U.S. patent application number 14/062185 was filed with the patent office on 2014-05-01 for tgf-beta receptor inhibitors to enhance direct reprogramming.
This patent application is currently assigned to THE GENERAL HOSPITAL CORPORATION. The applicant listed for this patent is THE GENERAL HOSPITAL CORPORATION. Invention is credited to Konrad Hochedlinger, Matthias Stadtfeld.
Application Number | 20140120621 14/062185 |
Document ID | / |
Family ID | 47045734 |
Filed Date | 2014-05-01 |
United States Patent
Application |
20140120621 |
Kind Code |
A1 |
Hochedlinger; Konrad ; et
al. |
May 1, 2014 |
TGF-BETA RECEPTOR INHIBITORS TO ENHANCE DIRECT REPROGRAMMING
Abstract
In general, iPS cells are produced by delivery of stem
cell-associated genes into adult somatic cells (e.g., fibroblasts).
Described herein are methods for enhancing the efficiency and rate
of induced pluripotent stem cell production by treating somatic
cells with a transforming growth factor-beta receptor (TGF.beta.R)
inhibitor. Also described herein are iPS cell compositions made
according to the methods described herein and iPS cell compositions
comprising an iPS cell in an admixture with a TGF.beta.R inhibitor.
Further described herein are kits for producing iPS cells using a
TGF.beta.R inhibitor.
Inventors: |
Hochedlinger; Konrad;
(Boston, MA) ; Stadtfeld; Matthias; (New York,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE GENERAL HOSPITAL CORPORATION |
BOSTON |
MA |
US |
|
|
Assignee: |
THE GENERAL HOSPITAL
CORPORATION
BOSTON
MA
|
Family ID: |
47045734 |
Appl. No.: |
14/062185 |
Filed: |
October 24, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13626575 |
Sep 25, 2012 |
8603818 |
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14062185 |
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12547022 |
Aug 25, 2009 |
8298825 |
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13626575 |
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61091574 |
Aug 25, 2008 |
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Current U.S.
Class: |
435/455 ;
435/377 |
Current CPC
Class: |
C12N 2501/15 20130101;
C12N 2506/1307 20130101; C12N 2501/603 20130101; C12N 2510/00
20130101; C12N 2501/606 20130101; C12N 2501/604 20130101; C12N
5/0696 20130101; C12N 2501/602 20130101 |
Class at
Publication: |
435/455 ;
435/377 |
International
Class: |
C12N 5/074 20060101
C12N005/074 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under Grant
Nos. OD003166 and OD003266 awarded by the National Institutes of
Health. The Government has certain rights in the invention.
Claims
1.-16. (canceled)
17. An in vitro method for producing an induced pluripotent stem
cell (iPSC) from a somatic cell, the method comprising: (a)
reprogramming a somatic cell to produce a pluripotent stem cell by
introducing a nucleic acid minimally encoding Oct3/4 and optionally
additionally encoding Sox2 and c-Myc into said somatic cell; (b)
contacting said somatic cell of step (a) with an inhibitor of
TGF-.beta. receptor activity; and (c) isolating a pluripotent stem
cell produced in step (b).
18. The method of claim 17, wherein said inhibitor of TGF-.beta.
receptor activity is selected from the group consisting of an
antibody that inhibits TGF-.beta. receptor activity, a small
molecule that inhibits TGF-.beta. receptor activity, and an RNA
interference molecule that inhibits TGF-.beta. receptor
activity.
19. The method of claim 18, wherein said small molecule is selected
from the group consisting of
2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 naphthyridine,
[3-(Pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and
3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole-
.
20. The method of claim 17, wherein said nucleic acid sequence is
introduced in a viral vector or a plasmid.
21. The method of claim 17, wherein production of said induced
pluripotent stem cell is confirmed by detection of a stem cell
marker.
22. An in vitro method for increasing efficiency of iPSC
production, the method comprising: (a) reprogramming a somatic cell
to produce a pluripotent stem cell by introducing a nucleic acid
minimally encoding Oct3/4 and optionally additionally encoding Sox2
and c-Myc into said somatic cell; and (b) contacting said somatic
cell of step (a) with an inhibitor of TGF-.beta. receptor activity,
wherein the efficiency of iPSC production is increased relative to
the efficiency of iPSC production by said reprogramming step (a)
that does not contact the somatic cell with said inhibitor.
23. An in vitro method for increasing the rate of iPSC production,
the method comprising: (a) reprogramming a somatic cell to produce
a pluripotent stem cell by introducing a nucleic acid minimally
encoding Oct3/4 and optionally additionally encoding Sox2 and c-Myc
into said somatic cell; and (b) contacting said somatic cell of
step (a) with an inhibitor of TGF-.beta. receptor activity, wherein
the rate of iPSC production is increased relative to the rate of
iPSC production by said reprogramming step (a) that does not
contact the somatic cell with said inhibitor.
24. A kit for producing iPSCs, the kit comprising: (a) nucleic acid
sequences encoding Oct3/4, and optionally encoding Sox2 and c-Myc;
(b) an inhibitor of TGF-.beta. receptor activity; and (c) packaging
material therefor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of co-pending
U.S. patent application Ser. No. 13/626,575 filed on Sep. 25, 2012,
which is a continuation application of U.S. patent application Ser.
No. 12/547,022 filed on Aug. 25, 2009 and issued as U.S. Pat. No.
8,298,825 on Oct. 30, 2012, which claims benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Application No. 61/091,574 filed
on Aug. 25, 2008, the contents of each of which are incorporated
herein by reference in their entireties.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Oct. 22, 2013, is named 030258-061933_SL and is 24,895 bytes in
size.
FIELD OF THE INVENTION
[0004] The present invention relates to the production of induced
pluripotent stem cells.
BACKGROUND
[0005] Reprogramming of cells by nuclear transfer (Wakayama et al.,
1998; Wilmut et al., 1997) and cell fusion (Cowan et al., 2005;
Tada et al., 2001) allows for the re-establishment of a pluripotent
state in a somatic nucleus (Hochedlinger and Jaenisch, 2006). While
the molecular mechanisms of nuclear reprogramming remain elusive,
cell fusion experiments have implied that reprogramming factors can
be identified in ES cells and be used to directly induce
reprogramming in somatic cells. Indeed, a rational approach
recently led to the identification of four transcription factors
whose expression enabled the induction of a pluripotent state in
adult fibroblasts (Takahashi and Yamanaka, 2006). Yamanaka and
colleagues demonstrated that retroviral expression of the
transcription factors Oct4, Sox2, c-Myc, and Klf4, combined with
genetic selection for Fbx 15 expression, gives rise to induced
pluripotent stem (iPS) cells directly from fibroblast cultures. Fbx
15-selected iPS cells contributed to diverse tissues in
mid-gestation embryos, however, these embryos succumbed at
mid-gestation, indicating a restricted developmental potential of
iPS cells compared with ES cells. Consistent with this observation,
only part of the ES cell transcriptome was expressed in iPS cells,
and methylation analyses of the chromatin state of the Oct4 and
Nanog promoters demonstrated an epigenetic pattern that was
intermediate between that of fibroblasts and ES cells.
[0006] These observations raised three fundamental questions about
the molecular and functional nature of directly reprogrammed cells:
(i) can selection for a gene that is essential for the ES cell
state generate pluripotent cells that are more similar to ES cells
than the previously described Fbx 15-selected iPS cells; (ii) does
the pluripotent state of iPS cells depend on continuous expression
of exogenous factors; and (iii) does transcription factor-induced
reprogramming reset the epigenetic landscape of a fibroblast genome
into that of a pluripotent cell.
[0007] Ectopic expression of the transcription factors Oct4, Sox2,
cMyc, and Klf4, as well as variants of this factor combination, are
sufficient to confer a pluripotent state upon several
differentiated cell types, generating induced pluripotent stem
cells (iPSCs) (Takahashi, K., and Yamanaka, S. (2006) Cell 126,
663-676; Takahashi, K., et al., (2007) Cell 131, 861-872; Yu, J.,
et al. (2007) Science 318, 1917-1920; Feng, B., et al., (2009) Nat
Cell Biol 11, 197-203; Eminli, S., et al., (2008); Hanna, J.,
(2008) Cell 133, 250-264; Stadtfeld, M., et al., (2008) Curr Biol
18, 890-894; Aoi, T., et al., (2008) Science 321, 699-702). The
derivation of iPSCs is a highly inefficient process with the
underlying mechanisms largely unknown. This low efficiency argues
for the existence of additional cooperative factors, whose
identification is critical for understanding the process of
reprogramming. Further, the therapeutic use of iPSCs relies on
developing efficient non-genetic means of factor delivery, and
while a handful of compounds that replace individual factors have
been identified, their use yields a further reduction to the
already low efficiency of reprogramming (Huangfu, D., et al.,
(2008) Nat. Biotechnol.; Shi, Y., (2008) Cell Stem Cell 3, 568-574;
Marson, A., (2008) Cell Stem Cell 3, 132-135). Thus, the
identification of compounds that enhance rather than solely replace
the function of the reprogramming factors will be of great use.
SUMMARY OF THE INVENTION
[0008] Induced pluripotent stem cells (iPSs) are a type of
pluripotent stem cell artificially derived from a somatic cell by
inserting or expressing stem cell-associated genes. iPS cells are
typically derived by viral delivery of stem cell-associated genes
into adult somatic cells (e.g., fibroblasts). Described herein are
methods for enhancing the efficiency and rate of induced
pluripotent stem cell production by treating somatic cells with a
transforming growth factor-beta receptor (TGF.beta.R) inhibitor.
Also described herein are iPS cell compositions comprising an iPS
cell in an admixture with a TGF.beta.R inhibitor, and kits for
producing iPS cells using a TGF.beta.R inhibitor.
[0009] One aspect described herein relates to a method for
producing an induced pluripotent stem cell from a somatic cell, the
method comprising: (a) treating a somatic cell to re-program it or
its progeny to a pluripotent stem cell phenotype; (b) contacting
the somatic cell or its progeny with an inhibitor of the TGF-.beta.
signaling pathway; and (c) isolating a pluripotent stem cell from
cells of step (b).
[0010] In one embodiment of this aspect and all other aspects
disclosed herein, the somatic cell comprises a human cell. In an
alternate embodiment, the cell is derived from a non-human
organism, such as a non-human mammal.
[0011] In another embodiment of this aspect and all other aspects
disclosed herein, treating comprises introducing a nucleic acid
sequence encoding one or more transcription factors selected from
the group consisting of Oct4, Sox2, c-MYC and Klf4 to a somatic
cell.
[0012] In another embodiment of this aspect and all other aspects
disclosed herein, the inhibitor of the TGF-.beta. signaling pathway
comprises an inhibitor of TGF-.beta. receptor activity. In another
embodiment of this aspect and all other aspects disclosed herein,
the inhibitor of TGF-.beta. receptor activity comprises an
inhibitor of ALK4, ALK5, or ALK7.
[0013] In another embodiment of this aspect and all other aspects
disclosed herein, the inhibitor of TGF-.beta. receptor activity is
selected from the group consisting of an antibody, a small
molecule, and an RNA interference molecule.
[0014] In another embodiment of this aspect and all other aspects
disclosed herein, the small molecule is selected from the group
consisting of 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5
napththyridine, [3-(Pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and
3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole-
.
[0015] In another embodiment of this aspect and all other aspects
disclosed herein, the nucleic acid sequences are comprised in a
viral vector or a plasmid.
[0016] In another embodiment of this aspect and all other aspects
disclosed herein, the viral vector is a retroviral vector.
[0017] In another embodiment of this aspect and all other aspects
disclosed herein, production of the induced pluripotent stem cell
is determined by detection of a stem cell marker.
[0018] In another embodiment of this aspect and all other aspects
disclosed herein, the stem cell marker is selected from the group
consisting of SSEA1, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3,
Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1.
[0019] Another aspect described herein relates to a method for
increasing the efficiency of induced pluripotent stem cell
production, the method comprising: (a) treating a somatic cell to
re-program it or its progeny to a pluripotent stem cell phenotype;
(b) contacting the somatic cells subjected to the process of step
(a) with an inhibitor of the TGF-.beta. signaling pathway, wherein
the efficiency of induced pluripotent stem cell generation is
increased relative to such generation occurring without
contacting.
[0020] Another aspect described herein relates to a method for
increasing the rate of induced pluripotent stem cell production,
the method comprising: (a) treating a somatic cell to re-program it
or its progeny to a pluripotent stem cell phenotype; and (b)
contacting the somatic cell with an inhibitor of the TGF-.beta.
signaling pathway, wherein the rate of induced pluripotent stem
cell generation is increased relative to the rate of induced
pluripotent stem cell generation occurring without contacting.
[0021] Another aspect described herein relates to an induced
pluripotent stem cell composition comprising an induced pluripotent
stem cell in an admixture with an inhibitor of the TGF-.beta.
signaling pathway. In one embodiment of this aspect and all other
aspects described herein, the induced pluripotent stem cell
composition consists essentially of an induced pluripotent stem
cell in an admixture with an inhibitor of the TGF-.beta. signaling
pathway. In another embodiment of this aspect and all other aspects
described herein, the induced pluripotent stem cell composition
consists of an induced pluripotent stem cell in an admixture with
an inhibitor of the TGF-.beta. signaling pathway.
[0022] Another aspect described herein relates to a kit for
producing induced pluripotent stem cells, the kit comprising: (a)
nucleic acid sequences encoding one or more transcription factors
selected from the group consisting of Oct4, Sox2, c-MYC and Klf4,
(b) an inhibitor of the TGF-.beta. signaling pathway, (c) packaging
materials therefor.
[0023] Another aspect described herein relates to a cell
composition derived from any combination of the methods described
herein.
DEFINITIONS
[0024] The term "pluripotent" as used herein refers to a cell with
the capacity, under different conditions, to differentiate to more
than one differentiated cell type, and preferably to differentiate
to cell types characteristic of all three germ cell layers.
Pluripotent cells are characterized primarily by the ability to
differentiate to more than one cell type, preferably to all three
germ layers, using, for example, a nude mouse teratoma formation
assay. Pluripotency is also evidenced by the expression of
embryonic stem (ES) cell markers, although the preferred test for
pluripotency is the demonstration of the capacity to differentiate
into cells of each of the three germ layers.
[0025] The term "re-programming" as used herein refers to the
process of altering the differentiated state of a
terminally-differentiated somatic cell to a pluripotent
phenotype.
[0026] By "differentiated primary cell" is meant any primary cell
that is not, in its native form, pluripotent as that term is
defined herein. It should be noted that placing many primary cells
in culture can lead to some loss of fully differentiated
characteristics. However, simply culturing such cells does not, on
its own, render them pluripotent. The transition to pluripotency
requires a re-programming stimulus beyond the stimuli that lead to
partial loss of differentiated character in culture. Re-programmed
pluripotent cells also have the characteristic of the capacity of
extended passaging without loss of growth potential, relative to
primary cell parents, which generally have capacity for only a
limited number of divisions in culture.
[0027] As used herein, the term "TGF-.beta. signaling pathway" is
used to describe the downstream signaling events attributed to
TGF-.beta. and TGF-.beta. like ligands. For example, in one
signaling pathway a TGF-.beta. ligand binds to and activates a Type
II TGF-.beta. receptor. The Type II TGF-.beta. receptor recruits
and forms a heterodimer with a Type I TGF-.beta. receptor. The
resulting heterodimer permits phosphorylation of the Type I
receptor, which in turn phosphorylates and activates a member of
the SMAD family of proteins. A signaling cascade is triggered,
which is well known to those of skill in the art, and ultimately
leads to control of the expression of mediators involved in cell
growth, cell differentiation, tumorigenesis, apoptosis, and
cellular homeostasis, among others. Other TGF-.beta. signaling
pathways are also contemplated for manipulation according to the
methods described herein.
[0028] The term "inhibitor of the TGF-.beta. signaling pathway" as
used herein, refers to inhibition of at least one of the proteins
involved in the signal transduction pathway for TGF-.beta.. It is
contemplated herein that an inhibitor of the TGF-.beta. signaling
pathway can be, for example, a TGF-.beta. receptor inhibitor (e.g.,
a small molecule, an antibody, an siRNA), a TGF-.beta. sequestrant
(e.g., an antibody, a binding protein), an inhibitor of receptor
phosphorylation, an inhibitor of a SMAD protein, or a combination
of such agents.
[0029] In one embodiment, the TGF-.beta. signaling pathway
inhibitor comprises or consists essentially of a TGF-.beta.
receptor inhibitor. One of skill in the art can easily test a
compound to determine if it inhibits TGF-.beta. receptor signaling
by assessing, for example, phosphorylation status of the receptor
or expression of downstream proteins controlled by TGF-.beta. in
cultured cells and comparing the results to cells not treated with
a TGF-.beta. receptor inhibitor. An agent is determined to be a
TGF-.beta. signaling pathway inhibitor if the level of
phosphorylation of the Type I TGF-.beta. receptor in a culture of
cells is reduced by at least 20% compared to the level of
phosphorylation of the Type I TGF-.beta. receptor in cells that are
cultured in the absence of a TGF-.beta. signaling pathway
inhibitor; preferably the level of phosphorylation is reduced by at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, at least 99%, or even
100% (no phosphorylation) in the presence of a TGF-.beta. signaling
pathway inhibitor.
[0030] As used herein, the term "Alk5" is used to denote a TGF-beta
receptor type I having serine/threonine protein kinase activity
(also referred to herein as TGF.beta.R-1). The term "TGF-beta
receptor" or "TGF.beta.R" is used herein to encompass all three
sub-types of the TGF.beta.R family (i.e., TGF.beta.R-1,
TGF.beta.R-2, TGF.beta.R-3). The TGF.beta. receptors are
characterized by serine/threonine kinase activity and exist in
several different isoforms that can be homo- or heterodimeric.
[0031] An "RNA interference molecule" as used herein, is defined as
any agent which interferes with or inhibits expression of a target
gene or genomic sequence by RNA interference (RNAi). Such RNA
interfering agents include, but are not limited to, nucleic acid
molecules including RNA molecules which are homologous to the
target gene or genomic sequence, or a fragment thereof, short
interfering RNA (siRNA), short hairpin or small hairpin RNA
(shRNA), microRNA (miRNA) and small molecules which interfere with
or inhibit expression of a target gene by RNA interference
(RNAi).
[0032] The term "vector" refers to a small carrier DNA molecule
into which a DNA sequence can be inserted for introduction into a
host cell. An "expression vector" is a specialized vector that
contains the necessary regulatory regions needed for expression of
a gene of interest in a host cell. In some embodiments the gene of
interest is operably linked to another sequence in the vector. The
term "operably linked" means that the regulatory sequences
necessary for expression of the coding sequence are placed in the
DNA molecule in the appropriate positions relative to the coding
sequence so as to effect expression of the coding sequence. This
same definition is sometimes applied to the arrangement of coding
sequences and transcription control elements (e.g. promoters,
enhancers, and termination elements) in an expression vector.
[0033] By "increasing the efficiency" of induced pluripotent stem
cell production is meant that the percentage of reprogrammed cells
in a given population is at least 5% higher in populations treated
with a TG.beta.R inhibitor than a comparable, control treated
population. It is preferred that the percentage of reprogrammed
cells in a TG.beta.R inhibitor treated population is at least 10%
higher, at least 20% higher, at least 30% higher, at least 40%
higher, at least 50% higher, at least 60% higher, at least 70%
higher, at least 80% higher, at least 90% higher, at least 1-fold
higher, at least 2-fold higher, at least 5-fold higher, at least 10
fold higher, at least 100 fold higher, at least 1000-fold higher or
more than a control treated population of comparable size and
culture conditions. The term "control treated population of
comparable size and culture conditions" is used herein to describe
a population of cells that has been treated with identical media,
viral induction, nucleic acid sequence, temperature, confluency,
flask size, pH, etc., with the exception of the TG.beta.R
inhibitor. To be clear, the only difference between a control
treated population and a TG.beta.R inhibitor treated cell
population is the condition of having been treated with a TG.beta.R
inhibitor.
[0034] By "increasing the rate" of induced pluripotent stem cell
production is meant that the amount of time for the induction of
induced pluripotent stem cells is at least 2 days less in a
TG.beta.R inhibitor treated cell population than in a control
treated population of comparable size and culture conditions;
preferably the time needed for pluripotent stem cell induction is
at least 3 days less, at least 4 days less, at least 5 days less,
at least 6 days less, at least 1 week less, at least 2 weeks less,
at least 3 weeks less or more, in the presence of a TG.beta.R
inhibitor than in a control treated population.
[0035] For simplicity, chemical moieties defined and referred to
throughout can be univalent chemical moieties (e.g., alkyl, aryl,
etc.) or multivalent moieties under the appropriate structural
circumstances clear to those skilled in the art. For example, an
"alkyl" moiety can be referred to a monovalent radical (e.g.
CH.sub.3--CH.sub.2--), or in other instances, a bivalent linking
moiety can be "alkyl," in which case those skilled in the art will
understand the alkyl to be a divalent radical (e.g.,
--CH.sub.2--CH.sub.2--), which is equivalent to the term
"alkylene." Similarly, in circumstances in which divalent moieties
are required and are stated as being "alkoxy", "alkylamino",
"aryloxy", "alkylthio", "aryl", "heteroaryl", "heterocyclic",
"alkyl" "alkenyl", "alkynyl", "aliphatic", or "cycloalkyl", those
skilled in the art will understand that the terms "alkoxy",
"alkylamino", "aryloxy", "alkylthio", "aryl", "heteroaryl",
"heterocyclic", "alkyl", "alkenyl", "alkynyl", "aliphatic", or
"cycloalkyl" refer to the corresponding divalent moiety.
[0036] The term "halo" refers to any radical of fluorine, chlorine,
bromine or iodine.
[0037] The term "acyl" refers to an alkylcarbonyl,
cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or
heteroarylcarbonyl substituent, any of which may be further
substituted by substituents. Exemplary acyl groups include, but are
not limited to, (C.sub.1-C.sub.6)alkanoyl (e.g., formyl, acetyl,
propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.),
(C.sub.3-C.sub.6)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl,
cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.),
heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl,
pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl,
piperazinylcarbonyl, tetrahydrofuranylcarbonyl, etc.), aroyl (e.g.,
benzoyl) and heteroaroyl (e.g., thiophenyl-2-carbonyl,
thiophenyl-3-carbonyl, furanyl-2-carbonyl, furanyl-3-carbonyl,
1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl,
benzo[b]thiophenyl-2-carbonyl, etc.). In addition, the alkyl,
cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl
group may be any one of the groups described in the respective
definitions.
[0038] The term "alkyl" refers to saturated non-aromatic
hydrocarbon chains that may be a straight chain or branched chain,
containing the indicated number of carbon atoms (these include
without limitation propyl, allyl, or propargyl), which may be
optionally inserted with N, O, S, SS, SO.sub.2, C(O), C(O)O, OC(O),
C(O)N or NC(O). For example, C.sub.1-C.sub.6 indicates that the
group may have from 1 to 6 (inclusive) carbon atoms in it.
[0039] The term "alkenyl" refers to an alkyl that comprises at
least one double bond. Exemplary alkenyl groups include, but are
not limited to, for example, ethenyl, propenyl, butenyl,
1-methyl-2-buten-1-yl and the like.
[0040] The term "alkynyl" refers to an alkyl that comprises at
least one triple bond.
[0041] The term "alkoxy" refers to an --O-alkyl radical.
[0042] The term "aminoalkyl" refers to an alkyl substituted with an
amino.
[0043] The term "mercapto" refers to an --SH radical.
[0044] The term "thioalkoxy" refers to an --S-alkyl radical.
[0045] The term "aryl" refers to monocyclic, bicyclic, or tricyclic
aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring
may be substituted by a substituent. Exemplary aryl groups include,
but are not limited to, phenyl, naphthyl, anthracenyl, azulenyl,
fluorenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl,
and the like.
[0046] The term "arylalkyl" refers to alkyl substituted with an
aryl.
[0047] The term "cyclyl" or "cycloalkyl" refers to saturated and
partially unsaturated cyclic hydrocarbon groups having 3 to 12
carbons, for example, 3 to 8 carbons, and, for example, 3 to 6
carbons, wherein the cycloalkyl group additionally may be
optionally substituted. Exemplary cycloalkyl groups include, but
are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,
cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl,
and the like.
[0048] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be
substituted by a substituent. Exemplary heteroaryl groups include,
but are not limited to, pyridyl, furyl or furanyl, imidazolyl,
benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, pyridazinyl,
pyrazinyl, quinolinyl, indolyl, thiazolyl, naphthyridinyl, and the
like.
[0049] The term "heteroarylalkyl" refers to an alkyl substituted
with a heteroaryl.
[0050] The term "heterocyclyl" refers to a nonaromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2 or 3 atoms of each ring may be
substituted by a substituent. Exemplary heterocyclyl groups
include, but are not limited to piperazinyl, pyrrolidinyl,
dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.
[0051] The term "haloalkyl" refers to an alkyl group having one,
two, three or more halogen atoms attached thereto. Exemplary
haloalkyl groups include, but are not limited to chloromethyl,
bromoethyl, trifluoromethyl, and the like.
[0052] The term "optionally substituted" means that the specified
group or moiety, such as an aryl group, heteroaryl group and the
like, is unsubstituted or is substituted with one or more
(typically 1-4 substituents) independently selected from the group
of substituents listed below in the definition for "substituents"
or otherwise specified.
[0053] The term "substituents" refers to a group "substituted" on
an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl,
heteroaryl, acyl, amino group at any atom of that group. Suitable
substituents include, without limitation, halo, hydroxy, oxo,
nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, aralkyl,
alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl,
aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkylthio,
CF.sub.3, N-morphilino, phenylthio, alkanesulfonyl, arenesulfonyl,
alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,
alkylcarbonyl, acyloxy, cyano or ureido. In some embodiments, a
substituent can itself be optionally substituted. In some cases,
two substituents, together with the carbons to which they are
attached to can form a ring.
[0054] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the invention, yet open to the
inclusion of unspecified elements, whether essential or not.
[0055] As used herein the term "consisting essentially of" refers
to those elements required for a given embodiment. The term permits
the presence of elements that do not materially affect the basic
and novel or functional characteristic(s) of that embodiment of the
invention.
[0056] The term "consisting of" refers to compositions, methods,
and respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
BRIEF DESCRIPTION OF THE FIGURES
[0057] FIG. 1:
[0058] 1A. Effect of Alk5 inhibitor treatment on the number of
Sox2-GFP+ iPS colonies
[0059] 1B. Example of untreated (top image) and Alk5
inhibitor-treated emerging iPS colonies. Note lack of GFP
expression in the untreated colony.
[0060] FIG. 2. An Alk5 Inhibitor Acts Cooperatively to Promote IPSC
Induction
[0061] 2A. Alkaline phosphatase stain for primary iPSC colonies,
treated with or without the Alk5 inhibitor (1 .mu.M) during dox
induction. MEFs were infected with four factors and colonies were
stained on day 12 (8 d+dox/4 d-dox).
[0062] 2B. Kinetics of reprogramming in MEFs infected with four
factors. Dox was applied for either 3, 4, or 5 days, with or
without the Alk5 inhibitor (2 .mu.M). Colonies were counted on day
16 based on morphology; all picked clones were capable of
generating dox-independent lines.
[0063] 2C. Pluripotency of iPSCs derived after four days of dox
induction using the Alk5 inhibitor. (i-iii) Immunostaining for
pluripotency markers, (i) Nanog, (ii) Oct4, and (iii) Sox2.
Colonies were stained after three passages without dox, thus
reflecting endogenous expression. (iv-yl) Teratoma formation,
demonstrating differentiation into lineages from all three germ
layers: (iv) neural tissue, (v) cartilage, (vi) gut-like
epithelium.
[0064] 2D. Dose-response curve for the Alk5 inhibitor in MEFs
induced with four factors. Dox and the inhibitor were applied for 8
days; colonies were quantified on day 12 based on Oct4
immunostaining.
[0065] 2E. Effect of TGF-.beta. ligands in reprogramming, using
four-factor infected MEFs (dox for 12 d; counts on day 16 based on
Oct4 immunostaining). TGF-.beta. concentrations: low, 1 ng/mL;
medium, 2.5 ng/mL; high, 5 ng/mL.
[0066] 2F. Timing of Alk5 inhibitor action. The Alk5 inhibitor (1
.mu.M) was applied in 4-day time intervals. Control=no inhibitor,
Full-time=inhibitor added days 1-16. Dox was withdrawn on day 12,
and colonies were quantified on day 16 based on Oct4
immunostaining.
[0067] 2G. Priming effect of the Alk5 inhibitor. Inhibitor (1
.mu.M) was applied for either 3 days before (-3 to 0 d) or during
the first 3 days of dox induction (0 to +3 d), or both (-3 d to +3
d). Control=no inhibitor. Dox was withdrawn on day 8 and colonies
were quantified on day 12 by October 4 immunostaining.
[0068] FIG. 3. An Alk5 Inhibitor Replaces the Individual Roles of
cMye and Sox2.
[0069] 3A. Kinetics of reprogramming in two secondary MEF lines.
Dox was applied for either 4, 6, 8, or 10 days, with or without the
Alk5 inhibitor (2 .mu.M). Colonies were quantified on day 16 based
on morphology and dox independence.
[0070] 3B. Dose-response curve for the Alk5 inhibitor in secondary
STEMCCA MEFs. Dox and the inhibitor were applied for 8 days, and
colonies were quantified on day 12 based on Oct4
immunostaining.
[0071] 3C. Replacement of the role of cMyc with the Alk5 inhibitor
(1 .mu.M). MEFs were infected with either three (OSK) or four
(OSMK) factors. Dox was withdrawn on day 8 and colonies were
quantified on day 12 based on morphology and dox independence.
[0072] 3D. Replacement of the role of Sox2 with the Alk5 inhibitor
(1 .mu.M). MEFs were infected with three (OMK) or two (OK) factors
and induced with dox for 16 days, then split on day 18 into media
without dox. Image depicts alkaline phosphatase stain of passage 1
cultures.
[0073] 3E. Pluripotency marker expression in an iPSC line made with
the Alk5 inhibitor in the absence of Sox2. Colonies were analyzed
after three passages without dox. (i) Nanog, (ii) Oct4, (iii),
Sox2.
[0074] 3F. Chimeric mice generated with OMK+inhibitor iPSC lines.
iPSCs were labeled with lentivirally-delivered tdTomato, injected
into diploid blastocysts, and harvested at E16.5. Three embryos are
shown at identical exposures, demonstrating varying degrees of
chimerism.
[0075] 3G. Adult chimera (6 weeks) derived from an OMK+inhibitor
iPSC line. Agouti coat color represents iPSC-derived cells.
[0076] FIG. 4. is a Table Depicting Reprogramming of Cells Using
Different Combinations of Nucleic Acid Sequences with or without an
Exemplary TGF-Beta Inhibitor.
[0077] FIG. 5. Dose-Response Curve for the Alk-4/5/7 Inhibitor
SB-431542.
[0078] Dox and the inhibitor were applied for 8 days in secondary
STEMCCA MEFs, and colonies were quantified on day 12 based on Oct4
immunostaining.
[0079] FIG. 6. Characterization of OMK+Inhibitor IPSCs.
[0080] 6A. Quantitative RT-PCR data demonstrating expression of
pluripotency genes (left) and silencing of viral genes (right).
Expression level was normalized to Gapdh. Three 3-factor lines
(OMK+inhibitor), a 4-factor line (OSMK), and a control ESC line
(v6.5) were included in the analysis. Left: primers were designed
to measure total Oct4 or Nanog; for Sox2, cMyc, and Klf4, primers
only amplified endogenous transcripts. Right: primers were designed
to only measure viral transcripts.
[0081] 6B. Teratoma derived from an OMK+inhibitor iPSC line
demonstrating differentiation into lineages from all three germ
layers. Left, keratinized epithelium; middle, cartilage; right,
gut-like epithelium.
[0082] 6C. Southern blot analysis for Sox2 integrations. Three
3-factor (OMK+inhibitor) iPSC lines were analyzed (3F), as well as
a control four-factor (OSMK) iPSC line, which showed an additional
band in both digests, and an ESC line (v6.5), which showed no
additional bands. Genomic DNA was digested with either BamHI or
XhoI, and the blot was probed with a Sox2 cDNA. Panels on the right
show the ethidium-bromide stained gels used for the blots.
[0083] FIG. 7. Cell Proliferation and Apoptosis During IPSC
Induction.
[0084] 7A. MEFs were infected with either three (OSK) or four
(OSMK) factors, then induced with dox in the presence or absence of
the Alk5 inhibitor (1 .mu.M). Cells were counted at each timepoint
noted, and values represent the fractional change in cell
number:(fraction of starting cell number at timepoint b-fraction of
starting cell number at timepoint a)/time between b and a.
[0085] 7B. Annexin V and propidium iodide (PI) staining to quantify
apoptosis during iPSC induction. Secondary cells carrying the
polycistronic STEMCCA construct were treated with dox for 2 days in
the presence or absence of the Alk5 inhibitor (1 .mu.M), then
stained and analyzed by flow cytometry. Annexin V-positive and
PI-negative cells represent the fraction of living cells in the
process of apoptosis.
[0086] FIG. 8. Induction of Sox2, cMyc, or Nanog with Alk5
Inhibitor Treatment.
[0087] MEFs were infected with various factor combinations, then
treated with dox for three days in the presence or absence of 1
mMeither three (OSK) or four (OSMK) factors, then induced with dox
in the presence or absence of Alk5 inhibitor (1 .mu.M). qPCR
analysis was done using primers that specifically detect endogenous
Sox2 or cMyc, as well as total Nanog. Experiments were done with
both biological and technical triplicates, and a Student's T-test
(paired, two-tailed) was used to assess statistical significance.
The Alk5 inhibitor led to a small (1.3-fold) but significant
induction of cMyc in cells infected with OSK (p=0.016); No other
genes showed a statistically significant change in expression
(Sox2: OMK, p=0.31; OSMK, p=0.43. cMyc: OSMK, p=0.21. Nanog: OMK,
p=0.25; OSK, p=0.55; OSMK, p=0.53).
DETAILED DESCRIPTION OF THE INVENTION
[0088] In general, iPS cells are produced by delivery of stem
cell-associated genes into adult somatic cells (e.g., fibroblasts).
Described herein are methods for enhancing the efficiency and rate
of induced pluripotent stem cell production by treating somatic
cells with a transforming growth factor-beta receptor (TGF.beta.R)
inhibitor. Also described herein are iPS cell compositions made
according to the methods described herein and iPS cell compositions
comprising an iPS cell in an admixture with a TGF.beta.R inhibitor,
and kits for producing iPS cells using a TGF.beta.R inhibitor.
Cells
[0089] While fibroblasts are preferred, essentially any primary
somatic cell type can be used. Some non-limiting examples of
primary cells include, but are not limited to, epithelial,
endothelial, neuronal, adipose, cardiac, skeletal muscle, immune
cells, hepatic, splenic, lung, circulating blood cells,
gastrointestinal, renal, bone marrow, and pancreatic cells. The
cell can be a primary cell isolated from any somatic tissue
including, but not limited to brain, liver, lung, gut, stomach,
intestine, fat, muscle, uterus, skin, spleen, endocrine organ,
bone, etc.
[0090] Where the cell is maintained under in vitro conditions,
conventional tissue culture conditions and methods can be used, and
are known to those of skill in the art. Isolation and culture
methods for various cells are well within the abilities of one
skilled in the art.
[0091] Further, the parental cell can be from any mammalian
species, with non-limiting examples including a murine, bovine,
simian, porcine, equine, ovine, or human cell. In one embodiment,
the cell is a human cell. In an alternate embodiment, the cell is
from a non-human organism such as e.g., a non-human mammal. The
parental cell should not express embryonic stem cell (ES) markers,
e.g., Nanog mRNA or other ES markers, thus the presence of Nanog
mRNA or other ES markers indicates that a cell has been
re-programmed. For clarity and simplicity, the description of the
methods herein refers to fibroblasts as the parental cells, but it
should be understood that all of the methods described herein can
be readily applied to other primary parent cell types.
[0092] Where a fibroblast is used, the fibroblast is flattened and
irregularly shaped prior to the re-programming, and does not
express Nanog mRNA. The starting fibroblast will preferably not
express other embryonic stem cell markers. The expression of
ES-cell markers can be measured, for example, by RT-PCR.
Alternatively, measurement can be by, for example,
immunofluorescence or other immunological detection approach that
detects the presence of polypeptides that are characteristic of the
ES phenotype.
Reprogramming
[0093] The production of iPS cells is generally achieved by the
introduction of nucleic acid sequences encoding stem
cell-associated genes into an adult, somatic cell. In general,
these nucleic acids are introduced using retroviral vectors and
expression of the gene products results in cells that are
morphologically and biochemically similar to pluripotent stem cells
(e.g., embryonic stem cells). This process of altering a cell
phenotype from a somatic cell phenotype to a stem cell-like
phenotype is termed "reprogramming".
[0094] Reprogramming can be achieved by introducing a combination
of stem cell-associated genes including, for example Oct3/4
(Pouf51), Sox, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2, Klf4,
Klf5, c-Myc, 1-Myc, n-Myc and LIN28. In general, successful
reprogramming is accomplished by introducing Oct-3/4, a member of
the Sox family, a member of the Klf family, and a member of the Myc
family to a somatic cell. In one embodiment of the methods
described herein, reprogramming is achieved by delivery of Oct-4,
Sox2, c-Myc, and Klf4 to a somatic cell (e.g., fibroblast). In one
embodiment, the nucleic acid sequences of Oct-4, Sox2, c-MYC, and
Klf4 are delivered using a viral vector, such as an adenoviral
vector, a lentiviral vector or a retroviral vector.
[0095] While it is understood that reprogramming is usually
accomplished by viral delivery of stem-cell associated genes, it is
also contemplated herein that reprogramming can be induced using
other delivery methods.
[0096] The efficiency of reprogramming (i.e., the number of
reprogrammed cells) can be enhanced by the addition of various
small molecules as shown by Shi, Y., et al (2008) Cell-Stem Cell
2:525-528, Huangfu, D., et al (2008) Nature Biotechnology
26(7):795-797, Marson, A., et al (2008) Cell-Stem Cell 3:132-135,
which are incorporated herein by reference in their entirety. It is
contemplated that the methods described herein can also be used in
combination with a single small molecule (or a combination of small
molecules) that enhances the efficiency of induced pluripotent stem
cell production. Some non-limiting examples of agents that enhance
reprogramming efficiency include soluble Wnt, Wnt conditioned
media, BIX-01294 (a G9a histone methyltransferase), PD0325901 (a
MEK inhibitor), DNA methyltransferase inhibitors, histone
deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine,
dexamethasone, suberoylanilide, hydroxamic acid (SAHA), and
trichostatin (TSA), among others. It is also contemplated herein
that inhibitors of the TGF-.beta. signaling pathway either alone or
in combination with another small molecule (or combination of small
molecules) can be used to replace one or more of the reprogramming
factors used for the production of iPS cells.
TGF-.beta. Receptors
[0097] The TGF-.beta. receptors contemplated for use in the methods
described herein can be any TGF-.beta. receptor including those
from the Activin-like kinase family (ALK), the Bone Morphogenic
Protein (BMP) family, the Nodal family, the Growth and
Differentiation Factors family (GDF), and the TGF-.beta. receptor
family of receptors. TGF-.beta. receptors are serine/threonine
kinase receptors that effect various growth and differentiation
pathways in the cell.
[0098] In one embodiment, the TGF-.beta. receptor useful for the
methods described herein is an ALK4, ALK5, or ALK7 receptor. In
another embodiment, the TGF-.beta. receptor inhibited by the
methods described herein is an ALK5 receptor. In another
embodiment, downstream effectors of any of the aforementioned
TGF-beta receptor signaling pathways can be targeted directly to
effect cell reprogramming with the methods described herein.
[0099] If desired, one of skill in the art can locate the protein
sequence of any of the TGF-.beta. receptors by simply searching
"transforming growth factor beta receptor" in a protein sequence
database such as NCBI. Some non-limiting examples of protein
sequence accession numbers for TGF-.beta. receptors are P36897.1
(SEQ ID NO:1), Q5T7S2 (SEQ ID NO:2), Q61R47 (SEQ ID NO:3), P37173
(SEQ ID NO:4), Q6A176 (not shown), Q706C0 (not shown), Q706C1 (not
shown), and Q03167.2 (SEQ ID NO:5), among others.
[0100] TGF-.beta.1 is a prototypic member of a family of cytokines
including the TGF-.beta.s, activins, inhibins, bone morphogenetic
proteins and Mullerian-inhibiting substance, that signal through a
family of single transmembrane serine/threonine kinase receptors.
These receptors can be divided into two classes, the type I or
activin like kinase (ALK) receptors and type H receptors. The ALK
receptors are distinguished from the type II receptors in that the
ALK receptors (a) lack the serine/threonine rich intracellular
tail, (b) possess serine/threonine kinase domains that are very
homologous between type I receptors, and (c) share a common
sequence motif called the GS domain, consisting of a region rich in
glycine and serine residues. The GS domain is at the amino terminal
end of the intracellular kinase domain and is critical for
activation by the type II receptor. Several studies have shown that
TGF-.beta. signalling requires both the ALK and type H receptors.
Specifically, the type II receptor phosphorylates the GS domain of
the type I receptor for TGF-.beta., ALK5, in the presence of
TGF-.beta.. The ALK5, in turn, phosphorylates the cytoplasmic
proteins Smad2 and Smad3 at two carboxy terminal serines. The
phosphorylated Smad proteins translocate into the nucleus and
activate genes that contribute to e.g., the production of
extracellular matrix.
[0101] Activin ligands transduce signals in a manner similar to
TGF-.beta. ligands. Activins bind to and activate ALK receptors,
which in turn phosphorylate Smad proteins such as Smad2 and Smad3.
The consequent formation of a hetero-Smad complex with Smad4
results in the activin-induced regulation of gene
transcription.
[0102] Smad proteins are exemplary downstream signal transduction
factors in the TGF-beta pathway and therefore can be activated or
inhibited directly to effect reprogramming (i.e., by treating a
cell with an activator or inhibitor of a Smad protein). In one
embodiment, an activator of Smad 7 is used to effect cell
reprogramming. In another embodiment, inhibition of Smad 2, 3, or 5
is used to effect cell reprogramming.
TGF-Beta Receptor (TGF.beta.R) Inhibitors
[0103] As used herein, the term "TGF-.beta. signaling inhibitor"
(also referred to as TGF-.beta. signal transduction inhibitor) is a
compound that inhibits TGF-.beta. signal transduction by inhibiting
any of the factors constituting the TGF-.beta. signal transduction
system pathway, such as TGF-.beta. ligand, TGF-.beta. Type I
receptors, TGF-.beta. Type II receptors, TGF-.beta. Type III
receptors (.beta.-glycan and endoglin), soluble forms of the
TGF-.beta. receptors, Smad proteins (1-8), antibodies against
receptors and ligands implicated in the signaling pathway, nucleic
acid based molecules (e.g., antisense, siRNA, aptamers and
ribozymes) targeting the pathway members, or a combination
thereof.
[0104] An "inhibitor" of a TGF.beta.R, as the term is used herein,
can function in a competitive or non-competitive manner, and can
function, in one embodiment, by interfering with the expression of
the TGF.beta.R polypeptide. A TGF.beta.R inhibitor includes any
chemical or biological entity that, upon treatment of a cell,
results in inhibition of a biological activity caused by activation
of the TGF.beta.R in response to binding of its natural ligand.
While any TGF-.beta. signaling pathway inhibitor can potentially be
used in the methods described herein, it is preferable that a
TGF-.beta. signaling pathway inhibitor is either selective for, or
specific for, a member of the TGF-.beta. signaling pathway. By
"specific" is meant that at the dose necessary for the inhibiting
agent to inhibit the TGF-.beta. signaling pathway, the inhibiting
agent does not have any other substantial pharmacological action in
the cell or host. By "selective" is meant that the dose of the
inhibitor necessary for inhibition of the TGF-.beta. signaling
pathway is at least 2-fold lower than the dose necessary for
activation or inhibition of another pharmacological action as
measured by the ED.sub.50 or EC.sub.50 of the agent for each
pharmacological effect; preferably the dose of inhibitor necessary
for TGF-.beta. pathway inhibition is at least 5-fold lower, at
least 10 fold lower, at least 20-fold lower, at least 30-fold
lower, at least 40-fold lower, at least 50-fold lower, at least
60-fold lower, at least 70-fold lower, at least 80-fold lower, at
least 90-fold lower, at least 100-fold lower, at least 500-fold
lower, at least 1000 fold lower or more, than the dose necessary
for another pharmacological action. Thus, to be clear, the agents
useful for the methods described herein primarily inhibit the
TGF-.beta. signaling pathway with only minor, if any, effects on
other pharmacological pathways, and the dose used for inhibition of
the TGF-.beta. signaling pathway is sub-clinical or sub-threshold
for other pharmacological responses.
[0105] Such an inhibitor can act by binding to the intracellular
domain of the receptor and blockade of its serine/threonine kinase
activity (e.g., ATP binding site). Alternatively, such an inhibitor
can act by occupying or sterically hindering the ligand binding
site (or a portion thereof) of the TGF.beta.R, thereby rendering
the receptor inaccessible to binding by the natural ligand, which
prevents activation by that ligand. In addition, the TGF.beta.R
inhibitor can also bind to a non-ligand binding site and, for
example, produce a conformational shift in the TGF.beta.R, such
that a ligand of the TGF.beta.R can no longer access the binding
site. An inhibitor can be, for example, a competitive inhibitor, a
non-competitive inhibitor, an inverse agonist or a partial agonist
of the TGF.beta.R.
[0106] Alternatively, such an inhibitor can act by modulating the
heterodimerization of TGF.beta.R polypeptides, the interaction of
TGF.beta.R with other proteins, or the ubiquitination or endocytic
degradation of the receptor. TGF.beta.R inhibitors, include, but
are not limited to small molecules, antibodies or antigen-binding
antibody fragments, antisense constructs, siRNAs and ribozymes.
[0107] The receptor activity of a TGF-.beta. receptor can be
measured, for example, as described by Laping, N J., et al (2002)
Molecular Pharmacology 62(1):58-64, which is herein incorporated by
reference in its entirety. In addition, the dose-response curve for
a TGF-.beta. receptor inhibitor can be determined by measuring
TGF-.beta. receptor activity over a variety of inhibitor
concentrations using the method of Laping, N J., et al (2002).
Small Molecule Inhibitors
[0108] As used herein, the term "small molecule" refers to a
chemical agent which can include, but is not limited to, a peptide,
a peptidomimetic, an amino acid, an amino acid analog, a
polynucleotide, a polynucleotide analog, an aptamer, a nucleotide,
a nucleotide analog, an organic or inorganic compound (i.e.,
including heteroorganic and organometallic compounds) having a
molecular weight less than about 10,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 5,000
grams per mole, organic or inorganic compounds having a molecular
weight less than about 1,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 500 grams per
mole, and salts, esters, and other pharmaceutically acceptable
forms of such compounds.
[0109] Some non-limiting examples of small molecule inhibitors of
TGF.beta.Rs include
2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 napththyridine,
[3-(Pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and
3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole-
, which can be purchased from Calbiochem (San Diego, Calif.). Other
small molecule inhibitors include, but are not limited to,
SB-431542 (see e.g., Halder et al., 2005; Neoplasia 7(5):509-521),
SM 16 (see e.g., Fu, K et al., 2008; Arteriosclerosis, Thrombosis
and Vascular Biology 28(4):665), and SB-505124 (see e.g., Dacosta
Byfield, S., et al., 2004; Molecular Pharmacology 65:744-52), among
others.
[0110] In one embodiment, the ALK5 inhibitor
2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 napththyridine is
used with the methods described herein. This inhibitor is also
referred to herein as ALK5 inhibitor II and is available
commercially from Calbiochem (Cat. No. 616452; San Diego, Calif.).
In one embodiment, the inhibitor is SB 431542, an ALK-4, -5, -7
inhibitor, commercially available from Sigma (product no. S4317;
Saint Louis, Mo.). SB 431542 is also referred to by the following
chemical names:
4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide,
4-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide-
, or
4-(5-benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamid-
e hydrate.
[0111] Small molecules inhibitors of TGF-.beta. signaling can be
classified based on the basic scaffold of the molecule. For
example, TGF-.beta. signaling inhibitors can be based on the
dihydropyrrlipyrazole-based scaffold, imidazole-based scaffold,
pyrazolopyridine-based scaffold, pyrazole-based scaffold,
imidazopyridine-based scaffold, triazole-based scaffold,
pyridopyrimidine-based scaffold, pyrrolopyrazole-based scaffold,
isothiazole-based scaffold and oxazole-based scaffold.
[0112] Inhibitors of TGF-.beta. signaling are described in
Callahan, J. F. et al., J. Med. Chem. 45, 999-1001 (2002); Sawyer,
J. S. et al., J. Med. Chem. 46, 3953-3956 (2003); Gellibert, F. et
al., J. Med. Chem. 47, 4494-4506 (2004); Tojo, M. et al., Cancer
Sci. 96: 791-800 (2005); Valdimarsdottir, G. et al., APMIS 113,
773-389 (2005); Petersen et al. Kidney International 73, 705-715
(2008); Yingling, J. M. et al., Nature Rev. Drug Disc. 3, 1011-1022
(2004); Byfield, S. D. et al., Mol. Pharmacol., 65, 744-752 (2004);
Dumont, N, et al., Cancer Cell 3, 531-536 (2003); WO Publication
No. 2002/094833; WO Publication No. 2004/026865; WO Publication No.
2004/067530; WO Publication No. 209/032667; WO Publication No.
2004/013135; WO Publication No. 2003/097639; WO Publication No.
2007/048857; WO Publication No. 2007/018818; WO Publication No.
2006/018967; WO Publication No. 2005/039570; WO Publication No.
2000/031135; WO Publication No. 1999/058128; U.S. Pat. No.
6,509,318; U.S. Pat. No. 6,090,383; U.S. Pat. No. 6,419,928; U.S.
Pat. No. 9,927,738; U.S. Pat. No. 7,223,766; U.S. Pat. No.
6,476,031; U.S. Pat. No. 6,419,928; U.S. Pat. No. 7,030,125; U.S.
Pat. No. 6,943,191; U.S. Publication No. 2005/0245520; U.S.
Publication No. 2004/0147574; U.S. Publication No. 2007/0066632;
U.S. Publication No. 2003/0028905; U.S. Publication No.
2005/0032835; U.S. Publication No. 2008/0108656; U.S. Publication
No. 2004/015781; U.S. Publication No. 2004/0204431; U.S.
Publication No. 2006/0003929; U.S. Publication No. 2007/0155722;
U.S. Publication No. 2004/0138188 and U.S. Publication No.
2009/0036382, the contents of each which are herein incorporated by
reference in their entirety.
[0113] Oligonucleotide based modulators of TGF-.beta. signaling,
such as siRNAs and antisense oligonucleotides, are described in
U.S. Pat. No. 5,731,424; U.S. Pat. No. 6,124,449; U.S. Publication
Nos. 2008/0015161; 2006/0229266; 2004/0006030; 2005/0227936 and
2005/0287128, each of which are herein incorporated by reference in
their entirety. Other antisense nucleic acids and siRNAs can be
obtained by methods known to one of ordinary skill in the art.
[0114] Exemplary inhibitors of TGF-.beta. signaling include, but
are not limited to, AP-12009 (TGF-3 Receptor type II antisense
oligonucleotide), Lerdelimumab (CAT 152, antibody against
TGF-.beta. Receptor type II) GC-1008 (antibody to all isoforms of
human TGF-.beta.), ID11 (antibody to all isoforms of murine
TGF-.beta.), soluble TGF-.beta., soluble TGF-.beta. Receptor type
II, dihydropyrroloimidazole analogs (e.g., SKF-104365),
triarylimidazole analogs (e.g., SB-202620
(4-(4-(4-fluorophenyl)-5-(pyridin-4-yl)-1H-imidazol-2-yl)benzoic
acid) and SB-203580 (4-(4-Fluorophenyl)-2-(4-methylsulfinyl
phenyl)-5-(4-pyridyl)-1H-imidazole)), RL-0061425, 1,5-naphthyridine
aminothiazole and pyrazole derivatives (e.g.,
4-(6-methyl-pyridin-2-yl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazole-2-amine
and
2-[3-(6-methyl-pyridin-2-yl)-1H-pyrazole-4-yl]-1,5-naphthyridine),
SB-431542
(4-(5-Benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-b-
enzamide), GW788388
(4-(4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)-N-(tetrahydro-2H-py-
ran-4-yl)benzamide), A-83-01
(3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carboth-
ioamide), Decorin, Lefty 1, Lefty 2, Follistatin, Noggin, Chordin,
Cerberus, Gremlin, Inhibin, BIO (6-bromo-indirubin-3'-oxime), Smad
proteins (e.g., Smad6, Smad7), and Cystatin C.
[0115] Inhibitors of TGF-.beta. signaling also include molecules
which inhibit TGF-.beta. Receptor type I. Inhibitors of TGF-.beta.
Receptor type I are described in Byfield, S. D., and Roberts, A.
B., Trends Cell Biol. 14, 107-111 (2004); Sawyer J. S. et al.,
Bioorg. Med. Chem. Lett. 14, 3581-3584 (2004); Sawyer, J. S. et
al., J. Med. Chem. 46, 3953-3956 (2003); Byfield, S. D. et al.,
Mol. Pharmacol. 65, 744-752 (2004); Gellibert, F. et al., J. Med.
Chem. 47, 4494-4506 (2004); Yingling, J. M. et al., Nature Rev.
Drug Disc. 3, 1011-1022 (2004); Dumont, N, et al., Cancer Cell 3,
531-536 (2003); Tojo, M. et al., Cancer Sci. 96: 791-800 (2005); WO
Publication No. 2004/026871; WO Publication No. 2004/021989; WO
Publication No. 2004/026307; WO Publication No. 2000/012497; U.S.
Pat. No. 5,731,424; U.S. Pat. No. 5,731,144; U.S. Pat. No.
7,151,169; U.S. Publication No. 2004/00038856 and U.S. Publication
No. 2005/0245508, contents of all of which are herein incorporated
in their entireties.
[0116] Exemplary inhibitors of TGF-.beta. Receptor type I include,
but are not limited to, soluble TGF-.beta. Receptor type I;
AP-11014 (TGF-.beta. Receptor type I antisense oligonucleotide);
Metelimumab (CAT 152, TGF-.beta. Receptor type I antibody);
LY550410; LY580276
(3-(4-fluorophenyl)-5,6-dihydro-2-(6-methylpyridin-2-yl)-4H-pyrrolo[1,2-b-
]pyrazole); LY364947
(4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-quinoline); LY2109761;
LY573636 (N-((5-bromo-2-thienyl)sulfonyl)-2,4-dichlorobenzamide);
SB-505124
(2-(5-Benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridi-
ne); SD-208
(2-(5-Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine);
SD-093; KI2689; SM16; FKBP12 protein;
3-(4-(2-(6-methylpyridin-2-yl)H-imidazo[1,2-a]pyridin-3-yl)quinolin-7-ylo-
xy)-N,N-dimethylpropan-1-amine; and
##STR00001##
[0117] In one aspect, the TGF-.beta. inhibitor has the structure
shown in formula (I):
##STR00002##
[0118] wherein
[0119] R.sup.1 cyclyl, heterocyclcyl, aryl or heteroaryl, each of
which can be optionally substituted;
[0120] R.sup.2 cyclyl, heterocyclcyl, aryl or heteroaryl, each of
which can be optionally substituted;
[0121] R.sup.3 is H, C.sub.1-C.sub.6 alkyl, arylC.sub.1-C.sub.6, or
a nitrogen protecting group, each of which can be optionally
substituted;
[0122] R.sup.4 is H, optionally substituted C.sub.1-C.sub.6 alkyl,
optionally substituted C.sub.2-C.sub.6 alkenyl, optionally
substituted C.sub.2-C.sub.6 alkynyl, or R.sup.3 and R.sup.4
together with the atoms they are attached to form a cyclyl,
heterocyclyl, aryl or heteroaryl, each of which can be optionally
substituted.
[0123] In some embodiments, R.sup.1 is aryl, e.g., a substituted
aryl. In some embodiments, R.sup.1 is substituted with two
substituents. In some embodiments, R.sup.1 is substituted with two
substituents, which together with the carbons to which they are
attached form a ring. In some embodiments, R.sup.1 is a substituted
phenyl. In some embodiments, R.sup.1 is a nitrogen containing
heteroaryl (e.g., including 1, 2, or 3 nitrogens). In some
embodiments, R.sup.1 is a bicyclic heteroaryl. In some embodiments,
R.sup.1 is a 6-6 fused heteroaryl. In some embodiments, R.sup.1 is
pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, quinolinyl,
naphthyridinyl (e.g., 1,5-naphthyridinyl), quinazolinyl,
5,6,7,8-tetrahydroquinazolinyl, 1,3-benzodioxlyl,
1,2,3-benzotriazolyl, benzoxazolyl, benzothiazolyl,
2,1,3-benzooxadiazole, imidazo[1,2-a]pyridinyl,
pyrazolo[1,5-a]pyridinyl, [1,2,4]triazolo[1,5-a]pyridinyl,
pyrazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl,
[1,2,3]triazolo[1,5-a]pyrimidinyl,
[1,2,4]triazolo[4,3-a]pyrimidinyl,
[1,2,4]triazolo[4,3-a]pyridazinyl. In some embodiments, R.sup.1
is
##STR00003##
In some embodiments, R.sup.1 is
##STR00004##
[0124] In some embodiments, R.sup.2 is aryl, e.g., a substituted
aryl. In some embodiments, R.sup.2 is a nitrogen comprising
heteroaryl (e.g., including 1, 2 or 3 nitrogens (e.g., 1 or 2)). In
some embodiments, R.sup.2 is an optionally substituted monocyclic
heteroaryl (e.g., a six membered heteroaryl such as pyridyl,
pyrimidyl, pyridazinyl or pyrazinyl). In some embodiments, R.sup.2
is substituted. Exemplary substituents include halo,
C.sub.1-C.sub.6 alkyl, haloC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6
alkoxy, OH, haloC.sub.1-C.sub.6alkoxy. In some embodiments, R.sup.2
is monosubstituted. In some embodiments, R.sup.2 is substituted
with methyl. In one embodiment, R.sup.2 is an optionally
substituted pyridyl. In some embodiments, R.sup.2 is
##STR00005##
In some embodiments, R.sup.2 is
##STR00006##
[0125] In one embodiment, R.sup.4 is H.
[0126] In one embodiment, the compound of formula (I) has the
structure shown in formula (Ia):
##STR00007##
wherein R.sup.5 is H, benzyl, aryl, heteroaryl,
C.sub.1-C.sub.6alkyl, alkenyl, alkynyl, halogen, amino,
--C(O)-amino, --SO.sub.2-alkyl, --O-alkyl or acyl, each of which
can be optionally substituted.
[0127] In some embodiments, R.sup.5 is H.
[0128] In one embodiment, the compound of formula (I) has the
structure shown in formula (Ib):
##STR00008##
wherein m is 1, 2 or 3.
[0129] Exemplary compounds of formula (I) include:
##STR00009## [0130]
4-[2-(6-Ethyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline;
[0131]
[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline--
7-carboxylic acid methyl ester, [0132]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-6-car-
boxylic acid methyl ester, [0133]
4-(5-Benzyl-2-pyridin-2-yl-pyrazolo[1,5-a]pyridin-3-yl)-quinoline-7-carbo-
xylic acid methyl ester, [0134]
3-(4-Fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridine-6-c-
arboxylic acid (2-dimethylamino-ethyl)-amide; [0135]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-6-car-
boxylic acid (2-dimethylamino-ethyl)-amide; [0136]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-car-
boxylic acid (2-dimethylamino-ethyl)-amide; [0137]
5-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-benzofuran-2-ca-
rboxylic acid (2-dimethylamino-ethyl)-amide; [0138]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-car-
boxylic acid [3-(4-methyl-piperazin-1-yl)-propyl]-amide; [0139]
4-[2-(6-Methoxy-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline,
4-[2-(6-Ethoxy-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline;
[0140]
3-(4-Fluoro-phenyl)-2-(6-methoxy-pyridin-2-yl)-pyrazolo[1,5-a]pyri-
dine; [0141]
2-(6-Ethoxy-pyridin-2-yl)-3-(4-fluoro-phenyl)-pyrazolo[1,5-a]pyridine;
[0142]
7-Benzyl-4-[2-(6-methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-
-quinoline; [0143]
3-{4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinolin-7-y-
l}-acrylic acid methyl ester; [0144]
3-{4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinolin-7-y-
l}-acrylic acid; [0145]
4-[2-(6-Ethylsulfanyl-pyridin-2-yl)-pyrazolo[1,5-a]-pyridin-3-yl]-quinoli-
ne; [0146]
4-[2-(6-Phenylsulfanyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-y-
l]-quinoline; [0147]
4-[2-(6-Morpholin-4-yl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoli-
ne; [0148]
3-(4-Fluoro-phenyl)-2-(6-methylsulfanyl-pyridin-2-yl)-pyrazolo[-
1,5-a]pyridine; [0149]
3-(4-Methylsulfanyl-phenyl)-2-(6-methylsulfanyl-pyridin-2-yl)-pyrazolo[1,-
5-a]pyridine; [0150]
Dimethyl-{4-[2-(6-methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quino-
lin-7-ylsulfanyl)}-ethyl)-amine; [0151]
2-(Pyridin-2-yl)-3-(quinolin-4-yl)-pyrazolo[1,5-a]pyridine-5-carboxylic
acid dimethylamide; [0152]
2-(Pyridin-2-yl)-3-(quinolin-4-yl)-pyrazolo[1,5-a]pyridine-6-carboxylic
acid dimethylamide; [0153]
4-[2-(6-Vinyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline,
6-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-imidazo[1,2-a]p-
yridin-2-yl-amine; [0154]
6-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-1H-benzoimidazo-
l-2-yl-amine; [0155]
[3-(4-Fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-6-y-
l]-methanol,
6-Allyloxymethyl-3-(4-fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-pyrazolo[1-
,5-a]pyridine; [0156]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-car-
boxylic acid (3-pyrrolidin-1-yl-propyl)-amide; [0157]
3-{4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinolin-7-y-
l}-propionamide; [0158]
3-(4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinolin-7-y-
l)-N-(3-pyrrolidin-1-yl-propyl)-propionamide; [0159]
N-(Dimethylamino-ethyl)-3-{4-[2-(6-methyl-pyridin-2-yl)-pyrazolo[1,5-a]py-
ridin-3-yl]-quinolin-7-yl)}-propionamide; [0160]
2-Pyridin-2-yl-3-quinolin-4-yl-pyrazolo[1,5-a]pyridine-5-carboxylic
acid (3-dimethylamino-propyl)-amide; [0161]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-car-
boxylic acid (2-hydroxy-ethyl)-amide; [0162]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-car-
boxylic acid hydrazide; [0163]
[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-carbo-
xylic acid (3-hydroxy-propyl)-amide; [0164]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-car-
boxylic acid methylamide; [0165]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-car-
boxylic acid (3-ethoxy-propyl)-amide; [0166]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-car-
boxylic acid (3-morpholin-4-yl-propyl)-amide; [0167]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-car-
boxylic acid (3-imidazol-1-yl-propyl)-amide; [0168]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-car-
boxylic acid (3-dimethylamino-propyl)-amide; [0169]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-car-
boxylic acid [2-(2-methoxy-phenyl)-ethyl]-amide; [0170]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-car-
boxylic acid (2-morpholin-4-yl-ethyl)-amide; [0171]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinoline-7-car-
boxylic acid amide; [0172]
Dimethyl-{4-[2-(6-methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quino-
lin-7-yloxy}-propyl)-amine; [0173]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-7-(2-morpholin--
4-yl-ethoxy)-quinoline; [0174]
Diisopropyl-(2-{4-[2-(6-methyl-pyridin-2-yl)-pyrazol][1,5-a]pyridin-3-yl]-
-quinolin-7-yloxy}-ethyl)-amine; [0175]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-7-(2-pyrrol-1-y-
l-ethoxy)-quinoline; [0176]
Dimethyl-(1-methyl-2-(4-[2-(6-methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-
-3-yl]-quinolin-7-yloxy)ethyl)-amine; [0177]
Methyl-(3-{4-[2-(6-methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quin-
olin-7-yl-oxy}-propyl)-amine; [0178]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-7-(2-piperidin--
1-yl-ethoxy)-quinoline; [0179]
Diethyl-(2-{4-[2-(6-methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-qui-
nolin-7-yloxy)}-ethyl)-amine; [0180]
Dimethyl-{3-[4-(2-pyridin-2-yl-pyrazolo[1,5-a]pyridin-3-yl)-quinolin-7-yl-
oxy]-propyl}-amine; [0181]
7-(2-Morpholin-4-yl-ethoxy)-4-(2-pyridin-2-yl-pyrazolo[1,5-a]pyridin-3-yl-
)-quinoline; [0182]
Diisopropyl-{2-[4-(2-pyridin-2-yl-pyrazolo[1,5-a]pyridin-3-yl)-quinolin-7-
-yloxy]-ethyl}-amine; [0183]
4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-7-(3-morpholin--
4-yl-propoxy)-quinoline; [0184]
1-(3-{4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridine-3-yl]-quinolin-
-7-yloxy}-propyl)-1,3-dihydro-benzoimidazol-2-one
3-{4-[2-(6-Methyl-pyridin-2-yl)-pyrazolo[1,5-a]pyridin-3-yl]-quinolin-7-y-
l}-propionic acid methyl ester, [0185]
Diethyl-3-[4-(2-pyridin-2-yl-pyrazolo[1,5-a]pyridin-3-yl)-quinolin-7-ylox-
y]-propyl}-amine; [0186]
Ethyl-methyl-{3-[4-(2-pyridin-2-yl-pyrazolo[1,5-a]pyridin-3-yl)-quinolin--
7-yloxy]-propyl}-amine; [0187]
4-(2-Pyridin-2-yl-pyrazolo[1,5-a]pyridin-3-yl)-7-(3-pyrrolidin-1-yl-propo-
xy)-quinoline; [0188]
7-(3-Piperidin-1-yl-propoxy)-4-(2-pyridin-2-yl-pyrazolo[1,5-a]pyridin-3-y-
l)-quinoline; [0189]
Diethyl-{2-[4-(2-pyridin-2-yl-pyrazolo[1,5-a]pyridin-3-yl)-quinolin-7-ylo-
xy]-ethyl}-amine; [0190]
Dimethyl-{2-[4-(2-pyridin-2-yl-pyrazolo[1,5-a]pyridin-3-yl)-quinolin-7-yl-
oxy]-ethyl}-amine; [0191]
6-Bromo-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quin-
oline; [0192]
3-Pyridin-4-yl-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole;
[0193]
2-(6-Methyl-pyridin-2-yl)-3-p-tolyl-5,6-dihydro-4H-pyrrolo[1,2-b]p-
yrazole; [0194]
4-[3-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-2-yl]-q-
uinoline; [0195]
2-(6-Methyl-pyridin-2-yl)-3-naphthalen-1-yl-5,6-dihydro-4H-pyrrolo[1,2-b]-
pyrazole; [0196]
(6-Methyl-pyridin-2-yl)-3-pyridin-3-yl-5,6-dihydro-4H-pyrrlo[1,2-b]pyrazo-
le; [0197]
3-(4-Fluoro-naphthalen-1-yl)-2-(6-methyl-pyridin-2-yl)-5,6-dihy-
dro-4H-pyrrolo[1,2-b]pyrazole; [0198]
3-(3,4-Difluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[-
1,2-b]pyrazole; [0199]
1-[2-(4-Methanesulfonyl-phenyl)-1-(6-methyl-pyridin-2-yl)-ethylideneamino-
]-pyrrolidin-2-one; [0200]
7-Methoxy-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-qu-
inoline; [0201]
7-Benzyloxy-6-methoxy-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyra-
zol-3-yl)-quinoline; [0202]
6-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline;
[0203]
6-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol--
3-yl]-quinoline; [0204]
3-Naphthalen-2-yl-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole;
[0205]
2-(6-Methyl-pyridin-2-yl)-3-naphthalen-2-yl-5,6-dihydro-4H-pyrrolo-
[1,2-b]pyrazole; [0206]
3-(4-Fluoro-phenyl)-2-(6-trifluoromethyl-pyridin-2-yl)-5,6-dihydro-4H-pyr-
rolo[1,2-b]pyrazole; [0207]
4-(Quinolin-4-yl)-3-(5-fluoropyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]p-
yrazole; [0208]
4-(7-Bromoquinolin-4-yl)-3-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]py-
razole; [0209]
(Quinolin-4-yl)-3-(2,4-difluorophenyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyraz-
ole; [0210]
4-(2-Pyrazin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline;
[0211]
4-(5-Methyl-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3--
yl)-quinoline; [0212]
6-Bromo-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-
-3-yl]-quinoline; [0213]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-6-
-trifluoromethyl-quinoline; [0214]
3-(3-Chloro-4-fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyr-
rolo[1,2-b]pyrazole; [0215]
3-(2-Chloro-4-fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyr-
rolo[1,2-b]pyrazole; [0216]
3-(4-Fluoro-3-trifluoromethyl-phenyl)-2-(6-methyl-pyridin-2-yl)-5,6-dihyd-
ro-4H-pyrrolo[1,2-b]pyrazole; [0217]
2-(6-Methyl-pyridin-2-yl)-3-(2,4,5-trifluoro-phenyl)-5,6-dihydro-4H-pyrro-
lo[1,2-b]pyrazole; [0218]
8-Fluoro-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazo-
l-3-yl]-quinoline; [0219]
7-Bromo-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-
-3-yl]-quinoline; [0220]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-6-
-trifluoromethoxy-quinoline; [0221]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-7-
-trifluoromethyl-quinoline; [0222]
7-Methoxy-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyraz-
ol-3-yl]-quinoline; [0223]
3-(2-Chloro-pyridin-4-yl)-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyr-
azole; [0224]
[2-(6-Methyl-pyridin-2-yl)-3-quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1,2-b]p-
yrazol-6-yl]-methanol; [0225]
[3-(7-Bromo-quinolin-4-yl)-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrro-
lo[1,2-b]pyrazol-6-yl]-methanol; [0226]
4-[2-(6-Chloro-pyridin-2-yl)-5-(4-fluorophenyl)-5,6-dihydro-4H-pyrrolo[1,-
2-b]pyrazol-3-yl]-quinoline; [0227]
4-[2-(6-Ethoxy-pyridin-2-yl)-5-(4-fluoro-phenyl)-5,6-dihydro-4H-pyrrolo[1-
,2-b]pyrazol-3-yl]-quinoline; [0228]
(S)-4-[6-Benzyloxymethyl-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo-
[1,2-b]pyrazol-3-yl]-7-chloro-quinoline; [0229]
(S)-4-[6-Benzyloxymethyl-2-(6-chloro-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo-
[1,2-b]pyrazol-3-yl]-quinoline; [0230]
4-[2-(6-Methyl-pyridin-2-yl)-3-quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1,2-b-
]pyrazol-5-yl]-benzoic acid ethyl ester; [0231]
3-(4-Fluoro-phenyl)-5,5-dimethyl-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-
-pyrrolo[1,2-b]pyrazole; [0232]
(R)-6-Benzyloxymethyl-3-(4-fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-5,6-d-
ihydro-4H-pyrrolo[1,2-b]pyrazole; [0233]
5-(4-Chloro-phenyl)-3-(4-fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-5,6-dih-
ydro-4H-pyrrolo[1,2-b]pyrazole; [0234]
4-[2-(3-Trifluoromethyl-phenyl)-4,5,6,7-tetrahydro-pyrazolo[1,5-a]pyridin-
-3-yl]-quinoline; [0235]
4-[2-(4-Trifluoromethyl-phenyl)-4,5,6,7-tetrahydro-pyrazolo[1,5-a]pyridin-
-3-yl]-quinoline; [0236]
4-[2-(4-Chloro-phenyl)-4,5,6,7-tetrahydro-pyrazolo[1,5-a]pyridin-3-yl]-qu-
inoline; [0237]
4-[2-(3-Chloro-phenyl)-4,5,6,7-tetrahydro-pyrazolo[1,5-a]pyridin-3-y]-qui-
noline; [0238]
4-[2-(3-Fluoro-5-trifluoromethyl-phenyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyr-
azol-3-yl]-quinoline; [0239]
4-[2-(3-Fluoro-5-trifluoromethyl-phenyl)-4,5,6,7-tetrahydro-pyrazolo[1,5--
a]pyridin-3-yl]-quinoline; [0240]
4-(2-Phenyl-4,5,6,7-tetrahydro-pyrazolo[1,5-a]pyridin-3-yl)-quinoline;
[0241]
4-(2-Pyridin-2-yl-4,5,6,7-tetrahydro-pyrazolo[1,5-a]pyridin-3-yl)--
[1,10]phenanthroline; [0242]
4-[2-(4-Fluoro-phenyl)-4,5,6,7-tetrahydro-pyrazolo[1,5-a]pyridin-3-yl]-qu-
inoline; [0243]
4-[2-(3-Trifluoromethoxy-phenyl)-4,5,6,7-tetrahydro-pyrazolo[1,5-a]pyridi-
n-3-yl]-quinoline; [0244]
4-[2-(2-Fluoro-phenyl)-4,5,6,7-tetrahydro-pyrazolo[1,5-a]pyridin-3-yl]-qu-
inoline; [0245]
4-(2-Quinolin-2-yl-4,5,6,7-tetrahydro-pyrazolo[1,5-a]pyridin-3-yl)-quinol-
ine; [0246]
4-[2-(4-Ethyl-pyridin-2-yl)-4,5,6,7-tetrahydro-pyrazolo[1,5-a]pyridin-3-y-
l]-quinoline; [0247]
4-(2-Quinolin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline;
[0248]
2-(3-Quinolin-4-yl-4,5,6,7-tetrahydro-pyrazolo[1,5-a]pyridin-2-yl)-
-[1,8]naphthyridine; [0249]
4-[5-(4-Fluoro-phenyl)-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazo-
l-3-yl]-quinoline; [0250]
4-(6-Hydroxymethyl-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3--
yl)-quinoline; [0251]
4-(3-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-2-yl)-quinoline;
[0252]
4-(4-Methyl-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3--
yl)-quinoline; [0253]
4-(5-Benzyl-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-qui-
noline; [0254]
4-(5-Phenethyl-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)--
quinoline; [0255]
4-(5-Phenyl-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-qui-
noline; [0256]
4-[2-(3-Trifluoromethylphenyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-
-quinoline; [0257]
4-[2-(4-Trifluoromethyl-phenyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl-
]-quinoline; [0258]
4-(2-Phenyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline;
[0259]
2-Chloro-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-qui-
noline; [0260]
6,8-Dimethoxy-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2b]py-
razol-3-yl]-quinoline; [0261]
4-[2-(6-Bromo-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-qu-
inoline; [0262]
6,8-Dimethoxy-4-[2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2b]pyrazol-3-yl]-
-quinoline; [0263]
3-(4-Fluorophenyl)-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole;
[0264]
3-(4-Methoxy-phenyl)-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]p-
yrazole; [0265]
3-(4-Fluorophenyl)-2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]-
pyrazole; [0266]
3-(4-Methoxyphenyl)-2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b-
]pyrazole; [0267]
4-(2-Thiophen-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)
quinoline; [0268]
4-[2-(6-Propylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl]-quinoline; [0269]
4-[2-(6-Isopropylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-
quinoline; [0270]
4-[2-(6-Ethyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]qui-
noline; [0271]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline; [0272]
4-[2-(3-Fluorophenyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-quinolin-
e; [0273]
4-[2-(2-Fluoro-phenyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl-
]-quinoline; [0274]
4-[2-(4-Fluoro-phenyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-quinoli-
ne; [0275]
4-[2-(3-Trifluoromethoxy-phenyl)-5,6-dihydro-4H-pyrrolo[1,2-b]p-
yrazol-3-yl]-quinoline; [0276]
4-[2-(4-Chloro-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline; [0277]
4-[2-(4-Fluoro-3-trifluoromethyl-phenyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyr-
azol-3-yl]quinoline; [0278]
4-[2-(2-Fluoro-3-trifluoromethyl-phenyl)-5,6-dihydro-4H-pyrrolo[1,2-b]-py-
razol-3-yl]-quinoline;
[0279]
4-[5-(3-Methoxy-phenyl)-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2--
b]pyrazol-3-yl]-quinoline; [0280]
4-[2-(4-Fluoro-3-trifluoromethyl-phenyl)-5-(3-methoxy-phenyl)-5,6-dihydro-
-4H-pyrrolo[1,2-b]pyrazol-3-yl]-quinoline; [0281]
4-(7-Chloro-quinolin-4-yl)-3-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrol-
o[1,2-b]pyrazole; [0282]
4-(7-Ethoxyquinolin-4-yl)-3-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo-
[1,2-b]pyrazole; [0283]
6-(3-Quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-2-yl)-pyridine-2--
carboxylic acid hydrochloride; [0284]
6,7-Difluoro-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]py-
razol-3-yl]-quinoline; [0285]
6,7-Dimethoxy-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]p-
yrazol-3-yl]-quinoline; [0286]
3-Benzo[1,3]dioxol-5-yl-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[-
1,2-b]pyrazole; [0287]
6-(4-Fluoro-phenyl)-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1-
,2-b]pyrazol-3-yl]-quinoline; [0288]
6-Benzo[1,3]dioxol-5-yl-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrro-
lo[1,2-b]pyrazol-3-yl]-quinoline; [0289]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-6-
-thiophen-2-yl-quinoline; [0290]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-6-
-phenyl-quinoline; [0291]
8-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline; [0292]
3-Benzo[b]thiophen-2-yl-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[-
1,2-b]pyrazole; [0293]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-6--
carboxylic acid methyl ester, [0294]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline-6-carboxylic acid methyl ester; [0295]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline-7-carboxylic acid methyl ester; [0296]
4-[2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-quinoline-7--
carboxylic acid methyl ester, [0297]
2-Pyridin-2-yl-3-quinolin-4-yl-pyrazolo[5,1-c]morpholine; [0298]
2-Pyridin-2-yl-3-quinolin-4-yl-pyrazolo[5,1-c]morpholin-4-one;
[0299]
Dimethyl-{3-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-
-quinolin-7-yloxy]-propyl}-amine; [0300]
{3-[6-Methoxy-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl-
)-quinolin-7-yloxy]-propyl}-dimethyl-amine; [0301]
Cyclopropylmethyl-propyl-{3-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-
-b]pyrazol-3-yl)-quinolin-7-yloxy]-propyl}-amine; [0302]
Diethyl-{3-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)--
quinolin-7-yloxy]-propyl}-amine; [0303]
Ethyl-methyl-{3-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl)-quinolin-7-yloxy]-propyl}-amine)jjjjj)3-[4-(2-Pyridin-2-yl-5,6-dihydr-
o-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin-7-yloxy]-propylamine;
[0304]
7-[3-(4-Methyl-piperazin-1-yl)-propoxy]-4-(2-pyridin-2-yl-5,6-dihydro-4H--
pyrrolo[1,2-b]pyrazol-3-yl)-quinoline; [0305]
Benzyl-methyl-{3-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol--
3-yl)-quinolin-7-yloxy]-propyl}-amine; [0306]
7-(3-Piperidin-1-yl-propoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-
-b]pyrazol-3-yl)-quinoline; [0307]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-7-(3-pyrroli-
din-1-yl-propoxy)-quinoline; [0308]
7-(3-Azepan-1-yl-propoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]-
pyrazol-3-yl)-quinoline; [0309]
7-(3-Imidazol-1-yl-propoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2--
b]pyrazol-3-yl)-quinoline; [0310]
7-(3-Pyrazol-1-yl-propoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b-
]pyrazol-3-yl)-quinoline; [0311]
1'-{3-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quino-
lin-7-yloxy]-propyl}-[1,4']bipiperidinyl; [0312]
Cyclopropyl-(1-methyl-piperidin-4-yl)-f3-[4-(2-pyridin-2-yl-5,6-dihydro-4-
H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin-7-yloxy]-propyl)}-amine;
[0313]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-7-(3-[1,2,3]-
triazol-1-yl-propoxy)-quinoline; [0314]
Dimethyl-(3-f4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]py-
razol-3-yl]-quinolin-7-yloxy)-propyl)-amine; [0315]
Diethyl-(3-{4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyr-
azol-3-yl]-quinolin-7-yloxy}-propyl)-amine; [0316]
Cyclopropylmethyl-(3-{4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo-
[1,2-b]pyrazol-3-yl]-quinolin-7-yloxy}-propyl)-propyl-amine; [0317]
Ethyl-methyl-(3-{4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2--
b]pyrazol-3-yl]-quinolin-7-yloxy}-propyl)-amine; [0318]
Dimethyl-{2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-
-quinolin-7-yloxy]-ethyl}-amine; [0319]
Diethyl-{2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)--
quinolin-7-yloxy]-ethyl}-amine; [0320]
7-(2-Piperidin-1-yl-ethoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2--
b]pyrazol-3-yl)-quinoline; [0321]
Ethyl-methyl-{2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl)-quinolin-7-yloxy]ethyl}-amine; [0322]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-7-(2-pyrroli-
din-1-yl-ethoxy)-quinoline; [0323]
7-[2-(4-Methyl-piperazin-1-yl)-ethoxy]-4-(2-pyridin-2-yl-5,6-dihydro-4H-p-
yrrolo[1,2-b]pyrazol-3-yl)-quinoline; [0324]
Dimethyl-{3-[1-oxy-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-
-3-yl)-quinolin-7-yloxy]-propyl}-amine; [0325]
7-Methylsulfanyl-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl)-quinoline; [0326]
7-Ethylsulfanyl-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3--
yl)-quinoline; [0327]
6-Methylsulfanyl-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl)-quinoline; [0328]
7-Benzylsulfanyl-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl)-quinoline; [0329]
3-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin--
7-yl sulfanyl]-propan-1-ol; [0330]
Dimethyl-{2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-
-quinolin-7-ylsulfanyl]-ethyl}-amine; [0331] Dimethyl
[6-(3-quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-2-yl)-pyridin-2--
yl-methyl]amine; [0332]
7-(2-Propoxy-ethoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyraz-
ol-3-yl)-quinoline; [0333]
N,N-Dimethyl-N'-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl)-pyridin-2-yl]-ethane-1,2-diamine; [0334]
N,N-Dimethyl-N'-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl)-pyridin-2-yl]-propane-1,3-diamine; [0335]
3-{3-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinol-
in-7-yloxy]-propyl}-oxazolidin-2-one; [0336]
1-{3-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinol-
in-7-yloxy]-propyl}-imidazolidin-2-one; [0337]
3-{3-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinol-
in-7-yloxy]-propyl}-3H-benzooxazol-2-one; [0338]
Dimethyl-(2-{4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]py-
razol-3-yl]-pyridin-2-ylsulfanyl)}-ethyl-amine; [0339]
4-(2-Pyridin-2-yl-5,6-dihydro-4H
pyrrolo[1,2-b]pyrazol-3-yl)-2pyrrolidin-1-yl-quinoline; [0340]
2-Phenylsulfanyl-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl)-quinoline; [0341]
2-Morpholin-4-yl-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl)-quinoline; [0342]
2-Ethylsulfanyl-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3--
yl)-quinoline; [0343]
Phenyl-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quin-
olin-2-yl]-amine; [0344]
2-Methoxy-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-qu-
inoline; [0345]
2-Ethoxy-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-qui-
noline; [0346]
4-[2-(6-Phenylsulfanyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-
-3-yl]-quinoline; [0347]
Phenyl-[6-(3-quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-2-yl)-pyr-
idin-2-yl]-amine; [0348]
4-(2-[6-(4-Methoxy-phenyl)-pyridin-2-yl]-5,6-dihydro-4H-pyrrolo[1,2-b]pyr-
azol-3-yl)-quinoline; [0349]
4-[2-(6-Phenyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline; [0350]
4-[2-(6-Morpholin-4-yl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-
-3-yl]-quinoline; [0351]
4-[2-(6-Pyrrolidin-1-yl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazo-
l-3-yl]-quinoline; [0352]
4-[2-(6-Methoxy-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]--
quinoline; [0353]
2-(3-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinol-
in-7-yloxy]-propyl)-isoindole-1,3-dione; [0354]
7-(3-Fluoro-propoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyraz-
ol-3-yl)-quinoline; [0355]
7-(3-Fluoro-propoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyraz-
ol-3-yl)-quinoline; [0356]
7-(3-Chloro-propoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyraz-
ol-3-yl)-quinoline; [0357]
7-(3-Chloro-propoxy)-6-methoxy-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1-
,2-b]pyrazol-3-yl)-quinoline; [0358]
7-(3-Chloro-propoxy)-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[-
1,2-b]pyrazol-3-yl]-quinoline; [0359]
(1-(3-[7-(2-Chloro-ethoxy)-quinolin-4-yl]-5,6-dihydro-4H-pyrrolo[1,2-b]py-
razol-2-yl)-propenyl)-methylene-amine; [0360]
N,N-Diethyl-2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-y-
l)-quinolin-7-yloxy]-acetamide; [0361]
7-[2-((2R)-1-Methyl-pyrrolidin-2-yl)-ethoxy]-4-(2-pyridin-2-yl-5,6-dihydr-
o-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline; [0362]
Dimethyl-{4-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-
-pyridin-2-yloxy]-butyl)}-amine; [0363]
1-{3-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-pyridi-
n-2-yloxy]-propyl)}-pyrrolidin-2-one; [0364]
7-(1-Methyl-piperidin-3-ylmethoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrro-
lo[1,2-b]pyrazol-3-yl)-quinoline; [0365]
7-(3-N,N-Dimethylamino-2-methyl-propyloxy)-4-(2-pyridin-2-yl-5,6-dihydro--
4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline; [0366]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-7-
-propoxy-quinoline; [0367]
4-[6-Benzyloxymethyl-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-
-b]pyrazol-3-yl]-quinoline; [0368]
{4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]--
quinolin-7-yloxy}-acetic acid methyl ester; [0369]
7-Isopropoxy-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]py-
razol-3-yl]-quinoline; [0370]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-7-
-(3-morpholin-4-yl-propoxy)-quinoline; [0371]
4-(6-Benzyloxymethyl-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol--
6-yl)-quinoline; [0372]
7-Benzyloxy-2-Pyridin-2-yl-3-quinolin-4-yl-pyrazolo[1,5-a]piperidine;
[0373]
2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-qu-
inolin-7-yloxy]-acetamide; [0374]
7-(5-Phenyl-[1,2,4]oxadiazol-3-ylmethoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4-
H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline; [0375]
7-(2,2-Difluoro-benzo[1,3]dioxol-5-ylmethoxy)-4-(2-pyridin-2-yl-5,6-dihyd-
ro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline; [0376]
7-[2-((259-1-Methyl-pyrrolidin-2-yl)-ethoxy]-4-(2-pyridin-2-yl-5,6-dihydr-
o-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline; [0377]
5-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin--
7-yloxymethyl]-pyrrolidin-2-one; [0378]
4-(6-Phenoxymethyl-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3--
yl)-quinoline; [0379]
4-(6-Methylene-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)--
quinoline; [0380]
3-(4-Fluoro-phenyl)-6-methylene-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H--
pyrrolo[1,2-b]pyrazole; [0381]
7-(1-Methyl-piperidin-2-ylmethoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrro-
lo[1,2-b]pyrazol-3-yl)-quinoline hydrochloride; [0382]
7-[2-(1-Methyl-pyrrolidin-2-yl)-ethoxy]-4-(2-pyridin-2-yl-5,6-dihydro-4H--
pyrrolo[1,2-b]pyrazol-3-yl)-quinoline hydrochloride; [0383]
4-[2-(6-Methyl-1-oxy-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl]-quinoline 1-oxide; [0384]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline 1-oxide; [0385]
4-[2-(6-Methyl-1-oxy-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl]-quinoline; [0386]
7-(3-Chloro-propoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyraz-
ol-3-yl)-quinoline 1-oxide; [0387]
7-Methanesulfonyl-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol--
3-yl)-quinoline; [0388]
3-(4-Fluoro-phenyl)-2-(6-methyl-1-oxy-pyridin-2-yl)-5,6-dihydro-4H-pyrrol-
o[1,2-b]pyrazole; [0389]
4-(Quinolin-N-1-oxide-4-yl)-3-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrro-
lo[1,2-b]pyrazole; [0390]
6-Methanesulfonyl-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol--
3-yl)-quinoline; [0391]
7-Ethanesulfonyl-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl)-quinoline; [0392]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-7-[3-(pyrimi-
dine-2-sulfonyl)-propoxy]-quinoline; [0393]
7-[3-(1-Methyl-1H-imidazole-2-sulfonyl)-propoxy]-4-(2-pyridin-2-yl-5,6-di-
hydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline; [0394]
7-[3-(4-Chloro-benzenesulfonyl)-propoxy]-4-(2-pyridin-2-yl-5,6-dihydro-4H-
-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline; [0395]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-7-[3-(pyridi-
n-2-ylmethanesulfonyl)-propoxy]-quinoline; [0396]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-7-[3-(pyridi-
n-2-ylmethanesulfinyl)-propoxy]-quinoline; [0397]
4-(Quinolin-1-N-oxide-4-yl)-3-(6-methylpyridin-2-yl-1-N-oxide)-5,6-dihydr-
o-4H-pyrrolo[1,2-b]pyrazole; [0398]
3-{4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl-
]-quinolin-7-yl}-acrylic acid methyl ester, [0399]
3-{4-[2-(6-Methylpyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]q-
uinolin-7-yl}-1-piperidin-1-yl-propenone; [0400]
3-{4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl-
]-quinolin-6-yl}-acrylic acid methyl ester, [0401]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-7-
-vinyl-quinoline; [0402]
4-[2-(6-Benzyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline; [0403]
7-Benzyl-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazo-
l-3-yl]-quinoline; [0404]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline-7-carboxylic acid; [0405]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline-6-carboxylic acid; [0406]
3-{4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl-
]-quinolin-7-yl}-acrylic acid; [0407]
3-{4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl-
]-quinolin-7-yl}-propionic acid; [0408]
4-[2-(6-Methyl-pyridin-2-yl)-3-quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1,2-b-
]pyrazol-5-yl]-benzoic acid; [0409]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid cyclopentylamide; [0410]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (2-morpholin-4-yl-ethyl)-amide; [0411]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid [2-(1H-imidazol-4-yl)-ethyl]-amide; [0412]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (2-methylamino-ethyl)-amide; [0413]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (3-methylamino-propyl)-amide; [0414]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (2-dimethylamino-ethyl)-amide; [0415] (4-M
ethyl-piperazin-1-yl)-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyr-
azol-3-yl)-quinolin-7-yl]-methanone;
[0416]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quino-
line-7-carboxylic acid cyclobutylamide; [0417]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid cyclopropylamide, [0418]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (1-ethyl-propyl)-amide; [0419]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid ethylamide; [0420]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid isobutyl-amide; [0421]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid tert-butylamide; [0422]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid isopropylamide; [0423]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid propylamide; [0424]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (2-methyl-butyl)-amide; [0425]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid ((2S)-2-methyl-butyl)-amide; [0426]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (2S)-sec-butylamide; [0427]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazo!-3-y!)-quinoline-7--
carboxylic acid (2R)-sec-butylamide; [0428]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid((IR)-1,2-dimethyl-propyl)-amide; [0429]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (pyridin-4-ylmethyl)-amide; [0430]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (pyridin-3-ylmethyl)-amide; [0431]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (pyridin-2-ylmethyl)-amide; [0432]
6-(3-Quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-2-yl)-pyridine-2--
carboxylic acid amide; [0433]
1-(4-Methyl-piperazin-1-yl)-2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1-
,2-b]pyrazol-3-yl)-quinolin-7-yloxy]-ethanone; [0434]
N-(2-dimethylamino-ethyl)-2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-
-b]pyrazol-3-yl)-quinolin-7-yloxy]-acetamide; [0435]
N-(2-dimethylamino-ethyl)-N-methyl-2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-py-
rrolo[1,2-b]pyrazol-3-yl)-quinolin-7-yloxy]-acetamide; [0436]
N,N-Dimethyl-3-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3--
yl)-quinolin-7-yloxy]-benzamide; [0437]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid amide; [0438]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-H]pyrazol-3-yl)-quinoline-7--
carboxylic acid (2-dimethylamino-ethyl)-methyl-amide; [0439]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-H]pyrazol-3-yl)-quinoline-7--
carboxylic acid (3-dimethylamino-propyl)-methyl-amide; [0440]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-H]pyrazol-3-yl)-quinoline-7--
carboxylic acid dimethylamide; [0441]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-H]pyrazol-3-yl)-quinoline-7--
carboxylic acid methylamide; [0442]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid pyridin-2-ylamide; [0443]
N-(2,2-Dimethylamino-ethyl)-N-methyl-3-{4-[2-(6-methyl-pyridin-2-yl)-5,6--
dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-quinolin-7-yl}-propionamide;
[0444]
2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-quin-
oline-6-carboxylic acid (2-dimethylamino-ethyl)-amide; [0445]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline-6-carboxylic acid (3-dimethylamino-propyl)-amide; [0446]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline-6-carboxylic acid (2-morpholin-4-yl-ethyl)-amide; [0447]
1-[2-(Quinolin-4-yl)-1-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-
-b]pyrazol-3-yl]quinoline-7-carboxylic acid
N,N-dimethylaminoethylamide; [0448]
4-[2-(6-Methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl]quinoline-7-carboxylic acid (2-piperidin-1-yl-ethyl)amide;
[0449]
N-(2-Dimethylamino-ethyl)-3-(4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H--
pyrrolo[1,2-b]pyrazol-3-yl]-quinolin-7-yl)-propionamide; [0450]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline-7-carboxylic acid (3-dimethylamino-propyl)-amide; [0451]
4-(2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline-7-carboxylic acid (3-pyrrolidin-1-yl-propyl)-amide; [0452]
4-(2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline-7-carboxylic acid (3-morpholin-4-yl-propyl)-amide; [0453]
3-{4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl-
]-quinolin-7-yl}-propionamide; [0454]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-6--
carboxylic acid (2-dimethylamino-ethyl)-amide; [0455]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-6--
carboxylic acid (2-morpholin-4-yl-ethyl)-amide; [0456]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-6--
carboxylic acid; [0457]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-6--
carboxylic acid hydrazide; [0458]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-6--
carboxylic acid amide; [0459]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-6--
carboxylic acid (3-methylamino-propyl)-amide; [0460]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-6--
carboxylic acid amide; [0461]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-6--
carboxylic acid (2-hydroxy-ethyl)-amide; [0462]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid hydrazide; [0463]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid hydroxyamide; [0464]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (2-amino-ethyl)-amide; [0465]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (2-hydroxy-ethyl)-amide; [0466]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
sulfonic acid amide; [0467]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
sulfonic acid methylamide; [0468]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
sulfonic acid dimethylamide; [0469]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
sulfonic acid (3-dimethylamino-propyl)-amide; [0470]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
sulfonic acid diethylamide; [0471]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
sulfonic acid (2-piperidin-1-yl-ethyl)-amide; [0472]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
sulfonic acid (2-hydroxy-ethyl)-amide; [0473]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin-7-y-
lamine; [0474]
2-Dimethylamino-N-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-
-3-yl)-quinolin-7-yl]-acetamide; [0475]
3-Dimethylamino-N-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-
-3-yl)-quinolin-7-yl]propionamide; [0476]
N-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin--
7-yl]-methanesulfonamide; [0477]
N-4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin-7-
-yl]-acetamide; [0478]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (2-acetylamino-ethyl)-amide; [0479]
N-{3-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinol-
in-7-yloxy]-propyl)}-methanesulfonamide; [0480]
1-methyl-1H-imidazole-4-sulfonic acid
{3-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin-
-7-yloxy]-propyl}-amide; [0481]
1-(2-Dimethylamino-ethyl)-3-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-
-b]pyrazol-3-yl)-quinolin-7-yl]-urea; [0482]
1-(3-Dimethylamino-propyl)-3-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,-
2-b]pyrazol-3-yl)-quinolin-7-yl]-urea; [0483]
1-(2-Hydroxy-ethyl)-3-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyr-
azol-3-yl)-quinolin-7-yl]-urea; [0484]
[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin-7--
yl]-carbamic acid methyl ester, [0485]
[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin-7--
yl]-carbamic acid 2-hydroxy-ethyl ester, [0486]
[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin-7--
yl]-carbamic acid 2-methoxy-ethyl ester; [0487]
1,3-Bis-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-qui-
nolin-7-yl]-urea; [0488] Dimethyl-carbamic acid
4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin-7-y-
l ester; [0489]
7-Bromo-2-isopropyl-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazo-
l-3-yl)-quinoline; [0490]
2-{4-(2-(6-Methyl-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-
-quinolin-6-yl}-propan-2-ol; [0491]
7-(3-Chloro-propylsulfanyl)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2--
b]pyrazol-3-yl)-quinoline; [0492]
7-Bromo-4-(4-chloro-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl)-quinoline; [0493]
8-Chloro-4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-qui-
nolin-7-ol; [0494]
8-Bromo-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quin-
olin-7-ol; [0495]
3-(7-Bromo-quinolin-4-yl)-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyr-
azol-4-ol; [0496]
7-Bromo-4-(4-methoxy-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol--
3-yl)-quinoline; [0497]
[3-(7-Bromo-quinolin-4-yl)-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]py-
razol-4-yl]-methyl-amine; [0498]
3-(7-Bromo-quinolin-4-yl)-2-pyridin-2-yl-5,6-dihydro-pyrrolo[1,2-b]pyrazo-
l-4-one; [0499]
3-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin--
7-yloxy]-benzamide; [0500]
N,N-Dimethyl-3-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3--
yl)-quinolin-7-yloxy]-thiobenzamide; [0501]
Dimethyl-{3-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-
-quinolin-7-yloxy]-benzyl}-amine; [0502]
4-(2-(6-Methyl-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-1H-
-quinolin-2-one; [0503]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin-7-o-
l; [0504]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazo-
l-3-yl]-quinolin-7-ol; [0505]
6-Methoxy-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-qu-
inolin-7-ol; [0506]
3-{4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl-
]-quinolin-7-yl}-propionic acid methyl ester, [0507]
4-(6-(Methyl-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-qu-
inoline; [0508]
3-{4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl-
]-quinolin-6-yl}-propionic acid methyl ester, [0509]
7-Amino-4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-
-3-yl]-quinoline; [0510]
N,N-Dimethyl-3-{4-(2-methyl-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyr-
azol-3-yl]-quinolin-7-yl}-propionamide; [0511]
N-{3-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinol-
in-7-yloxy]-propyl)}-acetamide; [0512]
N-Acetyl-N-{4-[2-(6-methyl-yridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyra-
zol-3-yl]-quinolin-7-yl}-acetamide,
2-Pyridin-2-yl-3-quinolin-4-yl-pyrazolo[1,5-a]piperidin-7-ol;
[0513]
7-Acetoxy-2-pyridin-2-yl-3-quinolin-4-yl-pyrazol[1,5-a]piperidine;
[0514]
Methyl-{3-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-q-
uinolin-7-yloxy]-propyl)}-amine; [0515]
7-(Piperidin-4-yloxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyra-
zol-3-yl)-quinoline; [0516]
4-(6-(Methyl-2-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline-7-carboxylic acid (2-amino-1,1-dimethyl-ethyl)-amide;
[0517]
16-[3-(4-Fluoro-phenyl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-2-yl]-pyridi-
n-2-yl)-methanol,rrrrrm-rrrrr)[6-(3-Quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1-
,2-b]pyrazol-2-yl)-pyridin-2-yl]-methanol; [0518]
4-(6-(Methyl-2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-ph-
enol; [0519]
7-(1-Methyl-pyrrolidin-3-ylmethoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrr-
olo[1,2-b]pyrazol-3-yl)-quinoline; [0520]
7-(1-Methyl-piperidin-4-ylmethoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrro-
lo[1,2-b]pyrazol-3-yl)-quinoline; [0521]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline-7-carboxylic acid
(2-dimethylamino-1,1-dimethyl-ethyl)-amide; [0522]
(S)-[3-(4-Fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H--
pyrrolo[1,2-b]pyrazol-6-yl]-methanol; [0523]
(R)-[3-(4-Fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo-
[1,2-b]pyrazol-6-yl]-methanol; [0524]
(S)-[3-(4-Fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo-
[1,2-b]pyrazol-6-yl]-acetonitrile; [0525]
(R)-[3-(4-Fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo-
[1,2-b]pyrazol-6-yl]-acetonitrile; [0526]
4-(3-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-2-yl)-quinoline;
[0527]
4-(6-Pyridin-2-yl-2,3-dihydro-pyrazolo[5,1-b]oxazol-7-yl)-quinolin-
e; [0528]
3-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)--
quinolin-7-yl]-oxazolidin-2-one; [0529]
1-[4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2b]pyrazol-3-yl)-quinolin-7-
-yl]-imidazolidin-2-one; [0530]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-7-(pyridin-4-
-ylmethoxy)-quinoline; [0531]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-7-(3-pyridin-
-3-yl-propoxy)-quinoline; [0532]
7-(4,5-Dihydro-1H-imidazol-2-yl)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo-
[1,2-b]pyrazol-3-yl)-quinoline; [0533]
4-[5-(4-Fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1-
,2-b]pyrazol-3-yl]-quinoline (Enantiomer A); [0534]
4-[5-(4-Fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1-
,2-b]pyrazol-3-yl]-quinoline (Enantiomer B); [0535]
2-Pyridin-2-yl-3-quinolin-4-yl-pyrazolo[5,1-c]morpholine; [0536]
4-[2-(6-Vinyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-qu-
inoline; [0537]
3-{4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl-
]-quinolin-6-yl}-acrylic acid; [0538]
7-(6-Methyl-pyridin-3-yloxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-
-b]pyrazol-3-yl)-quinoline; [0539]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-7-[4-(4-pyri-
midin-2-yl-piperazin-1-yl)-butoxy]-quinoline; [0540]
7-[3-[4-(2-Methoxy-phenyl)-piperazin-1-yl]-propoxy}-4-(2-pyridin-2-yl-5,6-
-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline; [0541]
Pyridin-2-yl-{3-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-
-yl)-quinolin-7-yloxy]-propyl)}-amine; [0542]
4-(6-(Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-qui-
noline-7-carboxylic acid
(2-dimethylamino-1-methyl-ethyl)-amide,rrrrnTn-rr)
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline-7-carboxylic acid amide; [0543]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline-7--
carboxylic acid (3-dimethylamino-propyl)-amide; [0544]
4-[2-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline-7-carboxylic acid (2-dimethylamino-ethyl)-methyl-amide;
[0545]
N,N-Dimethyl-3-{4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b-
]pyrazol-3-yl]-quinolin-7-yl}-acrylamide;
[0546]
4-(2-Pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quino-
line 1-oxide; [0547]
7-Benzyloxy-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyr-
azol-3-yl]-quinoline; [0548]
4-(2-(6-Chloro-6-dihydro-4H-pyrrololoro-pyridin-2-yl)-5[1,2-b]pyrazol-3-y-
l]-quinoline; [0549]
6-(3-Quinolin-4-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-2-yl)pyridine-2-c-
arboxylic acid methyl ester, [0550]
4-(7-Chloroquinolin-4-yl)-3-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2#b]p-
yrazole; [0551]
4-(2-Furan-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline;
[0552]
3-{4-(6-Methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol--
3-yl]-quinolin-6-yl}-acrylic acid methyl ester, [0553]
4-[2-(2-Methyl-thiazol-4-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-q-
uinoline; [0554]
3-(4-Fluoro-phenyl)-2-(2-methyl-thiazol-4-yl)-5,6-dihydro-4H-pyrrolo[1,2--
b]pyrazole; [0555]
4-[2-(2-Methyl-2H-pyrazol-3-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl-
]-quinoline; [0556]
4-(2-Thiazol-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline;
[0557]
4-[2-(1-Methyl-1H-imidazol-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyraz-
ol-3-yl]-quinoline; [0558]
6,7-Dichloro-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]py-
razol-3-yl]-quinoline; [0559]
(S)-6-Benzyloxymethyl-3-(4-fluoro-phenyl)-2-(6-methyl-pyridin-2-yl)-5,6-d-
ihydro-4H-pyrrolo[1,2-b]pyrazole; [0560]
N,N-Dimethyl-3-{4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b-
]pyrazol-3-yl]-quinolin-7-yl}-acrylamide; [0561]
3-methyl-6-[2-[6-methyl-(pyridin-2-yl)]-5,6-di-hydro-4H-pyrrolo[1,2-b]pyr-
azol-3-yl]-3H-quinazolin-4-one; [0562]
1-methyl-7-[2-[6-methyl-(pyridin-2-yl)]-5,6-di-hydro-4H-pyrrolo[1,2-b]pyr-
azol-3-yl]-1H-quinoxalin-2-one; [0563]
3-methyl-6-[2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-3-
H-quinazolin-4-one; [0564]
3-methyl-6-[2-[6-pentyl-(pyridin-2-yl)]-5,6-di-hydro-4H-pyrrolo[1,2-b]pyr-
azol-3-yl]-3H-quinazolin-4-one; [0565]
6-[2-[6-Methyl-(pyridin-2-yl)]-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-
-4H-benzo[1,4]oxazin-3-one; [0566]
3-(2-Chloro-ethyl)-6-[2-[6-methyl-(pyridin-2-yl)]-5,6-dihydro-4H-pyrrolo[-
1,2-b]pyrazol-3-yl]-3H-quinazolin-4-one; [0567]
6-[2-[6-methyl-(pyridin-2-yl)]-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-
-3-(2-morpholin-4-yl-ethyl)-3H-quinazolin-4-one; [0568]
3-(2-Dimethylamino-ethyl)-6-[2-[6-methyl-(pyridin-2-yl)]-5,6-dihydro-4H-p-
yrrolo[1,2-b]pyrazol-3-yl]-3H-quinazolin-4-one; [0569]
6-[2-[6-Methyl-(pyridin-2-yl)]-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-
-3-(2-piperidin-1-yl-ethyl)-3H-quinazolin-4-one; [0570]
6-[2-[6-Methyl-(pyridin-2-yl)]-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-
-3-(2-pyrrolidin-1-yl-ethyl)-3H-quinazolin-4-one; [0571]
3-(2-Azepan-1-yl-ethyl)-6-[2-[6-methyl-(pyri-din-2-yl)]-5,6-dihydro-4H-py-
rrolo[1,2-b]pyrazol-3-yl]-3H-quinazolin-4-one; [0572]
7-[2-[6-Methyl-(pyridin-2-yl)]-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-
-1-(2-pyrrolidin-1-yl-ethyl)-3,4-dihydro-1H-quinoxalin-2-one; and
[0573]
1-(2-Dimethylamino-ethyl)-7-[2-[6-methyl-(pyridin-2-yl)]-5,6-dihydro-4H-p-
yrrolo[1,2-b]pyrazol-3-yl]-3,4-dihydro-1H-quinoxalin-2-one.
[0574] In one aspect, the TGF-.beta. inhibitor has the structure
shown in formula (II)
##STR00010##
[0575] wherein
[0576] R.sup.1 is cyclyl, heterocyclcyl, aryl or heteroaryl, each
of which can be optionally substituted;
[0577] R.sup.2 is cyclyl, heterocyclcyl, aryl or heteroaryl, each
of which can be optionally substituted;
[0578] R.sup.3 is cyclyl, heterocyclcyl, aryl, heteroaryl or
--S(O)alkyl, each of which can be optionally substituted;
[0579] R.sup.4 is H, optionally substituted C.sub.1-C.sub.6 alkyl,
optionally substituted C.sub.2-C.sub.6 alkenyl, optionally
substituted C.sub.2-C.sub.6 alkynyl, or R.sup.3 and R.sup.4
together with the atoms they are attached to form a cyclyl,
heterocyclyl, aryl or heteroaryl, each of which can be optionally
substituted.
[0580] In some embodiments, R.sup.1 is aryl, e.g., a substituted
aryl. In some embodiments, R.sup.1 is substituted with two
substituents. In some embodiments, R.sup.1 is substituted with two
substituents, which together with the carbons to which they are
attached form a ring. In some embodiments, R.sup.1 is a substituted
phenyl. In some embodiments, R.sup.1 is a nitrogen containing
heteroaryl (e.g., including 1, 2, or 3 nitrogens (e.g., 1 or)). In
some embodiments, R.sup.1 is a bicyclic heteroaryl. In some
embodiments, R.sup.1 is a 6-6 fused heteroaryl. In some
embodiments, R.sup.1 is pyridyl, pyrimidyl, pyridazinyl, pyrazinyl,
quinolinyl, naphthyridinyl (e.g., 1,5-naphthyridinyl),
quinazolinyl, 5,6,7,8-tetrahydroquinazolinyl, 1,3-benzodioxlyl,
1,2,3-benzotriazolyl, benzoxazolyl, benzothiazolyl,
2,1,3-benzooxadiazole, imidazo[1,2-a]pyridinyl,
pyrazolo[1,5-a]pyridinyl, [1,2,4]triazolo[1,5-a]pyridinyl,
pyrazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl,
[1,2,3]triazolo[1,5-a]pyrimidinyl,
[1,2,4]trazolo[4,3-a]pyrimidinyl,
[1,2,4]triazolo[4,3-a]pyridazinyl. In some embodiments, R.sup.1
is
##STR00011##
[0581] In some embodiments, R.sup.2 is aryl, e.g., a substituted
aryl. In some embodiments, R.sup.2 is a nitrogen comprising
heteroaryl (e.g., including 1, 2 or 3 nitrogens (e.g., 1 or 2)). In
some embodiments, R.sup.2 is an optionally substituted monocyclic
heteroaryl (e.g., a six membered heteroaryl such as pyridyl,
pyrimidyl, pyridazinyl or pyrazinyl). In some embodiments, R.sup.2
is substituted. Exemplary substituents include halo,
C.sub.1-C.sub.6 alkyl, haloC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6
alkoxy, OH, haloC.sub.1-C.sub.6alkoxy. In some embodiments, R.sup.2
is monosubstituted. In some embodiments, R.sup.2 is substituted
with methyl. In one embodiment, R.sup.2 is an optionally
substituted pyridyl. In some embodiments, R.sup.2 is
##STR00012##
[0582] In some embodiments, R.sup.3 is aryl, e.g., a substituted
aryl. In some embodiments, R.sup.3 is substituted with two
substituents, which together with the carbons to which they are
attached form a ring. In some embodiments, R.sup.3 is a substituted
phenyl. In some embodiments, R.sup.3 is a nitrogen containing
heteroaryl (e.g., including 1, 2, or 3 nitrogens (e.g., 1 or)). In
some embodiments, R.sup.3 is a bicyclic heteroaryl. In some
embodiments, R.sup.3 is a 6-6 fused heteroaryl. In some
embodiments, R.sup.3 is pyridyl, pyrimidyl, pyridazinyl, pyrazinyl,
quinolinyl, naphthyridinyl (e.g., 1,5-naphthyridinyl),
quinazolinyl, 5,6,7,8-tetrahydroquinazolinyl, 1,3-benzodioxlyl,
1,2,3-benzotriazolyl, benzoxazolyl, benzothiazolyl,
2,1,3-benzooxadiazole, imidazo[1,2-a]pyridinyl,
pyrazolo[1,5-a]pyridinyl, [1,2,4]triazolo[1,5-a]pyridinyl,
pyrazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl,
[1,2,3]triazolo[1,5-a]pyrimidinyl,
[1,2,4]triazolo[4,3-a]pyrimidinyl,
[1,2,4]triazolo[4,3-a]pyridazinyl. In some embodiments, R.sup.3 is
monosubstituted. In some embodiments, R.sup.3 is an optionally
substituted phenyl. In some embodiments, R.sup.3 is
##STR00013##
[0583] In some embodiment, R.sup.4 is H.
[0584] In some embodiments, the compound of formula (II) has the
structure shown in formula (IIa):
##STR00014##
wherein z.sup.1-z.sup.4 are independently CR.sup.5 or N; R.sup.5 is
H, benzyl, aryl, heteroaryl, C.sub.1-C.sub.6alkyl, alkenyl,
alkynyl, halogen, amino, --C(O)-amino, --SO.sub.2-alkyl, --O-alkyl
or acyl, each of which can be optionally substituted, provided that
no two N are not next to each other.
[0585] In some embodiments, one of Z.sup.2 or Z.sup.3 is N.
[0586] In some embodiments, the compound of formula (II) has the
structure shown in formula (IIb):
##STR00015##
[0587] Exemplary compounds of formula (II) include
##STR00016## [0588]
6-(2-(6-methylpyridin-2-yl)H-imidazo[1,2-a]pyridin-3-yl)-N-(3-(piperidin--
1-yl)propyl)pyridin-2-amine; [0589]
3-isopropyl-6-(5-(6-methylpyridin-2-yl)-2H-1,2,3-triazol-4-yl)H-imidazo[1-
,2-a]pyridine; [0590]
1-(3-((pyridin-3-yl)methoxy)-4-carbamoylisothiazol-5-yl)-3-(3,5-dimethoxy-
benzyl)urea; [0591]
(2-Methoxy-ethyl)-{4-[2-(6-methyl-pyridin-2-yl)-imidazo-[1,2-a]pyridin-3--
yl]-pyrimidin-2-yl}-amine; [0592]
(3-{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2--
ylamino}-propyl)-carbamic acid tert-butyl ester, [0593]
(3-Imidazol-1-yl-propyl)-{4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyri-
din-3-yl]-pyrimidin-2-yl}-amine; [0594]
(4-Methoxy-benzyl)-{4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3--
yl]-pyrimidin-2-yl}-amine; [0595]
[2-(6-Methyl-pyridin-2-yl)-3-(2-methylsulfanyl-pyrimidin-4-yl)-imidazo[1,-
2-a]pyridin-6-yl]-methanol; [0596]
3-(2-Methanesulfonyl-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,-
2-a]pyridine; [0597]
(4-4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-y-
lamino}-butyl)-carbamic acid tert-butyl ester; [0598]
(4-Amino-benzyl)-{4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl-
]-pyrimidin-2-yl}-amine; [0599]
(5-{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2--
ylamino}-pentyl)-carbamic acid tert-butyl ester; [0600]
[3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyrid-
in-6-yl]-methanol; [0601]
[3-(2-amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyrid-
in-7-yl]-methanol; [0602]
[3-(2-Amino-pyrimidin-4-yl)-6-methyl-2-(6-methyl-pyridin-2-yl)-imidazo[1,-
2-a]pyridin-8-yl]-(2-morpholin-4-yl-ethyl)-amine; [0603]
[3-(2-Amino-pyrimidin-4-yl)-6-methyl-2-(6-methyl-pyridin-2-yl)-imidazo[1,-
2-a]pyridin-8-yl]-(2-pyridin-2-yl-ethyl)-amine; [0604]
[3-(2-Amino-pyrimidin-4-yl)-6-methyl-2-(6-methyl-pyridin-2-yl)-imidazo[1,-
2-a]pyridin-8-yl]-(2-pyridin-3-yl-ethyl)-amine; [0605]
[3-(2-methanesulfonyl-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1-
,2-a]pyridin-6-yl]-methanol; [0606]
[3-(2-Amino-pyrimidin-4-yl)-6-methyl-2-(6-methyl-pyridin-2-yl)-imidazo[1,-
2-a]pyridin-8-yl]-(2-pyridin-4-yl-ethyl)-amine; [0607]
[3-(2-Amino-pyrimidin-4-yl)-6-methyl-2-(6-methyl-pyridin-2-yl)-imidazo[1,-
2-a]pyridin-8-yl]-(3-morpholin-4-yl-propyl)-amine; [0608]
[3-(4-Methyl-piperazin-1-yl)-propyl]-{4-[2-(6-methyl-pyridin-2-yl)-imidaz-
o[1,2-a]pyridin-3-yl]-pyrimidin-2-yl}-amine; [0609]
[3-(4-Methyl-piperidin-1-yl)-propyl]-{4-[2-(6-methyl-pyridin-2-yl)-imidaz-
o[1,2-a]pyridin-3-yl]-pyrimidin-2-yl}-amine; [0610]
[4-(2-Pyridin-2-yl-imidazo[1,2-a]pyridin-3-yl)-pyrimidin-2-yl]-pyridin-3--
ylmethyl-amine; [0611]
{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-yl}-
-((R)-1-phenyl-ethyl)-amine; [0612]
{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-yl}-
-((S)-1-phenyl-ethyl)-amine; [0613]
{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-y]-pyrimidin-2-yl}--
(1H-tetrazol-5-yl)-amine; [0614]
{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-yl}-
-(2H-pyrazol-3-yl)-amine; [0615]
{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-yl}-
-(2-morpholin-4-yl-ethyl)-amine; [0616]
{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-yl}-
-(2-pyridin-2-yl-ethyl)-amine; [0617]
{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-yl}-
-(2-pyridin-3-yl-ethyl)-amine; [0618]
{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-yl}-
-(2-pyridin-4-yl-ethyl)-amine; [0619]
{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-yl}-
-(3-morpholin-4-yl-propyl)-amine; [0620]
{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-yl}-
-(3-piperidin-1-yl-propyl)-amine; [0621]
{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-yl}-
-[1,3,4]thiadiazol-2-yl-amine; [0622]
2-(6-Methyl-pyridin-2-yl)-3-(2-methylsulfanyl-pyrimidin-4-yl)-imidazo[1,2-
-a]pyridine; [0623]
2-(6-Methyl-pyridin-2-yl)-3-(2-methylsulfanyl-pyrimidin-4-yl)-imidazo[1,2-
-a]pyridine-6-carboxylic acid methyl ester; [0624]
2-(6-Methyl-pyridin-2-yl)-3-(2-methylsulfanyl-pyrimidin-4-yl)-imidazo[1,2-
-a]pyridine-7-carboxylic acid ethyl ester; [0625]
2-(6-Methyl-pyridin-2-yl)-3-(2-methylsulfanyl-pyrimidin-4-yl)-imidazo[1,2-
-a]pyrimidin-7-ylamine; [0626]
{7,7-Dimethyl-8-[5-(4-4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin--
3-yl]-pyrimidin-2-ylamino}-butylcarbamoyl)-pentyl]-2-oxo-4-trifluoromethyl-
-7,8-dihydro-2H-1-oxa-8-aza-anthracen-5-yl}-methanesulfonic acid;
[0627]
2-(2,7-Difluoro-6-hydroxy-3-oxo-9,9a-dihydro-3H-xanthen-9-yl)-3,5,6-trifl-
uoro-4-[(4-{4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrim-
idin-2-ylamino}-butylcarbamoyl)-methylsulfanyl]-benzoic acid;
[0628]
2-(6-Methyl-pyridin-2-yl)-3-(2-morpholin-4-yl-pyrimidin-4-yl)-imidazo[1,2-
-a]pyridine; [0629]
2-(6-Methyl-pyridin-2-yl)-3-(2-piperidin-1-yl-pyrimidin-4-yl)-imidazo[1,2-
-a]pyridine; [0630]
2-(6-Methyl-pyridin-2-yl)-3-(2-pyrrolidin-1-yl-pyrimidin-4-yl)-imidazo[1,-
2-a]pyridine; [0631]
2-(6-Methyl-pyridin-2-yl)-3-[2-(1H-tetrazol-5-yl)-pyrimidin-4-yl]-imidazo-
[1,2-a]pyridine; [0632]
2-(6-Methyl-pyridin-2-yl)-3-pyrimidin-4-yl-imidazo[1,2-a]pyridine;
[0633]
2-(6-Methyl-pyridin-2-yl)-3-pyrimidin-4-yl-imidazo[1,2-a]pyrimidin-7-ylam-
ine; [0634]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
n-8-ylamine; [0635]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carbonitrile; [0636]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carboxylic acid; [0637]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carboxylic acid ([1,4]dioxan-2-ylmethyl)-amide; [0638]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carboxylic acid ([1,4]dioxan-2-ylmethyl)-amide; [0639]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carboxylic acid (2-dimethylamino-ethyl)-amide; [0640]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carboxylic acid (2-methoxy-ethyl)-amide; [0641]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carboxylic acid (2-thiophen-2-yl-ethyl)-amide; [0642]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carboxylic acid [3-(4-methyl-piperazin-1-yl)-propyl]-amide;
[0643]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carboxylic acid amide; [0644]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carboxylic acid cyclopropylamide; [0645]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carboxylic acid ethylamide; [0646]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carboxylic acid hydroxyamide; [0647]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carboxylic acid methoxy-amide; [0648]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-6-carboxylic acid methyl ester; [0649]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid; [0650]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid [1,4]dioxan-2-ylmethyl)-amide; [0651]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid (2-amino-ethyl)-amide; [0652]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid (2-dimethylamino-ethyl)-amide; [0653]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid (2-hydroxy-ethyl)-amide; [0654]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid (2-oxo-2-pyridin-3-yl-ethyl)-amide; [0655]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid (2-thiophen-2-yl-ethyl)-amide; [0656]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid (piperidin-3-ylmethyl)-amide; [0657]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid 2,2-dimethylhydrazide; [0658]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid amide; [0659]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid cyclopropylamide; [0660]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid ethyl ester; [0661]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid ethylamide; [0662]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid hydroxyamide; [0663]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
ne-7-carboxylic acid methoxy-amide; [0664]
3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyrimi-
din-7-ylamine; [0665]
3-(2-Azetidin-1-yl-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2--
a]pyridine; [0666]
3-(2-Methanesulfonyl-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,-
2-a]pyridine-7-carboxylic acid ethyl ester; [0667]
3-(2-Methanesulfonyl-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,-
2-a]pyridine-6-carboxylic acid methyl ester; [0668]
3-(2-Methanesulfonyl-pyrimidin-4-yl)-7-methyl-2-(6-methyl-pyridin-2-yl)-i-
midazo[1,2-a]pyridine; [0669]
3-(2-Methanesulfonyl-pyrimidin-4-yl)-8-methyl-2-(6-methyl-pyridin-2-yl)-i-
midazo[1,2-a]pyridine; [0670]
3,3-Dimethyl-N-[2-(6-methyl-pyridin-2-yl)-3-(2-methylsulfanyl-pyrimidin-4-
-yl)-imidazo[1,2-a]pyrimidin-7-yl]-butyramide; [0671]
3-(2-Methanesulfonyl-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,-
2-a]pyridine-6-carbonitrile; [0672]
3-(2-Methylsulfanyl-pyrimidin-4-yl)-2-pyridin-2-yl-imidazo[1,2-a]pyridine-
; [0673]
3,6-Dichloro-N-(4-(4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyr-
idin-3-yl]-pyrimidin-2-ylamino)-butyl)-2-(2,4,5,7-Tetrachloro-6-hydroxy-3--
oxo-9,9a-dihydro-3H-xanthen-9-yl)-terephthalamic acid; [0674]
3-[2-(2-Methyl-aziridin-1-yl)-pyrimidin-4-yl]-2-(6-methyl-pyridin-2-yl)-i-
midazo[1,2-a]pyridine; [0675]
3-[2-(4-Methyl-piperazin-1-yl)-pyrimidin-4-yl]-2-(6-methyl-pyridin-2-yl)--
imidazo[1,2-a]pyridine; [0676]
3-([3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]py-
ridine-6-carbonyl]-amino)-propionic acid methyl ester; [0677]
3-([3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]py-
ridine-7-carbonyl]-amino)-propionic acid methyl ester; [0678]
3-(4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-y-
lamino)-phenol; [0679]
4-(2-{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin--
2-ylamino}-ethyl)-benzenesulfonamide; [0680]
4-(2-Pyridin-2-yl-imidazo[1,2-a]pyridin-3-yl)-pyrimidin-2-ylamine;
[0681]
4-[2-(6-Chloro-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-ylam-
ine; [0682]
4-[2-(6-Methyl-pyridin-2-yl)-7-trifluoromethyl-imidazo[1,2-a]pyridin-3-yl-
]-pyrimidin-2-ylamine; [0683]
4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-ylam-
ine; [0684]
4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidine-2-car-
bonitrile; [0685]
4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidine-2-car-
boxylic acid amide; [0686]
4-[6-Bromo-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidi-
n-2-ylamine; [0687]
4-[6-Chloro-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimid-
in-2-ylamine; [0688]
4-[6-Fluoro-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimid-
in-2-ylamine; [0689]
4-[6-Methyl-2-(6-methyl-pyridin-2-yl)-8-(2-morpholin-4-yl-ethylamino)-imi-
dazo[1,2-a]pyridin-3-yl]-pyrimidin-2-1; [0690]
4-[6-Methyl-2-(6-methyl-pyridin-2-yl)-8-(2-pyridin-2-yl-ethylamino)-imida-
zo[1,2-a]pyridin-3-yl]-pyrimidin-2-ol; [0691]
4-[6-Methyl-2-(6-methyl-pyridin-2-yl)-8-(2-pyridin-3-yl-ethylamino)-imida-
zo[1,2-a]pyridin-3-yl]-pyrimidin-2-ol; [0692]
4-[6-Methyl-2-(6-methyl-pyridin-2-yl)-8-(2-pyridin-4-yl-ethylamino)-imida-
zo[1,2-a]pyridin-3-yl]-pyrimidin-2-1; [0693]
4-[6-Methyl-2-(6-methyl-pyridin-2-yl)-8-morpholin-4-yl-imidazo[1,2-a]pyri-
din-3-yl]-pyrimidin-2-ol; [0694]
4-[6-Methyl-2-(6-methyl-pyridin-2-yl)-8-morpholin-4-yl-imidazo[1,2-a]pyri-
din-3-yl]-pyrimidin-2-ylamine; [0695]
4-[6-Methyl-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimid-
in-2-ylamine; [0696]
4-[7-Aminomethyl-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-py-
rimidin-2-ylamine; [0697]
4-[7-Methyl-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimid-
in-2-ylamine; [0698]
4-[8-Benzyloxy-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyri-
midin-2-ol; [0699]
4-[8-Benzyloxy-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyri-
midin-2-ylamine; [0700]
4-[8-Bromo-6-methyl-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-
-pyrimidin-2-ol; [0701]
4-[8-Methyl-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimid-
in-2-ylamine; [0702]
6-Chloro-3-(2-methanesulfonyl-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-i-
midazo[1,2-a]pyridine; [0703]
5-Dimethylamino-naphthalene-1-sulfonic acid
(4-(4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2--
ylamino)-butyl)-amide; [0704]
6-(2,7-Difluoro-6-hydroxy-3-oxo-3H-xanthen-9-yl)-N-(4-(4-[2-(6-methyl-pyr-
idin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-ylamino)-butyl)-isophth-
alamic acid; [0705]
6-Amino-9-[2-carboxy-5-(4-(4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyr-
idin-3-yl]-pyrimidin-2-ylamino)-butylcarbamoyl)-phenyl]-xanthen-3-ylidene--
ammonium; [0706]
6-Bromo-2-(6-methyl-pyridin-2-yl)-3-(2-methylsulfanyl-pyrimidin-4-yl)-imi-
dazo[1,2-a]pyridine; [0707]
6-Fluoro-2-(6-methyl-pyridin-2-yl)-3-(2-methylsulfanyl-pyrimidin-4-yl)-im-
idazo[1,2-a]pyridine; [0708]
7-Amino-4-methyl-3-[(4-{4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridi-
n-3-yl]-pyrimidin-2-ylamino}-butylcarbamoyl)-methyl]-2-oxo-2H-chromene-6-s-
ulfonic acid; [0709]
Cyclobutyl-{4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyri-
midin-2-yl}-amine; [0710]
Cyclopentyl-{4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyr-
imidin-2-yl}-amine; [0711]
Cyclopropyl-{4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyr-
imidin-2-yl}-amine; [0712]
Cyclopropyl-methyl-{4-[2-(6-methyl-pyridin-2-yl)-imidazo[,
2-a]pyridin-3-yl]-pyrimidin-2-yl}-amine; [0713]
Dimethyl-{4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimi-
din-2-yl}-amine; [0714]
Isopropyl-4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimi-
din-2-yl}-amine; [0715]
Methyl-{4-[2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidi-
n-2-yl}-amine; [0716]
N-(2-{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin--
2-ylamino}-ethyl)-acetamide; [0717]
N-(4-{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin--
2-ylamino}-butyl)-acetamide; [0718]
N,N-Dimethyl-N'-{4-[2-(6-methyl-pyridin-2-yl)-imidazo[,
2-a]pyridin-3-yl]-pyrimidin-2-yl}-ethane-1,2-diamine; [0719]
N-[2-(6-Methyl-pyridin-2-yl)-3-(2-methylsulfanyl-pyrimidin-4-yl)-imidazo[-
1,2-a]pyrimidin-7-yl]-3-pyridin-3-yl-propionamide;
[0720]
N-[2-(6-Methyl-pyridin-2-yl)-3-(2-methylsulfanyl-pyrimidin-4-yl)-i-
midazo[1,2-a]pyrimidin-7-yl]-nicotinamide; [0721]
N-[2-(6-Methyl-pyridin-2-yl)-3-(2-methylsulfanyl-pyrimidin-4-yl)-imidazo[-
1,2-a]pyrimidin-7-yl]-propionamide; [0722]
N-[3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyr-
idine-6-carbonyl]-methanesulfonamide; [0723]
N-[3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyr-
idine-7-carbonyl]-methanesulfonamide; [0724]
N-[3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyr-
imidin-7-yl]-2-(3-methoxy-phenyl)-acetamide; [0725]
N-[3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyr-
imidin-7-yl]-3,3-dimethyl-butyramide; [0726]
N-[3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyr-
imidin-7-yl]-3-pyridin-3-yl-propionamide; [0727]
N-[3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyr-
imidin-7-yl]-acetamide; [0728]
N-[3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyr-
imidin-7-yl]-nicotinamide; [0729]
N-[3-(2-Methanesulfonyl-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo-
[1,2-a]pyrimidin-7-yl]-2-(3-methoxy-phenyl)-acetamide; [0730]
N-[3-(2-Methanesulfonyl-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo-
[1,2-a]pyrimidin-7-yl]-3,3-dimethyl-butyramide; [0731]
N-[3-(2-Methanesulfonyl-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo-
[1,2-a]pyrimidin-7-yl]-3-pyridin-3-yl-propionamide; [0732]
N-[3-(2-Methanesulfonyl-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo-
[1,2-a]pyrimidin-7-yl]-nicotinamide; [0733]
N-[3-(2-Methanesulfonyl-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo-
[1,2-a]pyrimidin-7-yl]-propionamide; [0734]
N-[3-(2-Amino-pyrimidin-4-yl)-2-(6-methyl-pyridin-2-yl)-imidazo[1,2-a]pyr-
imidin-7-yl]-propionamide; [0735]
N-{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2-y-
l}-acetamide; [0736]
N1-{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2--
yl}-butane-1,4-diamine; [0737]
N1-{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin-2--
yl}-propane-1,3-diamine; [0738]
N-(4-{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin--
2-ylamino}-butyl)-(BODIPY FL)amide; and [0739]
N-(4-{4-[2-(6-Methyl-pyridin-2-yl)-imidazo[1,2-a]pyridin-3-yl]-pyrimidin--
2-ylamino}-butyl)-(Texas Red-X)amide.
[0740] In one aspect, the TGF-.beta. inhibitor has the structure
shown in formula (IIIa):
##STR00017##
or formula (IIIb)
##STR00018##
wherein:
[0741] R.sup.1 is cyclyl, heterocyclcyl, aryl or heteroaryl, each
of which can be optionally substituted;
[0742] R.sup.2 is cyclyl, heterocyclcyl, aryl or heteroaryl, each
of which can be optionally substituted;
[0743] R.sup.3 is R.sup.3 is H, C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, heteroaryl,
cyclyl, heterocyclyl, acyl or a nitrogen protecting group, each of
which can be optionally substituted.
[0744] In some embodiments, R.sup.1 is a nitrogen containing
heteroaryl (e.g., including 1, 2, or 3 nitrogens (e.g., 1 or)). In
some embodiments, R.sup.1 is a bicyclic heteroaryl. In some
embodiments, R.sup.1 is a 6-6 fused heteroaryl. In some
embodiments, R.sup.1 is pyridyl, pyrimidyl, quinolinyl,
naphthyridinyl (e.g., 1,5-naphthyridinyl), quinazolinyl,
5,6,7,8-tetrahydroquinazolinyl, 1,3-benzodioxlyl,
1,2,3-benzotriazolyl, benzoxazolyl, benzothiazolyl,
2,1,3-benzooxadiazole, imidazo[1,2-a]pyridinyl,
pyrazolo[1,5-a]pyridinyl, [1,2,4]triazolo[1,5-a]pyridinyl,
pyrazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl,
[1,2,3]triazolo[1,5-a]pyrimidinyl,
[1,2,4]triazolo[4,3-a]pyrimidinyl,
[1,2,4]triazolo[4,3-a]pyridazinyl. In some embodiments, R.sup.1
is
##STR00019##
[0745] In some embodiments, R.sup.1 is
##STR00020##
[0746] In some embodiments, R.sup.2 is aryl, e.g., a substituted
aryl. In some embodiments, R.sup.2 is a nitrogen comprising
heteroaryl (e.g., including 1, 2 or 3 nitrogens (e.g., 1 or 2)). In
some embodiments, R.sup.2 is an optionally substituted monocyclic
heteroaryl (e.g., a six membered heteroaryl such as pyridyl,
pyrimidyl, pyridazinyl or pyrazinyl). In some embodiments, R.sup.2
is substituted. Exemplary substituents include halo,
C.sub.1-C.sub.6 alkyl, haloC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6
alkoxy, OH, haloC.sub.1-C.sub.6alkoxy. In some embodiments, R.sup.2
is monosubstituted. In some embodiments, R.sup.2 is substituted
with methyl. In one embodiment, R.sup.2 is an optionally
substituted pyridyl. In some embodiments, R.sup.2 is
##STR00021##
In some embodiments, R.sup.2 is
##STR00022##
[0747] In one aspect, the TGF-.beta. inhibitor has the structure
shown in formula (IV):
##STR00023##
wherein: R.sup.1 is H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkenyl, C.sub.1-C.sub.6 alkynyl, aryl, heteroaryl, cyclyl,
optionally substituted heterocyclyl or acyl, each of which can be
optionally substituted; R.sup.2 is H, C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkynyl, aryl, heteroaryl,
cyclyl, optionally substituted heterocyclyl, acyl or amino (e.g.,
N(R.sup.3).sub.2), each of which can be optionally substituted;
[0748] R.sup.3 is independently for each occurrence H,
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkenyl, C.sub.1-C.sub.6
alkynyl, aryl, heteroaryl, cyclyl, optionally substituted
heterocyclyl or acyl, each of which can be optionally
substituted;
[0749] Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4 and Z.sup.5 are each
independently N or CR.sup.3, provided that at least two of Z.sup.2,
Z.sup.3, Z.sup.4 and Z.sup.5 are CR.sup.3, and further provided
that two adjacent Z positions are not N.
[0750] In one embodiment, R.sup.1 is aryl, e.g., a substituted
aryl. In one embodiment, R.sup.1 is phenyl. In one embodiment,
R.sup.1 is phenyl substituted with at least one halogen.
[0751] In one embodiment, R.sup.2 is NHR.sup.3.
[0752] Exemplary compounds of formula (IV) include: [0753]
2-phenyl-4-(4-pyridylamino)-quinazoline; [0754]
2-(2-bromophenyl)-4-(4-pyridylamino)-quinazoline; [0755]
2-(2-chlorophenyl)-4-(4-pyridylamino)-quinazoline; [0756]
2-(2-fluorophenyl)-4-(4-pyridylamino)-quinazoline; [0757]
2-(2-methylphenyl)-4-(4-pyridylamino)-quinazoline; [0758]
2-(4-fluorophenyl)-4-(4-pyridylamino)-quinazoline; [0759]
2-(3-methoxyanilyl)-4-(4-pyridylamino)-quinazoline; [0760]
2-(2,6-dichlorophenyl)-4-(4-pyridylamino)-quinazoline; [0761]
2-(2,6-dibromophenyl)-4-(4-pyridylamino)-quinazoline; [0762]
2-(2,6-difluorophenyl)-4-(4-pyridylamino)-quinazoline; [0763]
2-(2-fluorophenyl)-4-(6-pyridylamino)-6,7-dimethoxyquinazoline;
[0764]
2-(4-fluorophenyl)-4-(4-pyridylamino)-6,7-dimethoxyquinazoline;
[0765] 2-(2-fluorophenyl)-4-(4-pyridylamino)-6-nitroquinazoline;
[0766] 2-(2-fluorophenyl)-4-(4-pyridylamino-6-aminoquinazoline;
[0767] 2-(2-fluorophenyl)-4-(4-pyridylamino)-7-aminoquinazoline;
[0768]
2-(2-fluorophenyl)-4-(4-pyridylamino)-6-(3-methoxybenzylamino)-quinazolin-
e; [0769]
2-(2-fluorophen)-4-(4-pyridylamino)-6-(4-methoxybenzylamino)-qui-
nazoline; [0770]
2-(2-fluorophenyl)-4-(4-pyridylal7nino)-6-(2-isobutylamino)-quinazoline;
and [0771]
2-(2-fluorophenyl)-4-(4-pyridylamino)-6-(4-methylmercaptobenzylamino)-qui-
nazoline.
[0772] In one aspect, the disclosure features a method of producing
an iPS cell from a somatic cell, the method comprising:
[0773] In one aspect, the TGF-.beta. inhibitor has the structure
shown in formula (V):
##STR00024##
wherein:
[0774] R.sup.1 is H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkenyl, C.sub.1-C.sub.6 alkynyl, aryl, heteroaryl, cyclyl,
optionally substituted heterocyclyl or acyl, each of which can be
optionally substituted;
[0775] R.sup.2 is H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkenyl, C.sub.1-C.sub.6 alkynyl, aryl, heteroaryl, cyclyl,
optionally substituted heterocyclyl or acyl, each of which can be
optionally substituted;
[0776] R.sup.3 is H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkenyl, C.sub.1-C.sub.6 alkynyl, aryl, heteroaryl, cyclyl,
optionally substituted heterocyclyl, acyl or amino, each of which
can be optionally substituted.
[0777] In some embodiments, R.sup.1 is a nitrogen containing
heteroaryl (e.g., including 1, 2, or 3 nitrogens (e.g., 1 or)). In
some embodiments, R.sup.1 is a bicyclic heteroaryl. In some
embodiments, R.sup.1 is a 6-6 fused heteroaryl. In some
embodiments, R.sup.1 is pyridyl, pyrimidyl, pyridazinyl, pyrazinyl,
quinolinyl, naphthyridinyl (e.g., 1,5-naphthyridinyl),
quinazolinyl, 5,6,7,8-tetrahydroquinazolinyl, 1,3-benzodioxlyl,
1,2,3-benzotriazolyl, benzoxazolyl, benzothiazolyl,
2,1,3-benzooxadiazole, imidazo[1,2-a]pyridinyl,
pyrazolo[1,5-a]pyridinyl, [1,2,4]triazolo[1,5-a]pyridinyl,
pyrazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl,
[1,2,3]triazolo[1,5-a]pyrimidinyl,
[1,2,4]triazolo[4,3-a]pyrimidinyl,
[1,2,4]triazolo[4,3a]pyridazinyl. In some embodiments, R.sup.1
is
##STR00025##
In some embodiments, R.sup.1 is
##STR00026##
[0778] In some embodiments, R.sup.2 is aryl, e.g., a substituted
aryl. In some embodiments, R.sup.2 is a nitrogen comprising
heteroaryl (e.g., including 1, 2 or 3 nitrogens (e.g., 1 or 2)). In
some embodiments, R.sup.2 is an optionally substituted monocyclic
heteroaryl (e.g., a six membered heteroaryl such as pyridyl,
pyrimidyl, pyridazinyl or pyrazinyl). In some embodiments, R.sup.2
is substituted. Exemplary substituents include halo,
C.sub.1-C.sub.6 alkyl, haloC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6
alkoxy, OH, haloC.sub.1-C.sub.6alkoxy. In some embodiments, R.sup.2
is monosubstituted. In some embodiments, R.sup.2 is substituted
with methyl. In one embodiment, R.sup.2 is an optionally
substituted pyridyl. In some embodiments, R.sup.2 is
##STR00027##
In some embodiments, R.sup.2 is
##STR00028##
[0779] Exemplary compounds of formula (V) include: [0780]
4-(Pyridin-2-yl)-5-quinolin-4-yl-1,3-thiazol-2-amine; [0781]
4-(6-methylpyridin-2-yl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazol-2-amine;
[0782]
5-([1,5]Naphthyridin-2-yl)-4-pyridin-2-yl-1,3-thiazol-2-amine;
[0783]
5-[2-(4-Chlorophenyl)pyridin-4-yl]-4-pyridin-2-yl-1,3-thiazol-2-am-
ine; [0784]
5-[2-(4-Methoxyphenyl)pyridin-4-yl]-4-pyri-din-2-yl-1,3-thiazol-2-amine;
[0785]
5-[2-(4-Fluorophenyl)pyridin-4-yl]-4-pyridin-2-yl-1,3-thiazol-2-am-
ine; [0786]
5-[2-(4-Ethylphenyl)pyridin-4-yl]-4-pyridin-2-yl-1,3-thiazol-2-amine;
[0787]
5-[2-(4-Ethoxyphenyl)pyridin-4-yl]-4-pyridin-2-yl-1,3-thiazol-2-am-
ine; and [0788]
5-[2-(Thiophen-3-yl)pyridin-4-yl]-4-pyridin-2-yl-1,3-thiazol-2-amine.
[0789] In one aspect, the TGF-.beta. inhibitor has the structure
shown in formula (VI):
##STR00029##
wherein:
[0790] R.sup.1 is H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkenyl, C.sub.1-C.sub.6 alkynyl, aryl, heteroaryl, cyclyl,
heterocyclyl, acyl, amino, or amide (e.g., --CO.sub.2NH.sub.2),
each of which can be optionally substituted;
[0791] R.sup.2 is C.sub.1-C.sub.6 alkyl, --O-alkyl, amino, acyl,
aryl, heteroaryl, cyclyl or heterocyclyl, each of which can be
optionally substituted;
[0792] R.sup.3 is H, C.sub.1-C.sub.6 alkyl, --O-alkyl, amino,
amide, --NHC(O)NH-alkyl, acyl, aryl, heteroaryl, cyclyl,
heterocyclyl, each of which can be optionally substituted.
[0793] In some embodiments, R.sup.1 is optionally substituted
amide, e.g. --CO.sub.2NH.sub.2. In some embodiments, the amide is
substituted with C.sub.1-C.sub.6 alkyl, which can also be
optionally substituted,
[0794] In some embodiments, R.sup.1 is aryl, e.g., a substituted
aryl. In some embodiments, R.sup.1 is substituted with two
substituents. In some embodiments, R.sup.1 is substituted with two
substituents, which together with the carbons to which they are
attached form a ring. In some embodiments, R.sup.1 is a substituted
phenyl. In some embodiments, R.sup.1 is a nitrogen containing
heteroaryl (e.g., including 1, 2, or 3 nitrogens (e.g., 1 or)). In
some embodiments, R.sup.1 is a bicyclic heteroaryl. In some
embodiments, R.sup.1 is a 6-6 fused heteroaryl. In some
embodiments, R.sup.1 is pyridyl, pyrimidyl, pyridazinyl, pyrazinyl,
quinolinyl, naphthyridinyl (e.g., 1,5-naphthyridinyl),
quinazolinyl, 5,6,7,8-tetrahydroquinazolinyl, 1,3-benzodioxlyl,
1,2,3-benzotriazolyl, benzoxazolyl, benzothiazolyl,
2,1,3-benzooxadiazole, imidazo[1,2-a]pyridinyl,
pyrazolo[1,5-a]pyridinyl, [1,2,4]triazolo[1,5-a]pyridinyl,
pyrazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl,
[1,2,3]triazolo[1,5-a]pyrimidinyl,
[1,2,4]triazolo[4,3-a]pyrimidinyl,
[1,2,4]triazolo[4,3-a]pyridazinyl. In some embodiments, R.sup.1
is
##STR00030##
In some embodiments, R.sup.1 is
##STR00031##
[0795] In some embodiments, R.sup.2 is optionally substituted
--O-alkyl. Exemplary substituents include aryl, heteroaryl, cyclyl
and heterocyclyl, each of which can be optionally substituted. In
some embodiments, R.sup.2 is
##STR00032##
[0796] In some embodiments, R.sup.2 is aryl, e.g., a substituted
aryl. In some embodiments, R.sup.2 is a nitrogen comprising
heteroaryl (e.g., including 1, 2 or 3 nitrogens (e.g., 1 or 2)). In
some embodiments, R.sup.2 is an optionally substituted monocyclic
heteroaryl (e.g., a six membered heteroaryl such as pyridyl,
pyrimidyl, pyridazinyl or pyrazinyl). In some embodiments, R.sup.2
is substituted. Exemplary substituents include halo,
C.sub.1-C.sub.6 alkyl, haloC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6
alkoxy, OH, haloC.sub.1-C.sub.6alkoxy. In some embodiments, R.sup.2
is monosubstituted. In some embodiments, R.sup.2 is substituted
with methyl. In one embodiment, R.sup.2 is an optionally
substituted pyridyl. In some embodiments, R.sup.2 is
##STR00033##
[0797] In some embodiments, R.sup.3 is optionally substituted
--NHC(O)NH-alkyl. In some embodiments, substituents is aryl or
heteroaryl, each of which can be optionally substituted. In
OCH.sub.3 one embodiment,
##STR00034##
[0798] Exemplary compounds of formula (VI) include: [0799]
1-(3,5-dimethoxybenzyl)-3-(4-carbamoyl-3-(2-(pyridin-3-yl)ethyl)isothiazo-
l-5-yl)urea; [0800]
6-[3-(6-Methyl-pyridin-2-yl)-isoxazol-4-yl]-quinoxaline; [0801]
5-[3-(2-Cyclohex-1-enyl-ethyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazol-
e-4-carboxylic acid amide; [0802]
5-[3-(2,5-Dimethyl-benzyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazole-4--
carboxylic acid amide; [0803]
5-[3-(3,5-Dimethoxy-benzyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazole-4-
-carboxylic acid amide; [0804]
5-[3-(2-Ethoxy-benzyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazole-4-carb-
oxylic acid amide; [0805]
5-{3-[2-(2-Ethoxy-phenyl)-ethyl]-ureido}-3-(pyridin-3-ylmethoxy)-isothiaz-
ole-4-carboxylic acid amide; [0806]
5-{3-[2-(3,4-Dimethoxy-phenyl)-ethyl]-ureido}-3-(pyridin-3-ylmethoxy)-iso-
thiazole-4-carboxylic acid amide; [0807]
5-(3-Phenethyl-ureido)-3-(pyridin-3-yl-methoxy)-isothiazol-4-carboxylic
acid amide; [0808]
5-{3-[2-(3-Ethoxy-4-methoxy-phenyl)-ethyl]-ureido)}-3-(pyridin-3-ylmethox-
y)-isothiazole-4-carboxylic acid amide; [0809]
5-{3-[2-(4-Ethoxy-phenyl)-ethyl]-ureido)}-3-(pyridin-3-ylmethoxy)-isothia-
zole-4-carboxylic acid amide; [0810]
5-{3-[2-(4-Chloro-phenyl)-ethyl]-ureido)}-3-(pyridin-3-ylmethoxy)-isothia-
zole-4-carboxylic acid amide; [0811]
5-{3-[2-(3-Chloro-phenyl)-ethyl]-ureido)}-3-(pyridin-3-ylmethoxy)-isothia-
zole-4-carboxylic acid amide; [0812]
5-{3-[2-(3-Methoxy-phenyl)-ethyl]-ureido)}-3-(pyridin-3-ylmethoxy)-isothi-
azole-4-carboxylic acid amide; [0813]
5-{3-[2-(4-Methoxy-phenyl)-ethyl]-ureido)}-3-(pyridin-3-ylmethoxy)-isothi-
azole-4-carboxylic acid amide; [0814]
5-{3-[2-(3-Bromo-4-methoxy-phenyl)-ethyl]-ureido)}-3-(pyridin-3-ylmethoxy-
)-isothiazole-4-carboxylic acid amide; [0815]
5-{3-[2-(4-Bromo-phenyl)-ethyl]-ureido}-3-(pyridin-3-ylmethoxy)-isothiazo-
le-4-carboxylic acid amide; [0816]
5-{3-[2-(2-Chloro-phenyl)-ethyl]-ureido}-3-(pyridin-3-ylmethoxy)-isothiaz-
ole-4-carboxylic acid amide; [0817]
5-{3-[2-(3-Chloro-phenyl)-ethyl]-ureido}-3-(pyridin-3-ylmethoxy)-isothiaz-
ole-4-carboxylic acid amide; [0818]
5-(3-[2-(2-Fluoro-phenyl)-ethyl]-ureido)-3-(pyridin-3-ylmethoxy)-isothiaz-
ole-4-carboxylic acid amide; [0819]
5-(3-[2-(3-Fluoro-phenyl)-ethyl]-ureido)-3-(pyridin-3-ylmethoxy)-isothiaz-
ole-4-carboxylic acid amide; [0820]
5-(3-[2-(4-Fluoro-phenyl)-ethyl]-ureido)-3-(pyridin-3-ylmethoxy)-isothiaz-
ole-4-carboxylic acid amide; [0821]
5-{3-[2-(4-Ethoxy-3-methoxy-phenyl)-ethyl]-ureido)}-3-(pyridin-3-ylmethox-
y)-isothiazole-4-carboxylic acid amide; [0822]
5-{3-[2-(3-Ethoxy-4-methoxy-phenyl)-ethyl]-ureido)}-3-(pyridin-3-ylmethox-
y)-isothiazole-4-carboxylic acid amide; [0823]
5-{3-[2-(2,5-Dimethoxy-phenyl)-ethyl]-ureido)}-3-(pyridin-3-ylmethoxy)-is-
othiazole-4-carboxylic acid amide; [0824]
5-{3-[2-(3-Methoxy-phenyl)-ethyl]-ureido)}-3-(pyridin-3-ylmethoxy)-isothi-
azole-4-carboxylic acid amide; [0825]
5-[3-(2-Dinuoromethoxy-benzyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazol-
e-4-carboxylic acid amide; [0826]
5-[3-(2,6-Dimethoxy-benzyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazol-4--
carboxylic acid amide; [0827]
5-[3-(2,5-Dichloro-benzyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazol-4-c-
arboxylic acid amide; [0828]
5-[3-(3-Morpholin-4-yl-propyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazol-
e-4-carboxylic acid amide; [0829]
5-[3-(2-Morpholin-4-yl-ethyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazole-
-4-carboxylic acid amide; [0830]
5-[3-(2-Diethylamino-ethyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazol-4--
carboxylic acid amide; [0831]
5-[3-(3-Dimethylamino-propyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazole-
-4-carboxylic acid amide; [0832]
5-{3-[2-(1-Methyl-pyrrolidin-2-yl)-ethyl]-ureido)}-3-(pyridin-3-ylmethoxy-
)-isothiazole-4-carboxylic acid amide; [0833]
5-{3-[3-(2-Methyl-piperidin-1-yl)-propyl]-ureido)}-3-(pyridin-3-ylmethoxy-
)-isothiazole-4-carboxylic acid amide; [0834]
(R),(R)-5-[3-(2-Hydroxy-cycloheptyl-ethyl)-ureido]-3-(pyridin-3-ylmethoxy-
)-isothiazole-4-carboxylic acid amide; [0835]
(R),(R)-5-[3-(2-Hydroxy-cyclooctylmethyl)-ureido]-3-(pyridin-3-ylmethoxy)-
-isothiazole-4-carboxylic acid amide; [0836]
5-[3-(2-Hydroxy-ethyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazole-4-carb-
oxylic acid amide; [0837]
5-[3-(2-Hydroxy-butyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazole-4-carb-
oxylic acid amide; [0838]
5-{3-[3-(2-Oxo-pyrrolidin-1-yl)-propyl]-ureido}-3-(pyridin-3-ylmethoxy)-i-
sothiazole-4-carboxylic acid amide; [0839]
5-[3-(3-Imidazol-1-yl-propyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazole-
-4-carboxylic acid amide; [0840]
5-(3-Benzyl-ureido)-3-(pyridin-3-yl-methoxy)-isothiazole-4-carboxylic
acid amide; [0841]
5-[3-(2,5-Difluoro-benzyl)-ureido]-3-(pyridin-3-ylmethoxy)-isothiazole-4--
carboxylic acid amide; [0842]
3-(1-Pyridin-3-yl-ethoxy)-5-(3-pyridin-2-yl-methyl-ureido)-isothiazole-4--
carboxylic acid amide; [0843]
5-[3-(2,6-Dimethoxy-benzyl)-ureido]-3-(1-pyridin-3-yl-ethoxy)-isothiazole-
-4-carboxylic acid amide; [0844]
5-(3-Cyclopropylmethyl-ureido)-3-(pyridin-3-ylmethoxy)-isothiazole-4-carb-
oxylic acid amide; [0845]
5-(3-Methyl-ureido)-3-(pyridin-3-yl-methoxy)-isothiazole-4-carboxylic
acid amide; [0846]
5-(3-Methyl-ureido)-3-(1-pyridin-3-yl-ethoxy)-isothiazole-4-carboxylic
acid amide; and [0847]
5-[3-(3,5-Dichloro-benzyl)-ureido]-3-(pyri-din-3-ylmethoxy)-isothiazole-4-
-carboxylic acid amide.
Antibodies
[0848] Antibodies that can be used according to the methods
described herein include complete immunoglobulins, antigen binding
fragments of immunoglobulins, as well as antigen binding proteins
that comprise antigen binding domains of immunoglobulins. Antigen
binding fragments of immunoglobulins include, for example, Fab,
Fab', F(ab')2, scFv and dAbs. Modified antibody formats have been
developed which retain binding specificity, but have other
characteristics that may be desirable, including for example,
bispecificity, multivalence (more than two binding sites), and
compact size (e.g., binding domains alone).
[0849] Single chain antibodies lack some or all of the constant
domains of the whole antibodies from which they are derived.
Therefore, they can overcome some of the problems associated with
the use of whole antibodies. For example, single-chain antibodies
tend to be free of certain undesired interactions between
heavy-chain constant regions and other biological molecules.
Additionally, single-chain antibodies are considerably smaller than
whole antibodies and can have greater permeability than whole
antibodies, allowing single-chain antibodies to localize and bind
to target antigen-binding sites more efficiently. Furthermore, the
relatively small size of single-chain antibodies makes them less
likely to provoke an unwanted immune response in a recipient than
whole antibodies.
[0850] Multiple single chain antibodies, each single chain having
one VH and one VL domain covalently linked by a first peptide
linker, can be covalently linked by at least one or more peptide
linker to form multivalent single chain antibodies, which can be
monospecific or multispecific. Each chain of a multivalent single
chain antibody includes a variable light chain fragment and a
variable heavy chain fragment, and is linked by a peptide linker to
at least one other chain. The peptide linker is composed of at
least fifteen amino acid residues. The maximum number of linker
amino acid residues is approximately one hundred.
[0851] Two single chain antibodies can be combined to form a
diabody, also known as a bivalent dimer. Diabodies have two chains
and two binding sites, and can be monospecific or bispecific. Each
chain of the diabody includes a VH domain connected to a VL domain.
The domains are connected with linkers that are short enough to
prevent pairing between domains on the same chain, thus driving the
pairing between complementary domains on different chains to
recreate the two antigen-binding sites.
[0852] Three single chain antibodies can be combined to form
triabodies, also known as trivalent trimers. Triabodies are
constructed with the amino acid terminus of a VL or VH domain
directly fused to the carboxyl terminus of a VL or VH domain, i.e.,
without any linker sequence. The triabody has three Fv heads with
the polypeptides arranged in a cyclic, head-to-tail fashion. A
possible conformation of the triabody is planar with the three
binding sites located in a plane at an angle of 120 degrees from
one another. Triabodies can be monospecific, bispecific or
trispecific.
[0853] Thus, antibodies useful in the methods described herein
include, but are not limited to, naturally occurring antibodies,
bivalent fragments such as (Fab).sub.2, monovalent fragments such
as Fab, single chain antibodies, single chain Fv (scFv), single
domain antibodies, multivalent single chain antibodies, diabodies,
triabodies, and the like that bind specifically with an antigen
(e.g., a TGF.beta.R epitope).
[0854] Antibodies for use in the methods described herein can be
obtained from commercial sources such as AbCam (Cambridge, Mass.),
New England Biolabs (Ipswich, Mass.), Santa Cruz Biotechnologies
(Santa Cruz, Calif.), Biovision (Mountain View, Calif.), R&D
Systems (Minneapolis, Minn.), and Cell Signaling (Danvers, Mass.),
among others. Antibodies can also be raised against a polypeptide
or portion of a polypeptide by methods known to those skilled in
the art. Antibodies are readily raised in animals such as rabbits
or mice by immunization with the gene product, or a fragment
thereof. Immunized mice are particularly useful for providing
sources of B cells for the manufacture of hybridomas, which in turn
are cultured to produce large quantities of monoclonal antibodies.
Antibody manufacture methods are described in detail, for example,
in Harlow et al., Eds., Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York (1988), which is hereby
incorporated by reference in its entirety.
[0855] While both polyclonal and monoclonal antibodies can be used
in the methods described herein, it is preferred that a monoclonal
antibody is used where conditions require increased specificity for
a particular protein.
RNA Interference
[0856] "RNA interference (RNAi)" is an evolutionally conserved
process whereby the expression or introduction of RNA of a sequence
that is identical or highly similar to a target gene results in the
sequence specific degradation or specific post-transcriptional gene
silencing (PTGS) of messenger RNA (mRNA) transcribed from that
targeted gene (see Coburn, G. and Cullen, B., J. of Virology
76(18):9225 (2002)), thereby inhibiting expression of the target
gene. In one embodiment, the RNA is double stranded RNA (dsRNA).
This process has been described in plants, invertebrates, and
mammalian cells. In nature, RNAi is initiated by the dsRNA-specific
endonuclease Dicer, which promotes processive cleavage of long
dsRNA into double-stranded fragments termed siRNAs. siRNAs are
incorporated into a protein complex (termed "RNA induced silencing
complex," or "RISC") that recognizes and cleaves target mRNAs. RNAi
can also be initiated by introducing nucleic acid molecules, e.g.,
synthetic siRNAs or RNA interfering agents, to inhibit or silence
the expression of target genes. As used herein, "inhibition of
target gene expression" includes any decrease in expression or
protein activity or level of the target gene or protein encoded by
the target gene as compared to a situation wherein no RNA
interference has been induced. The decrease may be of at least 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared
to the expression of a target gene or the activity or level of the
protein encoded by a target gene which has not been targeted by an
RNA interfering agent.
[0857] "Short interfering RNA" (siRNA), also referred to herein as
"small interfering RNA" is defined as a nucleic acid-comprising
agent which functions to inhibit expression of a target gene, by
RNAi. An siRNA may be chemically synthesized, may be produced by in
vitro transcription, or may be produced within a host cell. In one
embodiment, siRNA is a double stranded RNA (dsRNA) molecule of
about 15 to about 40 nucleotides in length, preferably about 15 to
about 28 nucleotides, more preferably about 19 to about 25
nucleotides in length, and more preferably about 19, 20, 21, 22, or
23 nucleotides in length, and may contain a 3' and/or 5' overhang
on each strand having a length of about 0, 1, 2, 3, 4, or 5
nucleotides. The length of the overhang is independent between the
two strands, i.e., the length of the overhang on one strand is not
dependent on the length of the overhang on the second strand.
Preferably the siRNA is capable of promoting RNA interference
through degradation or specific post-transcriptional gene silencing
(PTGS) of the target messenger RNA (mRNA).
[0858] siRNAs also include small hairpin (also called stem loop)
RNAs (shRNAs). In one embodiment, these shRNAs are composed of a
short (e.g., about 19 to about 25 nucleotide) antisense strand,
followed by a nucleotide loop of about 5 to about 9 nucleotides,
and the analogous sense strand. Alternatively, the sense strand may
precede the nucleotide loop structure and the antisense strand may
follow. These shRNAs may be encoded by plasmids, retroviruses, and
lentiviruses and expressed from, for example, the pol III U6
promoter, or another promoter (see, e.g., Stewart, et al., RNA
April; 9(4):493-501 (2003), incorporated by reference herein in its
entirety).
[0859] The target gene or sequence of the RNA interfering agent may
be a cellular gene or genomic sequence, e.g. the TGF.beta.R
sequence. An siRNA may be substantially homologous to the target
gene or genomic sequence, or a fragment thereof. As used in this
context, the term "homologous" is defined as being substantially
identical, sufficiently complementary, or similar to the target
mRNA, or a fragment thereof, to effect RNA interference of the
target. In addition to native RNA molecules, RNA suitable for
inhibiting or interfering with the expression of a target sequence
include RNA derivatives and analogs. Preferably, the siRNA is
identical in sequence to its target.
[0860] The siRNA preferably targets only one sequence. Each of the
RNA interfering agents, such as siRNAs, can be screened for
potential off-target effects by, for example, expression profiling.
Such methods are known to one skilled in the art and are described,
for example, in Jackson et al., Nature Biotechnology 6:635-637
(2003). In addition to expression profiling, one may also screen
the potential target sequences for similar sequences in the
sequence databases to identify potential sequences which may have
off-target effects. For example, according to Jackson et al. (Id.)
15, or perhaps as few as 11 contiguous nucleotides, of sequence
identity are sufficient to direct silencing of non-targeted
transcripts. Therefore, one may initially screen the proposed
siRNAs to avoid potential off-target silencing using sequence
identity analysis by any known sequence comparison methods, such as
BLAST.
[0861] siRNA molecules need not be limited to those molecules
containing only RNA, but, for example, further encompasses
chemically modified nucleotides and non-nucleotides that effect RNA
interference, and also include molecules wherein a ribose sugar
molecule is substituted for another sugar molecule or a molecule
which performs a similar function. Moreover, a non-natural linkage
between nucleotide residues can be used, such as a phosphorothioate
linkage. The RNA strand can be derivatized with a reactive
functional group or a reporter group, such as a fluorophore.
Particularly useful derivatives are modified at a terminus or
termini of an RNA strand, typically the 3' terminus of the sense
strand. For example, the 2'-hydroxyl at the 3' terminus can be
readily and selectively derivatized with a variety of groups.
[0862] Other useful RNA derivatives incorporate nucleotides having
modified carbohydrate moieties, such as 2'O-alkylated residues or
2'-O-methyl ribosyl derivatives and 2'-O-fluoro ribosyl
derivatives. The RNA bases may also be modified. Any modified base
useful for inhibiting or interfering with the expression of a
target sequence may be used. For example, halogenated bases, such
as 5-bromouracil and 5-iodouracil can be incorporated. The bases
may also be alkylated, for example, 7-methylguanosine can be
incorporated in place of a guanosine residue. Non-natural bases
that yield successful inhibition can also be incorporated.
[0863] The most preferred siRNA modifications include
2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides
and RNA duplexes containing either phosphodiester or varying
numbers of phosphorothioate linkages. Such modifications are known
to one skilled in the art and are described, for example, in
Braasch et al., Biochemistry 42: 7967-7975 (2003). Most of the
useful modifications to the siRNA molecules can be introduced using
chemistries established for antisense oligonucleotide technology.
Preferably, the modifications involve minimal 2'-O-methyl
modification, preferably excluding such modification. Modifications
also preferably exclude modifications of the free 5'-hydroxyl
groups of the siRNA.
[0864] siRNAs useful for targeting TGF.beta.R or ALK5 expression
can be readily designed and tested. Chalk et al. (Nucl. Acids Res.
33: D131-D134 (2005)) describe a database of siRNA sequences and a
predictor of siRNA sequences. Linked to the sequences in the
database is information such as siRNA thermodynamic properties and
the potential for sequence-specific off-target effects. The
database and associated predictive tools enable the user to
evaluate an siRNA's potential for inhibition and non-specific
effects. The database is available at on the world wide web at
siRNA.cgb.ki.se.
[0865] Synthetic siRNA molecules, including shRNA molecules, can be
obtained using a number of techniques known to those of skill in
the art. For example, the siRNA molecule can be chemically
synthesized or recombinantly produced using methods known in the
art, such as using appropriately protected ribonucleoside
phosphoramidites and a conventional DNA/RNA synthesizer (see, e.g.,
Elbashir, S. M. et al., Nature 411:494-498 (2001); Elbashir, S. M.,
et al., Genes & Development 15:188-200 (2001); Harborth, J. et
al., J. Cell Science 114:4557-4565 (2001); Masters, J. R. et al.,
Proc. Natl. Acad. Sci., USA 98:8012-8017 (2001); and Tuschl, T. et
al., Genes & Development 13:3191-3197 (1999)). Alternatively,
several commercial RNA synthesis suppliers are available including,
but not limited to, Proligo (Hamburg, Germany), Dharmacon Research
(Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science,
Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes
(Ashland, Mass., USA), and Cruachem (Glasgow, UK). As such, siRNA
molecules are not overly difficult to synthesize and are readily
provided in a quality suitable for RNAi. In addition, dsRNAs can be
expressed as stem loop structures encoded by plasmid vectors,
retroviruses and lentiviruses (Paddison, P. J. et al., Genes Dev.
16:948-958 (2002); McManus, M. T. et al., RNA 8:842-850 (2002);
Paul, C. P. et al., Nat. Biotechnol. 20:505-508 (2002); Miyagishi,
M. et al., Nat. Biotechnol. 20:497-500 (2002); Sui, G. et al.,
Proc. Natl. Acad. Sci., USA 99:5515-5520 (2002); Brummelkamp, T. et
al., Cancer Cell 2:243 (2002); Lee, N. S., et al., Nat. Biotechnol.
20:500-505 (2002); Yu, J. Y., et al., Proc. Natl. Acad. Sci., USA
99:6047-6052 (2002); Zeng, Y., et al., Mol. Cell. 9:1327-1333
(2002); Rubinson, D. A., et al., Nat. Genet. 33:401-406 (2003);
Stewart, S. A., et al., RNA 9:493-501 (2003)).
[0866] In the methods described herein, the RNA interference
molecule is contacted with somatic cells in culture, thus
eliminating delivery problems inherent with administering e.g.,
siRNA in vivo to a patient in need thereof.
Confirming Pluripotency and Cell Reprogramming
[0867] To confirm the induction of pluripotent stem cells, isolated
clones can be tested for the expression of a stem cell marker. Such
expression identifies the cells as induced pluripotent stem cells.
Stem cell markers can be selected from the non-limiting group
including SSEA1, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4,
Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. Methods for
detecting the expression of such markers can include, for example,
RT-PCR and immunological methods that detect the presence of the
encoded polypeptides.
[0868] The pluripotent stem cell character of the isolated cells
can be confirmed by any of a number of tests evaluating the
expression of ES markers and the ability to differentiate to cells
of each of the three germ layers. As one example, teratoma
formation in nude mice can be used to evaluate the pluripotent
character of the isolated clones. The cells are introduced to nude
mice and histology is performed on a tumor arising from the cells.
The growth of a tumor comprising cells from all three germ layers
further indicates that the cells are pluripotent stem cells.
[0869] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural references
unless the context clearly dictates otherwise. Thus for example,
references to "the method" includes one or more methods, and/or
steps of the type described herein and/or which will become
apparent to those persons skilled in the art upon reading this
disclosure and so forth.
[0870] It is understood that the foregoing detailed description and
the following examples are illustrative only and are not to be
taken as limitations upon the scope of the invention. Various
changes and modifications to the disclosed embodiments, which will
be apparent to those of skill in the art, may be made without
departing from the spirit and scope of the present invention.
Further, all patents, patent applications, and publications
identified are expressly incorporated herein by reference for the
purpose of describing and disclosing, for example, the
methodologies described in such publications that might be used in
connection with the present invention. These publications are
provided solely for their disclosure prior to the filing date of
the present application. Nothing in this regard should be construed
as an admission that the inventors are not entitled to antedate
such disclosure by virtue of prior invention or for any other
reason. All statements as to the date or representation as to the
contents of these documents are based on the information available
to the applicants and do not constitute any admission as to the
correctness of the dates or contents of these documents.
[0871] The present invention may be as defined in any one of the
following numbered paragraphs. [0872] 1. A method for producing an
induced pluripotent stem cell from a somatic cell, the method
comprising: [0873] (a) treating a somatic cell to re-program it or
its progeny to a pluripotent stem cell phenotype; [0874] (b)
contacting the somatic cell or its progeny with an inhibitor of the
TGF-.beta. signaling pathway; and [0875] (c) isolating a
pluripotent stem cell from cells of step (b). [0876] 2. The method
of paragraph 1, wherein the treating comprises introducing a
nucleic acid sequence encoding one or more transcription factors
selected from the group consisting of Oct4, Sox2, c-MYC and Klf4 to
a somatic cell. [0877] 3. The method of paragraph 1, wherein the
inhibitor of the TGF-.beta. signaling pathway comprises an
inhibitor of TGF-.beta. receptor activity. [0878] 4. The method of
paragraph 3, wherein the inhibitor of TGF-.beta. receptor activity
comprises an inhibitor of ALK4, ALK5, or ALK7. [0879] 5. The method
of paragraph 3, wherein the inhibitor of TGF-.beta. receptor
activity is selected from the group consisting of an antibody, a
small molecule, and an RNA interference molecule. [0880] 6. The
method of paragraph 5, wherein the small molecule is selected from
the group consisting of
2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 napththyridine,
[3-(Pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and
3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole-
. [0881] 7. The method of paragraph 2, wherein the nucleic acid
sequences are comprised in a viral vector or a plasmid. [0882] 8.
The method of paragraph 1, wherein production of the induced
pluripotent stem cell is determined by detection of a stem cell
marker. [0883] 9. The method of paragraph 8, wherein the stem cell
marker is selected from the group consisting of SSEA1, CD9, Nanog,
Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3,
Rex1, Utf1, and Natl. [0884] 10. A method for increasing the
efficiency of induced pluripotent stem cell production, the method
comprising: [0885] (a) treating a somatic cell to re-program it or
its progeny to a pluripotent stem cell phenotype; and [0886] (b)
contacting the somatic cells subjected to the process of step (a)
with an inhibitor of the TGF-.beta. signaling pathway, [0887]
wherein the efficiency of induced pluripotent stem cell generation
is increased relative to such generation occurring without the
contacting. [0888] 11. The method of paragraph 10, wherein the
treating comprises introducing a nucleic acid sequence encoding one
or more transcription factors selected from the group consisting of
Oct4, Sox2, c-MYC and Klf4 to a somatic cell. [0889] 12. The method
of paragraph 10, wherein the inhibitor of the TGF-.beta. signaling
pathway comprises an inhibitor of TGF-.beta. receptor activity.
[0890] 13. The method of paragraph 12, wherein the inhibitor of
TGF-.beta. receptor activity comprises an inhibitor of ALK4, ALK5,
or ALK7. [0891] 14. The method of paragraph 12, wherein the
inhibitor of TGF-.beta. receptor activity is selected from the
group consisting of an antibody, a small molecule, and an RNA
interference molecule. [0892] 15. The method of paragraph 14,
wherein the small molecule is selected from the group consisting of
2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 napththyridine,
[3-(Pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and
3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole-
. [0893] 16. The method of paragraph 11, wherein the nucleic acid
sequences are comprised in a viral vector or a plasmid. [0894] 17.
The method of paragraph 10, wherein production of the induced
pluripotent stem cell is confirmed by detection of a stem cell
marker. [0895] 18. The method of paragraph 17, wherein the stem
cell marker is selected from the group consisting of SSEA1, CD9,
Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296,
Slc2a3, Rex1, Utf1, and Natl. [0896] 19. A method for increasing
the rate of induced pluripotent stem cell production, the method
comprising: [0897] (a) treating a somatic cell to re-program it or
its progeny to a pluripotent stem cell phenotype; and [0898] (b)
contacting the somatic cell with an inhibitor of the TGF-.beta.
signaling pathway, wherein the rate of induced pluripotent stem
cell generation is increased relative to the rate of the generation
occurring without the contacting. [0899] 20. The method of
paragraph 19, wherein the treating comprises introducing a nucleic
acid sequence encoding one or more transcription factors selected
from the group consisting of Oct4, Sox2, c-MYC and Klf4 to a
somatic cell. [0900] 21. The method of paragraph 19, wherein the
inhibitor of the TGF-.beta. signaling pathway comprises an
inhibitor of TGF-.beta. receptor activity. [0901] 22. The method of
paragraph 21, wherein the inhibitor of TGF-.beta. receptor activity
comprises an inhibitor of ALK4, ALK5, or ALK7. [0902] 23. The
method of paragraph 21, wherein the inhibitor of TGF-.beta.
receptor activity is selected from the group consisting of an
antibody, a small molecule, and an RNA interference molecule.
[0903] 24. The method of paragraph 23, wherein the small molecule
is selected from the group consisting of
2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 napththyridine,
[3-(Pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and
3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole-
. [0904] 25. The method of paragraph 20, wherein the nucleic acid
sequences are comprised in a viral vector or a plasmid. [0905] 26.
The method of paragraph 19, wherein production of the induced
pluripotent stem cell is confirmed by detection of a stem cell
marker. [0906] 27. The method of paragraph 26, wherein the stem
cell marker is selected from the group consisting of SSEA1, CD9,
Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296,
Slc2a3, Rex1, Utf1, and Natl. [0907] 28. An induced pluripotent
stem cell composition comprising an induced pluripotent stem cell
in an admixture with an inhibitor of the TGF-.beta. signaling
pathway. [0908] 29. The composition of paragraph 28, wherein the
inhibitor of the TGF-.beta. signaling pathway comprises an
inhibitor of TGF-.beta. receptor activity. [0909] 30. The
composition of paragraph 29, wherein the inhibitor of TGF-.beta.
receptor activity comprises an inhibitor of ALK4, ALK5, or ALK7.
[0910] 31. The composition of paragraph 29, wherein the inhibitor
of TGF-.beta. receptor activity is selected from the group
consisting of an antibody, a small molecule, and an RNA
interference molecule. [0911] 32. The composition of paragraph 31,
wherein the small molecule is selected from the group consisting of
2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 napththyridine,
[3-(Pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and
3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole-
. [0912] 33. The composition of paragraph 29, wherein the induced
pluripotent stem cell expresses a stem cell marker. [0913] 34. The
composition of paragraph 33, wherein the stem cell marker is
selected from the group consisting of SSEA1, CD9, Nanog, Fbx15,
Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1,
Utf1, and Natl. [0914] 35. A kit for producing induced pluripotent
stem cells, the kit comprising: [0915] (a) nucleic acid sequences
encoding one or more transcription factors selected from the group
consisting of Oct4, Sox2, c-MYC and Klf4, [0916] (b) an inhibitor
of the TGF-.beta. receptor signaling pathway, [0917] (c) packaging
materials therefor. [0918] 36. The kit of paragraph 35, wherein the
inhibitor of the TGF-.beta. signaling pathway comprises an
inhibitor of TGF-.beta. receptor activity. [0919] 37. The kit of
paragraph 35, wherein the inhibitor of TGF-.beta. receptor activity
comprises an inhibitor of ALK4, ALK5, or ALK7. [0920] 38. The kit
of paragraph 35, wherein the inhibitor of TGF-.beta. receptor
activity is selected from the group consisting of an antibody, a
small molecule, and an RNA interference molecule. [0921] 39. The
kit of paragraph 38, wherein the small molecule is selected from
the group consisting of
2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 napththyridine,
[3-(Pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and
3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole-
. [0922] 40. The kit of paragraph 35, wherein the nucleic acid
sequences are comprised in a viral vector or a plasmid. [0923] 41.
The kit of paragraph 35, comprising each of the transcription
factors selected from the group consisting of Oct4, Sox2, c-MYC and
Klf4. [0924] 42. A cell composition prepared by the method of any
one of paragraphs 1-27.
EXAMPLES
[0925] The following Examples indicate that inhibition of the
TGF-.beta. signaling pathway acts as a cooperative factor in the
reprogramming of murine fibroblasts, enabling more efficient and
faster induction of iPSCs in a dose-dependent manner, while
activation of TGF-.beta. signaling blocks reprogramming. In
addition to a strong cooperative effect, use of a TGF-.beta.
receptor inhibitor bypasses the requirement of exogenous cMyc or
Sox2, highlighting its dual role as both a cooperative and a
replacement factor. The identification of a highly characterized
pathway that operates in reprogramming will open up new avenues for
mechanistic dissection of the reprogramming process, as well as
facilitate the derivation of iPSCs using small molecules.
[0926] The TGF.beta. signaling pathway is identified as a
cooperative pathway in reprogramming during functional validation
of a constructed network that linked retroviral insertion sites
across several mouse iPSC lines (Varas, F., et al., (2008) Stem
Cells). Although the biological validation of network targets was
largely negative, in agreement with statistical analyses indicating
that the network did not differ from randomly constructed networks,
it was found that use of a TGF-.beta. receptor I
kinase/activin-like kinase 5 (Alk5) inhibitor enhances the
efficiency of iPSC derivation.
Example 1
[0927] Fibroblasts cultures were established from mice expressing
green-fluorescent protein (GFP) from the genomic locus of the
pluripotency gene Sox2. Sox2-GFP fibroblasts were then infected
with viruses expressing the reprogramming factors c-Myc, Klf4, Oct4
and Sox2 in the presence and absence of a chemical inhibitor highly
specific for the TGF-beta receptor Alk5 (ALK5 inhibitor II). GFP+
colonies with embryonic stem (ES) cell morphology were scored 20
days after infection. In the presence of Alk5 inhibitor II, 5-10
fold more GFP+ colonies were observed, which after picking could be
expanded like ES cells (see FIG. 1A). In addition, those colonies
that formed in the presence of the Alk5 inhibitor started
expressing GFP earlier than those that formed in the absence of the
chemical, indicating that inhibiting Alk5 accelerates the
activation of endogenous pluripotency genes (see FIG. 1B). These
observations indicate that using chemicals to interfere with
TGF-beta signaling is a way to increase both the kinetics and the
efficiency of reprogramming.
Example 2
[0928] To induce reprogramming, MEFs were infected with a reverse
tetracycline transactivator (rtTA) transgene with
doxycycline-inducible lentiviruses encoding the four reprogramming
factors as previously described (Stadtfeld, M., et al., (2008) Cell
Stem Cell 2, 230-240). Administration of the Alk5 inhibitor during
the course of doxycycline (dox) induction elicited a striking
increase in the number of iPSC colonies (FIG. 2A). These iPSC
clones could be expanded in the absence of dox, which is a strong
indicator of successful reprogramming (Stadtfeld, M., et al.,
(2008) Cell Stem Cell 2, 230-240; Maherali, N., et al., (2008) Cell
Stem Cell 3, 340-345). This robust increase in efficiency prompted
experiments examining whether the Alk5 inhibitor could also reduce
the temporal requirement of factor expression, thus reflecting an
increase in the kinetics of reprogramming. To test this,
doxycycline was applied for three, four, or five days on
four-factor infected MEFs, either in the presence or absence of the
Alk5 inhibitor (FIG. 2B). While iPSCs were not obtained with three
days of dox treatment in either condition, four days of dox
treatment was sufficient to give rise to iPSC colonies at a
frequency of 0.0013% in the inhibitor-treated condition. These
colonies were not immediately apparent and took at least one week
after dox withdrawal to emerge. No colonies were observed in the
control condition, demonstrating that the inhibitor promoted faster
induction of iPSCs. 4-day iPSCs expressed the pluripotency markers
Nanog, Oct4, and Sox2 (FIG. 2C), which was tested three passages
after dox withdrawal to ensure a lack of residual transgene
expression (Maherali, N., et al., (2008) Cell Stem Cell 3,
340-345). These cells were also competent to form lineages from all
three germ layers in the context of a teratoma (FIG. 2C), providing
a functional test of pluripotency and indicating that use of the
Alk5 inhibitor during reprogramming has no adverse effect on the
resulting iPSCs.
[0929] It was next sought to establish a dose-response curve for
the Alk5 inhibitor. Using four-factor infected MEFs, concentrations
of the Alk5 inhibitor were tested over a 1000-fold range, from 4 nM
to 4 .mu.M. Increasing concentrations of inhibitor led to a
progressive increase in the number of iPSC colonies obtained,
resulting in a 30-fold enhancement at the highest dose tested (FIG.
2D). To verify that manipulation of TGF-.beta. signaling affects
reprogramming, it was investigated whether pathway activation would
inhibit iPSC formation. Using four-factor infected MEFs, increasing
concentrations of TGF-.beta.1 or TGF-.beta.2 were applied during
the time of dox induction. Under control conditions, an average of
14 colonies per well (.about.0.017%) were obtained; however, upon
addition of 1 ng/mL TGF-.beta.1 or TGF-.beta.2, the efficiency was
reduced to one colony per well (.about.0.0013%), and at 2.5 ng/mL
and 5 ng/mL, iPSC formation was undetectable (FIG. 2E).
[0930] The opposing effects of pathway activation and inhibition
confirm the involvement of TGF-.beta. signaling in reprogramming.
To gain further insight into the mode of action of the Alk5
inhibitor, the temporal window during which the inhibitor elicited
its strongest effect was examined. In four-factor infected MEFs,
the inhibitor was applied in 4-day intervals from days 0-16 of
reprogramming. The largest increase in efficiency was observed
during the first four days of reprogramming, yielding an increase
comparable to full-time application of the inhibitor (FIG. 2F). A
strong enhancement was also observed with application during the
5-8 day interval, while after dox withdrawal (day 12), the
inhibitor appeared to have no effect. This early-acting effect
prompted experiments to investigate whether the Alk5 inhibitor was
acting to `prime` the cells, thus altering or destabilizing the
fibroblast state so that it is more amenable to reprogramming. To
test this, the inhibitor was applied in 3-day time intervals:
before dox administration (days -3 to 0) to test for a priming
effect, during the first 3 days of reprogramming (days 0 to +3) as
a control for the efficiency increase, during both intervals (days
-3 to +3) to test for a synergistic effect, and a no-inhibitor
control as a baseline measure (FIG. 1G).
[0931] Administration of the Alk5 inhibitor prior to expression of
the reprogramming factors (days -3 to 0) led to a slight reduction
in efficiency, thus arguing against a priming effect. In contrast,
addition of the inhibitor during the first 3 days of factor
expression led to .about.5-fold enhancement in efficiency,
consistent with previous findings. The increase in efficiency seen
in the -3 d/+3 d group was comparable to the 0 to +3d group,
indicating a lack of a synergistic effect. It therefore appears
that Alk5 inhibition must coincide with expression of the
reprogramming factors in order to elicit its effect. One limitation
of direct infection is that not every cell receives the full
complement of reprogramming factors, and there is a large amount of
heterogeneity in expression, as each cell harbors a unique pattern
of viral insertions. It is therefore difficult to ascertain whether
the increase in efficiency observed so far is due solely to a
cooperative effect, where the inhibitor enhances efficiency in the
presence of all four factors, or a replacement effect, where the
inhibitor substitutes for specific reprogramming factors. To this
end, we employed a "secondary system" consisting of MEFs isolated
from iPSC-derived chimeras. These MEFs contain a homogenous set of
inducible lentiviral integrations that were permissive for
conversion into primary iPSCs; thus, the levels and stoichiometry
of the re-expressed factors are unique to each MEF line (Maherali,
N., et al., (2008) Cell Stem Cell 3, 340-345; Wernig, M., et al.,
(2008) Nat Biotechnol 26, 916-924).
[0932] In two different secondary MEF lines, the effect of Alk5
inhibition on both the efficiency and kinetics of reprogramming was
tested. In this experiment, dox was applied for 4, 6, 8, or 10
days, either in the presence or absence of the Alk5 inhibitor, then
quantified iPSC colony number on day 16. The baseline levels of
reprogramming differed between the two lines (FIG. 2A), which was
not unexpected given that efficiency is dependent upon the levels
and stoichiometry of factor expression as previously reported
(Wernig, M., et al., (2008) Nat Biotechnol 26, 916-924). In both
lines, addition of the Alk5 inhibitor mediated an increase in both
efficiency and kinetics of reprogramming, consistent with a
cooperative effect (FIG. 3A). However, the degree to which
reprogramming was enhanced was different between the two lines,
with one line showing a 2-fold increase and the other a 30-fold
increase.
[0933] Without wishing to be bound by theory it was reasoned that a
purely cooperative action would produce a consistent fold-increase
between the different lines regardless of variation in factor
expression levels, which was not the case. Thus, to further discern
between a cooperative and a replacement effect, a polycistronic
construct was employed that linked all four reprogramming factors
on a single transcript ("STEMCCA") (Sommer, C. A., et al., (2009)
Stem Cells 27, 543-549), which in the context of a secondary system
enables expression of all factors in >95% of cells (data not
shown). In such a system, only a cooperative effect would be
revealed, as co-expression of all factors precludes factor
replacement.
[0934] Secondary STEMCCA MEFs were used to establish another
dose-response curve for the Alk5 inhibitor. In contrast to primary
infected MEFs, which demonstrated a steady increase across the
entire dose range tested (FIG. 2D), the STEMCCA MEFs reached a
maximum efficiency at 0.5 .mu.M, representing a .about.60-fold
increase, and began to decline at higher doses (FIG. 3B). A similar
pattern was also observed with another inhibitor, SB-431542, that
targets the type I Tgf.beta. receptors, Alk-4, -5, and -7 (FIG. 5).
These data corroborate the notion of a cooperative effect and also
introduce the possibility of a factor replacement effect that
operates at a different dose range, thus explaining the persistent
efficiency increase with higher doses of the Alk5 inhibitor in
primary infected MEFs, yet a decline in the secondary STEMCCA
MEFs.
[0935] Experiments were designed to investigate whether the Alk5
inhibitor could function to replace exogenous expression of any of
the reprogramming factors. To this end, primary MEFs were infected
with different combinations of the four factors in the presence or
absence of inhibitor (FIG. 4) and then scored for iPSC formation.
In the four-factor control infection (OSMK; O=Oct4, S=Sox2, M=cMyc,
K=Klf4), colonies were already apparent after 6 days; dox was
withdrawn on day 8 to avoid detrimental effects of prolonged factor
expression (Mikkelsen, T. S., et al., (2008) Nature 454, 49-55). At
this time colonies were observed in the cMyc replacement condition
(OSK+inhibitor), but none without the inhibitor; thus, dox was
withdrawn from these cultures on day 8 as well. Remarkably, use of
the Alk5 inhibitor (OSK+inhibitor) in place of cMyc resulted in a
2.5-fold higher efficiency than with the four factors alone
(OSK+M), indicating that Alk5 inhibition was more potent than the
action of cMyc in promoting reprogramming (FIG. 3C).
[0936] For all of the other replacement conditions, dox was
withdrawn on day 16, which would provide sufficient time for
reprogramming. While most conditions did not yield any obvious
colonies, the Sox2 replacement condition contained numerous
colonies morphologically similar to iPSCs. These colonies appeared
late in reprogramming, becoming readily apparent after 14 days.
Based on morphology, 50 colonies were observed in the
inhibitor-treated culture and 4 colonies in the control condition
that were identical to iPSCs (data not shown). Withdrawal of dox,
however, led to regression of many colonies in both conditions,
indicating that the cells were not independent of factor
expression. Nonetheless, colonies were picked from both conditions
and tested for their potential to form dox-independent lines. From
the control condition, 0/4 were capable of expansion, indicating
that infection with the three factors (OMK) was not sufficient to
induce reprogramming, while in the inhibitor-treated culture, 3/22
colonies formed dox-independent iPSC lines, demonstrating
successful replacement of Sox2. The efficiency of expansion was low
compared to that of four-factor iPSC clones, where all picked
colonies (6/6 in this set of experiments) were capable of forming
stable dox-independent lines.
[0937] Immunostaining of the OMK+inhibitor iPSC lines demonstrated
expression of Nanog, Oct4, and Sox2 (tested after three passages
without dox) (FIG. 3E). To further verify expression of
pluripotency genes, quantitative RT-PCR analysis was performed
using primer sets that distinguish endogenous and viral transcripts
(FIG. 6A), which revealed pluripotency gene activation and viral
gene silencing. To functionally assess pluripotency of the
OMK+inhibitor iPSCs, teratomas were generated, which contained
lineages from all three germ layers (FIG. 6B). The ability of these
cells to contribute to chimeric mice was tested; to this end, iPSCs
were labeled with a lentivirus constitutively expressing the
fluorescent protein, tdTomato, sorted the cells by flow cytometry,
and injected them into blastocysts. Mice harvested on embryonic day
E16.5 showed varying degrees of contribution (FIG. 3F).
[0938] As a more stringent test of pluripotency, the cells were
able to generate adult chimeras (FIG. 3G), though their potential
for germline transmission has not yet been evaluated. To ensure
that these iPSCs were free of Sox2 viral integrations, Southern
blot analysis was performed using a Sox2 cDNA probe. No extraneous
bands were observed in the OMK+inhibitor lines, confirming the
absence of exogenous Sox2 (FIG. 6C).
[0939] As a final test to determine whether the Alk5 inhibitor
enabled iPSC formation under any of the replacement conditions, the
primary cultures were passaged in the absence of dox, which permit
the amplification and selection of dox-independent cells that may
not have been visible in the initial culture. OSK and OSMK
conditions were excluded since these readily formed iPSCs.
Confirming the observations from the primary cultures, only the
Sox2 replacement condition (OMK+inhibitor) gave rise to
dox-independent colonies (FIG. 3D). The control condition
(OMK-inhibitor) also contained colonies that expressed alkaline
phosphatase (FIG. 3D, upper left); however, these colonies appeared
fibroblastic and did not grow, indicating that they were not iPSCs.
Interestingly, while the Alk5 inhibitor enabled colony formation in
the absence of either cMyc or Sox2, colonies could not be obtained
in the absence of both factors (OK; FIG. 3D), indicating that the
inhibitor performs distinct functions from Sox2 and cMyc but can
preferentially assume their role in the context of reprogramming
with the three remaining factors.
[0940] The results presented here demonstrate that TGF-.beta.
receptor I kinase inhibition enhances both the efficiency and
kinetics of reprogramming in a dose-dependent manner, while
activation of the TGF-.beta. signaling pathway blocks
reprogramming. The Alk5 inhibitor exerts its strongest effect
during the early stages of iPSC induction and acts in concert with
the reprogramming factors to mediate its effect, rather than
converting the fibroblasts to a state more amenable to
reprogramming. In addition to its cooperative action, the Alk5
inhibitor can replace the individual roles of cMyc or Sox2,
although it cannot replace them simultaneously. These results
provide the first defined pathway that produces both a strong
cooperative effect and can preferentially replace the roles of
specific reprogramming factors.
[0941] An important question that remains is how Alk5 inhibition
acts on a molecular level to enhance reprogramming. It was observed
that application of the inhibitor prior to factor expression was
unable to mediate an increase in efficiency, indicative of a
transient effect and/or one that is context-dependent, requiring
expression of the reprogramming factors to carry out its role. The
observation that the Alk5 inhibitor acts early in reprogramming
raises the question of whether it helps shut down the fibroblast
gene expression program. In support of this, it has been shown that
activation of TGF-.beta. signaling can promote an
epithelial-to-mesenchymal transition (Valcourt, U., et al., (2005)
Mol Biol Cell 16, 1987-2002); fibroblast reprogramming involves a
mesenchymal-to-epithelial conversion, which may be enhanced by
inhibition of the TGF-.beta. signaling pathway. An enhancement in
efficiency could also be mediated through increased proliferation.
This possibility was tested by examining proliferation rates during
reprogramming, but it was found that the inhibitor led to a
decrease in proliferation around day 6 (FIG. 7A). It was noted that
the inhibitor also mitigated the decrease in cell number that
normally accompanies reprogramming (day 2) (FIG. 7A); however, the
magnitude of this effect was unlikely to explain the large increase
in overall reprogramming efficiency. Further, quantification of
apoptosis with Annexin V staining showed a negligible difference
between inhibitor-treated and untreated cells (FIG. 7B).
[0942] As yet another possibility, the Alk5 inhibitor was tested in
the context of reprogramming by cell fusion (embryonic stem
cells+MEFs), which led to a negative result (data not shown). This
observation raises the question of whether Alk5 inhibition acts on
pathways in direct reprogramming that are already operational in
embryonic stem cells (ESCs), or whether the mechanisms of
reprogramming by direct factors or cell fusion are fundamentally
different. Without wishing to be bound by theory, the finding that
the Alk5 inhibitor could replace either cMyc or Sox2, but not both
together, indicates that its functions are distinct from these
reprogramming factors. It is possible that replacement of both
factors could occur with different doses of the inhibitor or with a
longer period of dox administration; however, the conditions used
that enabled replacement of the individual factors were unable to
elicit simultaneous replacement, indicating a unique mode of
action. It was tested whether the Alk5 inhibitor simply led to
induction of Sox2 or cMyc expression; however, analysis by qRT-PCR
revealed that only cMyc was significantly induced (1.3-fold) in
OSK+inhibitor treated cells (FIG. 8), and there was no significant
induction of Sox2 or cMyc in any of the other cells (OMK or OSMK).
A possibility is that the Alk5 inhibitor acts on a pathway that
completely bypasses the need for the individual reprogramming
factors; for instance, it has been shown that Sox2 is not required
for reprogramming by fusion of mESCs and human B lymphocytes
(Pereira, C. F., et al., (2008) PLoS Genet. 4, e11000170).
[0943] Several TGF-.beta. superfamily members represent important
contributors to the pluripotent state. For instance, BMP4 signaling
helps maintain mouse ESCs in an undifferentiated state (Ying, Q.
L., et al., (2003) Cell 115, 281-292), and use of an Alk5 inhibitor
in conjunction with GSK33 and MEK inhibitors facilitates rat and
human iPSC line propagation and supports a mouse ESC-like phenotype
(Li, W., et al., (2009) Cell Stem Cell 4, 16-19). In contrast to
its pluripotency-promoting effect in mouse ESCs, BMP4 induces
differentiation of human ESCs towards trophectoderm (Xu, R. H., et
al., (2002) Nat Biotechnol 20, 1261-1264), while Activin signaling
maintains pluripotency of hESCs (Xu, R. H., et al., (2008) Cell
Stem Cell 3, 196-206). The differential requirements of TGF-.beta.
signaling in mouse and human ESCs may explain why an increase in
the efficiency of human fibroblast reprogramming was not observed
(data not shown); it will be interesting to see whether activation
of other pathways that promote the undifferentiated state also
enhance reprogramming.
[0944] The highly characterized nature of the TGF-.beta. signaling
pathway makes it an attractive model for examining how interactions
between downstream targets and the four factors synergize to
enhance reprogramming. The identification of such pathways will
greatly facilitate our understanding of reprogramming at the
molecular level, potentially leading to the discovery of novel
targets that promote reprogramming and are of therapeutic
value.
Example 3
Exemplary Methods Useful for the Methods Described Herein Virus
Production
[0945] Vectors were constructed as previously described (Stadtfeld,
M., et al., (2008) Cell Stem Cell 2, 230-240). For all experiments,
cells were infected overnight at an MOI of 10 in the presence of
polybrene 6 .mu.g/mL, which was experimentally determined to infect
>90% of cells.
Cell Culture and iPSC Induction
[0946] Cells were obtained from mice harboring a reverse
tetracycline transactivator (rtTA) in the Rosa locus (Hochedlinger,
K., et al. (2005) Cell 121, 465-477). Fibroblasts were derived from
E14.5 mice; all experiments were conducted prior to passage 3. To
induce reprogramming, cells were infected with the viral cocktail,
then split two days later into ESC media (15% FBS, Invitrogen; 1000
U/mL LIF) with doxycycline (1 .mu.g/mL). Fibroblast feeder cells
were used only to maintain iPSC lines, not during reprogramming.
The Alk5 inhibitor (Calbiochem/EMD 616452) was used at a
concentration of 1 .mu.M, unless otherwise noted. The Alk-4/5/7
inhibitor, SB-431542, was obtained from Sigma; TGF-.beta. ligands
were obtained from Peprotech (TGF-.beta.1, 100-21; TGF-.beta.2,
100-35).
Alkaline Phosphatase and Immunostaining
[0947] Alkaline phosphatase (AP) staining was done using an AP
substrate kit according to manufacturer directions (VectorLabs,
SK-5100). The following antibodies were used for immunostaining:
.alpha.-Nanog (1:200, ab21603, Abcam), .alpha.-Oct4 (1:100sc-8628,
Santa Cruz Biotech), .alpha.-Sox2 (1:200, AB5603, Millipore).
Quantitative RT-PCR
[0948] RNA was extracted by using a Qiagen RNeasy kit (74104), then
converted to cDNA with the Superscript III First-Strand synthesis
system (Invitrogen) using oligo-dT primers. qRT-PCRs were carried
out using Brilliant II SYBR Green mix (Stratagene) and run on a
Stratagene MXPro400. Reactions were carried out in duplicate with
--RT controls, and data were analyzed using the delta-delta Ct
method.
Apoptosis Assay
[0949] Annexin V and propidium iodide staining was done using the
Annexin V-FITC kit (BD Pharmingen, 556547) according to
manufacturer directions. Cells were analyzed by flow cytometry on a
FACSCalibur (BD).
Teratomas
[0950] A 25 cm.sup.2 equivalent of confluent iPSCs grown on feeders
was harvested and injected subcutaneously into SCID mice. Teratomas
were harvested 3 weeks later and stained with
hematoxylin/eosin.
Blastocyst Injections
[0951] BDF1 females were superovulated with PMS and hCG, then mated
with BDF1 males. Blastocysts were flushed from uterine horns on day
3.5, and 10-20 labelled iPSCs were injected per blastocyst.
Swiss-webster mice were set up with vasectomized males and used as
recipients for the injected blastocysts.
Sequence CWU 1
1
51503PRTHomo sapiens 1Met Glu Ala Ala Val Ala Ala Pro Arg Pro Arg
Leu Leu Leu Leu Val 1 5 10 15 Leu Ala Ala Ala Ala Ala Ala Ala Ala
Ala Leu Leu Pro Gly Ala Thr 20 25 30 Ala Leu Gln Cys Phe Cys His
Leu Cys Thr Lys Asp Asn Phe Thr Cys 35 40 45 Val Thr Asp Gly Leu
Cys Phe Val Ser Val Thr Glu Thr Thr Asp Lys 50 55 60 Val Ile His
Asn Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro Arg 65 70 75 80 Asp
Arg Pro Phe Val Cys Ala Pro Ser Ser Lys Thr Gly Ser Val Thr 85 90
95 Thr Thr Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu Pro
100 105 110 Thr Thr Val Lys Ser Ser Pro Gly Leu Gly Pro Val Glu Leu
Ala Ala 115 120 125 Val Ile Ala Gly Pro Val Cys Phe Val Cys Ile Ser
Leu Met Leu Met 130 135 140 Val Tyr Ile Cys His Asn Arg Thr Val Ile
His His Arg Val Pro Asn 145 150 155 160 Glu Glu Asp Pro Ser Leu Asp
Arg Pro Phe Ile Ser Glu Gly Thr Thr 165 170 175 Leu Lys Asp Leu Ile
Tyr Asp Met Thr Thr Ser Gly Ser Gly Ser Gly 180 185 190 Leu Pro Leu
Leu Val Gln Arg Thr Ile Ala Arg Thr Ile Val Leu Gln 195 200 205 Glu
Ser Ile Gly Lys Gly Arg Phe Gly Glu Val Trp Arg Gly Lys Trp 210 215
220 Arg Gly Glu Glu Val Ala Val Lys Ile Phe Ser Ser Arg Glu Glu Arg
225 230 235 240 Ser Trp Phe Arg Glu Ala Glu Ile Tyr Gln Thr Val Met
Leu Arg His 245 250 255 Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Asn
Lys Asp Asn Gly Thr 260 265 270 Trp Thr Gln Leu Trp Leu Val Ser Asp
Tyr His Glu His Gly Ser Leu 275 280 285 Phe Asp Tyr Leu Asn Arg Tyr
Thr Val Thr Val Glu Gly Met Ile Lys 290 295 300 Leu Ala Leu Ser Thr
Ala Ser Gly Leu Ala His Leu His Met Glu Ile 305 310 315 320 Val Gly
Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser 325 330 335
Lys Asn Ile Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu 340
345 350 Gly Leu Ala Val Arg His Asp Ser Ala Thr Asp Thr Ile Asp Ile
Ala 355 360 365 Pro Asn His Arg Val Gly Thr Lys Arg Tyr Met Ala Pro
Glu Val Leu 370 375 380 Asp Asp Ser Ile Asn Met Lys His Phe Glu Ser
Phe Lys Arg Ala Asp 385 390 395 400 Ile Tyr Ala Met Gly Leu Val Phe
Trp Glu Ile Ala Arg Arg Cys Ser 405 410 415 Ile Gly Gly Ile His Glu
Asp Tyr Gln Leu Pro Tyr Tyr Asp Leu Val 420 425 430 Pro Ser Asp Pro
Ser Val Glu Glu Met Arg Lys Val Val Cys Glu Gln 435 440 445 Lys Leu
Arg Pro Asn Ile Pro Asn Arg Trp Gln Ser Cys Glu Ala Leu 450 455 460
Arg Val Met Ala Lys Ile Met Arg Glu Cys Trp Tyr Ala Asn Gly Ala 465
470 475 480 Ala Arg Leu Thr Ala Leu Arg Ile Lys Lys Thr Leu Ser Gln
Leu Ser 485 490 495 Gln Gln Glu Gly Ile Lys Met 500 2503PRTHomo
sapiens 2Met Glu Ala Ala Val Ala Ala Pro Arg Pro Arg Leu Leu Leu
Leu Val 1 5 10 15 Leu Ala Ala Ala Ala Ala Ala Ala Ala Ala Leu Leu
Pro Gly Ala Thr 20 25 30 Ala Leu Gln Cys Phe Cys His Leu Cys Thr
Lys Asp Asn Phe Thr Cys 35 40 45 Val Thr Asp Gly Leu Cys Phe Val
Ser Val Thr Glu Thr Thr Asp Lys 50 55 60 Val Ile His Asn Ser Met
Cys Ile Ala Glu Ile Asp Leu Ile Pro Arg 65 70 75 80 Asp Arg Pro Phe
Val Cys Ala Pro Ser Ser Lys Thr Gly Ser Val Thr 85 90 95 Thr Thr
Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu Pro 100 105 110
Thr Thr Val Lys Ser Ser Pro Gly Leu Gly Pro Val Glu Leu Ala Ala 115
120 125 Val Ile Ala Gly Pro Val Cys Phe Val Cys Ile Ser Leu Met Leu
Met 130 135 140 Val Tyr Ile Cys His Asn Arg Thr Val Ile His His Arg
Val Pro Asn 145 150 155 160 Glu Glu Asp Pro Ser Leu Asp Arg Pro Phe
Ile Ser Glu Gly Thr Thr 165 170 175 Leu Lys Asp Leu Ile Tyr Asp Met
Thr Thr Ser Gly Ser Gly Ser Gly 180 185 190 Leu Pro Leu Leu Val Gln
Arg Thr Ile Ala Arg Thr Ile Val Leu Gln 195 200 205 Glu Ser Ile Gly
Lys Gly Arg Phe Gly Glu Val Trp Arg Gly Lys Trp 210 215 220 Arg Gly
Glu Glu Val Ala Val Lys Ile Phe Ser Ser Arg Glu Glu Arg 225 230 235
240 Ser Trp Phe Arg Glu Ala Glu Ile Tyr Gln Thr Val Met Leu Arg His
245 250 255 Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Asn Lys Asp Asn
Gly Thr 260 265 270 Trp Thr Gln Leu Trp Leu Val Ser Asp Tyr His Glu
His Gly Ser Leu 275 280 285 Phe Asp Tyr Leu Asn Arg Tyr Thr Val Thr
Val Glu Gly Met Ile Lys 290 295 300 Leu Ala Leu Ser Thr Ala Ser Gly
Leu Ala His Leu His Met Glu Ile 305 310 315 320 Val Gly Thr Gln Gly
Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser 325 330 335 Lys Asn Ile
Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu 340 345 350 Gly
Leu Ala Val Arg His Asp Ser Ala Thr Asp Thr Ile Asp Ile Ala 355 360
365 Pro Asn His Arg Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu
370 375 380 Asp Asp Ser Ile Asn Met Lys His Phe Glu Ser Phe Lys Arg
Ala Asp 385 390 395 400 Ile Tyr Ala Met Gly Leu Val Phe Trp Glu Ile
Ala Arg Arg Cys Ser 405 410 415 Ile Gly Gly Ile His Glu Asp Tyr Gln
Leu Pro Tyr Tyr Asp Leu Val 420 425 430 Pro Ser Asp Pro Ser Val Glu
Glu Met Arg Lys Val Val Cys Glu Gln 435 440 445 Lys Leu Arg Pro Asn
Ile Pro Asn Arg Trp Gln Ser Cys Glu Ala Leu 450 455 460 Arg Val Met
Ala Lys Ile Met Arg Glu Cys Trp Tyr Ala Asn Gly Ala 465 470 475 480
Ala Arg Leu Thr Ala Leu Arg Ile Lys Lys Thr Leu Ser Gln Leu Ser 485
490 495 Gln Gln Glu Gly Ile Lys Met 500 3426PRTHomo sapiens 3Met
Glu Ala Ala Val Ala Ala Pro Arg Pro Arg Leu Leu Leu Leu Val 1 5 10
15 Leu Ala Ala Ala Ala Ala Ala Ala Ala Ala Leu Leu Pro Gly Ala Thr
20 25 30 Ala Leu Gln Cys Phe Cys His Leu Cys Thr Lys Asp Asn Phe
Thr Cys 35 40 45 Val Thr Asp Gly Leu Cys Phe Val Ser Val Thr Glu
Thr Thr Asp Lys 50 55 60 Val Ile His Asn Ser Met Cys Ile Ala Glu
Ile Asp Leu Ile Pro Arg 65 70 75 80 Asp Arg Pro Phe Val Cys Ala Pro
Ser Ser Lys Thr Gly Ser Val Thr 85 90 95 Thr Thr Tyr Cys Cys Asn
Gln Asp His Cys Asn Lys Ile Glu Leu Pro 100 105 110 Thr Thr Gly Leu
Pro Leu Leu Val Gln Arg Thr Ile Ala Arg Thr Ile 115 120 125 Val Leu
Gln Glu Ser Ile Gly Lys Gly Arg Phe Gly Glu Val Trp Arg 130 135 140
Gly Lys Trp Arg Gly Glu Glu Val Ala Val Lys Ile Phe Ser Ser Arg 145
150 155 160 Glu Glu Arg Ser Trp Phe Arg Glu Ala Glu Ile Tyr Gln Thr
Val Met 165 170 175 Leu Arg His Glu Asn Ile Leu Gly Phe Ile Ala Ala
Asp Asn Lys Asp 180 185 190 Asn Gly Thr Trp Thr Gln Leu Trp Leu Val
Ser Asp Tyr His Glu His 195 200 205 Gly Ser Leu Phe Asp Tyr Leu Asn
Arg Tyr Thr Val Thr Val Glu Gly 210 215 220 Met Ile Lys Leu Ala Leu
Ser Thr Ala Ser Gly Leu Ala His Leu His 225 230 235 240 Met Glu Ile
Val Gly Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp 245 250 255 Leu
Lys Ser Lys Asn Ile Leu Val Lys Lys Asn Gly Thr Cys Cys Ile 260 265
270 Ala Asp Leu Gly Leu Ala Val Arg His Asp Ser Ala Thr Asp Thr Ile
275 280 285 Asp Ile Ala Pro Asn His Arg Val Gly Thr Lys Arg Tyr Met
Ala Pro 290 295 300 Glu Val Leu Asp Asp Ser Ile Asn Met Lys His Phe
Glu Ser Phe Lys 305 310 315 320 Arg Ala Asp Ile Tyr Ala Met Gly Leu
Val Phe Trp Glu Ile Ala Arg 325 330 335 Arg Cys Ser Ile Gly Gly Ile
His Glu Asp Tyr Gln Leu Pro Tyr Tyr 340 345 350 Asp Leu Val Pro Ser
Asp Pro Ser Val Glu Glu Met Arg Lys Val Val 355 360 365 Cys Glu Gln
Lys Leu Arg Pro Asn Ile Pro Asn Arg Trp Gln Ser Cys 370 375 380 Glu
Ala Leu Arg Val Met Ala Lys Ile Met Arg Glu Cys Trp Tyr Ala 385 390
395 400 Asn Gly Ala Ala Arg Leu Thr Ala Leu Arg Ile Lys Lys Thr Leu
Ser 405 410 415 Gln Leu Ser Gln Gln Glu Gly Ile Lys Met 420 425
4567PRTHomo sapiens 4Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro
Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro
Pro His Val Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr
Asp Asn Asn Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe
Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys
Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln
Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90
95 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile
100 105 110 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys
Lys Lys 115 120 125 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser
Asp Glu Cys Asn 130 135 140 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn
Thr Ser Asn Pro Asp Leu 145 150 155 160 Leu Leu Val Ile Phe Gln Val
Thr Gly Ile Ser Leu Leu Pro Pro Leu 165 170 175 Gly Val Ala Ile Ser
Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn 180 185 190 Arg Gln Gln
Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys 195 200 205 Leu
Met Glu Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg 210 215
220 Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu
225 230 235 240 Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly
Arg Phe Ala 245 250 255 Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr
Ser Glu Gln Phe Glu 260 265 270 Thr Val Ala Val Lys Ile Phe Pro Tyr
Glu Glu Tyr Ala Ser Trp Lys 275 280 285 Thr Glu Lys Asp Ile Phe Ser
Asp Ile Asn Leu Lys His Glu Asn Ile 290 295 300 Leu Gln Phe Leu Thr
Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln 305 310 315 320 Tyr Trp
Leu Ile Thr Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr 325 330 335
Leu Thr Arg His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser 340
345 350 Ser Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro
Cys 355 360 365 Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys
Ser Ser Asn 370 375 380 Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu
Cys Asp Phe Gly Leu 385 390 395 400 Ser Leu Arg Leu Asp Pro Thr Leu
Ser Val Asp Asp Leu Ala Asn Ser 405 410 415 Gly Gln Val Gly Thr Ala
Arg Tyr Met Ala Pro Glu Val Leu Glu Ser 420 425 430 Arg Met Asn Leu
Glu Asn Val Glu Ser Phe Lys Gln Thr Asp Val Tyr 435 440 445 Ser Met
Ala Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val 450 455 460
Gly Glu Val Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu 465
470 475 480 His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp
Arg Gly 485 490 495 Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln
Gly Ile Gln Met 500 505 510 Val Cys Glu Thr Leu Thr Glu Cys Trp Asp
His Asp Pro Glu Ala Arg 515 520 525 Leu Thr Ala Gln Cys Val Ala Glu
Arg Phe Ser Glu Leu Glu His Leu 530 535 540 Asp Arg Leu Ser Gly Arg
Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp 545 550 555 560 Gly Ser Leu
Asn Thr Thr Lys 565 5850PRTHomo sapiens 5Met Thr Ser His Tyr Val
Ile Ala Ile Phe Ala Leu Met Ser Ser Cys 1 5 10 15 Leu Ala Thr Ala
Gly Pro Glu Pro Gly Ala Leu Cys Glu Leu Ser Pro 20 25 30 Val Ser
Ala Ser His Pro Val Gln Ala Leu Met Glu Ser Phe Thr Val 35 40 45
Leu Ser Gly Cys Ala Ser Arg Gly Thr Thr Gly Leu Pro Gln Glu Val 50
55 60 His Val Leu Asn Leu Arg Thr Ala Gly Gln Gly Pro Gly Gln Leu
Gln 65 70 75 80 Arg Glu Val Thr Leu His Leu Asn Pro Ile Ser Ser Val
His Ile His 85 90 95 His Lys Ser Val Val Phe Leu Leu Asn Ser Pro
His Pro Leu Val Trp 100 105 110 His Leu Lys Thr Glu Arg Leu Ala Thr
Gly Val Ser Arg Leu Phe Leu 115 120 125 Val Ser Glu Gly Ser Val Val
Gln Phe Ser Ser Ala Asn Phe Ser Leu 130 135 140 Thr Ala Glu Thr Glu
Glu Arg Asn Phe Pro His Gly Asn Glu His Leu 145 150 155 160 Leu Asn
Trp Ala Arg Lys Glu Tyr Gly Ala Val Thr Ser Phe Thr Glu 165 170 175
Leu Lys Ile Ala Arg Asn Ile Tyr Ile Lys Val Gly Glu Asp Gln Val 180
185 190 Phe Pro Pro Lys Cys Asn Ile Gly Lys Asn Phe Leu Ser Leu Asn
Tyr 195 200 205 Leu Ala Glu Tyr Leu Gln Pro Lys Ala Ala Glu Gly Cys
Val Met Ser 210 215 220 Ser Gln Pro Gln Asn Glu Glu Val His Ile Ile
Glu Leu Ile Thr Pro 225 230 235 240 Asn Ser Asn Pro Tyr Ser Ala Phe
Gln Val Asp Ile Thr Ile Asp Ile 245 250 255 Arg Pro Ser Gln Glu Asp
Leu Glu Val Val Lys Asn Leu Ile Leu Ile 260 265 270 Leu Lys Cys Lys
Lys Ser Val Asn Trp Val Ile Lys Ser Phe Asp Val 275 280 285 Lys Gly
Ser Leu Lys Ile Ile Ala Pro Asn Ser Ile Gly
Phe Gly Lys 290 295 300 Glu Ser Glu Arg Ser Met Thr Met Thr Lys Ser
Ile Arg Asp Asp Ile 305 310 315 320 Pro Ser Thr Gln Gly Asn Leu Val
Lys Trp Ala Leu Asp Asn Gly Tyr 325 330 335 Ser Pro Ile Thr Ser Tyr
Thr Met Ala Pro Val Ala Asn Arg Phe His 340 345 350 Leu Arg Leu Glu
Asn Asn Glu Glu Met Gly Asp Glu Glu Val His Thr 355 360 365 Ile Pro
Pro Glu Leu Arg Ile Leu Leu Asp Pro Gly Ala Leu Pro Ala 370 375 380
Leu Gln Asn Pro Pro Ile Arg Gly Gly Glu Gly Gln Asn Gly Gly Leu 385
390 395 400 Pro Phe Pro Phe Pro Asp Ile Ser Arg Arg Val Trp Asn Glu
Glu Gly 405 410 415 Glu Asp Gly Leu Pro Arg Pro Lys Asp Pro Val Ile
Pro Ser Ile Gln 420 425 430 Leu Phe Pro Gly Leu Arg Glu Pro Glu Glu
Val Gln Gly Ser Val Asp 435 440 445 Ile Ala Leu Ser Val Lys Cys Asp
Asn Glu Lys Met Ile Val Ala Val 450 455 460 Glu Lys Asp Ser Phe Gln
Ala Ser Gly Tyr Ser Gly Met Asp Val Thr 465 470 475 480 Leu Leu Asp
Pro Thr Cys Lys Ala Lys Met Asn Gly Thr His Phe Val 485 490 495 Leu
Glu Ser Pro Leu Asn Gly Cys Gly Thr Arg Pro Arg Trp Ser Ala 500 505
510 Leu Asp Gly Val Val Tyr Tyr Asn Ser Ile Val Ile Gln Val Pro Ala
515 520 525 Leu Gly Asp Ser Ser Gly Trp Pro Asp Gly Tyr Glu Asp Leu
Glu Ser 530 535 540 Gly Asp Asn Gly Phe Pro Gly Asp Met Asp Glu Gly
Asp Ala Ser Leu 545 550 555 560 Phe Thr Arg Pro Glu Ile Val Val Phe
Asn Cys Ser Leu Gln Gln Val 565 570 575 Arg Asn Pro Ser Ser Phe Gln
Glu Gln Pro His Gly Asn Ile Thr Phe 580 585 590 Asn Met Glu Leu Tyr
Asn Thr Asp Leu Phe Leu Val Pro Ser Gln Gly 595 600 605 Val Phe Ser
Val Pro Glu Asn Gly His Val Tyr Val Glu Val Ser Val 610 615 620 Thr
Lys Ala Glu Gln Glu Leu Gly Phe Ala Ile Gln Thr Cys Phe Ile 625 630
635 640 Ser Pro Tyr Ser Asn Pro Asp Arg Met Ser His Tyr Thr Ile Ile
Glu 645 650 655 Asn Ile Cys Pro Lys Asp Glu Ser Val Lys Phe Tyr Ser
Pro Lys Arg 660 665 670 Val His Phe Pro Ile Pro Gln Ala Asp Met Asp
Lys Lys Arg Phe Ser 675 680 685 Phe Val Phe Lys Pro Val Phe Asn Thr
Ser Leu Leu Phe Leu Gln Cys 690 695 700 Glu Leu Thr Leu Cys Thr Lys
Met Glu Lys His Pro Gln Lys Leu Pro 705 710 715 720 Lys Cys Val Pro
Pro Asp Glu Ala Cys Thr Ser Leu Asp Ala Ser Ile 725 730 735 Ile Trp
Ala Met Met Gln Asn Lys Lys Thr Phe Thr Lys Pro Leu Ala 740 745 750
Val Ile His His Glu Ala Glu Ser Lys Glu Lys Gly Pro Ser Met Lys 755
760 765 Glu Pro Asn Pro Ile Ser Pro Pro Ile Phe His Gly Leu Asp Thr
Leu 770 775 780 Thr Val Met Gly Ile Ala Phe Ala Ala Phe Val Ile Gly
Ala Leu Leu 785 790 795 800 Thr Gly Ala Leu Trp Tyr Ile Tyr Ser His
Thr Gly Glu Thr Ala Gly 805 810 815 Arg Gln Gln Val Pro Thr Ser Pro
Pro Ala Ser Glu Asn Ser Ser Ala 820 825 830 Ala His Ser Ile Gly Ser
Thr Gln Ser Thr Pro Cys Ser Ser Ser Ser 835 840 845 Thr Ala 850
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