup.2b, R.sup.2b),
--C(O)N(R.sup.2b, R.sup.2b) and --O-aryl, or if R.sup.1e groups are
present and if the two R.sup.2a groups are on adjacent ring atoms,
they are optionally taken together to form a functional group
including, but not limited to, --O--(CH.sub.2).sub.1-2--O--,
--O--C(CH.sub.3).sub.2CH.sub.2-- and --(CH.sub.2).sub.3-4--. Each
R.sup.2b group is independently selected and is a functional group
including, but not limited to, hydrogen and C.sub.1-4alkyl
[0061] In Formula I, R.sup.4 is a functional group including, but
not limited to, C.sub.1-4alkyl, C.sub.3-8cycloalkyl,
C.sub.1-4alkylhydroxy, C.sub.0-2alkylaryl, substituted with 0-2
R.sup.4a groups, and C.sub.0-2alkylheterocycle, optionally
substituted with C.sub.1-4alkyl. Each R.sup.4a group is
independently selected and is a functional group including, but not
limited to, hydrogen, halogen, C.sub.1-4alkyl, C.sub.1-4alkoxy and
aryl, or if R.sup.4a groups are present and if the two R.sup.4a
groups are on adjacent ring atoms, they are optionally taken
together to form --O--(CH.sub.2).sub.1-2--O--;
[0062] In Formula I, R.sup.5 is hydrogen, and R.sup.6 is a
functional group including, but not limited to, halogen,
C.sub.1-4alkyl, --C(O)--C.sub.1-4alkyl, --SO.sub.2--N(R.sup.2b,
R.sup.2b), C.sub.1-4alkylhalo, --O-aryl and --N(R.sup.7, R.sup.8),
or when R.sup.5 and R.sup.6 are on adjacent ring atoms they are
optionally taken together to form --O--(CH.sub.2).sub.1-2--O--.
R.sup.7 is a functional group including, but not limited to,
hydrogen, C.sub.1-4alkyl, C.sub.1-4alkylhydroxy, aryl and
--C(O)R.sup.7a, wherein R.sup.7a is a is a functional group
including, but not limited to, C.sub.1-4alkyl, C.sub.1-4alkylhalo,
C.sub.3-8cycloalkyl and aryl, and R.sup.8 is a functional group
including, but not limited to, H and C.sub.1-4alkyl, or R.sup.7 and
R.sup.8 are optionally taken together with the nitrogen to which
they are attached to form a heterocycle, optionally substituted
with C.sub.1-alkyl.
[0063] The compounds of the present invention include all
pharmaceutically acceptable salts, isomers, solvates, hydrates and
prodrugs thereof.
[0064] In a preferred embodiment, R.sup.1 is a member of the
following group of substituents:
##STR00003##
[0065] In a more preferred embodiment, R.sup.1 is a member of the
following group of substituents:
##STR00004##
[0066] In a most preferred embodiment, R.sup.1 is the
following:
##STR00005##
[0067] In another preferred embodiment, R.sup.4 is a member of the
following group:
##STR00006##
[0068] In a more preferred embodiment, R.sup.4 is a member of the
following group:
##STR00007##
[0069] In a most preferred embodiment, R.sup.4 is a cyclohexyl
group.
[0070] In another preferred embodiment, R.sup.5 is H and R.sup.6 is
a member of the group consisting of chloro, --CF.sub.3, --CH.sub.3,
--C(O)CH.sub.3, --SO.sub.2NH.sub.2, --NMe.sub.2, --NHMe,
--NHC(O)Me, --NHC(O)CF.sub.3,
##STR00008##
[0071] In a further preferred embodiment, R.sup.5 and R.sup.6 are
taken together along with the aniline to which they are attached to
form
##STR00009##
[0072] In a most preferred embodiment, R.sup.5 is hydrogen and
R.sup.6 is morpholino.
[0073] In a preferred embodiment,
[0074] R.sup.1 is
##STR00010##
[0075] R.sup.5 is H; and
[0076] R.sup.6 is morpholine.
[0077] In another preferred embodiment,
R.sup.1 is
##STR00011##
[0078] R.sup.5 is H;
[0079] R.sup.6 is morpholine; and R.sup.4 is a member selected from
the group consisting of:
##STR00012##
[0080] Preferred compounds of the present invention, include, but
are not limited to:
##STR00013## ##STR00014## ##STR00015## ##STR00016##
##STR00017##
[0081] In a most preferred embodiment, the compound of Formula I is
the following compound, herein referred to as Compound A:
##STR00018##
[0082] The compounds of Formula I can be readily screened for their
ability to induce osteogenesis using the in vitro and in vitro
screening methods set forth below and, in particular, in the
examples.
[0083] B. Preparation of Compounds
[0084] The compounds of the present invention can be prepared by
either solid-phase or solution-phase synthesis.
[0085] 1. Solid-Phase Synthesis
[0086] Methods directed to the solid-phase synthesis of the
compounds of Formula I are discussed herein in Example I, as well
as in U.S. Patent Application No. 60/328,763, filed Oct. 12, 2001,
U.S. Patent Application No. 60/331,835, filed Nov. 20, 2001, U.S.
Patent Application No. 60/346,480, filed Jan. 7, 2002, U.S. Patent
Application No. 60/348,089, filed Jan. 10, 2002, and U.S. patent
application Ser. No. 10/270,030, filed Oct. 12, 2002 (bearing
Attorney Docket No. 21288-000340).
[0087] In one aspect, the present invention provides a method for
synthesizing a substituted heteroaryl, the method comprising: (a)
providing a dihaloheteroaryl scaffold moiety; and (b) capturing the
dihaloheteroaryl scaffold moiety on a resin by nucleophilic
substitution of a first halogen by a resin-bound amine nucleophile
to afford a substituted heteroaryl, e.g., a resin-bound amine
substituted monohaloheteroaryl; (c) reacting the second halogen
with a suitably substituted amine or aryl alcohol to afford the
resin bound substituted heteroaryl; and (d) cleavage of the
substituted heteroaryl from the resin.
[0088] Suitable resins useful for the present invention include,
but are not limited to, PAL resin, Wang resin, and polystyrene
resin. Other suitable resins would be clear to a person of skill in
the art. In a preferred embodiment, the PAL resin is utilized.
[0089] In a preferred embodiment, the two halogens, i.e., halo
groups, of the dihaloheteroaryl scaffold moiety are independently
selected and include, but are not limited to, chloro, fluoro, bromo
and iodo. In a presently preferred embodiments, the two halogens
are chloro groups.
[0090] In a preferred embodiment, the method further comprises
substitution of the second halogen of the dihaloheteroaryl scaffold
moiety by nucleophilic displacement or, alternatively, by a
coupling reaction. In a presently preferred embodiment, a coupling
reaction is employed to carry out the substitution of the second
halogen of the dihaloheteroaryl scaffold moiety. In this
connection, the coupling reaction is preferably a
palladium-mediated coupling reaction.
[0091] It will be readily apparent to those of skill in the art
that the two halogens, i.e., halo groups, of the dihaloheteroaryl
scaffold moiety can be substituted with a number of different
functional groups. Suitable functional groups include, but are not
limited to, anilines, phenols, amines and boronic acids (see, Table
1). In a preferred embodiment, the functional group includes, but
is not limited to, aryl boronic acids, anilines and phenols.
[0092] In a preferred embodiment, the method further comprises
performing an initial substitution prior to substitution of the
first halogen of the dihaloheteroaryl scaffold moiety. In a
preferred embodiment, the initial substitution is carried out using
a reaction including, but not limited to, alkylation reactions,
acylation reactions and coupling reactions.
[0093] Numerous dihaloheteroaryl scaffold moieties can be used in
the methods of the present invention. Examples of suitable
dihaloheteroaryl scaffold moieties include, but not limited to,
purines, pyrimidines, quinazolines, pyrazines, phthalazines,
pyradazines and quinoxalines.
[0094] When a palladium-catalyzed coupling reaction is employed to
substitute the halo groups of the dihaloheteroaryl or the halo
group of the resin-bound amine substituted monohaloheteroaryl, the
palladium-catalyzed coupling reaction typically involves reacting
the dihaloheteroaryl or the resin-bound amine substituted
monohaloheteroaryl with a coupling agent in the presence of a
solvent, a palladium catalyst, a base and a carbene or phosphine
ligand. Suitable coupling agents include, but are not limited to,
boronic acids, amines and alcohols. In a presently preferred
embodiment, suitable coupling agents include, but are not limited
to, aryl boronic acids, anilines and phenols.
[0095] In the above methods, carbene or phosphine ligands can be
used. Examples of ligands suitable for use in the methods of the
present invention include, but are not limited to, the following
carbene and phosphine ligands:
##STR00019##
[0096] In a presently preferred embodiment, the ligand is a
phosphine ligand including, but not limited to, the following:
##STR00020##
[0097] A number of bases can be used in carrying out the methods of
the present invention. Examples of bases suitable for use in the
above method include, but are not limited to, cesium carbonate,
potassium carbonate, sodium carbonate, sodium bicarbonate,
potassium bicarbonate, cesium bicarbonate, potassium fluoride,
potassium phosphate, potassium tert-butyloxide, sodium
tert-butyloxide, and triethylamine.
[0098] A number of solvents can be used in carrying out the methods
of the present invention. Examples of solvents suitable for use in
the above method include, but are not limited to, 1,4-dioxane,
tetrahydrofuran, dimethoxyethane (DME), dimethylformamide (DMF),
benzene and toluene.
[0099] A number of palladium catalysts can be used in carrying out
the methods of the present invention. Typically, the oxidation
state of the palladium in the catalyst is (0) or (II). Examples of
palladium catalysts suitable for use in carrying out the methods of
the present invention include, but are not limited to,
Pd.sub.2(dba).sub.3, Pd(OAc).sub.2, Pd(PPh.sub.3).sub.4, Pd(0),
PdCl.sub.2(dppf) and PdCl.sub.2. Such catalysts are known to and
used by those of skill in the art and, thus, their structures are
known. In a preferred embodiment, the palladium catalyst is
Pd.sub.2(dba).sub.3.
[0100] In a preferred embodiment, the foregoing methods further
comprise cleaving the compound from the solid support. It will be
readily appreciated that the compounds of the present invention can
be readily cleaved from the solid support using standard methods
known to and used by those of skill in the art. Cleavage of a
resin-bound compound and liberation of the desired compound from
the resin is typically carried in the presence of an acid. Suitable
acids include, but are not limited to, an organic acid such as
formic acid, acetic acid, propionic acid, trichloroacetic acid,
trifluoroacetic acid and the like, and inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, hydrogen
chloride, etc., or the like. The reaction is usually carried out in
a solvent such as water, an alcohol such as methanol, ethanol, 1,4,
dioxane, methylene chloride, tetrahydrofuran, a mixture thereof or
any other solvent which does not adversely influence the
reaction.
[0101] In yet another aspect of the present invention, the
foregoing method is adapted to prepare a library (or an array) of
heteroaryl scaffold moieties. Typically, the library of substituted
scaffold moieties is prepared using a plurality of dihaloheteroaryl
scaffold moieties. As such, in another aspect, the present
invention provides a method for synthesizing a combinatorial
library of substituted heteroaryls (e.g., heterocycles), the method
comprising: providing a plurality of dihaloheterocyclic scaffold
moieties; and capturing the dichloroheterocyclic scaffold moieties
on a resin by nucleophilic substitution of a first chlorine by a
resin-bound amine nucleophile).
[0102] In a preferred embodiment, the two halogens, i.e., halo
groups, present in the dihaloheteroaryl scaffold moieties are
independently selected and include, but are not limited to, chloro,
fluoro, bromo and iodo. In a presently preferred embodiments, the
two halogens of the dihaloheteroaryl scaffold moieties are chloro
groups.
[0103] In a preferred embodiment, the method further comprises
substitution of the second halogen of the dihaloheteroaryl scaffold
moieties by nucleophilic displacement or, alternatively, by a
coupling reaction. In a presently preferred embodiment, a coupling
reaction is employed to carry out the substitution of the second
halogen of the dihaloheteroaryl scaffold moieties. In this
connection, the coupling reaction is preferably a
palladium-mediated coupling reaction.
[0104] It will be readily apparent to those of skill in the art
that the two halogens, i.e., halo groups, of the dihaloheteroaryl
scaffold moieties can be substituted with a number of different
functional groups, each of which is independently selected.
Suitable functional groups include, but are not limited to,
anilines, phenols, amines and boronic acids (see, Table I). In a
presently preferred embodiment, the functional groups include, but
are not limited to, aryl boronic acids, anilines and phenols.
[0105] In a preferred embodiment, the method further comprises
performing initial substitutions prior to substitution of the first
halogens of the dihaloheteroaryl scaffold moieties. In a preferred
embodiment, the initial substitution is carried out using a
reaction including, but not limited to, alkylation reactions,
acylation reactions and coupling reactions.
[0106] Numerous dihaloheteroaryl scaffold moieties can be used in
the methods of the present invention. Examples of suitable
dihaloheteroaryl scaffold moieties include, but not limited to,
purines, pyrimidines, quinazolines, pyrazines, phthalazines,
pyradazines and quinoxalines.
[0107] When a palladium-catalyzed coupling reaction is employed to
substitute the halo groups of the dihaloheteroaryl scaffold
moieties or the halo group of the resin-bound amine substituted
monohaloheteroaryls, the palladium-catalyzed coupling reaction
typically involves reacting the dihaloheteroaryl or the resin-bound
amine substituted monohaloheteroaryl with a coupling agent in the
presence of a solvent, a palladium catalyst, a base and a carbene
or phosphine ligand. Suitable coupling agents include, but are not
limited to, boronic acids, amines and alcohols. In a presently
preferred embodiment, suitable coupling agents include, but are not
limited to, aryl boronic acids, anilines and phenols. It is noted
that the foregoing discussions relating to the carbene or phosphine
ligands, bases, solvents, palladium catalysts and copper catalysts
set forth in connection with the methods for preparing a C-2
substituted purine compound or a 9-aryl substituted purine compound
are fully applicable to the methods for preparing a combinatorial
library or array of substituted heteroaryl compound and, thus, they
will not be repeated here.
[0108] 2. Solution-Phase Synthesis
[0109] The solution-phase synthesis of the compounds of Formula I
involves first substituting 2,6-dihaloheteroaryl with a suitable
substituent under appropriate reaction conditions known to one of
skill in the art. This is followed by substitution with a suitably
substituted aniline under appropriate reaction conditions known to
one of skill in the art. Finally, the heteroaryl is substituted by
reaction with a suitably substituted amine or arylalcohol using a
Pd catalyst under appropriate reaction conditions known to one of
skill in the art. It is noted that the foregoing discussions
relating to the carbene or phosphine ligands, bases, solvents and
palladium catalysts are set forth with the methods for the
preparing the compounds of Formula I via solid-support are fully
applicable to the methods for preparing the compounds of Formula I
via solution phase, and, thus, they will not be repeated here.
IV. Use of the Compounds/Compositions to Induce Osteogenesis
[0110] The compositions of the present invention can be used to
induce osteogenesis in mammalian cells. A mammalian cell is
contacted with a compound of Formula I, whereupon the mammalian
cell differentiates into a cell of an osteoblast lineage. The
mammalian cell can be contacted with a compound of Formula I (or a
composition thereof) either in vivo or in vitro.
[0111] A. In Vivo Induction of Osteogenesis
[0112] The compounds of Formula I as well as compositions thereof
can conveniently be used to induce osteogenesis in vivo. The
compounds and compositions of the present invention are
administered to an individual, e.g., a mammal such as a human, in
an amount effective to induce differentiation of mammalian cells
into cells of an osteoblast lineage. In view of their ability to
induce osteogenesis, the compounds of Formula I are useful for
treating bone disorders and diseases, such as osteoporosis,
rickets, osteomalacia, McCune-Albright syndrome, and Paget's
disease. In a preferred embodiment, the compounds and compositions
of the present invention are used to treat osteoporosis. In a
preferred embodiment, the compounds and compositions of the present
invention are used to increase bone density. In a particularly
preferred embodiment, the compounds and compositions of the present
invention are used to increase bone density and reduce bone
loss.
[0113] One of skill in the art will appreciate that the
compositions of the present invention can be used alone or in
combination with other compounds and therapeutic regimens to induce
osteogenesis. For example, a compound of Formula I may be
administered in conjunction with bone morphogenetic proteins or
anti-resorptive medications that affect the bone remodeling cycle.
Suitable bone morphogenetic proteins include, for example, BMP-2,
BMP-4, and BMP-7. Suitable anti-resorptive medications include, for
example, bisphosphonates such as, for example, alendronate sodium
and risedronate sodium; hormones, such as, for example, calcitonin
and estrogens, and selective estrogen receptor modulators, such as,
for example, raloxifene.
[0114] An effective amount of the composition will be determined by
the existence, nature, and extent of any adverse side-effects that
accompany the administration of the composition; the LD50 of the
composition; and the side-effects of the composition at various
concentrations. Typically, the amount of the composition
administered will range from about 0.01 to about 20 mg per kg, more
typically about 0.05 to about 15 mg per kg, even more typically
about 0.1 to about 10 mg per kg body weight.
[0115] The compositions can be administered, for example, by
intravenous infusion, orally, intraperitoneally, or subcutaneously.
Oral administration is the preferred method of administration. The
formulations of compounds can be presented in unit-dose or
multi-dose sealed containers, such as ampoules and vials.
[0116] The compositions of the present invention are typically
formulated with a pharmaceutically acceptable carrier before
administration to an individual or subject. Pharmaceutically
acceptable carriers are determined, in part, by the particular
composition being administered (e.g., Compound A), as well as by
the particular method used to administer the composition.
Accordingly, there are a wide variety of suitable formulations of
pharmaceutical compositions of the present invention (see, e.g.,
Remington's Pharmaceutical Sciences, 17th ed., 1989).
[0117] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the compound
of Formula I suspended in diluents, such as water, saline or PEG
400; (b) capsules, sachets or tablets, each containing a
predetermined amount of the active ingredient, as liquids, solids,
granules or gelatin; (c) suspensions in an appropriate liquid; and
(d) suitable emulsions. Tablet forms can include one or more of the
following: lactose, sucrose, mannitol, sorbitol, calcium
phosphates, corn starch, potato starch, microcrystalline cellulose,
gelatin, colloidal silicon dioxide, talc, magnesium stearate,
stearic acid, and other excipients, colorants, fillers, binders,
diluents, buffering agents, moistening agents, preservatives,
flavoring agents, dyes, disintegrating agents, and pharmaceutically
compatible carriers. Lozenge forms can comprise the active
ingredient in a flavor, e.g., sucrose, as well as pastilles
comprising the active ingredient in an inert base, such as gelatin
and glycerin or sucrose and acacia emulsions, gels, and the like
containing, in addition to the active ingredient, carriers known in
the art.
[0118] The compositions of the present invention may be in
formulations suitable for other routes of administration, such as,
for example, intravenous infusion, intraperitoneally, or
subcutaneously. The formulations include, for example, aqueous and
non-aqueous, isotonic sterile injection solutions, which can
contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic with the blood of the intended
recipient, and aqueous and non-aqueous sterile suspensions that can
include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives. Injection solutions and suspensions
can be prepared from sterile powders, granules, and tablets.
[0119] The dose administered to a patient, in the context of the
present invention should be sufficient to effect a beneficial
therapeutic response in the patient over time. For example, if the
compositions of the present invention are administered to treat or
prevent osteoporosis, the dose administered to the patient should
be sufficient to prevent, retard, or reverse decreases in bone
density. The dose will be determined by the efficacy of the
particular composition employed and the condition of the patient,
as well as the body weight or surface area of the patient to be
treated. The size of the dose also will be determined by the
existence, nature, and extent of any adverse side-effects that
accompany the administration of a particular composition in a
particular patient.
[0120] B. In Vitro Induction of Osteogenesis
[0121] The compositions of the present invention can conveniently
be used to induce osteogenesis in vitro. Mammalian cells are
contacted with the compositions, whereupon the mammalian cells
differentiates into cells of an osteoblast lineage.
[0122] 1. Suitable Cells
[0123] The mammalian cells can be stem cells, typically mesenchymal
stem cells (MSCs), pre-osteoblasts, or cells of other lineages such
as, for example, pre-adipocytes or myoblasts. Methods for isolation
and differentiation of human and animal MSCs have been described
(see, e.g., U.S. Pat. Nos. 5,942,225 and 5,486,359; and Pittenger
et al., Science 284:143 (1999)).
[0124] Human mesenchymal stem cells (MSC) may be obtained by
isolating pluripotent mesenchymal stem cells from other cells in
the bone marrow or other MSC source. Bone marrow cells may be
obtained from iliac crest, femora, tibiae, spine, rib or other
medullary spaces. Other sources of human mesenchymal stem cells
include embryonic yolk sac, placenta, umbilical cord, fetal and
adolescent skin, blood, adipose tissue, and muscle satellite cells.
Typically, cells from a tissue specimen containing mesenchymal stem
cells are cultured in growth medium containing growth factors that
(1) stimulate mesenchymal stem cell growth without differentiation,
and (2) allow for the selective adherence of only the mesenchymal
stem cells to a substrate surface. After culturing the cells for a
suitable amount of time, non-adherent matter is removed from the
substrate surface, thus providing an expanded population of
mesenchymal stem cells. Thus, homogeneous MSC populations are
obtained by positive selection of adherent marrow or periosteal
cells which are free of markers associated with either
hematopoietic cell or differentiated mesenchymal cells.
[0125] The cells to be differentiated into cells of an osteoblast
lineage can be derived from any suitable mammal. For example, the
cells may be obtained from a rodents such as, for example, mice,
rats, guinea pigs, and rabbits; non-rodent mammals such as, for
example, dogs, cats, pigs, sheep, horses, cows, and goats; primates
such as, for example, chimpanzees and humans. The cells to be
differentiated may be primary cells or may be cells maintained in
culture. If the cells are maintained in culture, they are typically
contacted with the compounds/compositions of the present invention
between the 12th and 15th passage in culture. Techniques and
methods for establishing a primary culture of cells for use in the
methods of the invention are known to those of skill in the art
(see, e.g., Humason, ANIMAL TISSUE TECHNIQUES, 4.sup.th ed., W.H.
Freeman and Company (1979), and Ricciardelli et al., (1989) In
Vitro Cell Dev. Biol. 25: 1016.
[0126] 2. General Culturing Methods
[0127] The mammalian cells may be contacted with Compound A alone
or with Compound A in the presence of other growth factors.
Typically, the additional growth factors are BMP-2, BMP-4, BMP-7,
or other members of the BMP family of proteins. Those of skill in
the art will appreciate that the amount of Compound A and growth
factors can be adjusted to facilitate induction of differentiation
in particular cell types. Typically, the amount of Compound A
contacted with the cells is from about 0.1 .mu.M (52 ng/ml) to
about 50 .mu.M (2.6 .mu.g/ml), more typically from about 0.25 .mu.M
to about 35 .mu.M, even more typically from about 0.5 .mu.M to
about 25 .mu.M, yet more typically from about 0.75 .mu.M to about
15 .mu.M, most typically at about 1 .mu.M. Typically the amount of
BMP protein contacted with the cells is from about 1 ng/ml to about
400 ng/ml, more typically from about 25 ng/ml to about 300 ng/ml,
even more typically from about 50 ng/ml to about 200 ng/ml, yet
more typically from about 75 ng/ml to about 125 ng/ml, most
typically at about 100 ng/ml.
[0128] This aspect of the present invention relies upon routine
techniques in the field of cell culture. Suitable cell culture
methods and conditions can be determined by those of skill in the
art using known methodology (see, e.g., Freshney et al., CULTURE OF
ANIMAL CELLS (3rd ed. 1994)). In general, the cell culture
environment includes consideration of such factors as the substrate
for cell growth, cell density and cell contract, the gas phase, the
medium, and temperature.
[0129] Incubation of cells is generally performed under conditions
known to be optimal for cell growth. Such conditions may include,
for example, a temperature of approximately 37.degree. C. and a
humidified atmosphere containing approximately 5% CO.sub.2. The
duration of the incubation can vary widely, depending on the
desired results. In general, incubation is preferably continued
until the cells express suitable Proliferation is conveniently
determined using .sup.3H thymidine incorporation or BrdU
labeling.
[0130] Plastic dishes, flasks, or roller bottles may be used to
culture cells according to the methods of the present invention.
Suitable culture vessels include, for example, multi-well plates,
petri dishes, tissue culture tubes, flasks, roller bottles, and the
like.
[0131] Cells are grown at optimal densities that are determined
empirically based on the cell type. Cells are typically passaged
12-5 times and discarded after 15 passages.
[0132] Cultured cells are normally grown in an incubator that
provides a suitable temperature, e.g., the body temperature of the
animal from which is the cells were obtained, accounting for
regional variations in temperature. Generally, 37.degree. C. is the
preferred temperature for cell culture. Most incubators are
humidified to approximately atmospheric conditions.
[0133] Important constituents of the gas phase are oxygen and
carbon dioxide. Typically, atmospheric oxygen tensions are used for
cell cultures. Culture vessels are usually vented into the
incubator atmosphere to allow gas exchange by using gas permeable
caps or by preventing sealing of the culture vessels. Carbon
dioxide plays a role in pH stabilization, along with buffer in the
cell media and is typically present at a concentration of 1-10% in
the incubator. The preferred CO.sub.2 concentration typically is
5%.
[0134] Defined cell media are available as packaged, premixed
powders or presterilized solutions. Examples of commonly used media
include MEM-A, DME, RPMI 1640, DMEM, Iscove's complete media, or
McCoy's Medium (see, e.g., GibcoBRL/Life Technologies Catalogue and
Reference Guide; Sigma Catalogue). Typically, MEM-.alpha. or DMEM
are used in the methods of the invention. Defined cell culture
media are often supplemented with 5-20% serum, typically heat
inactivated serum, e.g., human, horse, calf, and fetal bovine
serum. Typically, 10% fetal bovine serum is used in the methods of
the invention. The culture medium is usually buffered to maintain
the cells at a pH preferably from about 7.2 to about 7.4. Other
supplements to the media typically include, e.g., antibiotics,
amino acids, and sugars, and growth factors.
[0135] C. Detection of Osteogenesis
[0136] After administration of the compositions of the present
invention in vivo or in vitro, induction of osteogenesis can be
detected by detecting expression of osteoblast-specific proteins,
detecting expression of bone-specific transcription factors, and
detecting changes in bone density. Osteoblast-specific proteins
include, for example, alkaline phosphatase (ALP), collagen type I,
osteocalcin, and osteoponin (see, e.g., Olsen et al, Annu. Rev.
Cell. Dev. Biol. 16:191 (2000)). Typically, expression of alkaline
phosphatase is detected as an indicator of osteogenesis. Bone
specific transcription factors include, for example, Cbfa1/Runx2,
gsc, Dlx1, DlxS, Msx1, Cart1, Hoxa1, Hoxa2, Hoxa3, Hoxb1, rae28,
Twist, AP-2, Mf1, Pax1, Pax3, Pax9, TBX3, TBX4, TBXS, and Brachyury
(see, e.g., Olsen et al, 2000 supra). Typically, expression of
Cbfa1/Runx2 is detected as an indicator of osteogenesis.
[0137] 1. Detection of Osteoblast-Specific Proteins
[0138] Expression of osteoblast-specific proteins may be detected
by measuring the level of the osteoblast-specific protein or mRNA.
The level of particular osteoblast-specific proteins can
conveniently be measured using immunoassays such as
immunohistochemical staining, western blotting, ELISA and the like
with an antibody that selectively binds to the particular
osteoblast specific proteins or a fragment thereof. Detection of
the protein using protein-specific antibodies in immunoassays is
known to those of skill in the art (see, e.g., Harlow & Lane,
Antibodies: A Laboratory Manual (1988), Coligan, Current Protocols
in Immunology (1991); Goding, Monoclonal Antibodies: Principles and
Practice (2d ed. 1986); and Kohler & Milstein, Nature
256:495-497 (1975). For measurement of mRNA, amplification, e.g.,
PCR, LCR, or hybridization assays, e.g., northern hybridization,
RNAse protection, dot blotting, are preferred. The level of protein
or mRNA is detected, for example, using directly or indirectly
labeled detection agents, e.g., fluorescently or radioactively
labeled nucleic acids, radioactively or enzymatically labeled
antibodies. These assays are well-known to those of skill in the
art and described in, e.g., Ausubel, et al. ed. CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY (2001).
[0139] Typically, expression of the osteoblast specific-protein,
alkaline phosphatase, is used to detect differentiated osteoblasts.
Expression of alkaline phosphatase (ALP) is correlated with
osteogenesis. ALP hydrolyzes inorganic pyrophosphates to phosphates
and promotes the formation of hydroxyapatite crystals in bone
matrix. Deactivating mutations of ALP cause osteomalacia,
characterized by poorly mineralized bones and frequent bone
factures, indicating that ALP plays a significant role in bone
formation (see, e.g., Hessle et al., Proc. Natl. Acad. Sci. USA.
99:9445 (2002)). ALP is a highly active and stable enzyme, making
direct assays of its enzymatic activity convenient. In addition,
direct histochemical staining of cells can conveniently be used to
detect ALP.
[0140] a) Enzymatic Activity
[0141] For direct assays of ALP activity, cells are plated in
384-well plates and treated with an appropriate amount of a
compound of Formula I (e.g., Compound A), either alone or with
other growth factors (e.g., BMP-4) and then incubated at 37.degree.
C. in 5% CO.sub.2. After an appropriate incubation time, the media
is removed and lysis buffer is added into each well. After an
appropriate incubation time in lysis buffer, alkaline phosphatase
substrate solution (e.g.,
2'-[2'-benzothiazoyl]-6'-hydroxybenzothiazole phosphate (BBTP)) is
added to each well. After an appropriate incubation time at room
temperature, the plates are read on a plate reader using methods
known in the art.
[0142] b) Immunohistochemical Detection
[0143] For direct immunohistochemical staining of cells to detect
ALP, cells are seeded in 96-well assay plates at a suitable density
and treated with an appropriate amount of a compound of Formula I
(e.g., Compound A), either alone or with other growth factors
(e.g., BMP-4) for an appropriate time. Cells are then and fixed in
a 10% formalin solution. The fixed cells are washed again and
stained with a reagent specific for ALP (e.g., an antibody specific
for ALP or a colorimetric ALP substrate) using methods known to
those of skill in the art (see, e.g., Harlow & Lane, 1988,
supra; Coligan, 1991, supra; Goding, 1986, supra; and Kohler &
Milstein, 1975, supra). Photographic images of the cells are taken
and ALP positive cells are counted manually from the images.
[0144] 2. Detection of Bone-Specific Transcription Factors
[0145] Expression of bone-specific transcription factors can be
detected using reporter gene assays. These assays are well known to
those of skill in the art and are described in, e.g., Ausebel et
al., supra. Expression of the bone specific transcription factor
Cbfa1/Runx2 is typically used to detect osteogenesis. Cbfa1/Runx2
plays an essential role in osteoblast differentiation transgenic
mice lacking the Cbfa1/Runx2 gene die shortly after birth due to
loss in bone formation (see, e.g., Ducy et al., Cell 89:747 (1997)
and Komori et al., Cell 89:755 (1997)).
[0146] Reporter genes such as, for example, chloramphenicol
acetyltransferase, firefly luciferase, bacterial luciferase, or
.beta.-galactosidase can be used in the reporter gene assays. The
reporter construct is typically transiently or stably transfected
into a cell. The promoter region of the relevant gene is typically
amplified by PCR appropriate primers. The resulting PCR product is
inserted into a suitable cloning vector, amplified and sequenced.
The resulting plasmid is digested with appropriate restriction
enzymes and the resulting fragment is inserted into a vector
comprising a reporter gene.
[0147] a) Transiently Transfected Cells
[0148] For reporter gene assays with transiently transfected cells,
the cells are typically seeded in a 6-well plate at a density of
30,000 cells/well in 2 mL of growth medium an incubated overnight
or for a suitable time. Plasmid DNA is transfected into the cells
using a suitable transfection reagent. After 8 hours, the
transfected cells are plated into 96-well assay plates (e.g.,
Corning) and treated with an appropriate amount of a compound of
Formula I (e.g., Compound A). The cells are incubated for 4 days,
then the reporter gene activity in the cells is assayed using
methods known to those of skill in the art.
[0149] b) Stably Transfected Cells
[0150] For reporter gene assays with stably transfected cells, the
cells are typically seeded in a 6-well plate at a density of 30,000
cells/well in 2 mL of growth medium an incubated overnight or for a
suitable time. An appropriate amount of reporter plasmid and a
vector comprising a selectable marker (e.g., an antibiotic
resistance gene) are co-transfected into the cells using an
appropriate transfection reagent. After an appropriate incubation
time, cells are seeded in a 10 cm culture dish and an appropriate
amount of antibiotic is added to the culture medium. Fresh
antibiotic is added at appropriate intervals. The antibiotic
resistant colonies are pooled to yield the stably transfected
cells. The transfected cells are plated into 96-well assay plates
(e.g., Corning) and treated with an appropriate amount of a
compound of Formula I (e.g., Compound A). The cells are incubated
for 4 days, then the reporter gene activity in the cells is assayed
using methods known to those of skill in the art.
[0151] 3. Detection of Bone Density
[0152] To assess the effect of the compositions of the present
invention on bone density, a baseline measurement of bone density
in an individual who will receive treatment may taken. Bone density
is periodically measured at suitable intervals during and after
administration of the compounds of Formula I, e.g., Compound A.
Methods and devices for measuring bone density are well known in
the art and are described in, e.g., U.S. Pat. Nos. 6,436,042;
6,405,068; 6,320,931; 6,302,582; 6,246,745; 6,230,036; 6,213,934;
6,102,567; 6,058,157; 5,898,753; 5,891,033; 5,852,647; 5,817,020;
5,782,763; 5,778,045; 5,749,363; 5,745,544; 5,715,820; 5,712,892;
5,572,998; and 5,480,439.
[0153] 4. Administration of Differentiated Osteoblast Cells
[0154] Differentiated osteoblast cells can be administered to a
subject by any means known to those of skill in the art. In one
embodiment of the invention, differentiated osteoblast cells on an
intact solid support (e.g., a three-dimensional matrix or a planar
surface) can be administered to the subject, e.g., via surgical
implantation. Alternatively, the differentiated osteoblast cells
can be detached from the matrix, i.e., by treatment with a
protease, before administration to the subject, e.g., intravenous,
subcutaneous, or intraperitoneal.
[0155] In some embodiments of the present invention, mesenchymal
stem cells are extracted from a human and subsequently contacted
with a matrix for proliferation and differentiation into cells of
an osteoblastic cell lineage. Cells can be extracted from the
subject to be treated, i.e., autologous (thereby avoiding
immune-based rejection of the implant), or can be from a second
subject, i.e., heterologous. In either case, administration of
cells can be combined with an appropriate immunosuppressive
treatment.
[0156] Osteoblast cells differentiated according to the methods of
the present invention may be administered to a subject by any means
known in the art. Suitable means of administration include, for
example, intravenous, subcutaneous, intraperitoneal, and surgical
implantation.
[0157] The cells may be in formulations suitable for
administration, such as, for example, aqueous and non-aqueous,
isotonic sterile injection solutions, which can contain
antioxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood of the intended recipient, and
aqueous and non-aqueous sterile suspensions that can include
suspending agents, solubilizers, thickening agents, stabilizers,
and preservatives. Injection solutions and suspensions can be
prepared from sterile powders, granules, and tablets.
[0158] For surgical implantation, differentiated cells are
typically left on an intact solid support, e.g., a
three-dimensional matrix or planar surface. The matrix or planar
surface is surgically implanted into the appropriate site in a
subject. For example, a patient needing a bone graft can have
differentiated cells on an intact solid support surgically
implanted.
[0159] In determining the effective amount of the cells to be
administered in the treatment or prophylaxis of conditions owing to
diminished or aberrant osteoblasts, the physician evaluates cell
toxicity, transplantation reactions, progression of the disease,
and the production of anti-cell antibodies. For administration,
osteoblast cells differentiated according to the methods of the
present invention can be administered in an amount effective to
provide osteoblasts to the subject, taking into account the
side-effects of the osteoblasts at various concentrations, as
applied to the mass and overall health of the patient.
Administration can be accomplished via single or divided doses.
EXAMPLES
[0160] The following examples are offered to illustrate, but not to
limit, the claimed invention.
Example 1
Synthesis and Characterization of Compound A
[0161] The phosphine ligand for the Pd-catalyzed coupling was
purchased from Strem Chemicals. All the other chemicals were
purchased from Aldrich.
[0162] Solid Phase Synthesis
##STR00021##
[0163] The C6 position of 2,6-dichloro-9-substituted-purine was
substituted by suspending (4-formyl-3,5-dimethoxyphenoxy)methyl
polystyrene resin (PAL-resin) in dimethylformamide (DMF). A
suitably substituted aniline, acetic acid and sodium
triacetoxyborohydride were then added to the solution. The mixture
was shaken gently at room temperature for 12 hours. The resulting
aniline bound resin was then washed with DMF, methanol and
dichloromethane. The aniline bound resin was then reacted with
2,6-dichloro-9-substituted-purine and diisopropylethylamine in
1-butanol at 80.degree. C. for 12 hours. The resulting resin was
then washed as described above to afford the 2-chloro-6-resin bound
aniline-9-substituted purine.
[0164] The C2 position of the purine ring was subsequently
substituted by mixing the purine bound PAL resin with a suitable
amine or aryl alcohol, Pd.sub.2(dba).sub.3,
2-(di-t-butylphosphino)biphenyl and K.sub.3PO.sub.4 in a 10 mL
flame-dried Schlenk flask. The flask was then evacuated and
refilled with argon, and anhydrous toluene was added. The mixture
was heated at 80.degree. C. for 12 hours. The resulting resin was
then washed as described above and cleaved with
CH.sub.2Cl.sub.2:TFA:Me.sub.2S:H.sub.2O/45:45:5:5 at room
temperature for 2 hours. The solution was collected and dried in
vacuo to afford the desired crude product. The crude product was
then purified using preparative HPLC using H.sub.2O (with 0.1% TFA)
and acetonitrile (MeCN) as solvents. A linear gradient of 5% to 95%
MeCN over 5 min was used. The corresponding peak was collected and
lyophilized to afford the pure desired 2,6,9-substituted
purine.
Synthesis of
2-(1-Naphthoxy)-6-(4-morpholinoanilino)-9-cyclohexylpurine
(Compound A)
[0165] PAL-resin (1 g, 1.1 mmol) was suspended in DMF (4 mL) and
4-morpholinoaniline (196 mg, 5.5 mmole), acetic acid (0.65 mL, 1.13
mmol) and sodium triacetoxyborohydride (720 mg, 3.4 mmol) were then
added into the solution. The mixture was shaken gently at room
temperature for 12 hours. The resulting resin was then washed with
DMF (10 mL, 3 times), methanol (10 mL, 3 times) and dichloromethane
(10 mL, 3 times). The aniline bound resin was then reacted with
2,6-dichloro-9-cyclohexylpurine (598 mg, 2.2 mmole) and
diisopropylethylamine (0.5 mL, 3 mmol) in 1-butanol (5 mL) at
80.degree. C. for 12 hours. The resulting resin was then washed as
described above.
[0166] Purine bound PAL resin (100 mg, 0.1 mmol) was mixed with
1-naphthol (72 mg, 0.5 mmol), Pd.sub.2(dba).sub.3 (6.4 mg, 0.007
mmol), 2-(di-t-butylphosphino)biphenyl (8.3 mg, 0.028 mmol) and
K.sub.3PO.sub.4 (148 mg, 0.7 mmol) in a 10 mL flame-dried Schlenk
flask. The flask was then evacuated and refilled with argon, and
anhydrous toluene (1 mL) was added. The mixture was heated at
80.degree. C. for 12 hours. The resulting resin was then washed as
described above and cleaved with
CH.sub.2Cl.sub.2:TFA:Me.sub.2S:H.sub.2O/45:45:5:5 (0.5 mL) at room
temperature for 2 hours. The solution was collected and dried in
vacuo to afford the desired crude product. The crude product was
then purified using preparative HPLC using H.sub.2O (with 0.1% TFA)
and MeCN as solvents. A linear gradient of 5% to 95% MeCN over 5
min was used; the desired compound has the retention time of 3.9
min. The corresponding peak was collected and lyophilized to yield
pure 2-(1-Naphthoxy)-6-(4-morpholinoanilino)-9-cyclohexylpurine
(light yellow powder, 24 mg, overall 50% yield). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. (ppm) 1.34 (m, 1H), 1.58 (m, 2H), 1.75
(m, 2H), 1.85 (m, 1H), 2.01 (m, 2H), 2.33 (m, 2H), 3.21 (t, 4H,
J=4.8 Hz), 3.99 (t, 4H, J=4.9 Hz), 4.67 (m, 1H), 6.73 (d, 2H, J=9.1
Hz), 7.16 (2H, J=9.1 Hz), 7.33 (d, 1H, J=7.4 Hz), 7.44 (t, 1H,
J=8.3 Hz), 7.54 (m, 2H), 7.88 (d, 1H, J=8.3 Hz), 7.93 (d, 1H, J=8.2
Hz), 8.27 (s, 1H), 10.22 (s, 1H). High Resolution Mass Spectrometry
(MALDI-FTMS): calculated [MH.sup.+]
(C.sub.31H.sub.33N.sub.6O.sub.2) 521.2665, found 521.2670;
calculated [MNa.sup.+] (C.sub.31H.sub.32N.sub.6NaO.sub.2) 543.2484,
found 543.2488.
##STR00022##
[0167] Solution Phase Synthesis
##STR00023##
[0168] 2,6-dichloropurine was reacted with
diisopropylazodicarboxylate (DIAD), triphenylphosphine and a
suitable alcohol in anhydrous THF at -78.degree. C. The reaction
mixture was warmed up to room temperature slowly to yield the
2,6-dichloro-N-9-substituted purine. Sequentially, the
2,6-dichloro-9-substituted purine was then heated with a suitably
substituted aniline and diisopropylethylamine (DIEA) at 80.degree.
C. for 12 hours to yield 6-anilino-2-chloro-9-substituted purine.
This compound was then reacted with a suitable substituted amine or
arylalcohol along with a palladium catalyst to afford the desired
2,6,9-substituted purines.
Example 2
Cell Culture and Osteogenesis Detection Assays
[0169] Cell Culture: Mouse embryonic mesoderm fibroblasts C3H10T1/2
cells were obtained at the 10th passage from ATCC. Cells between
the 12th and 15th passages were used in these experiments. Cells
beyond 15th passage were discarded. Mouse embryonic mesoderm
fibroblasts C3H10T1/2 cells and mouse pre-osteoblasts MC3T3-E1
cells (from German collection of microorganisms and cell cultures)
were cultured at 37.degree. C. in 5% CO.sub.2 in MEM-.alpha. media
(Gibco) supplemented with 10% heat inactivated FBS (Gibco), 50 U/mL
penicillin and 50 .mu.g/mL of streptomycin (Gibco). When cells were
80% confluent, they were split a 1:5. Mouse pre-adipocytes 3T3-L1
cells (from ATCC) and mouse myoblasts C2C12 cells (from ATCC) were
cultured at 37.degree. C. in 5% CO.sub.2 in DMEM (Gibco)
supplemented with 10% heat inactivated FBS (Gibco), 50 U/mL
penicillin and 50 .mu.g/mL of streptomycin (Gibco).
[0170] Alkaline Phosphatase Assay: C3H10T1/2 cells were expanded in
T175 flasks; cells at the 13th passage were detached by
trypsin/EDTA and diluted in the growth media. The resulting cell
suspension was then plated into black clear bottom 384-well plates
(Greiner) with 2500 cells/well in 100 .mu.L growth media using a
Multi-drop.TM. liquid delivery system (Titertek Instruments). After
an overnight incubation, cells were attached to the bottom of the
wells. A stock solution of Compound A in DMSO (500 mL) was
delivered into corresponding well using a Mini Trak.TM.
multiposition dispenser system (Packard BioScience) to make the
final concentration of 5 .mu.M of each compound. Cells were then
incubated at 37.degree. C. in 5% CO.sub.2. After 4 days, the media
was removed and 10 .mu.L of Passive Lysis Buffer (Promega) was
added into each well. After 5 min, 10 .mu.L of alkaline phosphatase
substrate solution (1 mM in pH 10 buffer,
2'-[2'-benzothiazoyl]-6'-hydroxybenzothiazole phosphate (Promega)
was added to each well. After a 15 minute incubation at room
temperature, the plates were read on an Acquest high-throughput
plate reader (Molecular Devices) using the manufacturer's
protocol.
[0171] Cbfa1/Runx2 Reporter GeneAssay: The murine Cbfa1/Runx2 gene
promoter region (1818 bp) was amplified by PCR from mouse genomic
DNA library (Clontech) using the following primers:
5'-ACGCGTAAGAATCTTATGAACATGATTTCA (SEQ ID NO:1) and
5'-CTCGAGTCACACAATCCAAAAAAGCAAAA (SEQ ID NO:2)
[0172] The resulting PCR product was inserted into Topo cloning
vector (Invitrogen), amplified and sequenced. The resulting plasmid
was digested with restriction enzymes Mlu and XhoI (New England
Biolab) and the 1.8 kb fragment was inserted into the Mlu/XhoI
cloning sites in pGL3-BV luciferase reporter vector (Promega). For
transient transfection assays, cells were seeded in a 6-well plate
at a density of 30,000 cells/well in 2 mL of growth medium. After
an overnight incubation, the cells reached 80% confluency. Plasmid
DNA (1 .mu.g) was transfected into cells using 3 .mu.L of Fugene 6
transfection reagent (Roche) using the manufacturer's protocol.
After 8 hours, the transfected cells were plated into 96-well assay
plates (Corning) and treated with 300 ng/mL of recombinant human
bone morphogenetic protein 4 (BMP-4) (Sigma), 1% DMSO (Sigma) or
different concentrations of Compound A (dissolved in DMSO). The
cells were incubated for 4 days, then the luciferase activity in
the cells was assayed using the Bright-Glo luciferase assay kit
(Promega) using the manufacturer's protocol. Luminescence signals
were detected by Acquest AD (Molecular Devices) plate reader. Due
to the low transfection efficiency of MC3T3-E1 cells, the
Cbfa1/Runx2 reporter assay was carried out with stably transfected
MC3T3-E1 cells. Generally, MC3T3-E1 cells were plated in a 6-well
plate at 30,000 cells/well. After an overnight incubation, 1 .mu.g
of reporter plasmid and 0.2 .mu.g of pCMV-Tag2B vector comprising a
neomycin resistance gene were co-transfected into MC3T3-E1 cells
using 3.6 .mu.L of Fugene 6 transfection reagent (Roche) following
the manufacturer's protocol. After 8 hours, cells were detached by
treatment with trypsin/EDTA and seeded in a 10 cm culture dish.
After the cells attached to the dish, 200 .mu.g/mL of G418 (Gibco)
was added to the culture medium. Fresh G418 was added every 3 days.
After 14 days, the neomycin resistant colonies were pooled to yield
the stably transfected MC3T3-E1 cells.
[0173] Histochemical Staining for Endogenous Alkaline Phosphatase
Expression: C3H10T1/2 cells were seeded in 96-well assay plates
(Corning) at a density of 10,000 cells/well, and treated with 300
ng/mL of BMP-4, 1% DMSO, 2 .mu.M of Compound A, or 100 ng/mL of BMP
and 1 .mu.M of Compound A for 4 days. The cells were then washed 3
times with 200 .mu.L PBS and fixed by a 15 minute incubation in a
10% formalin solution (Sigma). The fixed cells were washed again
with 3 times with 200 .mu.L PBS and stained with the Alkaline
Phosphatase Staining Kit 86R (Sigma Diagnostics) using the
manufacturer's protocol. The images were taken on a Nikon Eclipse
TE300 microscope, and ALP positive cells were counted manually from
the images.
Example 3
Identification of Compound A as a Compound with Osteogenesis
Inducing Activity
[0174] A heterocycle combinatorial library of approximately 50,000
compounds with purine, pyrimidine, quinazoline, pyrazine,
phthalazine, pyrazine and quinoxaline-based scaffolds was screened
to identify small molecules with osteogenesis inducing activity
(see, e.g., Ding et al., J. Am. Chem. Soc. 124:1594 (2002); Gray et
al., Science 281:533 (1998); Rosania et al. Nat. Biotechnol. 18:304
(2000); and Rosania et al., Proc. Natl. Acad. Sci. USA. 96:4797
(1999). One 2,6,9-trisubstituted purine was found to have
significant activity in the ALP enzymatic assay. This compound,
which has morpholinoaniline substitution at the C6 position of
purine nucleus, was named Compound A. Further studies indicated
that the EC50 for Compound A is 1 .mu.M in C3H10T1/2 cells;
moreover, this compound can lead to greater than a 50 fold increase
in ALP after treatment for 4 days as compared with 1% DMSO
treatment, and is more effective than BMP-4 in inducing the
expression of ALP (FIG. 1).
Example 4
Compound A is a Potent Inducer of Osteogenesis in Multipotent
C3H10T1/2 Cells
[0175] Mouse embryonic mesoderm fibroblast C3H10T1/2 cells were
used for this study. C3H10T1/2 cells, like MSCs, are multipotent
mesenchymal progenitor cells which can differentiate into various
mesenchymal cells and have been widely used as a model system for
studies of osteoblast differentiation (see, e.g., 3. Taylor, et
al., Cell 17:771 (1979) and Aubin and Liu, F., PRINCIPLES OF BONE
BIOLOGY; Bilezikian, et al. ed.; Academic Press, San Diego: pp.
51-67). Upon treatment with bone morphogenetic protein 4 (BMP-4),
C3H10T1/2 cells differentiate into osteoblasts (see, e.g., Piccolo,
et al., Cell 86:589 (1996)).
[0176] C3H10T1/2 cells were treated with DMSO (control), BMP-4(300
ng/mL), BMP-4 (100 ng/mL) and Compound A (1 .mu.M), or 0.5 .mu.M, 1
.mu.M, 1.2 .mu.M, 1.5 .mu.M, 1.8 .mu.M, 2 .mu.M, 5 .mu.M, or 10
.mu.M Compound A. Alkaline phosphatase (ALP) activity was measured
as described in Example 2 above, after two, four, and six days of
treatment. The results are shown in FIG. 1.
Example 5
Compound A is a Potent Inducer of Osteogenesis in C3H10T1/2 Cells,
3T3-L1, MC-3T3E1 Cells, and C2C12 Cells
[0177] C3H10T1/2 cells, 3T3-L1, MC-3T3E1 cells, and C2C12 cells
were treated with DMSO (control), BMP-4(300 ng/mL), BMP-4 (100
ng/mL) and Compound A (1 .mu.M), or 1 .mu.M, 2 .mu.M, or 10 .mu.M
Compound A. Cbfa1/Runx2 reporter gene activity was assayed as
described in Example 2 above after four days of treatment.
[0178] Treatment of C3H10T1/2 cells with Compound A for 4 days led
to a more than 6 fold increase in the reporter activity in
transient transfection experiments indicating Cbfa1/Runx2 gene is
up-regulated. This result is consistent with the differentiation of
C3H10T1/2 cells into an osteoblast lineage. These results are shown
in FIG. 2.
[0179] Mouse MC3T3-E1 cells are progenitor cells committed to an
osteoblast lineage. Both BMP-4 and Compound A promote the terminal
differentiation of MC3T3-E1 cells. Since MC3T3-E1 cells already
have high levels of endogenous Cbfa1/Runx2, slight up-regulation of
this gene may be sufficient to promote terminal differentiation
(see, e.g., Xiao et al., J. Cell. Biochem. 74:596 (1999) and Wang
et al., J. Bone. Miner. Res. 14:893 (1999)). The change of
Cbfa1/Runx2 reporter activity in stably transfected MC3T3-E1 cells
is not dramatic when treated with Compound A.
Example 6
Compound A is a Potent Inducer of Osteogenesis in 3T3-L1 Cells,
C2C12 Cells, and MC-3T3E1 Cells
[0180] 3T3-L1 cells, C2C12 cells, and MC-3T3E1 cells were treated
with DMSO (control), BMP-4(300 ng/mL), BMP-4 (100 ng/mL) and
Compound A (1 .mu.M), or 0.1 .mu.M, 0.5 .mu.M, 1 .mu.M, 5 .mu.M, or
10 .mu.M Compound A. ALP activity was measured as described in
Example 1 above, after four days of treatment. The results are
shown in FIG. 3.
Example 7
Histochemical Staining of ALP Expression in C3H10T1/2 Cells Shows
that Compound A is a Potent Inducer of Osteogenesis
[0181] C3H10T1/2 cells were treated with DMSO (a), 300 ng/mL of
BMP-4 (b), 2 .mu.M of Compound A (c), and 100 ng/mL of BMP and 1
.mu.M of Compound A (d) for 4 days and stained for ALP activity.
ALP positive cells were stained red and the cell nuclei were
stained blue.
[0182] Histochemical staining of endogenous ALP indicated that more
than 80% of the cells expressed ALP after Compound A treatment (2
.mu.M for 4 days), while only 40% of BMP-4 treated cells stained
positive for ALP. Interestingly, although fewer cells were induced
by BMP-4, those induced cells had higher expression level of ALP
compared to cells treated with Compound A. Our observations suggest
that Compound A induces the majority of the cell population to
commit into an osteoblast lineage, while BMP-4 may be more potent
in promoting the maturation of osteoblasts. Compound A also showed
a synergistic effect with BMP-4 in inducing the differentiation of
C3H10T1/2 cells. When cells were treated with both BMP-4 and
Compound A, the induction for ALP activity was approximately 3 fold
greater than the simple additive effect of the individual
molecules, suggesting that Compound A does not act as a BMP-4
analogue.
Example 8
Compound A can Induce Transdifferentiation of Non-Osteoblast
Lineage Cells into Osteoblasts
[0183] 3T3-L1 pre-adipocyte cells, which are progenitor cells
committed to an adipocyte lineage, can be transdifferentiated into
osteoblast lineage when treated with Compound A and BMP-4. Compound
A induces Cbfa1/Runx2 expression in pre-adipocyte cells, and
increases the ALP level 9 fold at an optimal concentration of 5
.mu.M, while BMP-4 induces ALP expression more than 40 fold.
Compound A (1 .mu.M) and BMP-4 (100 ng/mL) together increase ALP
activity more than 90 fold in 3T3-L1 cells (FIG. 2). Similarly,
C2C12 cells, which are progenitor cells committed to a skeletal
muscle lineage, can be induced by Compound A to express the
Cbfa1/Runx2 gene (FIG. 2). This observation is consistent with a
previous report that transient up-regulation of Cbfa1/Runx2 is
required, but not sufficient, to transdifferentiate C2C12 cells
into osteoblasts (see, e.g., Lee et al., J. Cell. Biochem. 73:114
(1999)).
Example 9
Morphology of C3H10T1/2 Cells and C2C12 Cells after Treatment with
Compound A
[0184] C3H10T1/2 cells were treated with DMSO (1%) and Compound A
(purmorphamine) (2 .mu.M) for 4 days. C2C12 cells were treated with
DMSO (1%), Compound A (purmorphamine) (2 .mu.M) or BMP-4 (300
ng/mL) for 4 days. The morphology of the cells was examined under
light microscopy. The morphology of the mouse embryonic mesoderm
fibroblast C3H10T1/2 cells treated with Compound A changed from
fibroblast (long and spindle-shaped) to osteocyte (small and
round).
Example 10
Compound A is a Potent Inducer of Osteogenesis
[0185] C3H10T1/2 cells were treated with DMSO alone (control);
BMP-4 alone; and Compound A alone (2 .mu.M). Alkaline phosphatase
(ALP) activity was measured after two, four, and six days of
treatment. FIG. 4A illustrates results demonstrating that Compound
A is a potent inducer of osteogenesis in multipotent C3H10T1/2
cells.
[0186] FIG. 4B illustrates that Compound A induces Cbfa1 gene
upregulation in C3H10T1/2 cells, indicating that Compound A induces
the differentiation of C3H10T1/2 cells into cells of an osteoblast
lineage.
Example 11
Morphological and Transcriptional Analysis of Osteogenesis Induced
by Compound A
[0187] FIG. 5 illustrates morphological and transcriptional
analysis of osteogenesis induced by Compound A. FIG. 5A indicates
that Compound A induces expression of the following proteins:
alkaline phosphatase, collagen type I and osteoponin, all of which
indicate that osteogenesis is induced by Compound A. In addition,
FIG. 5B sets forth a DNA microarray analysis indicating that
Compound A induces expression of a number of transcription factors
and proteins.
Example 12
Analysis of Additional Compounds for Osteogenesis-Inducing
Activity
[0188] Additional compounds of Formula I were synthesized and
tested for osteogenesis inducing activity using the alkaline
phophastase assays and Cbfa1/Runx2 reporter gene assays described
in Example 2, above.
[0189] For the compounds tested, R.sup.1 was
##STR00024##
[0190] R.sup.5 was H, R.sup.6 was morpholine and the substituent at
R.sup.4 was varied.
[0191] Typical EC.sub.50 about 1 .mu.M, 2 .mu.M, 2.5 .mu.M, 5
.mu.M, 7 .mu.M, or 10 .mu.M.
[0192] The foregoing examples unequivocally establish that the
compounds of the present invention (such as Compound A) induce
osteogenesis of pre-osteoblast cells (such as MC-3T3E1 cells). In
addition, combined with BMP-4, the compounds of the present
invention (such as Compound A) transdifferentiate pre-adipocyte
(such as 3T3-L1 cells) and myoblast (such as C2C12) cells into
osteoblasts.
[0193] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0194] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
Sequence CWU 1
1
2130DNAArtificial SequenceDescription of Artificial Sequencemurine
Cbfa1/Runx2 gene promoter region PCR amplification primer
1acgcgtaaga atcttatgaa catgatttca 30229DNAArtificial
SequenceDescription of Artificial Sequencemurine Cbfa1/Runx2 gene
promoter region PCR amplification primer 2ctcgagtcac acaatccaaa
aaagcaaaa 29
* * * * *