Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Mediators of Hedgehog Sighalihg PatlZwaysa
Compositions ahd Uses Related Thereto
Bacl~round of the Invention
Pattern formation is the activity by which embryonic cells form ordered
spatial arrangements of differentiated tissues. The physical complexity of
higher
organisms arises during embryogenesis through the interplay of cell-intrinsic
lineage
and cell-extrinsic signaling. Inductive interactions are essential to
embryonic
patterning in vertebrate development from the earliest establishment of the
body
plan, to the patterning of the organ systems, to the generation of diverse
cell types
during tissue differentiation (Davidson, E., (1990) Development 108: 365-389;
Gtudon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al., (1992) Cell 68:
257-
270). The effects of developmental cell interactions are varied. Typically,
responding cells are diverted from one route of cell differentiation to
another by
inducing cells that differ from both the uninduced and induced states of the
responding cells (inductions). Sometimes cells induce their neighbors to
differentiate
lilce themselves (homeogenetic induction); in other cases a cell inhibits its
neighbors
from differentiating lilce itself. Cell interactions in early development may
be
sequential, such that an initial induction between two cell types leads to a
progressive amplification of diversity. Moreover, inductive interactions occur
not
only in embryos, but in adult cells as well, and can act to establish and
maintain
moiphogenetic patterns as well as induce differentiation (J.B. Gurdon (1992)
Cell
68:185-199).
Members of the Hedgehog family of signaling molecules mediate many
important short- and long-range patterning processes during invertebrate and
vertebrate development. In the fly, a single hedgehog gene regulates segmental
and
imaginal disc patterning. In contrast, in vertebrates, a hedgehog gene family
is
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involved in the control of left-right asymnetiy, polarity in the CNS, somites
and
limb, organogenesis, chondrogenesis and spermatogenesis.
The first hedgehog gene was identified by a genetic screen in the fruitfly
D~oso~hila i~aelahogastef° (Niisslein-Volhard, C. and Wieschaus, E.
(1980) Nature
287, 795-801). This screen identified a number of mutations affecting
embryonic
and larval development. In 1992 and 1993, the molecular nature of the Df
osophila
hedgehog (hh) gene was reported (C.F., Lee et al. (1992) Gell 71, 33-50), and
since
then, several hedgehog homologues have been isolated from various vertebrate
species. While only one hedgehog gene has been found in D~oso~hila and other
invertebrates, multiple Hedgehog genes are present in vertebrates.
The vertebrate family of hedgehog genes includes at least four members, e.g.,
paralogs of the single drosophila hedgehog gene. Exemplary hedgehog genes and
proteins are described in PCT publications WO 95/18856 and WO 96/17924. Three
of these members, herein referred to as Desert hedgelzog (Dhh), Sonic hedgehog
(Shh) and Indian hedgehog (Ihh), apparently exist in all vertebrates,
including fish,
birds, and mammals. A fourth member, herein referred to as tiggie-wiude
hedgehog
(TIZh), appears specific to fish. Desert hedgehog (Dhh) is expressed
principally in the
testes, both in mouse embryonic development and in the adult rodent and human;
Indian hedgehog (Ihh) is involved in bone development during embryogenesis and
in
bone formation in the adult; and, Shh, which as described above, is primarily
involved in morphogenic and neuroinductive activities. Given the critical
inductive
roles of hedgehog polypeptides in the development and maintenance of
vertebrate
organs, the identification of hedghog interacting proteins is of paramount
significance in both clinical and research contexts.
The various Hedgehog proteins consist of a signal peptide, a highly
conserved N-terminal region, and a more divergent C-terminal domain. In
addition
to signal sequence cleavage in the secretory pathway (Lee, J.J. et al. (1992)
Cell
71:33-50; Tabata, T. et al. (1992) Genes Dev. 2635-2645; Chang, D.E. et al.
(1994)
Development 120:3339-3353), Hedgehog precursor proteins undergo an internal
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autoproteolytic cleavage which depends on conserved sequences in the C-
terminal
portion (Lee et al. (1994) Science 266:1528-1537; Porter et al. (1995) Nature
374:363-366). This autocleavage leads to a 19 1cD N-terminal peptide and a C-
terminal peptide of 26-28 1D (Lee et al. (1992) supra; Tabata et al. (1992)
supra;
S Chang et al. (1994) supra; Lee et al. (1994) supra; Bumcrot, D.A., et al.
(1995) Mol.
Cell. Biol. 15:2294-2303; Porter et al. (1995) supra; Eldcer, S.C. et al.
(1995) Curr.
Biol. 5:944-955; Lai, C.J. et al. (1995) Development 121:2349-2360). The N-
terminal peptide stays tightly associated with the surface of cells in which
it was
synthesized, while the C-terminal peptide is freely diffusible both i~z vitro
and in vivo
(Porter et al. (1995) Nature 374:363; Lee et al. (1994) supra; Bumcrot et al.
(1995)
supra; Marti, E. et al. (1995) Development 121:2537-2547; Roelinlc, H. et al.
(1995)
Cell 81:445-455). Interestingly, cell surface retention of the N-terminal
peptide is
dependent on autocleavage, as a truncated form of HH encoded by an RNA which
terminates precisely at the normal position of internal cleavage is diffusible
ih vita°o
(Porter et al. (1995) su ra and in vivo (Porter, J.A. et al. (1996) Cell 86,
21-34).
Biochemical studies have shown that the autoproteolytic cleavage of the HH
precursor protein proceeds through an internal thioester intermediate which
subsequently is cleaved in a nucleophilic substitution. It is likely that the
nucleophile
is a small lipophilic molecule which becomes covalently bound to the C-
terminal
end of the N-peptide (Porter et al. (1996) supra), tethering it to the cell
surface. The
biological implications are profound. As a result of the tethering, a high
local
concentration of N-terminal Hedgehog peptide is generated on the surface of
the
Hedgehog producing cells. It is this N-terminal peptide which is both
necessary and
sufficient for short- and long-range Hedgehog signaling activities in
Dy°osophila and
vertebrates (Porter et al. (1995) supra; El~lcer et al. (1995) su ra~ Lai et
al. (1995)
suRra; Raelinlc, H. et al. (1995) Cell 81:445-455; Porter et al. (1996) su ra~
Fietz,
M.J. et al. (1995) Curr. Biol. 5:643-651; Fan, C.-M. et al. (1995) Cell 81:457-
465;
Marti, E., et al. (1995) Nature 375:322-325; Lopez-Martinez et al. (1995)
Curr. Biol
5:791-795; Elder, S.C. et al. (1995) Development 121:2337-2347; Forbes, A.J.
et
a1.(1996) Development 122:1125-1135).
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HH has been implicated in short- and long-range patterning processes at
various sites during D~osophila development. In the establishment of segment
polarity in early embryos, it has short-range effects which appear to be
directly
mediated, while in the patterning of the imaginal discs, it induces long range
effects
via the induction of secondary signals.
In vertebrates, several hea'gehog genes have been cloned in the past few
years. Of these genes, Shh has received most of the experimental attention, as
it is
expressed in different organizing centers which are the sources of signals
that pattern
neighboring tissues. Recent evidence indicates that Shh is involved in these
interactions.
The expression of Shh starts shortly after the onset of gastrulation in the
presumptive midline mesoderm, the node in the mouse (Chang et al. (1994)
supra;
Echelard, Y. et al. (1993) Cell 75:1417-1430), the rat (Roelink, H. et al.
(1994) Cell
76:761-775) and the chick (Riddle, R.D. et al. (1993) Cell 75:1401-1416), and
the
shield in the zebrafish (Eldcer et al. (1995) supra; I~rauss, S. et a1.(1993)
Cell
75:1431-1444). In chick embyros, the Shh expression pattern in the node
develops a
left-right asymmetry, which appears to be responsible for the left-right situs
of the
heart (Levin, M. et al. (1995) Cell 82:803-814).
In the CNS, Shh from the notochord and the floorplate appears to induce
ventral cell fates. When ectopically expressed, Shh leads to a ventralization
of large
regions of the mid- and hindbrain in mouse (Echelard et al. (1993) supra;
Goodrich,
L.V. et al. (1996) Genes Dev. 10:301-312), XesZOpus (Roelinlc, H. et al.
(1994)
supra; Ruiz i Altaba, A. et al. (1995) Mol. Cell. Neurosci. 6:106-121), and
zebrafish
(Eldter et al. (1995) supra; Krauss et al. (1993) su ra; Hammerselunidt, M.,
et al.
(1996) Genes Dev. 10:647-658). In explaaits of intermediate neuroectoderm at
spinal
cord levels, Shh protein induces floorplate and motor neuron development with
distinct concentration thresholds, floor plate at high and motor neurons at
lower
concentrations (Roeliuc et al. (1995) su ra; Marti et al. (1995) supra;
Tanabe, Y. et
al. (1995) Curr. Biol. 5:651-658). Moreover, antibody blocking suggests that
Shh
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produced by the notochord is required for notochord-mediated induction of
motor
neuron fates (Marti et al. (1995) sera). Thus, high concentration of Shh on
the
surface of Shh-producing midline cells appears to account for the contact-
mediated
induction of flooiplate observed in vitro (Placzelc, M. et al. (1993)
Development
117:205-218), and the midline positioning of the floorplate immediately above
the
notochord i~ vivo. Lower concentrations of Shh released from the notochord and
the
floorplate presumably induce motor neurons at more distant ventrolateral
regions in
a process that has been shown to be contact-independent i~ vits o (Yamada, T.
et al.
(1993) Cell 73:673-686). In.explants taken at midbrain and forebrain levels,
Shh also
induces the appropriate ventrolateral neuronal cell types, dopaminergic
(Heynes, M.
et al. (1995) Neuron 15:35-44; Wang, M.Z. et al. (1995) Nature Med. 1:1184-
1188)
arid cholinergic (Ericson, J. et al. (1995) Cell 81:747-756) precursors,
respectively,
indicating that Shh is a common inducer of ventral specification over the
entire
length of the CNS. These observations raise a question as to how the
differential
response to Shh is regulated at particular anteroposterior positions.
Shh from the midline also patterns the paraxial regions of the vertebrate
embryo, the somites in the trunk (Fan et al. (1995) supra) and the head
mesenchyme
rostral of the somites (Hammerschmidt et al. (1996) supra). In chick and mouse
paraxial mesoderm explants, Shh promotes the expression of sclerotome specific
markers lilce Paxl and Twist, at the expense of the dermamyotomal marker Pax3.
Moreover, filter barrier experiments suggest that Shh mediates the induction
of the
sclerotome directly xather than by activation of a secondary signaling
mechanism
(Fan, C.-M. and Tessier-Lavigne, M. (1994) Cell 79, 1175-1186).
Shh also induces myotomal gene expression (Hammersclunidt et al. (1996)
supra; Johnson, R.L. et al. (1994) Cell 79:1165-1173; Miinsterberg, A.E. et
al.
(1995) Genes Dev. 9:2911-2922; Weinberg, E.S. et al. (1996) De-velopment
122:271-280), although recent experiments indicate that members of the WNT
faanily, vertebrate homologues of D~~osophila wingless, are required in
concert
(Miinsterberg et al. (1995) supra). Puzzlingly, myotomal induction in chicks
requires
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higher Shh concentrations than the induction of sclerotomal markers
(Miinsterberg et
al. (1995) su ra), although the sclerotome originates from somitic cells
positioned
much closer to the notochord. Similar results were obtained in the zebrafish,
where
high concentrations of Hedgehog induce myotomal and repress sclerotomal marker
gene expression (Hammerschmidt et al. (1996) s, u~). In contrast to amniotes,
however, these observations are consistent with the architecture of the fish
embryo,
as here, the myotome is the predominant and more axial component of the
somites.
Thus, modulation of Shh signaling and the acquisition of new signaling factors
may
have modified the somite structure during vertebrate evolution.
h1 the vertebrate limb buds, a subset of posterior mesenchymal cells, the
"Zone of polarizing activity" (ZPA), regulates anteroposterior digit identity
(reviewed in Honig, L.S. (1981) Nature 291:72-73). Ectopic expression of Shh
or
.application of beads soaked in Shh peptide mimics the effect of anterior ZPA
grafts,
generating a mirror image duplication of digits (Chang et al. (1994) supra;
Lopez-
Martinet et al. (1995) supy~a; Riddle et al. (1993) supra) (Fig. 2g). Thus,
digit
identity appears to depend primarily on Shh concentration, although it is
possible
that other signals may relay this information over the substantial distances
that
appear to be required for AP patterning (100-150 ~,m). Similar to the
interaction of
HH and DPP in the D~osophila imaginal discs, Shh in the vertebrate limb bud
activates the expression of Bmp2 (Francis, P.H. et al. (1994) Development
120:209-
218), a dpp homologue. However, unlike DPP in D~~osophila, B~ap2 fails to
mimic
the polarizing effect of Shh upon ectopic application in the chick limb bud
(Francis
et al. (1994) s_upra). In addition to anteroposterior patterning, Slzh also
appears to be
involved in the regulation of the proximodistal outgrowth of the limbs by
inducing
the synthesis of the fibroblast growth factor FGF4 in the posterior apical
ectodermal
ridge (Laufer, E. et al. (1994) Cell 79:993-1003; Niswander, L. et a1.(1994)
Nature
371:609-612).
The close relationship between Hedgehog proteins and BMPs is lilcely to
have been conserved at many, but probably not all sites of vertebrate Hedgehog
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expression. For example, in the chick lundgut, Shh has been shown to induce
the
expression of Bmp4, another vertebrate dpp homologue (Robeus, D.J. et al.
(1995)
Develo ip nent 121:3163-3174). Fux-thermore, Shh and B~np2, 4, or 6 show a
strilcing
correlation in their expression in epithelial and mesenchymal cells of the
stomach,
the urogenital system, the lung, the tooth buds and the hair follicles
(Bitgood, M.J.
and McMahon, A.P. (1995) Dev. Biol. 172:126-138). Further, Ihh, one of the two
other mouse Hedgehog genes, is expressed adjacent to Bmp expressing cells in
the
gut and developing cartilage (Bitgood and McMahon (1995) supfa).
Recent evidence suggests a model in which Ihh plays a crucial role in the
regulation of chondrogenic development (Roberts et al. (1995) supra). During
cartilage formation, chondrocytes proceed from a proliferating state via an
intermediate, prehypertrophic state to differentiated hypertrophic
chondrocytes. Ihh
is expressed in the prehypertrophic chondrocytes and initiates a signaling
cascade
that leads to the blockage of chondrocyte differentiation. Its direct target
is the
perichondrium around the Ihh expression domain, which responds by the
expression
of Gli and Patched (Ptc), conserved transcriptional targets of Hedgehog
signals (see
below). Most likely, this leads to secondary signaling resulting in the
synthesis of
parathyroid hormone-related protein (PTHrP) in the periarticular
perichondrium.
PTHrP itself signals back to the prehypertrophic chondrocytes, blocking their
further
differentiation. At the same time, PTHrP represses expression of Ihh, thereby
forming a negative feedback loop that modulates the rate of chondrocyte
differentiation.
Patched was originally identified in Drosophila as a segment polarity gene,
one of a group of developmental genes that affect cell differentiation within
the
individual segments that occur in a homologous series along the anterior-
posterior
axis of the embryo. See Hooper, J.E. et al. (1989) Cell 59:751; and Nalcano,
Y. et al.
(1989) Nature 341:508. Patterns of expression of the vertebrate homologue of
patehed suggest its involvement in the development of neural tube, skeleton,
limbs,
craniofacial structure, and slcin.
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Genetic and functional studies demonstrate that patched is part of the
hedgehog signaling cascade, an evolutionarily conserved pathway that regulates
expression of a number of downstream genes. See Perrimon, N. (1995) Cell
80:517;
a~ld Perrimon, N. (1996) Cell 86:513. Patched participates in the constitutive
transcriptional repression of the target genes; its effect is opposed by a
secreted
glycoprotein, encoded by hedgehog, or a vertebrate homologue, which induces
transcriptional activation. Genes under control of this pathway include
members of
the Wnt and TGF-beta families.
Patched proteins possess two large extracellular domains, twelve
transmembrane segments, and several cytoplasmic segments. See Hooper, supra;
Nalcano, su ra; Johnson, R.L. et al. (1996) Science 272:1668; and Hahn, H. et
al.
(1996) Cell 85:841. The biochemical role of patched in the hedgehog signaling
pathway is unclear. Direct interaction with the hedgehog protein has, however,
been
reported (Chen, Y. et al. (1996) Cell 87:553), and patched may participate in
a
hedgehog receptor complex along with another transmembrane protein encoded by
the s~zoothe~ed gene. See Perrimon, supra; and Chen, suura.
The human homologue of patched was recently cloned and mapped to
chromosome 9q22.3. See Johnson, supra; and Hahn, su ra. This region has been
implicated in basal cell nevus syndrome (BCNS), which is characterized by
developmental abnormalities including rib and craniofacial alterations,
abnormalities
of the hands and feet, and spina bifida.
BCNS also predisposes to multiple tumor types, the most frequent being
basal cell carcinomas (BCC) that occur in many locations on the body and
appear
within the first two decades of life. Most cases of BCC, however, are
unrelated to
the syndrome and arise sporadically in small numbers on sun-exposed sites of
middle-aged or older people of northern European ancestry.
Recent studies in BCNS-related and sporadic BCC suggest that a functional
loss of both alleles of patched leads to development of BCC. See Johnson,
supra;
Hahn, supra; and Gailani, M.R. et al. (1996) Nature Genetics 14:78. Single
allele
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deletions of chromosome 9q22.3 occur frequently in both sporadic and
hereditary
BCC. Linkage analysis revealed that the defective inherited allele was
retained and
the normal allele was lost in tumors from BCNS patients.
Sporadic tumors also demonstrated a loss of both functional alleles of
patched. Of twelve tumors in which patched mutations were identified with a.
single
strand conformational polymorphism screening assay, nine had chromosomal
deletion of the second allele and the other three had inactivating mutations
in both
alleles (Gailani, supra). The alterations did not occur in the corresponding
germline
DNA.
Most of the identified mutations resulted in premature stop codons or frame
shifts. Lench, N.J., et al., Hu~rz. Genet. 1997 Oct; 100(5-6): 497-502.
Several,
however, were point mutations leading to amino acid substitutions in either
extracellular or cytoplasmic domains. These sites of mutation may indicate
functional importance for interaction with extracellular proteins or with
cytoplasmic
members of the downstream signaling pathway.
The involvement of patched in the inlubition of gene expression and the
occurrence of frequent allelic deletions of patched in BCC support a tumor
suppressor function for this gene. Its role in the regulation of gene families
known to
be involved in cell signaling and intercellular communication provides a
possible
mechanism of tumor suppression.
Summary of the Invention
The present invention malces available methods and reagents for inhibiting
activation of the hedgehog signaling pathway, e.g., to inhibit aberrant growth
states
resulting from phenotypes such as ptc loss-of function, hedgehog gain-of
function,
or smoothened gain-of function, comprising contacting the cell with an agent,
such
as a small molecule, in a sufficient amount to agonize a normal ptc activity,
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antagonize a normal hedgehog activity, or antagonize smoothefZed activity,
e.g., to
reverse or control the aberrant growth state.
Brief Description of the Drawings
Figuxes 1-31 depict reactions useful for synthesizing compounds according to
the present invention.
Figure 32a-o illustrates representative compounds according to the present
invention.
Figure 33A shows gli-1 mRNA expression in cells treated with vehicle
(Lane 1); 5 ~,M jervine, the positive control compound (Lane 2); and 1 ~.M D
(Lane
3). Compared with vehicle, D and jervine significantly decreased the
expression of
gli-1 mRNA.
Figure 33B demonstrates that D and jemine inhibited the gli-1 mRNA levels
as measured by quantitative real-time PCR.
Figure 34A shows that adding Shh protein to cultured skin explants resulted
in ptc activation as indicated by the blue staining of these cultures (X-gal).
Histology
samples show intensely stained cells with basophilic nuclei and a high nucleus
to
cytoplasm ratio (H&E [10x] and H&E [40x]). These structures resemble BCCs in
that they are arranged in clusters throughout the dermal layer and are
separated by
palisades of normal appearing dermal cells. Blue staining indicates that the
Patched
pathway was active in cells within the BCC-lilce structures (Eosin+X-gal).
Figure 34B illustrates that BCC-life clusters, one of which is indicated by
the
arrow, in the mouse skin punch expressed keratin-14 (brown reaction product),
a
marlcer of undifferentiated lceratinocytes. Undifferentiated basal cells in
the
epidermis were also keratin-14-positive. Human BCCs are reported to express
keratin-14.
Figure 35A demonstrates that increasing concentrations of D are associated
with a dose-dependent decrease in the amount of lacZ reporter enzyme activity.
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Lower levels of lacZ activity are indicative of decreased Patched pathway
activity in
the presence of Shh protein.
Figure 35B shows staining of D-treated explants and demonstrates that 0.2
~.M D decreased X-gal staining compared with the intense X-gal staining of
skin
punches treated with Shh protein alone, indicating the downregulation of the
expression of the ptc gene.
Figure 35C portrays histology samples of slcin punches treated with D
(bottom row), suggesting that treatment inhibited the appearance of Shh-
induced
BCC-like structures.
Figure 36 depicts that slcin punches treated for 6 days with exogenous Shh
protein alone showed intense X-gal staining compared with those treated with
vehicle alone (top row). Slcin punches pretreated with D at 10, 20 aald 50 ~.M
for
5 hours before being exposed to exogenous Shh protein demonstrated complete
inhibition of Shh protein-induced upregulation of the Patched pathway (bottom
row-3 slides on the right). No inhibition was seen when the skin punches
pretreated
with vehicle were exposed to exogenous Shh protein, as shown by intense X-gal
staining (bottom row on the left). The short period of pretreatment was
essentially
equivalent to 6-day exposure to D in terms of the level of ptc inhibition
(compare top
and bottom rows).
Figure 37A shows that D, at either 1 or 5 ~M, significantly reduced the size
and number of Shh-induced BCC-lilce structures in treated skin punches, as
compared with vehicle treated explants.
Figure 37B illustrates that after 2 days of exposure to 5 ~.M D (right) or
vehicle (left), apoptotic nuclei, indicated by the brown color in the slides
on the
right, appeared within the BCC-like structures.
Figure 38A demonstrates that short-term treatment with D reduced the
amount of X-gal staining, suggesting a downregulation of pathway activity,
compared with vehicle.
Figure 38B shows that even at a concentration of 1 ~,M, D induced the
regression of X-gal-positive BCC-life structures compared with vehicle.
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Figure 38C portratys that shout-term treatment with D completely
downregulated gli-1 transcription (left). This effect appeared to be specific
to the
Patched pathway and was not due simply to general cytotoxicity, as shown by
the
fairly constant mRNA levels of a housekeeping enzyme, GAPDH (right).
Figure 39A: X-gal staining of the treated explants showed that skin punches
cultured in the presence of vehicle alone developed intensely stained blue
foci
indicative of an upregulation of the Patched pathway and BCC structures.
Compared
with vehicle, 5 ~.M D, lilce the jervine positive control, greatly decreased
the number
and size of BCC structures (blue spots).
, Figure 39B: Histology samples showed that 5 ~M D reduced the number of
ultraviolet-induced BCC structures, as compared with the vehicle control.
Figure 39C: In skin punches from transgenic mice D, at concentrations of 1
and 5 ~,M, siguficantly inhibited the level of gli-1 mRNA compared with skin
punches from mice treated with vehicle alone (left). This inhibition did not
appear to
be caused by non-specific cytotoxicity, as statistical comparison (using
ANOVA) of
the mRNA levels of the gene that encodes the housekeeping GAPDH enzyme among
groups showed no significant difference in general cellular metabolic activity
(right).
Figure 40A: The morphological features characteristic of BCCs, such as
islands of undifferentiated basal cells, and in some cases, palisading of
peripheral
cells and stromal clefting were maintained when cultures were stained with
H&E.
Figure 40B: The GLII gene, a pivotal indicator of Patched signaling,
remained active at high levels, as indicated in red.
Figure 41: Quantitative in situ hybridization shows that the level of GLI 1
expression is reduced in the D-treated samples as compared to vehicle-treated
controls.
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Detailed Description of the Invention
I. Overview
The present invention relates to the discovery that signal transduction
pathways regulated by hedgehog, patched (ptc), gli and/or sf~2oothehed can be
inhibited, at least in part, by small molecules. While not wishing to bound by
any
particular theory, the activation of a receptor may be the mechanism by which
these
agents act. For example, the ability of these agents to inhibit proliferation
of patched
loss-of function (ptclof) cells may be due to the ability of such molecules to
interact
with hedgehog, patched, or smoothened, or at least to interfere with the
ability of
those proteins to activate a hedgehog, ptc, and/or stnoothehed-mediated signal
transduction pathway.
It is, therefore, specifically contemplated that these small molecules which
intefere with aspects of hedgehog, ptc, or sf~2oothened signal transduction
activity
will likewise be capable of inhibiting proliferation (or other biological
consequences) in normal cells and/or cells having a patched loss-of function
phenotype, a hedgehog gain-of function phenotype, or a smoothened gain-of
function phenotype. Thus, it is contemplated that in certain embodiments,
these
compounds may be useful for inhibiting hedgehog activity in normal cells,
e.g.,
which do not have a genetic mutation that activates the hedgehog pathway. In
preferred embodiments, the subject inhibitors are organic molecules having a
molecular weight less than 2500 amu, more preferably less thaxi 1500 amu, and
even
more preferably less than 750 amu, and are capable of inhibiting at least some
of the
biological activities of hedgehog proteins, preferably specifically in target
cells.
Thus, the methods of the present invention include the use of small
molecules which agonize ptc inhibition of hedgehog signalling, such as by
inhibiting
activation of smoothev~ed or downstream components of the signal pathway, in
the
regulation of repair and/or functional performance of a wide range of cells,
tissues
and organs, including normal cells, tissues, and organs, as well as those
having the
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phenotype of ptc loss-of function, hedgehog gain-of function, or sozzoothe~ed
gain-
of function. For instance, the subject method has therapeutic and cosmetic
applications ranging from regulation of neural tissues, bone and cartilage
formation
and repair, regulation of spermatogenesis, regulation of smooth muscle,
regulation of
lung, liver and other organs arising from the primative gut, regulation of
hematopoietic function, regulation- of skin and hair growth, etc. Moreover,
the
subject methods can be performed on cells which axe provided in culture (in
vita°o),
or on cells in a whole animal (ire vivo). See, for example, PCT publications
WO
95/18856 and WO 96/17924 (the specifications of which are expressly
incorporated
by reference herein).
In a preferred embodiment, the subject method can be to treat epithelial cells
having a phenotype of ptc loss-of function, hedgehog gain-of function, or
smoothexzed gain-of function. For instance, the subject method can be used in
treating or preventing basal cell carcinoma or other hedgehog pathway-related
disorders.
In certain embodiments, a subject antagonist may inhibit activation of a
hedgehog pathway by binding to srzzoothehed. In certain embodiments, a subject
antagonist may inhibit activation of a hedgehog pathway by binding to patched.
In another preferred embodiment, the subject method can be used as part of a
treatment regimen for malignant medulloblastoma and other primary CNS
malignant
neuroectodermal tumors.
In another aspect, the present invention provides pharmaceutical preparations
comprising, as an active ingredient, a hedgehog antagonist, ptc agonist, or
smoothened antagonist such as described herein, formulated in an amount
sufficient
to inhibit, izz vivo, proliferation or other biological consequences of ptc
loss-of
function, hedgehog gain-of function, or smoothened gain-of function.
The subject treatments using hedgehog antagonists, patched agonists, or
szzzoothezzed antagonists can be effective for both human and animal subjects.
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Animal subjects to which the invention is applicable extend to both domestic
animals and livestoclc, raised either as pets or for commercial purposes.
Examples
are dogs, cats, cattle, horses, sheep, hogs, and goats.
II. Definitions
For convenience, certain terms employed in the specification, examples, and
appended claims are collected here.
The phrase "aberrant modification or mutation" of a gene refers to such
genetic lesions as, for example, deletions, substitution or addition of
nucleotides to a
gene, as well as gross chromosomal rearrangements of the gene and/or abnormal
methylation of the gene. Lilcewise, mis-expression of a gene refers to
aberrant levels
of transcription of the gene relative to those levels in a normal cell under
similar
conditions, as well as non-wild-type splicing of mRNA transcribed from the
gene.
"Basal cell carcinomas" exist in a variety of clinical and histological forms
such as nodular-ulcerative, superficial, pigmented, morphealilce,
fibroepithelioma
and nevoid syndrome. Basal cell carcinomas are the most common cutaneous
neoplasms found in humans. The majority of new cases of nonmelanoma shin
cancers fall into this category.
"Burn wounds" refer to cases where large surface areas of skin have been
removed or lost from an individual due to heat and/or chemical agents.
The term "carcinoma" refers to a malignant new growth made up of epithelial
cells tending to infiltrate surrounding tissues and to give rise to
metastases.
Exemplary carcinomas include: "basal cell carcinoma", which is an epithelial
tumor
of the skin that, while seldom metastasizing, has potentialities for local
invasion and
destruction; "squamous cell carcinoma", which refers to carcinomas arising
from
squamous epithelium and having cuboid cells; "carcinosaxcoma", which include
malignant tumors composed of carcinomatous and sarcomatous tissues;
"adenocystic
carcinoma", carcinoma marked by cylinders or bands of hyaline or mucinous
stroma
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separated or surrounded by nests or cords of small epithelial cells, occurring
in the
mammary and salivary glands, and mucous glands of the respiratory tract;
"epidermoid carcinoma", which refers to cancerous cells which tend to
differentiate
in the same way as those of the epidermis; i.e., they tend to form priclde
cells and
undergo cornification; "nasopharyngeal carcinoma", which refers to a malignant
tumor arising in the epithelial lining of the space behind the nose; and
"renal cell
carcinoma", which pertains to carcinoma of the renal parenchyma composed of
tubular cells in varying arrangements. Other carcinomatous epithelial growths
are
"papillomas", which refers to benign tumors derived from epithelium and having
a
papillomavirus as a causative agent; and "epidermoidomas", which refers to a
cerebral or meningeal tumor formed by inclusion of ectodermal elements at the
time
of closure of the neural groove.
The "corium" or "dermis" refers to the layer of the shin deep to the
epidermis, consisting of a dense bed of vascular connective tissue, and
containing
the nerves and terminal organs of sensation. The hair roots, and sebaceous and
sweat
glands are structures of the epidermis which are deeply embedded in the
dermis.
"Dental tissue" refers to tissue in the mouth which is similar to epithelial
tissue, for example gum tissue. The method of the present invention is useful
for
treating periodontal disease.
"Dermal skin ulcers" refer to lesions on the skin caused by superficial loss
of
tissue, usually with inflammation. Dermal shin ulcers which can be treated by
the
method of the present invention include decubitus ulcers, diabetic ulcers,
venous
stasis ulcers and arterial ulcers. Decubitus wounds refer to chronic ulcers
that result
from pressure applied to areas of the skin for extended periods of time.
Wounds of
this type are often called bedsores or pressure sores. Venous stasis ulcers
result from
the stagnation of blood or other fluids from defective veins. Auterial ulcers
refer to
necrotic shin in the area around arteries having poor blood flow.
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The term "EDsp" means the dose of a drug which produces 50% of its
maximum response or effect.
An "effective amount" of, e.g., a hedgehog antagonist, with respect to the
subject method of treatment, refers to an amount of the antagonist in a
preparation
which, when applied as part of a desired dosage regimen brings about, e.g., a
change
in the rate of cell proliferation and/or the state of differentiation of a
cell and/or rate
of survival of a cell according to clinically acceptable standards for the
disorder to be
treated or the cosmetic purpose.
The terms "epithelia", "epithelial" and "epithelium" refer to the cellular
covering of internal and external body surfaces (cutaneous, mucous and
serous),
including the glands and other structures derived therefrom, e.g., corneal,
esophegeal, epidermal, and hair follicle epithelial cells. Other exemplary
epithelial
tissue includes: olfactory epithelium, which is the pseudostratified
epithelium lining
the olfactory region of the nasal cavity, and containing the receptors for the
sense of
smell; glandular epithelium, which refers to epithelium composed of secreting
cells;
squamous epithelium, which refers to epithelium composed of flattened plate-
lilce
cells. The term epithelium can also refer to transitional epithelium, like
that which is
characteristically found lining hollow organs that are subject to great
mechanical
change due to contraction and distention, e.g., tissue which represents a
transition
between stratified squamous and columnar epithelium.
The term "epithelialization" refers to healing by the growth of epithelial
tissue over a denuded surface.
The term "epidermal gland" refers to an aggregation of cells associated with
the epidermis and specialized to secrete or excrete materials not related to
their
ordinary metabolic needs. For example, "sebaceous glands" are holocrine glands
in
the corium that secrete an oily substance and sebmn. The term "sweat glands"
refers
to glands that secrete sweat, situated in the corium or subcutaneous tissue,
opening
by a duct on the body surface.
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The term "epidermis" refers to the outermost and nonvascular layer of the
skin, derived from the embryonic ectoderm, varying in thickness from 0.07-1.4
mm.
On the palmar and plantar surfaces it comprises, from within outward, five
layers:
basal layer composed of columnar cells arranged perpendicularly; prickle-cell
or
spinous layer composed of flattened polyhedral cells with short processes or
spines;
granular layer composed of flattened granular cells; clear layer composed of
several
layers of clear, transparent cells in which the nuclei are indistinct or
absent; a~zd
horny layer composed of flattened, cornified non-nucleated cells. In the
epidermis of
the general body surface, the clear layer is usually absent.
"Excisional wounds" include tears, abrasions, cuts, punctures or lacerations
in the epithelial layer of the skin and may extend into the dermal layer and
even into
subcutaneous fat and beyond. Excisional wounds can result from surgical
procedures
or from accidental penetration of the shin.
The "growth state" of a cell refers to the rate of proliferation of the cell
and/or the state of differentiation of the cell. An "altered growth state" is
a growth
state characterized by an abnormal rate of proliferation, e.g., a cell
exhibiting an
increased or decreased rate of proliferation relative to a normal cell.
The term "hair" refers to a threadlike structure, especially the specialized
epidermal structure composed of keratin and developing from a papilla sunk in
the
corium, produced only by mammals and characteristic of 'that group of animals.
Also, "hair" may refer to the aggregate of such hairs. A "hair follicle"
refers to one
of the tubular-invaginations of the epidermis enclosing the hairs, and from
which the
hairs grow. "Hair follicle epithelial cells" refers to epithelial cells which
surrouaid the
dermal papilla in the hair follicle, e.g., stem cells, outer root sheath
cells, matrix
cells, and inner root sheath cells. Such cells may be normal non-malignant
cells, or
transformed/immortalized cells.
The term "hedgehog antagonist" refers to an agent which potentiates or
recapitulates the bioactivity of patched, such as to repress transcription of
target
genes. Preferred hedgehog antagonists can be used to overcome a ptc loss-of
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function and/or a szzzoothezzed gain-of function, the latter also being
refered to as
szzzootlZezzed antagonists. The term 'hedgehog antagonist' as used herein
refers not
only to any agent that may act by directly inhibiting the normal function of
the
hedgehog protein, but also to any agent that inhibits the hedgehog signalling
pathway, and thus recapitulates the function of ptc.
The term "hedgehog gain-of function" refers to an aberrant modification or
mutation of a ptc gene, hedgehog gene, or smoothened gene, or a decrease (or
loss)
in the level of expression of such a gene, which results in a phenotype which
resembles contacting a cell with a hedgehog protein, e.g., aberrant activation
of a
hedgehog pathway. The gain-of function may include a loss of the ability of
the ptc
gene product to regulate the level of expression of Ci genes, e.g., Glil,
Gli2, and
Gli3. The term 'hedgehog gain-of function' is also used herein to refer to any
similar
cellular phenotype (e.g., exhibiting excess proliferation) which occurs due to
an
alteration anywhere in the hedgehog signal transduction pathway, including,
but not
limited to, a modification or mutation of hedgehog itself. For example, a
tumor cell
with an abnormally high proliferation rate due to activation of the hedgehog
signalling pathway would have a 'hedgehog gain-of function' phenotype, even if
hedgehog is not mutated in that cell.
As used herein, "immortalized cells" refers to cells which have been altered
via chemical and/or recombinant means such that the cells have the ability to
grow
through an indefinite number of divisions in culture.
"Internal epithelial tissue" refers to tissue inside the body which has
characteristics similar to the epidermal layer in the shin. Examples include
the lining
of the intestine. The method of the present invention is useful for promoting
the
healing of certain internal wounds, for example wounds resulting from surgery.
The term "lceratosis" refers to proliferative shin disorder characterized by
hyperplasia of the horny layer of the epidermis. Exemplary lceratotic
disorders
include l~eratosis follicularis, lceratosis palmaris et plantaris, lceratosis
pharyngea,
lceratosis pilaris, and actinic lceratosis.
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The term "LDsp" means the dose of a drug which is lethal in 50% of test
subj ects.
The term "nail" refers to the horny cutaneous plate on the dorsal surface of
the distal end of a finger or toe.
The term "patched loss-of function" refers to an aberrant modification or
mutation of a ptc gene, or a decreased level of expression of the gene, which
results
in a phenotype which resembles contacting a cell with a hedgehog protein,
e.g.,
aberrant activation of a hedgehog pathway. The loss-of function may include a
loss
of the ability of the ptc gene product to regulate the level of expression of
Ci genes,
e.g., Glil, Gli2 and Gli3. The term 'ptc loss-of function' is also used herein
to refer
to any similar cellular phenotype (e.g., exhibiting excess proliferation)
which occurs
due to an alteration anywhere in the hedgehog signal transduction pathway,
including, but not limited to, a modification or mutation of ptc itself. For
example, a
tumor cell with an abnormally high proliferation rate due to activation of the
hedgehog signalling pathway would have a 'ptc loss-of function' phenotype,
even if
ptc is not mutated in that cell.
A "patient" or "subject" to be treated by the subject method can mean either a
human or non-human animal.
The term "prodrug" is intended to encompass compounds which, under
physiological conditions, are converted into the therapeutically active agents
of the
present invention. A common method for malting a prodrug is to include
selected
moieties which are hydrolyzed under physiological conditions to reveal the
desired
molecule. In other embodiments, the prodrug is converted by an enzymatic
activity
of the host animal.
As used herein, "proliferating" and "proliferation" refer to cells undergoing
mitosis.
Throughout this application, the term "proliferative shin disorder" refers to
any disease/disorder of the skin marked by unwanted or aberrant proliferation
of
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cutaneous tissue. These conditions are typically characterized by epidermal
cell
proliferation or incomplete cell differentiation, and include, for example, X-
linced
ichthyosis, psoriasis, atopic dermatitis, allergic contact dermatitis,
epidermolytic
hyperlceratosis, and seborrheic dermatitis. For example, epidennodysplasia is
a form
of faulty development of the epidermis. Another example is "epidennolysis",
which
refers to a loosened state of the epidermis with formation of blebs and bullae
either
spontaneously or at the site of trauma.
As used herein, the term "psoriasis" refers to a hyperproliferative shin
disorder which alters the shin's regulatory mechanisms. In particular, lesions
axe
formed which involve primary and secondary alterations in epidermal
proliferation,
inflammatory responses of the skin, and an expression of regulatory molecules
such
as lympholcines and inflammatory factors. Psoriatic shin is morphologically
characterized by an increased turnover of epidermal cells, thiclcened
epidermis,
abnormal lceratinization, inflammatory cell infiltrates into the dermis layer
and
polymorphonucleax leulcocyte infiltration into the epidermis layer resulting
in an
increase in the basal cell cycle. Additionally, hyperl~eratotic and
paralceratotic cells
axe present.
The term "slcin" refers to the outer protective covering of the body,
consisting
of the corium and the epidermis, and is understood to include sweat and
sebaceous
glands, as well as hair follicle structures. Throughout the present
application, the
adjective "cutaneous" may be used, and should be understood to refer generally
to
attributes of the slcin, as appropriate to the context in which they are used.
The term "s~rzoothened gain-of function" refers to an aberrant modification or
mutation of a s~rzo gene, or an increased level of expression of the gene,
which
results in a phenotype which resembles contacting a cell with a hedgehog
protein,
e.g., aberrant activation of a hedgehog pathway. While not wishing to be bound
by
any particular theory, it is noted that ptc may not signal directly into the
cell, but
rather interact with s~zoothehed, another membrane-bound protein located
downstream of ptc in hedgehog signaling (Marigo et al., (1996) Nature 384: 177-
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179). The gene smo is a segment-polarity gene required for the coiTect
patterning of
every segment in Drosophila (Alcedo et al., (1996) Cell 86: 221-232). Human
homologs of smo have been identified. See, for example, Stone et al. (1996)
Nature
384:129-134, and GenBanlc accession U84401. The smoot7zefZed gene encodes an
integral membrane protein with characteristics of heterotrimeric G-protein-
coupled
receptors; i.e., 7-transmembrane regions. This protein shows homology to the
Drosoplula F~~izzled (Fz) protein, a member of the wif~gless pathway. It was
originally thought that smo encodes a receptor of the Hh signal. However, this
suggestion was subsequently disproved, as evidence for ptc being the Hh
receptor
was obtained. Cells that express Smo fail to bind Hh, indicating that snzo
does not
interact directly with Hh (Nusse, (1996) Nature 384: 119-120). Rather, the
binding
of Sohie hedgehog (SHH) to its receptor, PTCH, is thought to prevent normal
inhibition by PTCH of smoothened (SMO), a seven-span transmembrane protein.
Recently, it has been reported that activating smoothehed mutations occur in
sporadic basal cell carcinoma, Xie et al. (1998) Nature 391: 90-2, and
primitive
neuroectodermal tumors of the central nervous system, Reifenberger et al.
(1998)
Cancer Res 58: 1798-803.
The term "therapeutic index" refers to the therapeutic index of a drug defined
as LDso/EDso.
As used herein, "transformed cells" refers to cells which have spontaneously
converted to a state of unrestrained growth, i.e., they have acquired the
ability to
grow through an indefinite number of divisions in culture. Transformed cells
may be
characterized by such terms as neoplastic, anaplastic and/or hyperplastic,
with
respect to their loss of growth control.
The term "acylamino" is art-recognized and refers to a moiety that can be
represented by the general formula:
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0
R 11
R9
wherein R9 is as defined above, and R'l l represents a hydrogen, an allcyl, an
allcenyl
or -(CH2)m-Rg, where m and Rg are as defined above.
Herein, the term "aliphatic group" refers to a straight-chain, branched-chain,
or cyclic aliphatic hydrocarbon group and includes saturated and unsaturated
aliphatic groups, such as an alkyl group, an allcenyl group, and an allcynyl
group.
The terms "allcenyl" and "allcynyl" refer to unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls described above,
but that
contain at least one double or triple bond respectively.
The terms "allcoxyl" or "allcoxy" as used herein refers to an alkyl group, as
defined above, having an oxygen radical attached thereto. Representative
allcoxyl
groups include methoxy, ethoxy, propyloxy, tent-butoxy and the like. An
"ether" is
two hydrocarbons covalently linked by an oxygen. Accor dingly, the substituent
of an
alkyl that renders that alkyl an ether is or resembles an allcoxyl, such as
can be
represented by one of -O-alkyl, -O-allcenyl, -O-allcynyl, -O-(CH2)m-Rg, where
m
and Rg are described above.
The term "alkyl" refers to the radical of saturated aliphatic groups,
including
straight-chain alkyl groups, branched-chain alkyl groups, cycloallcyl
(alicyclic)
groups, alkyl-substituted cycloallcyl groups, and cycloallcyl-substituted
alkyl groups.
In preferred embodiments, a straight chain or branched chain alkyl has 30 or
fewer
carbon atoms in its backbone (e.g., C1-C3p for straight chains, C3-C30 for
branched
chains), and more preferably 20 or fewer. Likewise, preferred cycloallcyls
have from
3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7
carbons
in the ring structure.
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Moreover, the term "alkyl" (or "lower alkyl") as used throughout the
specification, examples, and claims is intended to include both "unsubstituted
alkyls" and "substituted alkyls", the latter of wluch refers to alkyl moieties
having
substituents replacing a hydrogen on one or more carbons of the hydrocarbon
baclcbone. Such substituents can include, for example, a halogen, a hydroxyl,
a
carbonyl (such as a carboxyl, an allcoxycarbonyl, a formyl, or an acyl), a
thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an
allcoxyl, a
phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amide, an
amidine, an imine, a cyano, a nitre, an azido, a sulfhydryl, an allcylthio, a
sulfate, a
sulfonate, a sulfamoyl, a sulfonamide, a sulfonyl, a heterocyclyl, an
arallcyl, or an
aromatic or heteroaromatic moiety. It will be understood by those skilled in
the art
that the moieties substituted on the hydrocarbon chain can themselves be
substituted,
if appropriate. For instance, the substituents of a substituted alkyl may
include
substituted and unsubstituted forms of amino, azido, imino, amide, phosphoryl
(including phosphonate and phosphinate), sulfonyl (including sulfate,
sulfonamide,
sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios,
carbonyls
(including lcetones, aldehydes, carboxylates, and esters), -CF3, -GN and the
like.
Exemplary substituted alkyls are described below. Cycloallcyls can be furtlier
substituted with alkyls, allcenyls, allcoxys, alkylthios, aminoallcyls,
carbonyl
substituted alkyls, -CF3, -CN, and the lilce.
Unless the number of carbons is otherwise specified, "lower alkyl" as used
herein means an allcyl group, as defined above, but having from one to ten
carbons,
more preferably from one to six carbon atoms in its backbone structure.
Likewise,
"lower allcenyl" and "lower allcynyl" have similar chain lengths. Throughout
the
application, preferred allcyl groups are lower alkyls. In preferred
embodiments, a
substituent designated herein as alkyl is a lower alkyl.
The term "allcylthio" refers to a~i alkyl group, as defined above, having a
sulfur radical attached thereto. In preferred embodiments, the "allcylthio"
moiety is
represented by one of -S-allcyl, -S-allcenyl, -S-allcynyl, and -S-(CH2)m-Rg,
wherein
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m and Rg are defined above. Representative allcylthio groups include
methylthio,
ethylthio, and the like.
The terms "amine" and "amino" are art-recognized and refer to both
mlsubstituted and substituted amines, e.g., a moiety that can be represented
by the
general formula:
R'
io
~RZO ~ +
-i'-Rlo
R9 R
9
wherein R9, Rl p and R' l0 each independently represent a hydrogen, an alkyl,
an
alkenyl, -(CH2)m-Rg, or Rg and Rlp taken together with the N atom to which
they
are attached complete a heterocycle having from 4 to 8 atoms in the ring
structure;
Rg represents an aryl, a cycloallcyl, a cycloallcenyl, a heterocycle or a
polycycle; and
m is zero or an integer in the range of 1 to 8. In preferred embodiments, only
one of
R9 or Rl0 can be a carbonyl, e.g., Rg, Rl0 and the nitrogen together do not
form an
imide. In even more preferred embodiments, R9 and R10 (and optionally R' l0)
each
independently represent a hydrogen, an allcyl, an alkenyl, or -(CH2)m-Rg.
Thus, the
term "alkylamine" as used herein means an amine group, as defined above,
having a
substituted or unsubstituted all~yl attached thereto, i.e., at least one of R9
and Rl p is
an allcyl group.
The term "amido" is art-recognized as an amino-substituted carbonyl and
includes a moiety that can be represented by the general formula:
O
R9
N
Rio
wherein R9, Rl p are as defined above. Preferred embodiments of the amide will
not
include imides which may be unstable.
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The term "arallcyl", as used herein, refers to an allcyl group substituted
with
an aryl group (e.g., an aromatic or heteroaromatic group).
The term "aryl" as used herein includes 5-, 6-, and 7-membered single-ring
aromatic groups that may include from zero to four heteroatoms, for example,
benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,
pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups
having
heteroatoms in the ring structure may also be referred to as "aryl
heterocycles" or
"heteroaromatics." The aromatic ring can be substituted at one or more ring
positions
with such substituents as described above, for example, halogen, azide, alkyl,
arallcyl, alkenyl, allcynyl, cycloallcyl, hydroxyl, allcoxyl, amino, nitre,
sulfhydryl,
imino, amide, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl,
ether,
allcylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl,
aromatic or
heteroaromatic moieties, -CF3, -CN, or the like. The term "aryl" also includes
polycyclic ring systems having two or more cyclic rings in which two or more
carbons are common to two adjoining rings (the rings are "fused rings")
wherein at
least one of the rings is aromatic, e.g., the other cyclic rings can be
cycloalkyls,
cycloallcenyls, cycloallcynyls, aryls and/or heterocyclyls.
The term "carbocycle", as used herein, refers to an aromatic or non-aromatic
ring in which each atom of the ring is carbon.
The term "carbonyl" is art-recognized and includes such moieties as can be
represented by the general formula:
0 0
~X-Ri1 ~ or-X~R,
11
wherein X~ is a bond or represents an oxygen or a sulfur, and R11 represents a
hydrogen, an alkyl, an allcenyl, -(CH2)m Rg or a pharmaceutically acceptable
salt,
R' 11 represents a hydrogen, an alkyl, an allcenyl or -(CH2)m-Rg, where m and
Rg
are as defined above. Where X is an oxygen and Rl 1 or R' 11 is not hydrogen,
the
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formula represents an "ester". Where X is an oxygen, and Rl1 is as defined
above,
the moiety is referred to herein as a carboxyl group, and particularly when
Rl1 is a
hydrogen, the formula represents a "carboxylic acid". Where X is an oxygen,
and
R'11 is hydrogen, the formula represents a "formate". In general, where the
oxygen
atom of the above formula is replaced by sulfiu, the formula represents a
"thiocarbonyl" group. Where X is a sulfur and Rll or R'11 is not hydrogen, the
formula represents a "thioester." Where X is a sulfur and R11 is hydrogen, the
formula represents a "thiocarboxylic acid." Where X is a sulfur and Rll' is
hydrogen, the formula represents a "thiolformate." On the other hand, where X
is a
bond, and Rll is not hydrogen, the above formula represents a "l~etone" group.
Where X is a bond, and Rll is hydrogen, the above formula represents an
"aldehyde" group.
The term "heteroatom" as used herein means an atom of any element other
than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen,
phosphorus, sulfur and selenium.
The terms "heterocyclyl" or "heterocyclic group" refer to 3- to 10-membered
ring structures, more preferably 3- to 7-membered rings, whose ring structures
include one to four heteroatoms. Heterocycles can also be polycycles.
Heterocyclyl
groups include, for example, thiophene, thianthrene, fiuan, pyran,
isobenzofuran,
chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isotluazole,
isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,
indole,
indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,
naphthyridine,
quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,
phenanthridine,
acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine,
furazan, phenoxazine, pyTOlidine, oxolane, thiolane, oxazole, piperidine,
piperazine,
morpholine, lactones, lactams such as azetidinones and pyrrolidinones,
sultams,
sultones, and the like. The heterocyclic ring can be substituted at one or
more
positions with such substituents as described above, as for example, halogen,
allcyl,
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arallcyl, allcenyl, allcynyl, cycloallcyl, hydroxyl, amino, nitro, sulfhydryl,
imino,
amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,
allsylthio, sulfonyl, lcetone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, -CF3, -CN, or the like.
As used herein, the term "nitro" means -N02; the term "halogen" designates -
F, -Cl, -Br or -l; the term "sulfhydryl" means -SH; the term "hydroxyl" means -
OH;
and the term "sulfonyl" means -S02-.
A "phosphonamidite" can be represented in the general formula:
R4s Rne
I I
-~2 I -O- O.r ~2 I - OR46
N ~ R9 ~ Rlo N ~ R9 ~ Rio
wherein R9 and Rlp are as defined above, Q2 represents O, S or N, and Rq.g
represents a lower alkyl or an aryl, Q~ represents O, S or N.
A "phosphoramidite" can be represented in the general formula:
0 0
n n
-QZ p-0- -Q2 p- 0846
or I
N ~ R91 Rio N ~ R9 ~ Rio
wherein R9 and Rlp are as defined above, and Q2 represents O, S or N.
A "phosphoryl" can in general be represented by the formula:
Qz
-p
I
084 6
wherein Q1 represented S or O, and Rq.6 represents hydrogen, a lower allcyl or
an
aryl. When used to substitute, for example, an alltyl, the phosphoryl group of
the
phosphorylallcyl can be represented by the general formula:
28
CA 02424785 2003-04-03
WO 02/30421 PCT/USO1/32054
y y
-Q2 i -p- or-Q~ i - OR4s
OR46 ' ORQs
wherein Q1 represented S or O, and each Rq.6 independently represents
hydrogen, a
lower alkyl or an aryl, Q~ represents O, S or N. When Q 1 is a~1 S, the
phosphoryl
moiety is a "phosphorothioate".
The terms "polycyclyl" or "polycyclic group" refer to two or more rings (e.g.,
cycloallcyls, cycloallcenyls, cycloallcynyls, aryls and/or heterocyclyls) in
which two or
more carbons are common to two adjoining rings, e.g., the rings are "fused
rings".
Rings that are joined through non-adjacent atoms are termed "bridged" rings.
Each
of the rings of the polycycle can be substituted with such substituents as
described
above, as for example, halogen, allcyl, arallcyl, allcenyl, alkynyl,
cycloalkyl, hydroxyl,
amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate,
carbonyl, carboxyl, silyl, ether, allcylthio, sulfonyl, lcetone, aldehyde,
ester, a
heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
The phrase "protecting group" as used herein means temporary substituents
which protect a potentially reactive functional group from undesired chemical
transformations. Examples of such protecting groups include esters of
carboxylic
acids, silyl ethers of alcohols, and acetals and lcetals of aldehydes and
ketones,
respectively. The field of protecting group chemistry has been reviewed
(Greene,
T.W.; Wuts, P.G.M. Protective Groups irc O~gahic Syv~thesis, 2"d ed.; Wiley:
New
York, 1991 ).
A "selenoallcyl" refers to an alkyl group having a substituted seleno group
attached thereto. Exemplary "selenoethers" which may be substituted on the
alkyl are
selected from one of -Se-alkyl, -Se-allcenyl, -Se-allcynyl, and -Se-(CH2)m-Rg,
m and
Rg being defined above.
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As used herein, the term "substituted" is contemplated to include all
permissible substituents of organic compounds. In a broad aspect, the
permissible
substituents include acyclic and cyclic, branched and unbranched, carbocyclic
and
heterocyclic, aromatic and nonaromatic substituents of organic compounds.
Illustrative substituents include, for example, those described herein above.
The
permissible substituents can be one or more and the same or different for
appropriate
organic compounds. For purposes of this invention, the heteroatoms such as
nitrogen
may have hydrogen substituents andlor any permissible substituents of organic
compounds described herein which satisfy the valences of the heteroatoms. This
invention is not intended to be limited in any manner by the permissible
substituents
of organic compounds.
It will be understood that "substitution" or "substituted with" includes the
implicit proviso that such substitution is in accordance with permitted
valence of the
substituted atom and the substituent, and that the substitution results in a
stable
compound, e.g., which does not spontaneously undergo transformation such as by
rearrangement, cyclization, elimination, etc.
The ternz "sulfamoyl" is art-recognized and includes a moiety that can be
represented by the general formula:
Rio
-S-N
II \
R9
in which R9 and R10 are as defined above.
The term "sulfate" is art recognized and includes a moiety that can be
represented by the general formula:
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O
I I
-0- ~-OR41
0
in which Rq.l is as defined above.
The term "sulfonamido" is art recognized and includes a moiety that can be
represented by the general formula:
O
I 1
~I R~ m
R O
9
in which R9 and R'11 axe as defined above.
The term "sulfonate" is axt-recognized and includes a moiety that can be
represented by the general formula:
0
I I
- i-OR4i
0
in which Rq.l is an electron pair, hydrogen, all~yl, cycloalkyl, or aryl.
The terms "sulfoxido" or "sulfinyl", as used herein, refers to a moiety that
can be represented by the general formula:
O
I I
-s-R44
4
in which Rq.q. is selected from the group consisting of hydrogen, allcyl,
allcenyl,
allcynyl, cycloallcyl, heterocyclyl, arallcyl, or aryl.
Analogous substitutions can be made to allcenyl and allcynyl groups to
produce, for example, aminoallcenyls, aminoallcynyls, amidoalkenyls,
amidoallcynyls,
iminoallcenyls, iminoallcynyls, thioallcenyls, thioall~yryls, carbonyl-
substituted
allcenyls or allcynyls.
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As used herein, the definition of each expression, e.g., alkyl, m, n, etc.,
when
it occurs more than once in airy structure, is intended to be independent of
its
definition elsewhere in the same structure.
The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to
trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, and
nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate,
mesylate,
and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester,
p-
toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate
ester
functional groups and molecules that contain said groups, respectively.
The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl,
trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl and
methanesulfonyl, respectively. A more comprehensive list of the abbreviations
utilized by organic chemists of ordinary skill in the au appears in the first
issue of
each volume of the Jou~hal of O~ga~ic Chemistry; this list is typically
presented in a
table entitled Standard List of Abbreviations. The abbreviations contained in
said
list, and all abbreviations utilized by organic chemists of ordinary skill in
the art are
hereby incorporated by reference.
Certain compounds of the present invention may exist in particular geometric
or stereoisomeric forms. The present invention contemplates all such
compounds,
including cis- and traps-isomers, R- and S-enantiomers, diastereomers, (D)-
isomers,
(L)-isomers, the racemic mixtures thereof, annd other mixtures thereof, as
falling
within the scope of the invention. Additional asynunetric carbon atoms may be
present in a substituent such as an alkyl group. All such isomers, as well as
mixtures
thereof, are intended to be included in this invention.
If, for instance, a particular enantiomer of a compound of the present
invention is desired, it may be prepared by asymmetric synthesis, or by
derivation
with a chiral auxiliary, where the resulting diastereomeric mixture is
separated and
the auxiliary group cleaved to provide the pure desired ena~ltiomers.
Alternatively,
where the molecule contains a basic functional group, such as amino, or an
acidic
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functional group, such as carboxyl, diastereomeric salts may be formed with an
appropriate optically active acid or base, followed by resolution of the
diastereomers
thus formed by fractional crystallization or chromatographic means well lcnovm
in
the art, and subsequent recovery of the pure enantiomers.
Contemplated equivalents of the compounds described above include
compounds which otherwise correspond thereto, and which have the same general
properties thereof (e.g., the ability to inhibit hedgehog signaling), wherein
one or
more simple variations of substituents are made which do not adversely affect
the
efficacy of the compound. In general, the compounds of the present invention
may
be prepared by the methods illustrated in the general reaction schemes as, for
example, described below, or by modifications thereof, using readily available
stauting materials, reagents and conventional synthesis procedures. In these
reactions, it is also possible to make use of variants which are in themselves
known,
but are not mentioned here.
For purposes of this invention, the chemical elements are identified in
accordance with the Periodic Table of the Elements, CAS version, Handbook of
Chemistry and Physics, 67th Ed., 1986-87, inside cover. Also for purposes of
this
invention, the term "hydrocarbon" is contemplated to include all permissible
compounds having at least one hydrogen and one carbon atom. In a broad aspect,
the
permissible hydrocarbons include acyclic and cyclic, branched and unbranched,
carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds which
can be substituted or unsubstituted.
III. Exemplary Compounds of the Ihve~ctio~.
As described in further detail below, it is contemplated that the subject
methods can be carried out using a variety of different small molecules which
can be
readily identified, for example, by such drug screening assays as described
herein.
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For example, compounds useful in the subject methods include compounds may be
represented by general formula (I):
LR~
Formula I
wherein, as valence and stability permit,
Rt, R2, R3, and R4, independently for each occurrence, represent H, lower
alkyl, -(CH~)naryl (e.g., substituted or unsubstituted), or -(CH2)nheteroaryl
(e.g.,
substituted or unsubstituted);
L, independently for each occurrence, is absent or represents -(CH2)n-,
allcenyl-, -allcynyl-, -(CH~)nallcenyl-, -(CH~)nallcynyl-, -(CH2)n0(CH~)p-,
(CH2)nNRs(CH2)p-~ -(CH2)nS(CH2)p-~ -(CH2)nallcenyl(CH2)p-, _
(CH~,)nallcynyl(CH2)p-, -O(CH2)11-, -NRg(CH2)n-, or -S(CH~)n-;
X and D, independently, can be selected from -N(Rg)-, -O-, -S-, -(Rg)N-
N(Rg)-, -ON(Rg)-, or a direct bond;
Y and Z, independently, can be selected from O or S;
E represents O, S, or NRS, wherein RS represents LR8 or -(C=O)LRB.
Rg, independently for each occurrence, represents H, lower alkyl, -
(CH2)naryl (e.g., substituted or unsubstituted), -(CH2)nheteroaryl (e.g.,
substituted or
unsubstituted), or two Rg talcen together may form a 4- to 8-membered ring;
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p' represents, independently for each occurrence, an integer from 0 to 10,
preferably from 0 to 3;
n, individually for each occurrence, represents an integer from 0 to 10,
preferably from 0 to 5; and
q and r represent, independently for each occurrence, an integer from 0-2.
In certain embodiments, D does not represent N-lower alkyl. In certain
embodiments, D represents an arallcyl- or heteroaralkyl-substituted amine.
In certain embodiments, Rl represents a lower alkyl group, such as a
branched alkyl, a cycloallcyl, or a cycloallcylallcyl, for example,
cyclopropyl,
cyclopropylmethyl, neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl,
cyclobutylmethyl, etc.
In certain embodiments, ~ and Z are O.
In certain embodiments, the sum of q and r is less than 4, e.g., is 2 or 3.
In certain embodiments, XLR4, taken together, include a cyclic amine, such
as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
In certain embodiments, at least one of Rl, R2, and R3 includes an aryl or
heteroaryl group. In certain related embodiments, at least two of Rl, R2, and
R3
include an aryl or heteroaryl group. In certain embodiments, R1 is lower
alkyl.
In certain embodiments, L attached to Rl represents O, S, or NRB, such as
NH.
In certain embodiments, E is NRB. In certain embodiments, E represents an
arallcyl- or heteroarallcyl-substituted amine, e.g., including polycyclic R8.
In certain embodiments, X is not NH. In certain embodiments, X is included
in a ring, or, taken together with -C(='S~-, represents a tertiary amide.
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In certain embodiments, compounds useful in the present invention may be
represented by general formula (II):
M R3
Z
LR~
Formula II
wherein, as valence and stability permit,
Rl, R2, R3, R4, Rg, L, X, Y, Z, n, p, q, and r are as defined above;
M is absent or represents L, -S02L-, or -(C=O)L-; and
s represents, independently for each occurrence, an integer from 0-2.
In certain embodiments, Y and Z are O.
In certain embodiments, Rl represents a lower allcyl group, such as a
branched alkyl, a cycloallcyl, or a cycloallcylalkyl, for example,
cyclopropyl,
cyclopropylmethyl, neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl,
cyclobutylmethyl, etc.
In certain embodiments, the sum of q, r, and s is less than 5, e.g., is 2, 3,
or 4.
In certain embodiments, XLR4, taken together, include a cyclic amine, such
as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
In certain embodiments, L attached to Rl represents O, S, or NRB, such as
NH.
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In certain embodiments, at least one of Rl, R2, a~.ld R3 includes an aryl or
heteroaxyl group. In certain related embodiments, at least two of RI, R2, and
R3
include an aryl or heteroasyl group.
In certain embodiments, M is absent.
In certain embodiments, X is not NH. In certain embodiments, X is included
in a ring, or, taken together with -C(=Y)-, represents a tertiary amide.
In certain embodiments, compounds useful in the present invention may be
represented by general formula (III):
Formula III
wherein, as valence and stability permit,
Rl, R~, R3, R4, Rg, L, M, X, Y, Z, n, p, q, and r are as defined above.
In certain embodiments, Y and Z are O.
In certain embodiments, Rl represents a lower alkyl group, preferably a
branched alkyl, a cycloalkyl, or a cycloallcylalkyl, for example, cyclopropyl,
cyclopropylmethyl, neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl,
cyclobutylmethyl, etc.
In certain embodiments, the sum of q and r is less than 4, e.g., is 2 or 3.
37
re2t LR1
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In certain embodiments, XLR4, taken together, include a cyclic amine, such
as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
In certain embodiments, at least one of Rl, Ra, and R3 includes an aryl or
heteroaryl group. In certain related embodiments, at least two of R~, R2, and
R3
include an aryl or heteroaryl group. In certain embodiments, Rl is lower
alkyl.
In certain embodiments, L attached to Rl represents Q, S, or NRB, such as
NH.
In certain embodiments, M is absent.
In certain embodiments, X is not NH. In certain embodiments, X is included
in a ring, or, taken together with -C(=~-, represents a tertiary amide.
In certain embodiments, compounds useful in the present invention may be
represented by general formula (IV):
Formula IV
wherein, as valence and stability permit,
Rl, R2, R3, R4, Rg, L, M, X, n, and p are as defined above.
In certain embodiments, XLR4, taken together, include a cyclic amine, such
as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
In certain embodiments, Rl represents a lower alkyl group, preferably a
branched alkyl, a cycloallcyl, or a cycloallcylalkyl, for example,
cyclopropyl,
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cyclopropylmethyl, neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl,
cyclobutylmethyl, etc.
In certain embodiments, at least one of Rl, R2, and R3 includes an aryl or
heteroaryl group. In certain related embodiments, at least two of Rl, R2, and
R3
include an aryl or heteroaryl group. In certain embodiments, Rl is lower
alkyl.
In certain embodiments, L attached to Rl repxesents O, S, or NRg, such as
NH.
In certain embodiments, M is absent.
In certain embodiments, X is not NH. In certain embodiments, X is included
in a ring, or, taken together with -C(=Y)-, represents a tertiary amide.
In certain embodiments L represents a direct bond for all occurrences.
In certain embodiments, compounds useful in the present invention may be
represented by general formula (V):
'r
N Z'
R~ vR5
Formula V
wherein, as valence and stability permit,
Y, n, p, q, and r are as defined above;
Z' represents -C(=O)-, -C(=S)-, -C(=NH)-, 502, or SO, preferably -C(=O)-, -
C(=S)-;
V is absent or represents O, S, or NRs;
G is absent or represents -C(=O)- or -S02-;
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J, independently for each occurrence, represents H or substituted or
unsubstituted lower alkyl or allcylene, such as methyl, ethyl, methylene,
ethylene,
etc., attached to NC(=~, such that both occurrences of N adjacent to J axe
linked
through at least one occurrence of J, and
R9, independently for each occurrence, is absent or represents H or lower
allcyl, or two occurrences of J or one occurrence of J taken together with one
occurrence of R9, forms a ring of from 5 to 7 members, which ring includes one
or
both occurrences of N;
RS represents substituted or unsubstituted alkyl (e.g., branched or
unbranched), allcenyl (e.g., branched or unbranched), allcynyl (e.g., branched
or
unbranched), cycloallcyl, or cycloallcylallryl;
R~ represents substituted or unsubstituted axyl, arallcyl, heteroatyl,
heteroarallcyl, heterocyclyl, heterocyclylallcyl, cycloallcyl, or
cycloallcylalkyl,
including polycyclic groups; and
R7 represents substituted or unsubstituted aryl, aralkyl, heteroaiyl, or
heteroarallcyl.
In certain embodiments, Y is O. In certain embodiments, Z' represents 502, -
C(=O)-, or -C(=S)-. a
In certain embodiments, the sum of q and r is less than 4.
In certain embodiments, NJ2N, tal~en together, represent a cyclic diamine,
such as a piperazine, etc., which may be substituted or unsubstituted, e.g.,
with one
or more substitutents such as oxo, lower allcyl, lower alkyl ether, etc. In
certain other
embodiments, NJ2 or NJR9 taken together represent a substituted or
unsubstituted
heterocyclic ring to which the other occurrence of N is attached. In certain
embodiments, one or both occurrences of J are substituted with one or more of
lower
alkyl, lower allcyl ether, lower alkyl thioether, amido, oxo, etc. In certain
CA 02424785 2003-04-03
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embodiments, a heterocyclic ring which comprises an occmTence of J has from 5
to
8 members.
In certain embodiments, RS represents a branched allcyl, cycloallcyl, or
cycloalkylallcyl.
In certain embodiments, R6 includes at least one heterocyclic ring, such as a
thiophene, furan, oxazole, benzodioxane, benzodioxole, pyrrole, indole, etc.
In certain embodiments, R7 represents a phenyl alkyl, such as a benzyl group,
optionally substituted with halogen, hydroxyl, lower alkyl, nitro, cyano,
lower alkyl
ether (e.g., optionally substituted, such as CHF2CFZO), or lower alkyl
thioether (e.g.,
optionally substituted, such as CF3S).
In certain embodiments, R8, when it occurs in V, represents H or lower allcyl,
preferably H.
In certain embodiments, compounds useful in the present invention may be
represented by general formula (VI):
(R9)NJ2
VR5
Formula VI
wherein, as valence and stability permit,
R5, R6, R7, Rg, R9, Rlo, G, J, V, Y, Z', n, and p are as defined above.
In certain embodiments, Y is O. In certain embodiments, Z' represents SOz, -
C(=O)-, or -C(=S)-.
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In certain embodiments, NJ2N, talcen together, represent a heterocyclic ring,
such as a piperazine, etc., which may be substituted or unsubstituted, e.g.,
with one
or more substitutents such as oxo, lower alkyl, lower allcyl ether, etc. In
certain other
embodiments, NJa or NJR9 taken together represent a substituted or
unsubstituted
heterocyclic ring to which the other occurrence of N is attached. In certain
embodiments, one or both occurrences of J are substituted with one or more of
lower
alkyl, lower alkyl ether, lower alkyl thioether, amido, oxo, etc. In certain
embodiments, a heterocyclic ring which comprises an occurrence of J has from 5
to
8 members.
In certain embodiments, RS represents a branched alkyl, cycloallcyl, or
cycloalkylallcyl.
In certain embodiments, R6 includes at least one heterocyclic ring, such as a
thiophene, furan, oxazole, benzodioxane, benzodioxole, pyrrole, indole, etc.
In certain embodiments, R7 represents a phenyl alkyl, such as a benzyl group,
optionally substituted with halogen, hydroxyl, lower alkyl, nitro, cyano,
lower alkyl
ether (e.g., optionally substituted, such as CHF~,CF20), or lower alkyl
thioether (e.g.,
optionally substituted, such as CF3S).
In certain embodiments, R8, when it occurs in V, represents H or lower alkyl,
preferably H.
In certain embodiments, the subject compound is selected from the
compounds depicted in Figure 32.
In certain embodiments, the subject antagonists can be chosen on the basis of
their selectively for the hedgehog pathway. This selectivity can be for the
hedgehog
pathway versus other pathways, or for selectivity between particular hedgehog
pathways, e.g., ptc-l, ptc-2, etc.
In certain preferred embodiments, the subject inhibitors inhibit ptc loss-of
function, hedgehog gain-of function, or smoothened gain-of function mediated
signal transduction with an EDsp of 1 mM or less, more preferably of 1 ~,M or
less,
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and even more preferably of 1 nM or less. Similarly, in certain preferred
embodiments, the subject inhibitors inhibit activity of the hedgehog pathway
with a
K; less than 10 nM, preferably less than 1 nM, even more preferably less than
0.1
nM.
In particular embodiments, the small molecule is chosen for use because it is
more selective for one patched isoform over the next, e.g., 10-fold, and more
preferably at least 100- or even 1000-fold more selective for one hatched
pathway
(ptc-l,~te-2) over another.
In certain embodiments, a compound which is an antagonist of the hedgehog
pathway is chosen to selectively antagonize hedgehog activity over protein
l~inases
other than PISA, such as PI~C, e.g., the compound modulates the activity of
the
hedgehog pathway at least an order of magnitude more strongly than it
modulates the
activity of a~.a.other protein lunase, preferably at least two orders of
magnitude more
strongly, even more preferably at least three orders of magnitude more
strongly.
Thus, for example, a preferred inhibitor of the hedgehog pathway may inhibit
hedgehog activity with a I~; at least an order of magnitude lower than its I~;
for
inhibition of PI~C, preferably at least two orders of magnitude lower, even
more
preferably at least three orders of magnitude lower. In certain embodiments,
the K;
for PKA inlubition is less than 10 nM, preferably less than 1 nM, even more
preferably less than 0.1 nM.
Methods of Preparation of Subject Compounds
The present invention further provides methods for preparing the subject
compounds, as set forth above. For example, in one embodiment, a compound of
Formula X may be transformed according to the following scheme:
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Y Y
B B
A s N/ A A 7s N/
9 ,. 9 r
X GH XI LG
B
y Y
B B
~N/ C A s N/
E
XIII XII
NHS Ns
wherein q, s, and r each represent, independently, an integer in the range of-
0 to 2,
such that the sum of q+s+r is an integer in the range of 2-4;
LG represents a leaving group, such as a halogen (e.g., Cl, Br, or I) or a
sulfonate ester (e.g., tosylate, mesylate, triflate, etc.);
A represents an oxygen or sulfur bound to an acid-protecting group or a
group having the formula XLRq;
B represents a nitrogen-protecting group or a group having the formula MR3;
R3 and R4, independently for each occurrence, represent H, lower alkyl, -
(CH2)na~yl (e.g., substituted or unsubstituted), or -(CH~)nheteroaryl (e.g.,
substituted
or unsubstituted);
Y can be selected from O and S;
X is be selected from -N(Rg)-, -O-, -S-, or a direct bond;
M is absent or represents L, -S02L-, or -(C=O)L-;
L, independently for each occurrence, is absent ~or represents -(CH~)nallcyl-,
-
allcenyl-, -allcynyl-, -(CH~)"all~enyl-, -(CH2)nall~ynyl-, -(CH2)n0(CH2)p-, -
(CH2)nNRs(CH2)p-~ -(CH2)ns(CH?)p-, -(CH2)nallcenyl(CH2)p-, _
(CH2)nallcynyl(CH~)p-, -O(CH2)n-, -NRg(CH~)n-, or -S(CH2)n-;
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Rg, independently for each occurrence, represents H, lower alkyl, -
(CH2)naryl (e.g., substituted or unsubstituted), -(CH~)"heteroaryl (e.g.,
substituted or
unsubstituted), or two Rg taken together may form a 4- to 8-membered ring;
p represents, independently for each occurrence, an integer from 0 to 10,
preferably from 0 to 3; and
n, individually for each occmTence, represents an integer from 0 to 10,
preferably from 0 to 5,
and wherein step A includes converting the hydroxyl to a leaving group,
step B includes displacing the leaving group with an azide, and
step C includes reducing the azide to an amine.
In certain embodiments, converting the hydroxyl to a leaving group may be
performed by reacting the hydroxyl with a sulfonyl halide to generate a
sulfonate
ester, e.g., using tosyl chloride or tosyl anhydride to generate a tosylate,
mesyl
chloride or mesyl anhydride to generate a mesylate, or triflyl chloride or
triflyl
anhydride to generate a triflate, etc. In certain other embodiments,
converting the
hydroxyl to a leaving group may be performed by reacting the hydroxyl with an
halogenating reagent such as a thionyl halide, a phosphorous trihalide,
phosphorous
pentahalide, phosphorous oxyhalide, etc. Other techniques for converting a
hydroxyl
group to a leaving group are well known in the art and may be used in step A.
In certain embodiments, step A further includes displacing a first leaving
group with a second leaving group and inverting the stereochemistry of the
leaving
group-bearing carbon. Thus, for example, if the hydroxyl of the compound of
Formula X has a cis stereochemical relationship with the group bearing Y and
A,
reaction of this compound with mesyl chloride will generate a mesylate in a
cis
stereochemical relationship with the group bearing Y and A. Reaction of this
mesylate with a nucleophilic halide reagent, such as NaI, will result in
displacement
of the mesylate with iodide, generating a compound of Formula XI wherein the
leaving group, iodine, and the group bearing Y and A have a tocrhs
stereochemical
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relationship. Use of this technique permits compounds having either cis or
t~a~s
stereochemistry, selectively, from a diastereomerically pure starting
material, e.g., a
pure compound having a cis stereochemical relationship between the hydroxyl
and
the group bearing Y and A.
In certain embodiments, displacing the leaving group with an azide may be
performed using an allcali or alkaline earth metal salt of azide anion, such
as sodium
azide, using a silyl azide reagent, such as trimethylsilyl azide, or using any
other
azide reagent, e.g., a nucleophilic azide source, as is well known in the art.
In certain embodiments, reducing the azide to an amine may be performed
using a hydride reagent, such as lithium aluminum hydride, lithium
triallcylborohydride, etc., using a reducing metal and an acid source, such as
zinc
metal or samarium diiodide with acetic acid, using catalytic hydrogenation,
such as
hydrogen and a transition metal catalyst such as platinum or palladium, or by
any
other suitable means.
In certain embodiments, q+s+r is an integer from 2 to 3. In certain
embodiments, s is 0. In certain embodiments, q and r each represent 1.
In certain embodiments, A represents an oxygen bound to an acid-protecting
group. For example, the acid protecting group may be a substituted or
unsubstituted
alkyl, allcenyl, alkynyl, aryl, or arallcyl group. Examples of such groups
include
methyl, ethyl, trimethylsilylethyl, methylthiomethyl, allyl, benzyl, p-
nitrobenzyl,
tetrahydropyranyl (THP), t-butyl, or any other suitable group. A wide variety
of acid-
protecting groups are known in the art and may be employed in this method
without
departing from the scope and spirit of the invention. In other embodiments, A
represents an allcylthio group.
In certain embodiments, B represents a nitrogen-protecting group, such as a
substituted or unsubstituted acyl, alkyl, allcenyl, alkynyl, aryl, or arallcyl
group, or a
group which, when taken together with N, forms a carbamate. Common nitrogen-
protecting groups include benzyl, allyl, p-methoxybenzyl, acetyl,
trifluoroacetyl, t-
butoxycarbonyl, benzyloxycarbonyl, etc. A wide variety of nitrogen-protecting
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groups are known in the art and may be employed in this method without
departing
from the scope and spirit of the invention.
In certain embodiments, Y is O.
In certain embodiments, A represents XLR4, which may, taken together,
include a cyclic amine, such as a piperazine, a morpholine, a piperidine, a
pyrrolidine, etc.
In certain embodiments, R3 includes an aryl or heteroa~yl group.
In certain embodiments, M is absent.
In certain embodiments, X is NRB, and preferably is not NH. In certain
embodiments, X is
included in a ring, or, taken together with-C(=Y)-, represents a tertiary
amide.
In certain embodiments, the compound of Formula XIII is enriched for the
isomer wherein the amine and the substituent including Y and A have a eis
relationship, e.g., >75%, >85%, or even >95% of the cis isomer. In other
embodiments, the compound of Formula XIII is enriched for the isomer wherein
the
two substituents have a t~a~s relationship, e.g., >75%, >85%, or even >95% of
the
t~~aszs isomer. Preferably, such enrichment results from employing an
isomerically
enriched starting material, e.g., the compound of Formula X is enriched for,
>75%,
>85%, or even >95% of the cis or tnahs isomer prior to beginning step A.
Similarly, in another embodiment, a compound of Formula XIV may be
transformed according to the following scheme:
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0 0
l
A N A p N
'r ~ ~ 'r
XIV 9 XV ~1
OH LG
O O
B B
A N A N
E C
~r ~ 'r
XVII 9 NHZ XVI ~ Ns
wherein q and r each represent, independently, an integer in the range of 0 to
2, such
that the sum of q+r is an integer in the range of 2-4;
LG represents a leaving group, such as a halogen (e.g., Cl, Br, or I) or a
sulfonate ester (e.g., tosylate, mesylate, triflate, etc.);
A represents an oxygen or sulfur bound to an acid-protecting group or a
group having the formula NJ2N(R9)2;
B represents a nitrogen-protecting group or a group having the formula GR~;
G is absent or represents -C(=O)-, -C(=S)-, or -S02-;
J, independently for each occurrence, represents H or substituted or
unsubstituted lower alkyl or allcylene, such as methyl, ethyl, etc., attached
to
NC(=~, such that both occurrences of N adjacent to 3 are linked through at
least
one occurrence of J, and
R9, independently for each occurrence, is absent or represents H or lower
alkyl, or two occurrences of J or one occurrence of J talcen together with one
occurrence of R9, forms a ring of from S to 7 members, which ring includes one
or
both occurrences of N;
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R6 represents substituted or unsubstituted aryl, arallcyl, heteroa~.yl,
heteroarallcyl, heterocyclyl, heterocyclylallcyl, cycloallcyl, or
cycloallcylallcyl,
including polycyclic groups; and
Y can be selected from O and S;
and wherein step A includes converting the hydroxyl to a leaving group,
step B includes displacing the leaving group with an azide, and
step C includes reducing the azide to an amine.
In certain embodiments, converting the hydroxyl to a leaving group may be
performed by reacting the hydroxyl with a sulfonyl halide to generate a
sulfonate
ester, e.g., using tosyl chloride or tosyl anhydride to generate a tosylate,
mesyl
chloride or mesyl anhydride to generate a mesylate, or triflyl chloride or
triflyl
anhydride to generate a triflate, etc. In certain other embodiments,
converting the
hydroxyl to a leaving group may be performed by reacting the hydroxyl with an
halogenating reagent such as a thionyl halide, a phosphorous trihalide,
phosphorous
pentahalide, phosphorous oxyhalide, etc. Other techniques for converting a
hydroxyl
group to a leaving group are well known in the art and may be used in step A.
In certain embodiments, step A further includes displacing a first leaving
group with a second leaving group and inverting the stereochemistry of the
leaving
group-bearing carbon. Thus, for example, if the hydroxyl of the compound of
Formula XIV has a cis stereochemical relationship with the group beaxing Y and
A,
reaction of this compound with mesyl chloride will generate a mesylate in a
cis
stereochemical relationship with the group bearing Y and A. Reaction of this
mesylate with a nucleophilic halide reagent, such as NaI, will result in
displacement
of the mesylate with iodide, generating a compound of Formula XV wherein the
leaving group, iodine, and the group bearing Y and A have a t~a~s
stereochemical
relationship. Use of this technique permits compounds having either cis or
tans
stereochemistry, selectively, from a diastereomerically pure starting
material, e.g., a
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pw-e compound having a cis stereachemical relationship between the hydroxyl
and
the group bearing Y and A.
In certain embodiments, displacing the leaving group with an azide may be
performed using an alkali or allcaline earth metal salt of azide anion, such
as sodium
azide, using a silyl azide reagent, such as trimethylsilyl azide, or using any
other
azide reagent, e.g., a nucleoplulic azide source, as is well known in the art.
In certain embodiments, reducing the azide to an aanine may be performed
using a hydride reagent, such as lithium aluminum hydride, lithium
triallcylborohydride, etc., using a reducing metal and an acid source, such as
zinc
metal or samarium diiodide with acetic acid, using catalytic hydrogenation,
such as
hydrogen and a transition metal catalyst such as platinum or palladium, or by
any
other suitable means.
In certain embodiments, q+r is an integer from 2 to 3. In certain
embodiments, q and r each represent 1.
In certain embodiments, A represents an oxygen bound to an acid-protecting
group. For example, the acid protecting group may be a substituted or
unsubstituted
alkyl, allcenyl, alleynyl, aryl, or arallcyl group. Examples of such groups
include
methyl, ethyl, trimethylsilylethyl, methylthiomethyl, allyl, benzyl, p-
nitrobenzyl,
tetrahydropyranyl (THP), t-butyl, or any other suitable group. A wide variety
of acid-
protecting groups are known in the art and may be employed in this method
without
departing from the scope and spirit of the invention. In other embodiments, A
represents an allcylthio group.
In certain embodiments, B represents a nitrogen-protecting group, such as a
substituted or unsubstituted acyl, alkyl, alkenyl, allcynyl, aryl, or aralkyl
group, or a
group which, when taken together with N, forms a carbamate. Common nitrogen
protecting groups include benzyl, allyl, p-methoxybenzyl, acetyl,
trifluoroacetyl, t-
butoxycarbonyl, benzyloxycarbonyl, etc. A wide variety of nitrogen-protecting
groups are lcnown in the art and may be employed in this method without
departing
from the scope and spirit of the invention.
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In certain embodiments, Y is O.
In certain embodiments, B is GR~, wherein R~ includes at least one
heterocyclic ring, such as a thiophene, furan, oxazole, benzodioxane,
benzodioxole,
pyrrole, indole, etc.
In certain embodiments, A represents NJ2N, which, talcen together, may
represent a cyclic diamine, such as a piperazine, etc., which may be
substituted or
unsubstituted, e.g., with one or more substitutents such as oxo, lower allcyl,
lower
alkyl ether, etc. W certain other embodiments, NJ2 or NJR~ taken together
represent a
substituted or unsubstituted heterocyclic ring to which the other occurrence
of N is
attached. In certain embodiments, one or both occurrences of J are substituted
with
one or more of lower alkyl, lower alkyl ether, lower alkyl thioether, amido,
oxo, etc.
hl certain embodiments, a heterocyclic ring which comprises an occurrence of J
has
from 5 to 8 members.
In certain embodiments, the compound of Formula XVII is enriched for the
isomer wherein the amine and the substituent including Y and A have a eas
relationship, e.g., >75%, >85%, or even >95% of the cis isomer. In other
embodiments, the compound of Formula XVII is enriched for the isomer wherein
the
two substituents have a t~ahs relationship, e.g., >75%, >85%, or even >95% of
the
t~°a~s isomer. Preferably, such enriclunent results from employing an
isomerically
enriched starting material, e.g., the compound of Formula XIV is enriched for,
>75%, >85%, or even >95% of the cis or t~aas isomer prior to beginning step A.
In certain embodiments, an amine having a structure of Formula XIII or XVII
may be further transformed, e.g., by performing additional steps towards
generating
a compound of at least one of Formulae I-VI. Thus, for example, a method
according
to the present invention might include one or more of the following steps:
D) coupling to the exocyclic amine a group -C(=Z)LRl or -Z' VRS;
E) coupling to the exocyclic amine a group -R~ or -LR2;
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F) coupling to the group bearing Y a group NJ2N(R~)2 or -XLRa;
G) coupling to the nitrogen in the ring a group -MR3 or -GR~;
H) removing a protecting group from the nitrogen in the ring;
I) removing a protecting group from the group bearing Y;
J) placing a ntrogen-protecting group on the exocyclic amine;
K) removing a protecting group from the exocyclic amine,
wherein L, J, R~, M, R3, and R~ are as defined above,
Rl, R2, R3, and R4, independently for each occurrence, represent H, lower
allcyl, -(CH2)na~.yl (e.g., substituted or unsubstituted), or -
(CH2)nheteroaryl (e.g.,
substituted or unsubstituted);
Z is O or S;
Z' absent or represents -SO2-, -(C=S)-, or -(C=O)-;
V is absent or represents O, S, or NRB;
RS represents substituted or unsubstituted alkyl (e.g.; branched or
unbranched), allcenyl (e.g., branched or unbranched), allcynyl (e.g., branched
or
unbranched), cycloallcyl, or cycloallcylallcyl; and
R7 represents substituted or unsubstituted aryl, arallcyl, heteroaryl, or
heteroarall~yl.
Any of steps D through I~, as may be selected, may be performed in any
order, depending on the various reactions and protecting groups used, as is
well
understood in the art. Various protecting groups suitable for use in the
present
method have been outlined above, and are well known in the art, as are
numerous
techniques for attaching and removing such protecting groups, and any of these
may
be employed in the present method without departing from the scope and spirit
of
the present invention.
5~,
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In certain embodiments, step D may be performed by reacting the exocyclic
amine with an acylating agent, such as an acid halide, an isocyanate, an
isothiocyanate, a haloformate, a halothioformate, aaz anhydride, a
dicarbonate, a
sulfonyl halide, a sulfmyl halide, a carbamyl chloride, a thiocarbamyl
chloride, or an
activated acylating moiety prepared ivy situ. An acylating agent may be
prepared iyz
situ, for example, by reacting a carboxylic acid with a~.z activating agent,
such as a
carbodiimide (e.g., diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide, etc.), phosphorous-based reagents
(such
as~BOP-Cl, PyBROP, etc.), oxalyl chloride, phosgene, triphosgene, or any other
reagent that reacts with a carboxylic acid group resulting in a reactive
intermediate
having an increased susceptibility, relative to the carboxylic acid, towards
coupling
with an amine. A wide variety of such reagents are well lcnown in the art of
orgaaiic
synthesis, especially peptide coupling. Similarly, a primary amine or alcohol
can be
treated with a phosgene equivalent, such as carbonyl diimidazole, phosgene,
triphosgene, diphosgene, etc., or a thiophosgene equivalent, such as
thiophosgene,
thiocarbonyldiimidazole, etc., to generate an acylating agent (e.g., an
isocyanate,
isothiocyanate, chloroformamide, or chlorothioformamide, for example) capable
of
reacting with an amine to form a urea or thiourea, without necessitating
isolation or
purification of the acylating agent.
In embodiments wherein M or G represents 502, C=O, or C=S, step G may
be performed using reagents and techniques such as those described for step D,
above. In embodiments wherein M or G is absent, step G may be performed by
reacting the endocyclic amine with an electrophile, such as a~.z alkyl halide
or
sulfonate, an arallcyl halide or sulfonate, a heteroarallcyl halide or
sulfonate, a
cycloallcyl halide or sulfonate, a cycloallcylallcyl halide or sulfonate, a
heterocyclyl
halide or sulfonate, or a heterocyclylall~yl halide or sulfonate.
Alternatively, step G
may be performed by reductive allcylation, e.g., reacting the endocyclic amine
with
an appropriately substituted aldehyde in the presence of a reducing agent,
such as
sodium borohydride.
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In certain embodiments, step E may be performed using reductive allcylation
or by reacting the exocyclic amine with an electrophile, such as a halide or
sulfonate.
In certain embodiments, step F may be performed by reacting an ester,
thioester, or xanthate with a compound having the formula, for example, of
S HNJZN(Rg)2 Or HXLR4, e.g., in the presence of a Lewis acid, at an elevated
temperature,.etc. In other embodiments, step F may be performed by reacting a
carboxylic acid with an activating agent, such as a carbodiimide (e.g.,
diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide, etc.), a phosphorous-based reagent (such as BOP-Cl, PyBROP,
etc.), oxalyl chloride, phosgene, triphosgene, or any other reagent that
reacts with a
carboxylic acid group resulting in a reactive intermediate having an increased
susceptibility, relative to the carboxylic acid, towards coupling with a
nucleophile.
Other techniques for coupling a nucleophile with a carboxylic acid or
derivative
thereof (such as an ester, thioester, etc.) are well known in the a~.-t and
may be
substituted for those specifically enumerated here.
h1 certain embodiments, Y and Z are O.
In certain embodiments, Rl represents a lower alkyl group, such as a
branched alkyl, a cycloallcyl, or a cycloallcylallcyl, for example,
cyclopropyl,
cyclopropylmethyl, neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl,
cyclobutylmethyl, etc.
In certain embodiments, XLR4, taken together, include a cyclic amine, such
as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
In certain embodiments, L attached to Rl represents O, S, or NRg, such as
NH.
In certain embodiments, at least one of Rl, R2, and R3 includes an aryl or
heteroaryl group. In certain related embodiments, at least two of Rl, R2, and
R3
include an aryl or heteroaryl group.
In certain embodiments, M is absent.
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In certain embodiments, X is not NH. In certain embodiments, X is included
in a ring, or, talcen together with -C(=Y)-, represents a tertiary amide.
In certain embodiments, NJ2N, taken together, represent a cyclic diamine,
such as a piperazine, etc., which may be substituted or unsubstituted, e.g.,
with one
or more substitutents such as oxo, lower alkyl, lower alkyl ether, etc. In
certain other
embodiments, NJ2 or NJR9 taken together represent a substituted or
unsubstituted
heterocyclic ring to which the other occurrence of N is attached. In certain
embodiments, one or both occurrences of J are substituted with one or more of
lower
alkyl, lower alkyl ether, lower alkyl thioether, amido, oxo, etc. In certain
embodiments, a heterocyclic ring which comprises an occurrence of J has from 5
to
8 members.
In certain embodiments, RS represents a branched alkyl, cycloallcyl, or
cycloallcylallcyl.
In certain embodiments, RG includes at least one heterocyclic ring, such as a
thiophene, fitran, oxazole, benzodioxane, benzodioxole, pyrrole, indole, etc.
In certain embodiments, R7 represents a phenyl alkyl, such as a benzyl group,
optionally substituted with halogen, hydroxyl, lower allcyl, nitro, cyano,
lower alkyl
ether (e.g., optionally substituted, such as CHF2CF20), or lower alkyl
thioether (e.g.,
optionally substituted, such as CF3S).
In certain embodiments, R8, when it occurs in V, represents H or lower alkyl,
preferably H.
ITl Exe~zpla~y Applications of Method and Compositions
Another aspect of the present invention relates to a method of modulating a
differentiated state, survival, andlor proliferation of a cell having a ptc
loss-of
function, hedgehog gain-of function, or smoothened gain-of function, by
contacting
the cells with a hedgehog antagonist according to the subject method and as
the
circumstances may warrant.
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For instance, it is contemplated by the invention that, in light of the
findings
of an apparently broad involvement of hedgehog, ptc, and smoothened in the
formation of ordered spatial arrangements of differentiated tissues in
vertebrates, the
subject method could be used as part of a process for generating and/or
maintaining
an array of different vet-tebrate tissue both ih vitro and ih vivo. The
hedgehog
antagonist, whether inductive or anti-inductive with respect proliferation or
differentiation of a given tissue, can be, as appropriate, any of the
preparations
described above.
For example, the present method is applicable to cell culture techniques
wherein, whether for genetic or biochemical reasons, the cells have a ptc loss-
of
function, hedgehog gain-of function, or smoothened gain-of function phenotype.
In
vitf~o neuronal culture systems have proved to be fundamental and
indispensable
tools for the study of neural development, as well as the identification of
neurotrophic factors such as nerve growth factor (NGF), ciliary trophic
factors
(CNTF), and brain derived neurotrophic factor (BDNF). One use of the present
method may be in cultures of neuronal stem cells, such as in the use of such
cultures
for the generation of new neurons and glia. In such embodiments of the subject
method, the cultured cells can be contacted with a hedgehog antagonist of the
present invention in order to alter the rate of proliferation of neuronal stem
cells in
the culture and/or alter the rate of differentiation, or to maintain the
integrity of a
culture of certain terminally differentiated neuronal cells. In an exemplaay
embodiment, the subject method can be used to culture, for example, sensory
neurons or, alternatively, motor neurons. Such neuronal cultures can be used
as
convenient assay systems as well as sources of implantable cells for
therapeutic
treatments.
According to the present invention, large numbers of non-tumorigenic neural
progenitor cells can be perpetuated in vits°o and their rate of
proliferation and/or
differentiation can be affected by contact with hedgehog antagonists of the
present
invention. Generally, a method is provided comprising the steps of isolating
neural
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progenitor cells fiom an animal, perpetuating these cells i~ vitro or i~ vivo,
preferably in the presence of growth factors, and regulating the
differentiation of
these cells into particular neural phenotypes, e.g., neurons and glia, by
contacting the
cells with a hedgehog antagonist.
Progeutor cells are thought to be under an inhibitory influence which
maintains the progenitors in a suppressed state until their differentiation is
required.
However, recent techniques have been provided which permit these cells to be
proliferated, and unlike neurons which are terminally differentiated and
therefore
non-dividing, they can be produced in unlimited number and are highly suitable
for
transplantation into heterologous and autologous hosts with neurodegenerative
diseases.
By "progenitor" it is meant an oligopotent or multipotent stem cell which is
able to divide without limit and, under specific conditions, can produce
daughter
cells which terminally differentiate such as into neurons and glia. These
cells can be
used for transplantation into a heterologous or autologous host. By
heterologous is
meant a host other than the animal from which the progenitor cells were
originally
derived. By autologous is meant the identical host from which the cells were
originally derived.
Cells can be obtained from embryonic, post-natal, juvenile or adult neural
tissue from any animal. By any animal is meant any multicellular animal which
contains nervous tissue. More particularly, is meant any fish, reptile, bird,
amphibian
or mammal and the lilce. The most preferable donors are mammals, especially
mice
and humans.
In the case of a non-human heterologous donor animal, the animal may be
euthanized, and the brain and specific area of interest removed using a
sterile
procedure. Brain areas of particular interest include any area from which
progenitor
cells can be obtained which will serve to restore function to a degenerated
area of the
host's brain. These regions include areas of the central nervous system (CNS)
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including the cerebral cortex, cerebellum, midbrain, brainstem, spinal cord
and
ventricular tissue, and areas of the peripheral nervous system (PNS) including
the
carotid body and the adrenal medulla. More particularly, these areas include
regions
in the basal ganglia, preferably the striatum which consists of the caudate
a~zd
putamen, or various cell groups such as the globus pallidus, the subthalamic
nucleus,
the nucleus basalis which is found to be degenerated in Alzheimer's Disease
patients,
or the substantia nigra gars compacta which is found to be degenerated in
Parkinson's Disease patients.
Human heterologous neural progeW for cells may be derived from fetal tissue
obtained from elective abortion, or from a post-natal, juvenile or adult organ
donor.
Autologous neural tissue can be obtained by biopsy, or from patients
undergoing
neurosurgery in which neural tissue is removed, in particular during epilepsy
surgery, and more particularly during temporal lobectomies and
hippoca~.npalectomies.
Cells can be obtained from donor tissue by dissociation of individual cells
from the connecting extracellular matrix of the tissue. Dissociation can be
obtained
using any knovm procedure, including treatment with enzymes such as trypsin,
collagenase and the like, or by using physical methods of dissociation such as
with a
blunt instrument or by mincing with a scalpel to a allow outgrowth of specific
cell
types from a tissue. Dissociation of fetal cells can be carried out in tissue
culture
medium, while a preferable medium for dissociation of juvenile and adult cells
is
artificial cerebral spinal fluid (aCSF). Regular aCSF contains 124 mM NaCI, 5
mM
ICI, 1.3 mM MgCl2, 2 mM CaCl2, 26 mM NaHC03, and 10 mM D-glucose. Low
Ca2+ aCSF contains the same ingredients except for MgCl2 at a concentration of
3.2
mM and CaCl2 at a concentration of 0.1 mM.
Dissociated cells can be placed into any known culture medium capable of
supporting cell growth, including MEM, DMEM, RPMI, F-12, and the like,
containing supplements which axe required for cellular metabolism such as
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glutamine and other amino acids, vitamins, minerals and useful proteins such
as
transferrin and the like. Medium may also contain antibiotics to prevent
contamination with yeast, bacteria and fungi such as penicillin, streptomycin,
gentamicin and the like. In some cases, the medium may contain serum derived
from
bovine, equine, chicken and the like. A particularly preferable medium for
cells is a
mixture of DMEM and F-12.
Conditions for culturing should be close to physiological conditions. The pH
of the culture media should be close to physiological pH, preferably between
pH 6-8,
more preferably close to pH 7, even more particularly about pH 7.4. Cells
should be
cultured at a temperature close to physiological temperature, preferably
between 30
°C-40 °C, more preferably between 32 °C-38 °C, and
most preferably between 35
°C-37 °C.
Cells can be grown in suspension or on a fixed substrate, but proliferation of
the progenitors is preferably done in suspension to generate large numbers of
cells
by formation of "neurospheres" (see, for example, Reynolds et al. (1992)
Science
255:1070-1709; and PCT Publications W093/01275, W094/09119, W094/10292,
and WO94/16718). In the case of propagating (or splitting) suspension cells,
flasks
are shaken well and the neurospheres allowed to settle on the bottom corner of
the
flash. The spheres axe then transferred to a 50 ml centrifuge tube and
centrifuged at
low speed. The medium is aspirated, the cells resuspended in a small amount of
medium with growth factor, and the cells mechanically dissociated and
resuspended
in separate aliquots of media.
Cell suspensions in culture medium are supplemented with any growth factor
which allows for the proliferation of progenitor cells and seeded in any
receptacle
capable of sustaining cells, though as set out above, preferably in culture
flaslcs or
roller bottles. Cells typically proliferate within 3-4 days in a 37 °C
incubator, and
proliferation can be reinitiated at any time after that by dissociation of the
cells and
resuspension in fresh medium containing growth factors.
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In the absence of substrate, cells lift off the floor of the flaslc and
continue to
proliferate in suspension forming a hollow sphere of undifferentiated cells.
After
approximately 3-10 days ih vitro, the proliferating clusters (neurospheres)
are fed
every 2-7 days, and more particularly every 2-4 days by gentle centrifugation
and
resuspension in medium containing growth factor.
After 6-7 days iu vitro, individual cells in the neurospheres can be separated
by physical dissociation of the neurospheres with a blunt instrument, more
pat-ticularly by triturating the neurospheres with a pipette. Single cells
from the
dissociated neurospheres are suspended in culture medium containing growth
factors, and differentiation of the cells can be control in culture by plating
(or
resuspending) the cells in the presence of a hedgehog antagonist.
To further illustrate other uses of the subject hedgehog antagonists, it is
noted that intracerebral grafting has emerged as an additional approach to
central
nervous system therapies. For example, one approach to repairing damaged brain
tissues involves the transplantation of cells from fetal or neonatal animals
into the
adult brain (Dunnett et al. (1987) JExp Biol 123:265-2~9; and Freund et al.
(195) J
Neu~ ~sei 5:603-616). Fetal neurons from a variety of brain regions can be
successfully incorporated into the adult brain, and such grafts can alleviate
behavioral defects. For example, movement disorder induced by lesions of
dopaminergic projections to the basal ganglia can be prevented by grafts of
embryonic dopaminergic neurons. Complex cognitive functions that are impaired
after lesions of the neocortex can also be partially restored by grafts of
embryonic
cortical cells. The subject method can be used to regulate the growth state in
the
culture, or where fetal tissue is used, especially neuronal stem cells, can be
used to
regulate the rate of differentiation of the stem cells.
Stem cells useful in the present invention are generally known. For example,
several neural crest cells have been identified, some of which are multipotent
and
likely represent uncommitted neural crest cells, and others of which can
generate
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only one type of cell, such as sensory neurons, and likely represent committed
progenitor cells. The role of hedgehog antagonists employed in the present
method
to culture such stem cells can be to regulate differentiation of the
uncommitted
progenitor, or to regulate further restriction of the developmental fate of a
committed
progenitor cell towards becoming a terminally difFerentiated neuronal cell.
For
example, the present method can be used ivy uit~o to regulate the
differentiation of
neural crest cells into glial cells, schwann cells, chromaffm cells,
cholinergic
sympathetic or parasympathetic neurons, as well as peptidergic and
serotonergic
neurons. The hedgehog antagonists can be used alone, or can be used in
combination
with other neurotrophic factors which act to more particularly enhance a
pa~~ticular
differentiation fate of the neuronal progenitor cell.
In addition to the implantation of cells cultured in the presence of the
subject
hedgehog antagonists, yet another aspect of the present invention concerns the
therapeutic application of a hedgehog antagonist to regulate the growth state
of
neurons and other neuronal cells in both the central nervous system and the
peripheral nervous system. The ability of ptc, hedgehog, and s~zoothefzed to
regulate
neuronal differentiation during development of the nervous system and also
presumably in the adult state indicates that, in certain instances, the
subject
hedgehog antagonists can be expected to facilitate control of adult neurons
with
regard to maintenance, functional performance, and aging of normal cells;
repair and
regeneration processes in chemically or mechanically lesioned cells; and
treatment of
degeneration in certain pathological conditions. In light of this
understanding, the
present invention specifically contemplates applications of the subj ect
method to the
treatment protocol of (prevention andlor reduction of the severity ofj
neurological
conditions deriving from: (i) acute, subacute, or chronic injury to the
nervous
system, including traumatic injury, chemical injury, vascular injury and
deficits
(such as the ischemia resulting from stroke), together with
infectious/inflammatory
and tumor-induced injury; (ii) aging of the nervous system including
Alzheimer's
disease; (iii) chronic neurodegenerative diseases of the nervous system,
including
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Parl~inson's disease, Huntington's chorea, amylotrophic lateral sclerosis and
the like,
as well as spinocerebellar degenerations; and (iv) chronic immunological
diseases of
the nervous system or affecting the nervous system, including multiple
sclerosis.
As appropriate, the subject method can also be used in generating nerve
prostheses for the repair of central and peripheral nerve damage. In
particular, where
a crushed or severed axon is intubulated by use of a prosthetic device,
hedgehog
antagonists can be added to the prosthetic device to regulate the rate of
growth and
regeneration of the dendridic processes. Exemplary nerve guidance chamlels are
described in U.S. patents 5,092,71 and 4,955,892.
liz another embodiment, the subject method can be used in the treatment of
neoplastic or hyperplastic transformations such as may occur in the central
nervous
system. For instance, the hedgehog antagonists can be utilized to cause such
transformed cells to become either post-mitotic or apoptotic. The present
method
may, therefore, be used as part of a treatment for, e.g., malignant gliomas,
meningiomas, medulloblastomas, neuroectodermal tumors, and ependymomas.
In a preferred embodiment, the subject method can be used as pa~.-t of a
treatment regimen for malignant medulloblastoma and other primary CNS
malignant
neuroectodermal tumors.
In certain embodiments, the subject method is used as part of treatment
program for medulloblastoma. Medulloblastoma, a primary brain tumor, is the
most
common brain tumor in children. A medulloblastoma is a primitive
neuroectodermal
tumor arising in the posterior fossa. They account for approximately 25% of
all
pediatric brain tumors (Miller). Histologically, they are small round cell
tumors
commonly arranged in true rosettes, but may display some differentiation to
astrocytes, ependymal cells or neurons (Rorlee; I~leihues). PNET's may arise
in other
areas of the brain including the pineal gland (pineoblastoma) and cerebrum.
Those
arising in the supratentorial region generally fare worse than their PF
counterparts.
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Medulloblastoma/PNET's are l~nown to recur anywhere in the CNS after
resection, and can even metastasize to bone. Pretreatment evaluation should
therefore include an examination of the spinal cord to exclude the possibility
of
"dropped metastases". Gadolinium-enhanced MRI has largely replaced myelography
for this purpose, and CSF cytology is obtained postoperatively as a routine
procedure.
In other embodiments, the subject method is used as part of treatment
program for ependymomas. Ependymomas account for approximately 10% of the
pediatric brain tumors in children. Grossly, they are tumors that arise from
the
ependymal Bung of the ventricles and microscopically form rosettes, canals,
acid
perivascular rosettes. In the CHOP series of 51 children reported with
ependymomas, 3/4 were histologically benign. Approximately 2/3 arose from the
region of the 4th ventricle. One third presented in the supratentorial region.
Age at
presentation peaks between birth and 4 years, as demonstrated by SEER data as
well
as data from CHOP. The median age is about 5 years. Because so many children
with this disease are babies, they often require multimodal therapy.
Yet another aspect of the present invention concerns the observation in the
art that ptc, hedgehog, and/or smoothened are involved in morphogenic signals
involved in other vertebrate organogenic pathways in addition to neuronal
differentiation as described above, having apparent roles in other endodermal
patterning, as well as both mesodermal and endodermal differentiation
processes.
Thus, it is contemplated by the invention that compositions comprising
hedgehog
antagonists can also be utilized for both cell culture and therapeutic methods
involving generation and maintenance of non-neuronal tissue.
In one embodiment, the present invention makes use of the discovery that
ptc, hedgehog, and smoothened axe apparently involved in controlling the
development of stem cells responsible for formation of the digestive tract,
liver,
lungs, and other organs which derive from the primitive gut. Shh serves as an
inductive signal from the endoderm to the mesoderm, which is critical to gut
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morphogenesis. Therefore, for example, hedgehog antagonists of the instant
method
can be employed for regulating the development and maintenance of an
artificial
liver which can have multiple metabolic functions of a normal liver. In an
exemplary
embodiment, the subject method can be used to regulate the proliferation and
differentiation of digestive tube stem cells to form hepatocyte cultures which
can be
used to populate extracellular matrices, or which can be encapsulated in
biocompatible polymers, to form both implantable and extracorporeal artificial
livers.
In another embodiment, therapeutic compositions of hedgehog antagonists
can be utilized in conjunction with transplantation of such artificial livers,
as well as
embryonic liver structures, to regulate uptal~e of intraperitoneal
implantation,
vascularization, and ih vivo differentiation and maintenance of the engrafted
liver
tissue.
In yet another embodiment, the subject method can be employed
therapeutically to regulate such organs after physical, chemical or
pathological
insult. For instance, therapeutic compositions comprising hedgehog antagonists
can
be utilized in liver repair subsequent to a partial hepatectomy.
The generation of the pancreas and small intestine from the embryonic gut
depends on intercellular signalling between the endodermal and mesodermal
cells of
the gut. In particular, the differentiation of intestinal mesoderm into smooth
muscle
has been suggested to depend on signals from adjacent endodermal cells. One
candidate mediator of endodermally derived signals in the embryonic hindgut is
Sonic hedgehog. See, for example, Apelqvist et al. (1997) Curr Biol 7:801-4.
The
Shh gene is expressed throughout the embryonic gut endoderm with the exception
of
the pancreatic bud endoderm, which instead ~ expresses high levels of the
homeodomain protein Ipfl/Pdxl (insulin promoter factor 1/pancreatic and
duodenal
homeobox 1), an essential regulator of early pancreatic development. Apelqvist
et
al., supra, have examined whether the differential expression of S1W in the
embryonic gut tube controls the differentiation of the surrounding mesoderm
into
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specialised mesoderm derivatives of the small intestine and pancreas. To test
this,
they used the promoter of the Ipfl/Pdxl gene to selectively express Shh in the
developing pancreatic epithelium. In Ipfl/Pdxl- S1W transgenic mice, the
pancreatic
mesoderm developed into smooth muscle and interstitial cells of Cajal,
characteristic
of the intestine, rather than into pancreatic mesenchyme and spleen. Also,
pancreatic
explants exposed to Shh underwent a similar program of intestinal
differentiation.
These results provide evidence that the differential expression of
endodermally
derived Shh controls the fate of adjacent mesoderm at different regions of the
gut
tube.
In the context of the present invention, it is contemplated therefore that the
subject hedgehog antagonists can be used to control or regulate the
proliferation
and/or differentiation of pancreatic tissue both ih vivo and in vita°o.
There axe a wide variety of pathological cell proliferative and
differentiative
conditions for which the inhibitors of the present invention may provide
therapeutic
benefits, with the general strategy being, for example, the correction of
aberrant
insulin expression, or modulation of differentiation. More generally, however,
the
present invention relates to a method of inducing and/or maintaining a
differentiated
state, enhancing survival and/or affecting proliferation of pancreatic cells,
by
contacting the cells with the subject inhibitors. For instance, it is
contemplated by
the invention that, in light of the apparent involvement of ptc, hedgehog, and
smoothehed in the formation of ordered spatial arrangements of pancreatic
tissues,
the subject method could be used as part of a technique to generate and/or
maintain
such tissue both i~ vitr o and ivy vivo. For instance, modulation of the
function of
hedgehog can be employed in both cell culture and therapeutic methods
involving
generation and maintenance [3-cells and possibly also for non-pancreatic
tissue, such
as in controlling the development and maintenance of tissue from the digestive
tract,
spleen, lungs, urogenital organs (e.g., bladder), and other organs which
derive from
the primitive gut.
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In an exemplary embodiment, the present method can be used in the
treatment of hyperplastic and neoplastic disorders effecting pancreatic
tissue,
particularly those characterized by aberrant proliferation of pancreatic .
cells. For
instance, pancreatic cancers are marked by abnormal proliferation of
pancreatic cells
which can result in alterations of insulin secretory capacity of the pancreas.
For
instance, certain pancreatic hyperplasias, such as pancreatic carcinomas, can
result in
hypoinsulinemia due to dysfunction of (3-cells or decreased islet cell mass.
To the
extent that aberrant ptc, hedgehog, and smoothened signaling may be indicated
in
disease progression, the subject inhibitors, can be used to enhance
regeneration of
the tissue after anti-tumor therapy.
Moreover, manipulation of hedgehog signaling properties at different points
may be useful as part of a strategy for reshaping/repairing pancreatic tissue
both in
vivo and in vitf°o. In one embodiment, the present invention malces use
of the
apparent involvement of ptc, hedgehog, and smootheized in regulating the
development of pancreatic tissue. In general, the subject method can be
employed
therapeutically to regulate the pancreas after physical, chemical or
pathological
insult. In yet another embodiment, the subject method can be applied to to
cell
culture techniques, and in particular, may be employed to enhance the initial
generation of prosthetic pancreatic tissue devices. Manipulation of
proliferation and
differentiation of pancreatic tissue, for example, by altering hedgehog
activity, can
provide a means for more carefully controlling the characteristics of a
cultured
tissue. In an exemplary embodiment, the subject method can be used to augment
production of prosthetic devices which require (3-islet cells, such as may be
used in
the encapsulation devices described in, for example, the Aebischer et al. U.S.
Patent
No. 4,892,538, the Aebischer et al. U.S. Patent No. 5,106,627, the Lim U.S.
Patent
No. 4,391,909, and the Sefton U.S. Patent No. 4,353,888. Early progenitor
cells to
the pancreatic islets are multipotential, and apparently coactivate all the
islet-specific
genes from the time they first appear. As development proceeds,. expression of
islet-
specific hormones, such as insulin, becomes restricted to the pattern of
expression
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characteristic of mature islet cells. The phenotype of mature islet cells,
however, is
not stable in culture, as reappearence of embryonal traits in matl~re (3-cells
can be
observed. By utilizing the subject hedgehog antagonists, the differentiation
path or
proliferative index of the cells can be regulated.
Furthermore, manipulation of the differentiative state of pancreatic tissue
can
be utilized in conjunction with transplantation of aa-tificial pancreas so as
to promote
implantation, vascularization, and in vivo differentiation and maintenance of
the
engrafted tissue. For instance, manipulation of hedgehog function to affect
tissue
differentiation can be utilized as a means of maintainng graft viability.
Bellusci et al. (1997) Development 124:53 report that Sonic hedgehog
regulates lung mesenchymal cell proliferation in vivo. Accordingly, the
present
method can be used to regulate regeneration of lung tissue, e.g., in the
treatment of
emphysema.
Fujita et al. (1997) Biochem Biophys Res Co~z~rzuv~ 238:658 reported that
Sonic hedgehog is expressed in human lung squamous carcinoma and
adenocarcinoma cells. The expression of Sonic hedgehog was also detected in
the
human lung squamous carcinoma tissues, but not in the normal lung tissue of
the
same patient. They also observed that Sonic hedgehog stimulates the
incorporation
of BrdU into the carcinoma cells and stimulates their cell growth, while anti-
Shh-N
inhibited their cell growth. These results suggest that a ptc, Izedgehog,
and/or
smoothef2ed is involved in the cell growth of such transformed lung tissue and
therefore indicates that the subject method can be used as part of a treatment
of lung
carcinoma and adenocarcinomas, and other proliferative disorders involving the
lung
epithelia. .
Many other tumors may, based on evidence such as involvement of the
hedgehog pathway in these tumors, or detected expression of hedgehog or its
receptor in these tissues during development, be affected by treatment with
the
subject compounds. Such tumors include, but are by no means limited to, tumors
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related to Gorlin's syndrome (e.g., basal cell carcinoma, medulloblastoma,
meningioma, etc.), tumors evidenced in pct laioclc-out mice (e.g., hemangioma,
rhabdomyosarcoma, etc.), tumors resulting fiom gli-1 amplification (e.g.,
glioblastoma, sarcoma, etc.), tumors connected with TRCB, a ptc homolog (e.g.,
renal carcinoma, thyroid carcinoma, etc.), Ext-1-related tumors (e.g., bone
cancer,
etc.), S1W -induced tumors (e.g., lung cancer, chondrosarcomas, etc.), and
other
tumors (e.g., breast cancer, urogenital cancer (e.g., lcidney, bladder,
ureter, prostate,
etc.), adrenal cancer, gastrointestinal cancer (e.g., stomach, intestine,
etc.), etc.).
In still another embodiment of the present invention, compositions
comprising hedgehog antagonists can be used in the isz vitro generation of
skeletal
tissue, such as from slceletogenic stem cells, as well as the ivy vivo
treatment of
slceletal tissue deficiencies. The present invention particularly contemplates
the use
of hedgehog antagonists to regulate the rate of chondrogenesis and/or
osteogenesis.
By "skeletal tissue deficiency", it is meant a deficiency in bone or other
slceletal
connective tissue at any site where it is desired to restore the bone or
connective
tissue, no matter how the deficiency originated, e.g. whether as a result of
surgical
intervention, removal of tumor, ulceration, implant, fracture, or other
traumatic or
degenerative conditions.
For instance, the method of the present invention can be used as part of a
regimen for restoring cartilage function to a connective tissue. Such methods
are
useful in, for example, the repair of defects or lesions in cartilage tissue
which is the
result of degenerative wear such as that which results in arthritis, as well
as other
mechanical derangements which may be caused by trauma to the tissue, such as a
displacement of torn meniscus tissue, meniscectomy, a Taxation of a joint by a
torn
ligament, malignment of joints, bone fracture, or by hereditary disease. The
present
reparative method is also useful for remodeling cartilage matrix, such as in
plastic or
reconstructive surgery, as well as periodontal surgery. The present method may
also
be applied to improving a previous reparative procedure, for example,
following
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surgical repair of a meniscus, ligament, or cartilage. Furthermore, it may
prevent the
onset or exacerbation of degenerative disease if applied early enough after
trauma.
In one embodiment of the present invention, the subject method comprises
treating the afflicted connective tissue with a therapeutically su~cient
amount of a
hedgehog antagonist, particularly an antagonist selective for Indian
Izedgelaog signal
transduction, to regulate a cartilage repair response in the comlective tissue
by
managing the rate of differentiation and/or proliferation of chondrocytes
embedded
in the tissue. Such connective tissues as articular cartilage, interarticular
cartilage
(menisci), costal cartilage (connecting the true ribs and the sternum),
ligaments, and
tendons are particularly amenable to treatment in reconstructive and/or
regenerative
therapies using the subject method. As used herein, regenerative therapies
include
treatment of degenerative states which have progressed to the point of which
impairment of the tissue is obviously manifest, as well as preventive
treatments of
tissue where degeneration is in its earliest stages or imminent.
In an illustrative embodiment, the subject method can be used as part of a
therapeutic intervention in the treatment of cartilage of a diarthroidal
joint, such as a
l~nee, an anlde, an elbow, a hip, a wrist, a l~nuclcle of either a finger or
toe, or a
tempomandibular joint. The treatment can be directed to the meniscus of the
joint, to
the articular cartilage of the joint, or both. To further illustrate, the
subject method
can be used to treat a degenerative disorder of a knee, such as which might be
the
result of traumatic injury (e.g., a sports injury or excessive wear) or
osteoarthritis.
The subject antagonists may be administered as an injection into the joint
with, for
instance, an arthroscopic needle. In some instances, the injected agent can be
in the
form of a hydrogel or other slow release velv.cle described above in order to
permit a
more extended and regular contact of the agent with the treated tissue.
The present invention further contemplates the use of the subject method in
the field of cartilage transplantation and prosthetic device therapies.
However,
problems arise, for instance, because the characteristics of cartilage acid
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fibrocartilage varies between different tissue: such as between aoticular,
meniscal
cartilage, ligaments, and tendons, between the two ends of the same ligament
or
tendon, and between the superficial and deep pax-ts of the tissue. The zonal
arrangement of these tissues may reflect a gradual change in mechanical
properties,
and failure occurs when implanted tissue, which has not differentiated under
those
conditions, lacks the ability to appropriately respond. For instance, when
meniscal
cartilage is used to repair anterior cruciate ligaments, the tissue undergoes
a
metaplasia to pure fibrous tissue. By regulating the rate of chondrogenesis,
the
subject method can be used to particularly address this problem, by helping to
adaptively control the implanted cells in the new environment and effectively
resemble hypertrophic chondrocytes of an earlier developmental stage of the
tissue.
In similar fashion, the subject method can be applied to enhancing both the
generation of prosthetic cartilage devices and to their implantation. The need
for
improved treatment has motivated research aimed at creating new cartilage that
is
based on collagen-glycosaminoglycan templates (Stone et al. (1990) C'lih
O~thop
Relat Red 252:129), isolated chondrocytes (Grande et al. (1989) J O~thop Res
7:208;
and Takigawa et al. (1987) Bohe Minef° 2:449), and chondrocytes
attached to natural
or synthetic polymers (Walitani et al. (1989) JBoue Jt Suing 71B:74; Vacanti
et al.
(1991) Plast Reco~cstr Surg 88:753; von Schroeder et al. (1991) JBiomed
Mate~° Res
25:329; Freed et al. (1993) J Biomed Mater Res 27:11; and the Vacanti et al.
U.S.
Patent No. 5,041,138). For example, chondrocytes can be grown in culture on
biodegradable, biocompatible highly porous scaffolds formed from polymers such
as
polyglycolic acid, polylactic acid, agarose gel, or other polymers which
degrade over
time as function of hydrolysis of the polymer backbone into innocuous
monomers.
The matrices are designed to allow adequate nutrient and gas exchange to the
cells
until engraftment occurs. The cells can be cultured In Vitl"O 1111tH adequate
cell
volume and density has developed for the cells to be implanted. One advantage
of
the matrices is that they can be cast or molded into a desired shape on an
individual
basis, so that the final product closely resembles the patient's own ear or
nose (by
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way of example), or flexible matrices can be used which allow for manipulation
at
the time of implantation, as in a joint.
In one embodiment of the subject method, the implants are contacted with a
hedgehog antagonist during certain stages of the culturing process in order to
manage the rate of differentiation of chondrocytes and the formation of
hypertrophic
chrondrocytes in the culture.
In another embodiment, the implanted device is treated with a hedgehog
antagonist in order to actively remodel the implanted matrix and to make it
more
suitable for its intended function. As set out above with respect to tissue
transplants,
the artificial transplants suffer from the same deficiency of not being
derived in a
setting which is comparable to the actual mechanical environment in which the
matrix is implanted. The ability to regulate the chondrocytes in the matrix by
the
subject method can allow the implant to acquire characteristics similar to the
tissue
for which it is intended to replace.
In yet another embodiment, the subject method is used to enhance attachment
of prosthetic devices. To illustrate, the subject method can be used in the
implantation of a periodontal prosthesis, wherein the treatment of the
surrolmding
connective tissue stimulates formation of periodontal ligament about the
prosthesis.
In still further embodiments, the subject method can be employed as part of a
regimen for the generation of bone (osteogenesis) at a site in the animal
where such
skeletal tissue is deficient. Indian hedgehog is particularly associated with
the
hypertrophic chondrocytes that are ultimately replaced by osteoblasts. For
instance,
administration of a hedgehog antagonists of the present invention can be
employed
as part of a method for regulating the rate of bone loss in a subject. For
example,
preparations comprising hedgehog antagonists can be employed, for example, to
control endochondral ossification in the formation of a "model" for
ossification.
In yet another embodiment of the present invention, a hedgehog antagonist
can be used to regulate spermatogenesis. The hedgehog proteins, particularly
Dhh,
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have been shown to be involved in the differentiation and/or proliferation and
maintenance of testicular germ cells. Dhh expression is initiated in Seutoli
cell
precursors shortly after the activation of Sry (testicular determining gene)
and
persists in the testis into the. adult. Males are viable but infertile, owing
to a complete
absence of mature sperm. Examination of the developing testis in different
genetic
backgrounds suggests that Dhh regulates both early and late stages of
spermatogenesis. Bitgoad et al. (1996) Curr Biol 6:298. In a preferred
embodiment,
the hedgehog antagonist can be used as a contraceptive. In similar fashion,
hedgehog
antagonists of the subject method are potentially useful for modulating normal
ovarian function.
The subject method also has wide applicability to the treatment or
prophylaxis of disorders afflicting epithelial tissue, as well as in cosmetic
uses. In
general, the method can be characterized as including a step of administering
to an
animal an amount of a hedgehog antagonist effective to alter the growth state
of a
treated epithelial tissue. The mode of administration and dosage regimens will
vary
depending on the epithelial tissues) which is to be treated. For example,
topical
formulations will be preferred where the treated tissue is epidermal tissue,
such as
dermal or mucosal tissues.
A method which "promotes the healing of a wound" results in the wound
healing more quiclcly as a result of the treatment than a similar wound heals
in the
absence of the treatment. "Promotion of wound healing" can also mean that the
method regulates the proliferation and/or growth of, inter alia,
lceratinocytes, or that
the wound heals with less scarring, less wound contraction, less collagen
deposition
and more superficial surface area. In certain instances, "promotion of wound
healing" can also mean that certain methods of wound healing have improved
success rates, (e.g., the take rates of skin grafts,) when used together with
the method
of the present invention.
Despite significant progress in reconstructive surgical techniques, scarring
can be an important obstacle in regaining normal function and appearance of
healed
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skin. This is particularly true when pathologic scarring such as lceloids or
hypertrophic scars of the hands or face causes functional disability or
physical
deformity. In the severest circumstances, such scarring may precipitate
psychosocial
distress and a life of economic deprivation. Wound repair includes the stages
of
hemostasis, inflammation, proliferation, and remodeling. The proliferative
stage
involves multiplication of fibroblasts and. endothelial and epithelial cells.
Through
the use of the subject method, the rate of proliferation of epithelial cells
in and
proximal to the wound can be controlled in order to accelerate closure of the
wound
and/or minimize the formation of scar tissue.
The present treatment can also be effective as part of a therapeutic regimen
for treating oral and paraoral ulcers, e.g. resulting from radiation and/or
chemotherapy. Such ulcers commonly develop within days after chemotherapy or
radiation therapy. These ulcers usually begin as small, painful irregularly
shaped
lesions usually covered by a delicate gray necrotic membrane and sunowlded by
inflammatory tissue. In many instances, laclc of treatment results in
proliferation of
tissue around the periphery of the lesion on an inflammatory basis. For
instance, the
epithelium bordering the ulcer usually demonstrates proliferative activity,
resulting
in loss of continuity of surface epithelium. These lesions, because of their
size and
loss of epithelial integrity, dispose the body to potential secondary
infection. Routine
ingestion of food and water becomes a very painful event and, if the ulcers
proliferate throughout the alimentary canal, diarrhea usually is evident with
all its
complicating factors. According to the present invention, a treatment for such
ulcers
which includes application of an hedgehog antagoust can reduce the abnormal
proliferation and differentiation of the affected epithelium, helping to
reduce the
severity of subsequent inflammatory events.
The subject method and compositions can also be used to treat womlds
resulting from dermatological diseases, such as lesions resulting from
autoimmune
disorders such as psoriasis. Atopic dermititis refers to slcin trauma
resulting from
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allergies associated with an immune response caused by allergens such as
pollens,
foods, dander, insect venoms and plant toxins.
In other embodiments, antiproliferative preparations of hedgehog antagonists
can be used to inhibit lens epithelial cell proliferation to prevent post-
operative
complications of extracapsular cataract extraction. Cataract is am intractable
eye
disease and various studies on a treatment of cataract have been made. But at
present, the treatment of cataract is attained by surgical operations.
Cataract surgery
has been applied for a long time and various operative methods have been
examined.
Extracapsular lens extraction has become the method of choice for removing
cataracts. The major medical advantages of this technique over intracapsular
extraction are lower incidence of aphalcic cystoid macular edema and retinal
detachment. Extracapsular extraction is also required for implantation of
posterior
chamber type intraocular lenses which are now considered to be the lenses of
choice
in most cases.
However, a disadvantage of extracapsular cataract extraction is the high
incidence of posterior lens capsule opacification, often called after-
cataract, which
can occur in up to 50% of cases within three years after surgery. After-
cataract is
caused by proliferation of equatorial and anterior capsule lens epithelial
cells which
remain after extracapsular lens extraction. These cells proliferate to cause
Sommerling rings, and along with fibroblasts which also deposit and occur on
the
posterior capsule, cause opacification of the posterior capsule, which
interferes with
vision. Prevention of after-cataract would be preferable to treatment. To
inhibit
secondary cataract formation, the subject method provides a means for
inhibiting
proliferation of the remaining lens epithelial cells. For example, such cells
can be
induced to remain quiescent by instilling a solution containing an hedgehog
antagonist preparation into the anterior chamber of the eye after lens
removal.
Furthermore, the solution can be osmotically balanced to provide minimal
effective
dosage when instilled into the anterior chamber of the eye, thereby inhibiting
subcapsular epithelial growth with some specificity.
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The subject method can also be used in the treatment of corneopathies
maxlced by corneal epithelial cell proliferation, as for exaanple in ocular
epithelial
disorders such as epithelial downgrowth or squamous cell carcinomas of the
ocular
surface.
Levine et aI. (1997) J Neurosci 17:6277 show that hedgehog proteins can
regulate mitogenesis and photoreceptor differentiation in the vertebrate
retina, and
Ihh is a candidate factor from the pigmented epithelium to promote retinal
progenitor
proliferation ' and photoreceptor differentiation. Likewise, Jensen et al.
(1997)
Development 124:363 demonstrated that treatment of cultures of perinatal mouse
retinal cells with the amino-terminal fragment of Sonic hedgehog protein
results in
an increase in the proportion of cells that incorporate bromodeoxuridine, in
total cell
numbers, and in rod photoreceptors, amacrine cells and Muller glial cells,
suggesting
that Sonic hedgehog promotes the proliferation of retinal precursor cells.
Thus, the
subject method can be used in the treatment of proliferative diseases of
retinal cells
and regulate photoreceptor differentiation.
Yet another aspect of the present invention relates to the use of the subject
method to control hair growth. Hair is basically composed of keratin, a tough
and
insoluble protein; its chief strength lies in its disulphide bond of cystine.
Each
individual hair comprises a cylindrical shaft and a root, and is contained in
a follicle,
a flask-lilce depression in the skin. The bottom of the follicle contains a
finger-life
projection termed the papilla, which consists of connective tissue from which
hair
grows, and through which blood vessels supply the cells with nourishment. The
shaft
is the part that extends outwards from the skin surface, whilst the root has
been
described as the buried part of the hair. The base of the root expands into
the hair
bulb, which rests upon the papilla. Cells from which the hair is produced grow
in the
bulb of the follicle; they are extruded in the form of fibers as the cells
proliferate in
the follicle. Hair "growth" refers to the formation and elongation of the hair
fiber by
the dividing cells.
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As is well known in the ai-t, the common hair cycle is divided into three
stages: anagen, catagen and telogen. During the active phase (anagen), the
epidermal
stem cells of the dermal papilla divide rapidly. Daughter cells move upward
and
differentiate to form the concentric layers of the hair itself. The
transitional stage,
catagen, is marked by the cessation of mitosis of the stem cells in the
follicle. The
resting stage is known as telogen, where the hair is retained within the scalp
for
several weeks before an emerging new hair developing below it dislodges the
telogen-phase shaft from its follicle. From this model it has become clear
that the
larger the pool of dividing stem cells that differentiate into hair cells, the
more hair
growth occurs. Accordingly, methods for increasing or reducing hair growth can
be
carried out by potentiating or inhibiting, respectively, the proliferation of
these stem
cells.
In certain embodiments, the subject method can be employed as a way of
reducing the growth of human hair as opposed to its conventional removal by
cutting, shaving, or depilation. For instance, the present method can be used
in the
treatment of trichosis characterized by abnormally rapid or dense growth of
hair, e.g.
hypertrichosis. In an exemplary embodiment, hedgehog ailtagonists can be used
to
manage hirsutism, a disorder marked by abnormal hairiness. The subject method
can
also provide a process for extending the duration of depilation.
Moreover, because a hedgehog antagonist will often be cytostatic to
epithelial cells, rather than cytotoxic, such agents can be used to protect
hair follicle
cells from cytotoxic agents which require progression into S-phase of the cell-
cycle
for efficacy, e.g. radiation-induced death. Treatment by the subject method
can
provide protection by causing the hair follicle cells to become quiescent,
e.g., by
inhibiting the cells from entering S phase, and thereby preventing the
follicle cells
from undergoing mitotic catastrophe or programmed cell death. For instance,
hedgehog antagonists can be used for patients undergoing chemo- or radiation-
therapies which ordinarily result in hair loss. By inhibiting cell-cycle
progression
during such therapies, the subject treatment can protect hair follicle cells
from death
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which might otherwise result from activation of cell death programs. After the
therapy has concluded, the instant method can also be removed with
concommitant
relief of the inhibition of follicle cell proliferation. ,
The subject method can also be used in the treatment of folliculitis, such as
folliculitis decalvans, folliculitis ulerythematosa reticulata or lceloid
folliculitis. For
example, a cosmetic prepration of an hedgehog antagonist can be applied
topically in
the treatment of pseudofolliculitis, a chronic disorder occurring most often
in the
submandibular region of the neck and associated with shaving, the
characteristic
lesions of which are erythematous papules and pustules containing buried
hairs.
In another aspect of the invention, the subject method can be used to induce
differentiation and/or inhibit proliferation of epithelially derived tissue.
Such forms
of these molecules can provide a basis fox differentiation therapy for the
treatment of
hyperplastic and/or neoplastic conditions involving epithelial tissue. For
example,
such preparations can be used for the treatment of cutaneous diseases in which
there
is abnormal proliferation or growth of cells of the skin.
For instance, the pharmaceutical preparations of the invention are intended
for
the treatment of hyperplastic epidermal conditions, such as lceratosis, as
well as for
the treatment of neoplastic epidermal conditions such as those characterized
by a
high proliferation rate for various skin cancers, as for example basal cell
carcinoma
or squamous cell carcinoma. The subject method can also be used in the
treatment of
autoimmune diseases affecting the skin, in particular, of dermatological
diseases
involving morbid proliferation and/or lceratinization of the epidermis, as for
example, caused by psoriasis or atopic dermatosis.
Many common diseases of the skin, such as psoriasis, ~ squamous cell
carcinoma, lceratoacanthoma ~ and actinic lceratosis are characterized by
localized
abnormal proliferation and growth. For example, in psoriasis, which is
characterized
by scaly, red, elevated plaques on the skin, the lceratinocytes aie known to
proliferate
much more rapidly than normal and to differentiate less completely.
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In one embodiment, the preparations of the present invention are suitable for
the treatment of dermatological ailments linlced to lceratinization disorders
causing
abnormal proliferation of skin cells, which disorders may be marlced by either
inflaanmatory or non-inflammatory components. To illustrate, therapeutic
preparations of a hedgehog antagonist, e.g., which promotes quiescence or
differentiation can be used to treat varying forms of psoriasis, be they
cutaneous,
mucosal or ungual. Psoriasis, as described above, is typically characterized
by
epidermal lceratinocytes which display marked proliferative activation and
differentiation along a "regenerative" pathway. Treatment with an
antiproliferative
embodiment of the subject method can be used to reverse the pathological
epidermal
activiation and can provide a basis for sustained remission of the disease.
A variety of other lceratotic lesions are also candidates for treatment with
the
subject method. Actinic lceratoses, for example, are superficial inflammatory
premalignant tumors arising on sun-exposed and irradiated slcin. The lesions
are
erythematous to brown with variable scaling. Current therapies include
excisional
and cryosurgery. These treatments are painful, however, and often produce
cosmetically unacceptable scarring. Accordingly, treatment of lceratosis, such
as
actinic keratosis, can include application, preferably topical, of a hedgehog
antagonist composition in amounts sufficient to inhibit hyperproliferation of
epidermal/epidermoid cells of the lesion.
Acne represents yet another dermatologic ailment which may be treated by
the subject method. Acne vulgaris, for instance, is a multifactorial disease
most
commonly occurring in teenagers and young adults, and is characterized by the
appearance of inflammatory and noninflammatory lesions on the face and upper
trunk. The basic defect which gives rise to acne vulgaris is
hypercornification of the
duct of a hyperactive sebaceous gland. Hypercornification blocks the normal
mobility of slcin and follicle microorganisms, and in so doing, stimulates the
release
of lipases by Pj°opinobacte~ium aches and Staphylococcus
epidef°midis bacteria and
Pit~ospo~um ovale, a yeast. Treatment with an antiproliferative hedgelTOg
antagonist,
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particularly topical preparations, may be useful for preventing the
transitional
features of the ducts, e.g. hypercornification, which lead to lesion
formation. The
subject treatment may further include, for example, antibiotics, retinoids and
antiandrogens.
The present invention also provides a method for treating various forms of
dermatitis. Dermatitis is a descriptive term referring to poorly demarcated
lesions
which are either pruritic, erythematous, scaley, blistered, weeping, fissured
or
crusted. These lesions arise from any of a wide variety of causes. The most
common
types of dermatitis are atopic, contact and diaper dermatitis. For instance,
seborrheic
dermatitis is a chronic, usually pruritic, dermatitis with erythema, dry,
moist, or
greasy scaling, and yellow crusted patches on various areas, especially the
scalp,
with exfoliation of an excessive amount of dry scales. The subject method can
also
be used in the treatment of stasis dermatitis, an often chronic, usually
eczematous
dermatitis. Actinic dermatitis is dermatitis that due to exposure to actinic
radiation
_ 15 such as that from the sun, ultraviolet waves or x- or gamma-radiation.
According to
the present invention, the subject method can be used in the treatment and/or
prevention of certain symptoms of dermatitis caused by unwanted proliferation
of
epithelial cells. Such therapies for these various forms of dermatitis can
also include
topical and systemic corticosteroids, antipuritics, and antibiotics.
For example, it is contemplated that the subject method could be used to
inhibit angiogenesis. Hedgehog is known to stimulate angiogenesis. MATRIGEL"
plugs impregnated with hedgehog protein and inserted into mice evince
substantial
neovascularization, whereas MATRIGEL° plugs not carrying hedgehog show
comparatively little vascularization. Hedgehog protein is also capable of
increasing
vascularization of the normally avascular mouse cornea. The ptc-1 gene is
expressed
in normal vascular tissues, including the endothelial cells of the aorta,
vascular
smooth muscle cells, adventitial fbroblasts of the aorta, the coronary
vasculature
and cardiomyocytes of the atria and ventricles. These tissues are also
sensitive to A
hedgehog protein. Treatment with exogenous hedgehog causes upregulation of ptc-
1
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expression. In addition, hedgehog proteins stimulate proliferation of vascular
smooth
muscle cells in vivo. Hedgehog proteins also cause fibroblasts to increase
expression
of angiogenic growth factors such as VEGF, bFGF, Ang-1 and Ang-2. Lastly,
hedgehog proteins are known to stimulate recovery fiom ischemic injury and
S stimulate formation of collateral vessels.
Given that hedgehog promotes angiogenesis, hedgehog antagonists are
expected to act as angiogenesis inhibitors, particularly in situations where
some level
of hedgehog signaling is necessary for angiogenesis.
Angiogenesis is fundamental to many disorders. Persistent, unregulated
angiogenesis occurs in a range of disease states, tumor metastases and
abnormal
growths by endothelial cells. The vasculature created as a result of
angiogenic
processes supports the pathological damage seen in these conditions. The
diverse
pathological states created due to unregulated angiogenesis have been grouped
together as angiogenic dependent or angiogenic associated diseases. Therapies
directed at control of the angiogenic processes could lead to the abrogation
or
mitigation of these diseases.
Diseases caused by, supported by or associated with angiogenesis include
ocular neovascular disease, age-related macular degeneration, diabetic
retinopathy,
retinopathy of prematurity, corneal graft rejection, neovascular glaucoma,
retrolental
fibroplasia, epidemic lceratoconjunctivitis, Vitamin A deficiency, contact
lens
overwear, atopic lceratitis, superior limbic lceratitis, pteiygium l~eratitis
sicca,
Sjogren's, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections,
lipid
degeneration, chemical burns, bacterial ulcers, ftmgal ulcers, Herpes simplex
infections, Herpes zoster infections, protozoan infections, I~aposi sarcoma,
Mooren
ulcer, Terrien's marginal degeneration, marginal lceratolysis, rheumatoid
arthritis,
systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis,
Stevens
Johnson disease, periphigoid radial lceratotomy, corneal graph rejection,
rheumatoid
arthritis, osteoarthritis chronic inflammation (eg., ulcerative colitis or
Crohn's
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disease), hemangioma, Osler-Weber-Rendu disease, and hereditary hemorrhagic
telangiectasia.
In addition, angiogenesis plays a critical role in cancer. A tumor cannot
expand without a blood supply to provide nutrients and remove cellular wastes.
Tumors in which angiogenesis is important include solid tumors such as
rhabdomyosarcomas, retinoblastoma, Ewing sarcoma, neuroblastoma, and
osteosarcoma, and benign tumors such as acoustic neuroma, neurofibroma,
trachoma
and pyogenic granulomas. Angiogenic factors have been found associated with
several solid tumors. Prevention of angiogenesis could halt the growth of
these
tumors and the resultant damage to the animal due to the presence of the
tumor.
A~zgiogenesis is also associated with blood-born tumors such as leulcemias,
any of
various acute or chronic neoplastic diseases of the bone marrow in which
unrestrained proliferation of white blood cells occurs, usually accompanied by
anemia, impaired blood clotting, and enlargement of the lymph nodes, liver,
and
spleen. It is believed that angiogenesis plays a role in the abnormalities in
the bone
marrow that give rise to leul~emia-lilce tumors.
In addition to tumor growth, angiogenesis is important in metastasis.
Initially, angiogenesis is important is in the vascularization of the tumor
which
allows cancerous cells to eater the blood stream and to circulate throughout
the
body. After the tumor cells have left the primary site, and have settled into
the
secondary, metastasis site, angiogenesis must occur before the new tumor can
grow
and expand. Therefore, prevention of angiogenesis could lead to the prevention
of
metastasis of tumors and possibly contain the neoplastic growth at the primary
site.
Angiogenesis is also involved in normal physiological processes such as
reproduction and wound healing. Angiogenesis is an important step in ovulation
and
also in implantation of the blastula after fertilization. Prevention of
angiogenesis
could be used to induce amenorrhea, to block ovulation or to prevent
implantation
by the blastula. '
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It is anticipated that the invention will be useful for the treatment andlor
prevention of respiratory distress syndrome or other disorders resulting from
inappropriate lung surface tension. Respiratory distress syndrome results from
insufficient surfactant in the alveolae of the lungs. The lungs of vertebrates
contain
surfactant, a complex mixture of lipids and protein which causes surface
tension to
rise during lung inflation and decrease during lung deflation. During burg
deflation,
surfactant decreases such that there axe no surface forces that would
otherwise
promote alveolar collapse. Aerated alveoli that have not collapsed during
expiration
permit continuous oxygen and carbon dioxide transport between blood and
alveolar
gas and require much less force to inflate during the subsequent inspiration.
During
inflation, lung surfactant increases surface tension as the alveolar surface
area
increases. A rising surface tension in expanding alveoli opposes over-
inflation in
those airspaces and tends to divert inspired air to less well-aerated alveoli,
thereby
facilitating even lung aeration.
Respiratory distress syndrome is particularly prevalent among premature
infants. Lung surfactant is normally synthesized at a very low rate until the
last six
weeks of fetal life. Human infants born more than six weeks before the normal
term
of a pregnancy have a high risk of being born with inadequate amounts of lung
surfactant and inadequate rates of surfactant synthesis. The more prematurely
an
infant is born, the more severe the surfactant deficiency is lil~ely to be.
Severe
surfactant deficiency can lead to respiratory failure within a few minutes or
hours of
birth. The surfactant deficiency produces progressive collapse of alveoli
(atelectasis)
because of the decreasing ability of the lung to expand despite maximum
inspiratory
effort. As a result, inadequate amounts of oxygen reach the infant's blood.
RDS can
occur in adults as well, typically as a consequence of failure in surfactant
biosynthesis.
Lung tissue of premature infants shows high activity of the hedgehog
signaling pathway. Inhibition of this pathway using hedgehog antagonists
increases
the formation of lamellar bodies and increases the expression of genes
involved in
surfactant biosynthesis. Lamellar bodies are subcellular structures associated
with
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surfactant biosynthesis. For these reasons, treatment of premature infaazts
with a
hedgehog antagonist should stimulate surfactant biosynthesis and ameliorate
RDS.
In cases where adult RDS is associated with hedgehog pathway activation,
treatment
with hedgehog antagonists should also be effective.
It is further contemplated that the use of hedgehog antagonists may be
specifically targeted to disorders where the affected tissue and/or cells
evince high
hedgehog pathway activation. Expression of gli genes is activated by the
hedgehog
signaling pathway, including gli-1, gli-2 and gli-3. gli-1 expression is most
consistently correlated with hedgehog signaling activity across a wide range
of
tissues and disorders, while gli-3 is somewhat less so. The gli genes encode
transcription factors that activate expression of many genes needed to elicit
the full
effects of hedgehog signaling. However, the Gli-3 transcription factor can
also act as
a repressor of hedgehog effector genes, and therefore, expression of gli-3 can
cause a
decreased effect of the hedgehog signaling pathway. Whether Gli-3 acts as a
transcriptional activator or repressor depends on post-translational events,
and
therefore it is expected that methods for detecting the activating form
(versus the
repressing form) of Gli-3 protein would also be a reliable measure of hedgehog
pathway activation. gli-~ gene expression is expected to provide a reliable
marker
for hedgehog pathway activation. The gli-1 gene is strongly expressed in a
wide
array of cancers, hyperplasias and immature lungs, and serves as a marker for
the
relative activation of the hedgehog pathway. In addition, tissues, such as
immature
lung, that have high gli gene expression are strongly affected by hedgehog
inhibitors.
Accordingly, it is contemplated that the detection of gli gene expression may
be used
as a powerful predictive tool to identify tissues and disorders that will
particularly
benefit from treatment with a hedgehog antagonist.
In preferred embodiments, gli-1 expression levels are detected, either by
direct detection of the transcript or by detection of protein levels or
activity.
Transcripts may be detected using any of a wide range of techniques that
depend
primarily on hybridization of probes to the gli-1 transcripts or to cDNAs
synthesized
therefrom. Well l~nown techniques include northern blotting, reverse-
transcriptase
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PCR and microarray analysis of transcript levels. Methods for detecting Gli
protein
levels include western blotting, immunoprecipitation, two-dimensional
polyacrylamide gel electrophoresis (2D SDS-PAGE)(preferably compared against a
standard wherein the position of the Gli proteins has been determined), and
mass
spectroscopy. Mass spectroscopy may be coupled with a series of purification
steps
to allow high-throughput identification of many different protein levels in a
particular sample. Mass spectroscopy and 2D SDS-PAGE can also be used to
identify post-transcriptional modifications to proteins including proteolytic
events,
ubiquitination, phosphorylation, lipid modification etc. Gli activity may also
be
assessed by analyzing binding to substrate DNA or in vitro transcriptional
activation
of target promoters. Gel shift assays, DNA footprinting assays and DNA-protein
crosslinl~ing assays are all methods that may be used to assess the presence
of a
protein capable of binding to Gli binding sites on DNA.
In preferred embodiments, gli transcript levels are measured and diseased or
disordered tissues showing abnormally high gli levels are treated with a
hedgehog
antagonist. Premature lung tissue, lung cancers (e.g., adenocarcinomas,
broncho-
alveolar adenocarcinomas, small cell carcinomas), breast cancers (e.g.,
inferior
ductal carcinomas, inferior lobular carcinomas, tubular carcinomas), prostate
caazcers
(e.g., adenocarcinomas), and benign prostatic hyperplasias all show strongly
elevated
gli-1 expression levels in certain cases. Accordingly, gli-1 expxession levels
are a
powerful diagnostic device to determine which of these tissues should be
treated
with a hedgehog antagonist. In addition, there is substantial correlative
evidence that
cancers of urothelial cells (e.g., bladder cancer, other urogenital cancers)
will also
have elevated gli-1 levels in certain cases. For example, it is lcnown that
loss of
heterozygosity on chromosome 9q22 is common in bladder cancers. The ptc-I gene
is located at this position and ptc-I loss of function is probably a partial
cause of
hyperproliferation, as in many other cancer types. Accordingly, such cancers
would
also show high gli expression and would be particularly amenable to treatment
with
a hedgehog antagonist.
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Expression of ptc-1 and ptc-2 is also activated by the hedgehog signaling
pathway, but these genes are inferior to the gli genes as marlcexs of hedgehog
pathway activation. In certain tissues only one of ptc-1 or ptc-2 is expressed
although the hedgehog pathway is highly active. For example, in testicular
development, Indian hedgehog plays an important role and the hedgehog pathway
is
activated, but only ptc-2 is expressed. Accordingly, these genes are
individually
unreliable as markers for hedgehog pathway activation, although simultaneous
measurement of both genes are contemplated as a useful indicator for tissues
to be
treated with a hedgehog antagonist.
Ailments which may be treated by the subject method are disorders specific
to non-humans, such as mange.
In still another embodiment, the subject method can be used in the treatment
of human cancers, particularly basal cell carcinomas and other tumors of
epithelial
tissues such as the skin. For example, hedgehog antagonists can be employed,
in the
subject method, as part of a treatment for basal cell nevus syndrome (BCNS),
and
other other human carcinomas, adenocarcinomas, sarcomas and the like.
In a preferred embodiment, the subject method is used as part of a treatment
of prophylaxis regimen for treating (or preventing) basal cell carcinoma. The
deregulation of the hedgehog signaling pathway may be a general feature of
basal
cell carcinomas caused by ptc mutations. Consistent overexpression of human
ptc
mRNA has been described in tumors of familial and sporadic BCCs, determined by
in situ hybridization. Mutations that inactivate ptc may be expected to result
in
overexpression of mutant Ptc, because ptc displays negative autoregulation.
Prior
research demonstrates that overexpression of hedgehog proteins can also lead
to
tumorigenesis. That sonic hedgehog (Shh) has a role in tumorigenesis in the
mouse
has been suggested by research in which transgenic mice overexpressing Shh in
the
skin developed features of BCNS, including multiple BCC-like epidermal
proliferations over the entire skin surface, after only a few days of skin
development.
A mutation in the Shh human gene from a BCC was also described; it was
suggested
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that Shh or other Hh genes in humans could act as dominant oncogenes in
humans.
Sporadic ptc mutations have also been observed in BCCs from otherwise normal
individuals, some of which are UV-signature mutations. In one recent study of
sporadic BCCs, five UV-signature type mutations, either CT or CCTT changes,
were
found out of fifteen tumors determined to contain ptc mutations. Another
recent
analysis of sporadic ptc mutations in BCCs and neuroectodermal tumors revealed
one CT change in one of three ptc mutations found in.the BCCs. See, for
example,
Goodrich et al. (1997) Science 277:1109-13; Xie et al. (1997) Cancer Res
57:2369-
72; Oro et al. (1997) Science 276:817-21; Xie et al. (1997) Genes Chromosomes
Cancer 18:305-9; Stone et al. (1996) Nature 384:129-34; and Johnson et al.
(1996)
Science 272:1668-71.
The subject method can also be used to treatment patients with BCNS, e.g.,
to prevent BCC or other effects of the disease which may be the result of ptc
loss-of
function, hedgehog gain-of function, or smoothened gain-of function. Basal
cell
nevus syndrome is a rare autosomal dominant disorder characterized by multiple
BCCs that appear at a young age. BCNS patients are very susceptible to the
development of these tumors; in the second decade of life, large nmnbers
appear,
mainly on sun-exposed areas of the skin. This disease also causes a number of
developmental abnormalities, including rib, head and face alterations, and
sometimes polydactyly, syndactyly, and spina bifida. They also develop a
number of
tumor types in addition to BCCs: fibromas of the ovaries and heart, cysts of
the shin
and jaws, and in the central nervous system, medulloblastomas and meningiomas.
The subject method can be used to prevent or treat such tumor types in BCNS
and
non-BCNS patients. Studies of BCNS patients show that they have both genomic
and sporadic mutations in the ptc gene, suggesting that these mutations are
the
ultimate cause of this disease.
In another aspect, the present invention provides pharmaceutical preparations
comprising hedgehog antagonists. The hedgehog antagonists for use in the
subject
method may be conveniently formulated for administration with a biologically
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acceptable medium, such as water, buffered saline, polyol (for example,
glycerol,
propylene glycol, liquid polyethylene glycol and the like) or suitable
mixtures
thereof. The optimum concentration of the active ingredients) in the chosen
medium
can be determined empirically, according to procedures well known to medicinal
chemists. As used herein, "biologically acceptable medium" includes any and
all
solvents, dispersion media, and the like which may be appropriate for the
desired
route of administration of the pharmaceutical preparation. The use of such
media for
pharmaceutically active substances is known in the art. Except insofar as any
conventional media or agent is incompatible with the activity of the hedgehog
antagonist, its use in the pharmaceutical preparation of the invention is
contemplated. Suitable vehicles and their formulation inclusive of other
proteins are
described, for example, in the boolc Remihgton's Phaf°maceutical
Sciences
(Remington°s Pharmaceutical Sciences. Maclc Publishing Company, Easton,
Pa.,
USA 1985). These vehicles include injectable "deposit formulations".
Pharmaceutical formulations of the present invention can also include
veterinary compositions, e.g., pharmaceutical preparations of the hedgehog
antagonists suitable for veterinary uses, e.g., for the treatment of live
stoclc or
domestic animals, e.g., dogs.
Methods of introduction may also be provided by rechargeable or
biodegradable devices. Various slow release polymeric devices have been
developed
and tested in vivo in recent years for the controlled delivery of drugs,
including
proteinacious biopharmaceuticals. A variety of biocompatible polymers
(including
hydrogels), including both biodegradable and non-degradable polymers, can be
used
to form an implant for the sustained release of a hedgehog antagonist at a
particular
target site.
The preparations of the present invention may be given orally, parenterally,
topically, or rectally. They are of course given by forms suitable for each
administration route. For example, they axe administered in tablets or capsule
form,
by injection, inhalation, eye lotion, ointment, suppository, controlled
release patch,
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etc. administration by injection, infusion or inhalation; topical by lotion or
ointment;
and rectal by suppositories. Oral and topical administrations are preferred.
The phrases "parenteral administration" and "administered parenterally" as
used herein means modes of administration other than enteral and topical
administration, usually by injection, and includes, without limitation,
intravenous,
intralnuscular, intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac,
intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal
injection and
111fLlSioll.
The phrases "systemic , administration," "administered systemically,"
"peripheral administration" and "administered peripherally" as used herein
mean the
administration of a compound, drug or other material other than directly into
the
central nervous system, such that it enters the patient's system and, thus, is
subject to
metabolism and other like processes, for example, subcutaneous administration.
These compounds may be administered to humans and other animals for
therapy by any suitable route of administration, including orally, nasally, as
by, for
example, a spray, rectally, intravaginally, parenterally, intracisternally and
topically,
as by powders, ointments or drops, including buccally and sublingually.
Regardless of the route of administration selected, the compounds of the
present invention, which may be used in a suitable hydrated form, and/or the
pharmaceutical compositions of the present invention, are formulated into
pharmaceutically acceptable dosage forms such as described below or by other
conventional methods lcrlown to those of skill in the al-t.
Actual dosage levels of the active ingredients in the pharmaceutical
compositions of this invention may be varied so as to obtain an amount of the
active
ingredient which is effective to achieve the desired therapeutic response for
a
particular patient, composition, and mode of administration, without being
toxic to
the patient.
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The selected dosage level will depend upon a variety of factors including the
activity of the particular compound of the present invention employed, or the
ester,
salt or amide thereof, the route of administration, the time of
administration, the rate
of excretion of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in combination with
the
particular hedgehog antagonist employed, the age, sex, weight, condition,
general
health and prior medical history of the patient being treated, and lilce
factor s well
known in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily
determine and prescribe the effective amount of the pharmaceutical composition
required. For example, the physician or veterinarian could start doses of the
compounds of the invention employed in the pharmaceutical composition at
levels
lower than that required in order to achieve the desired therapeutic effect
and
gradually increase the dosage until the desired effect is achieved.
In general, a suitable daily dose of a compound of the invention will be that
amount of the compound which is the lowest dose effective to produce a
therapeutic
effect. Such an effective dose will generally depend upon the factors
described
above. Generally, intravenous, intracerebroventricular and subcutaneous doses
of the
compounds of this invention for a patient will range from about 0.0001 to
about 100
mg per kilogram of body weight per day.
If desired, the effective daily dose of the active compound may be
administered as two, three, four, five, six or more sub-doses administered
separately
at appropriate intervals throughout the day, optionally, in unit dosage forms.
The term "treatment" is intended to encompass also prophylaxis, therapy and
cure.
The patient receiving this . treatment is any animal in need, including
primates, in particular humans, aald other mammals such as equines, cattle,
swine
and sheep; and poultry and pets in general.
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The compound of the invention can be administered as such or in admixtures
with pharmaceutically acceptable and/or sterile carriers and can also be
administered
in conjunction with other antimicrobial agents such as penicillins,
cephalosporins,
aminoglycosides and glycopeptides. Conjunctive therapy, thus includes
sequential,
simultaneous and separate administration of the active compound in a way that
the
therapeutical effects of the first administered one is not entirely
disappeared when
the subsequent is administered.
Tl Pharmaceutical Comzaositiohs
While it is possible for a compound of the present invention to be
administered alone, it is preferable to administer the compound as a
pharmaceutical
formulation (composition). The hedgehog antagonists according to the invention
may be formulated for administration in any convenient way for use in human or
veterinary medicine. In certain embodiments, the compound included in the
pharmaceutical preparation may be active itself, or may be a prodrug, e.g.,
capable of
being converted to an active compound in a physiological setting.
Thus, another aspect of the present invention provides pharmaceutically
acceptable compositions comprising a therapeutically effective amount of one
or
more of the compounds described above, formulated together with one or more
pharmaceutically acceptable carriers (additives) and/or diluents. As described
in
detail below, the pharmaceutical compositions of the present invention may be
specially formulated for administration in solid or liquid form, including
those
adapted for the following: (1) oral administration, for example, drenches
(aqueous or
non-aqueous solutions or suspensions), tablets, boluses, powders, granules,
pastes
for application to the tongue; (2) parenteral administration, for example, by
subcutaneous, intramuscular or intravenous injection as, for example, a
sterile
solution or suspension; (3) topical application, for example, as a cream,
ointment or
spray applied to the slcin; or (4) intravaginally or intrarectally, for
example, as a
pessary, cream or foam. However, in certain embodiments the subject compounds
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may be simply dissolved or suspended in sterile water. In certain embodiments,
the
pharmaceutical preparation is non-pyrogenic, i.e., does not elevate the body
temperature of a patient.
The phrase "therapeutically effective amount" as used herein means that
amount of a compound, material, or composition comprising a compound of the
present invention which is effective for producing some desired therapeutic
effect by
overcoming a ptc loss-of function, hedgehog gain-of function, or s~raoother~ed
gain
of function in at least a sub-population of cells in an animal and thereby
blocl~ing the
biological consequences of that pathway in the treated cells, at a reasonable
benefit/rislc ratio applicable to any medical treatment.
The phrase "pharmaceutically acceptable" is employed herein to refer to
those compounds, materials, compositions, andlor dosage forms Which are,
within
the scope of sound medical judgment, suitable for use in contact with the
tissues of
human beings and animals without excessive toxicity, irritation, allergic
response, or
other problem or complication, commensurate with a reasonable benefit/risl~
ratio.
The phrase "pharmaceutically acceptable carrier" as used herein means a
pharmaceutically acceptable material, composition or vehicle, such as a liquid
or
solid filler, diluent, excipient, solvent or encapsulating material, involved
in carrying
or transporting the subject antagonists from one organ, or portion of the
body, to
a~lother organ, or portion of the body. Each carrier must be "acceptable" in
the sense
of being compatible with the other ingredients of the formulation and not
injurious
to the patient. Some examples of materials which can serve as pharmaceutically
acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose;
(2)
starches, such as corn starch and potato starch; (3) cellulose, and its
derivatives, such
as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as
cocoa
butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil,
safflower
oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as
propylene
glycol; (11) polyols, such as glycerin, sorbitol, mamiitol and polyethylene
glycol;
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(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering
agents,
such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl
alcohol;
(20) phosphate buffer solutions; and (21) other non-toxic compatible
substances
employed in pharmaceutical formulations.
As set out above, certain embodiments of the present hedgehog a~.ltagonists
may contain a basic functional group, such as amino or allcylamino, and are,
thus,
capable of forming pharmaceutically acceptable salts with pharmaceutically
acceptable acids. The term "pharmaceutically acceptable salts" in this
respect, refers
to the relatively non-toxic, inorganic and organic acid addition salts of
compounds of
the present invention. These salts can be prepared in situ during the final
isolation
and purification of the compounds of the invention, or by separately reacting
a
purified compound of the invention in its free base form with a suitable
organic or
inorganic acid, and isolating the salt thus formed. Representative salts
include the
hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,
valerate,
oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,
citrate,
maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate,
lactobionate, and laurylsulphonate salts and the lilce. (See, for example,
Berge et al.
(1977) "Pharmaceutical Salts", J. Pha~~a. Sci. 66:1-19)
The pharmaceutically acceptable salts of the subject compowds include the
conventional nontoxic salts or quaternary ammonium salts of the compounds,
e.g.,
from non-toxic organic or inorganic acids. For example, such conventional
nontoxic
salts include those derived from inorganic acids such as hydrochloride,
hydrobromic,
sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared
from
organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic,
malic,
tartaric, citric, ascorbic, palmitic, malefic, hydroxymaleic, phenylacetic,
glutamic,
benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, isothionic, and the lilce.
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In other cases, the compounds of the present invention may contain one or
more acidic functional groups and, thus, are capable of forming
pharmaceutically
acceptable salts with pharmaceutically acceptable bases. The term
"pharmaceutically
acceptable salts" in these instances refers to the relatively non-toxic,
inorganic and
organic base addition salts of compounds of the present invention. These salts
can
likewise be prepared in situ during the final isolation and pl~rification of
the
compounds, or by separately reacting the purified compound in its free acid
form
with a suitable base, such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically acceptable metal ration, with ammonia, or with a
pharmaceutically
acceptable organic primary, secondary or tertiary amine. Representative alkali
or
alkaline earth salts include the lithium, sodium, potassium, calcium,
magnesium, and
aluminum salts and the like. Representative organic amines useful for the
formation
of base addition salts include ethylamine, diethylamine, ethylenediamine,
ethanolamine, diethanolamine, piperazine and the like. (See, for example,
Berge et
al., suy°a)
Wetting agents, emulsifiers and lubricaazts, such as sodium lauryl sulfate and
magnesium stearate, as well as coloring agents, release agents, coating
agents,
sweetening, flavoring and perfuming agents, preservatives and antioxidants can
also
be present in the compositions.
Examples of pharmaceutically acceptable antioxidants include: (1) water
soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate,
sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble
antioxidants, such
as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene
(BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic acid
(EDTA),
sorbitol, tartaric acid, phosphoric acid, and the like.
Formulations of the present invention include those suitable for oral, nasal,
topical (including buccal and sublingual), rectal, vaginal and/or parenteral
administration. The formulations may conveniently be presented in unit dosage
form
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and may be prepared by any methods well known in the art of pharmacy. The
amount of active ingredient which can be combined with a carrier material to
produce a single dosage form will vary depending upon the host being treated,
the
particular mode of administration. The amount of active ingredient which can
be
combined with a caxrier material to produce a single dosage form will
generally be
that amount of the compound which produces a therapeutic effect. Generally,
out of
one hundred per cent, this amount will ra~zge from about 1 per cent to about
ninety-
nine percent of active ingredient, preferably from about 5 per cent to about
70 per
cent, most preferably from about 10 per cent to about 30 per cent.
Methods of preparing these formulations or compositions include the step of
bringing into association a compound of the present invention with the carrier
and,
optionally, one or more accessory ingredients. In general, the formulations
are
prepared by uniformly and intimately bringing into association a compound of
the
present invention with liquid caa.Tiers, or finely divided solid carriers, or
both, and
then, if necessary, shaping the product.
Formulations of the invention suitable for oral administration may be in the
form of capsules, cachets, pills, tablets, lozenges (using a flavored basis,
usually
sucrose and acacia or tragacanth), powders, granules, or as a solution or a
suspension
in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil
liquid
emulsion, or as an elixir or syrup, or as pastilles (using an inept base, such
as gelatin
and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each
containing a predetermined amount of a compound of the present invention as an
active ingredient. A compound of the present invention may also be
administered as
a bolus, electuary or paste.
In solid dosage forms of the invention for oral administration (capsules,
tablets, pills, dragees, powders, granules and the like), the active
ingredient is mixed
with one or more pharmaceutically acceptable carriers, such as sodium citrate
or
dicalcium phosphate, and/or any of the following: (1) fillers or extenders,
such as
starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2)
binders, such as,
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for example, carboxymethylcellulose, alginates, gelatin, polyvinyl
pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating
agents,
such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid,
certain
silicates, acid sodium carbonate; (5) solution retarding agents, such as
paraffin; (6)
absorption accelerators, such as quaternary ammonium compowids; (7) wetting
agents, such as, for example, cetyl alcohol and glycerol monostearate; (8)
absorbents, such as lcaolin and bentonite clay; (9) lubricants, such a talc,
calciwn
stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and
mixtures thereof; and (10) coloring agents. W the case of capsules, tablets
and pills,
the pharmaceutical compositions may also comprise buffering agents. Solid
compositions of a similar type may also be employed as fillers in soft and
hard-filled
gelatin capsules using such excipients as lactose or mills sugars, as well as
high
molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or
more accessory ingredients. Compressed tablets may be prepared using binder
(for
example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent,
preservative, disintegrant (for example, sodium starch glycolate or cross-
linked
sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded
tablets
may be made by molding in a suitable machine a mixture of the powdered
compound moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions
of the present invention, such as dragees, capsules, pills and granules, may
optionally be scored or prepared with coatings and shells, such as enteric
coatings
and other coatings well known in the pharmaceutical-formulating art. They may
also
be formulated so as to provide slow or controlled release of the active
ingredient
therein using, for example, hydroxypropylmethyl cellulose in varying
proportions to
provide the desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration through a
bacteria-
retaining filter, or by incorporating sterilizing agents in the form of
sterile solid
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compositions which can be dissolved in sterile water, or some other sterile
injectable
medium immediately before use. These compositions may also optionally contain
opacifying agents and may be of a composition that they release the active
ingredients) only, or preferentially, in a ceutain portion of the
gastrointestinal tract,
optionally, in a delayed manner. Examples of embedding compositions which can
be
used include polymeric substances and waxes. The active ingredient can also be
in
micro-encapsulated form, if appropriate, with one or more of the above-
described
excipients.
Liquid dosage forms for oral administration of the compounds of the
invention include pharmaceutically acceptable emulsions, microemulsions,
solutions, suspensions, syrups and elixirs. In addition to the active
ingredient, the
liquid dosage forms may contain inert diluents commonly used in the art, such
as, for
example, water or other solvents, solubilizing agents and emulsifiers, such as
ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,
benzyl
benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed,
groundnut, corn, germ, olive, castor and sesame oils), glycerol,
tetrahydrofuryl
alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures
thereof.
Besides inert diluents, the oral compositions can also include adjuvants such
as wetting agents, emulsifying and suspending agents, sweetening, flavoring,
coloring, perfuming and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending
agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene
sorbitol and
sorbitan esters, microcrystalline cellulose, ahuninum metahydroxide,
bentonite,
agar-agar and tragacanth, and mixtures thereof.
Formulations of the pharmaceutical compositions of the invention for rectal
or vaginal administration may be presented as a suppository, which may be
prepared
by mixing one or more compounds of the invention with one or more suitable
nonirritating excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which is solid at
room
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temperature, but liquid at body temperature and, therefore, will melt in the
rectum or
vaginal cavity and release the active hedgehog antagonist.
Formulations of the present invention which are suitable for vaginal
administration also include pessaries, tampons, creams, gels, pastes, foams or
spray
formulations containing such carriers as are lcnov~m in the art to be
appropriate.
Dosage forms for the topical or transdermal administration of a compound of
this invention include powders, sprays, ointments, pastes, creams, lotions,
gels,
solutions, patches and inhalants. The active compound may be mixed under
sterile
conditions with a pharmaceutically acceptable carrier, aald with any
preservatives,
buffers, or propellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to an active
compound of this invention, excipients, such as animal and vegetable fats,
oils,
waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene
glycols,
silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to a compound of this invention,
excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium
silicates
a~ld polyamide powder, or mixtures of these substances. Sprays can
additionally
contain customary propellants, such as chlorofluorohydrocarbons and volatile
unsubstituted hydrocarbons, such as butane and propane.
Transdermal patches have the added advantage of providing controlled
delivery of a compound of the present invention to the body. Such dosage forms
can
be made by dissolving or dispersing the hedgehog antagonists in the proper
medium.
Absorption enhancers can also be used to increase the flux of the 7zedgehog
antagonists across the skin. The rate of such flux can be controlled by either
providing a rate controlling membrane or dispersing the compound in a polymer
matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the life, are
also contemplated as being within the scope of this invention.
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Pharmaceutical compositions of this invention suitable for parenteral
achninistration comprise one or more compounds of the invention in combination
with one or more pharmaceutically acceptable sterile isotonic aqueous or
nonaqueous solutions, dispersions, suspensions or emulsions, or sterile
powders
which may be reconstituted into sterile injectable solutions or dispersions
just prior
to use, which may contain antioxidants, buffers, bacteriostats, solutes which
render
the formulation isotonic with the blood of the intended recipient or
suspending or
thickening agents.
Examples of suitable aqueous and nonaqueous carriers which may be
employed in the pharmaceutical compositions of the invention include water,
ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and
the
lilce), and suitable mixtures thereof, vegetable oils, such as olive oil, and
injectable
organic esters, such as ethyl oleate. Proper fluidity can be maintained, for
example,
by the use of coating materials, such as lecithin, by the maintenance of the
required
particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives,
wetting agents, emulsifying agents and dispersing agents. Prevention of the
action of
microorganisms may be ensured by the inclusion of various antibacterial and
antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid,
and the
like. It may also be desirable to include isotonic agents, such as sugars,
sodium
chloride, and the lilce into the compositions. In addition, prolonged
absorption of the
injectable pharmaceutical form may be brought about by the inclusion of agents
which delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to
slow
the absorption of the drug from subcutaneous or intramuscular injection. This
may
be accomplished by the use of a liquid suspension of crystalline or amorphous
material having poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend upon crystal
size and
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crystalline form. Alternatively, delayed absorption of a parenterally
administered
drug form is accomplished by dissolving or suspending the drug in a~1 oil
vehicle.
lnjectable depot forms are made by forming microencapsule matrices of the
subject compounds in biodegradable polymers such as polylactide-polyglycolide.
Depending on the ratio of drug to polymer, and the nature of the particular
polymer
employed, the rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot
injectable formulations are also prepared by entrapping the drug in liposomes
or
microemulsions which are compatible with body tissue.
When the compounds of the present invention are administered as
pharmaceuticals, to humans and animals, they can be given per se or as a
pharmaceutical composition containing, for example, 0.1 to 99.5% (more
preferably,
0.5 to 90%) of active ingredient in combination with a pharmaceutically
acceptable
carver.
The addition of the active compound of the invention to animal feed is
preferably accomplished by preparing an appropriate feed premix containing the
active compound in an effective amount and incorporating the premix into the
complete ration.
Alternatively, an intermediate concentrate or feed supplement containing the
active ingredient can be blended into the feed. The way in which such feed
premixes
and complete rations can be prepared and administered are described in
reference
books (such as "Applied Animal Nutrition", W.H. Freedman and CO., San
Francisco, U.S.A., 1969 or "Livestock Feeds and Feeding" O and B boobs,
Corvallis,
Ore., U.S.A., 1977).
Tn certain embodiments, a subject compound, such as compound D or a salt
thereof, may be formulated in an aqueous solution, e.g., for topical
application.
Suitable salts include salts of subject compounds with hydrochloric acid,
hydrobromic acid, hydroiodic acid, succinic acid, tartaric acid, lactic acid,
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methanesulfonic acid, malefic acid, or any other suitable acid, such as one
that forms
a pharmaceutically acceptable anion in the presence of an amine base.
In certain embodiments, the aqueous solution may contain a
pharmaceutically acceptable salt, such as a salt including a canon selected
from
sodium, potassium, magnesium, and calcium, and an anion selected from acetate,
citrate, phosphate, chloride, any other suitable ions, or combinations thereof
In certain embodiments, the aqueous solution may, additionally or
alternatively, include dextrose, lactose, mannitol, or another
polyhydroxylated
compound, such as a pharmaceutically acceptable carbohydrate, such as a mono-
or
di-saccharide, or polyol.
In certain embodiments, an aqueous solution may contain solutes to result in
an osmolarity between 200 and 400 mOsm, preferably between 250 and 350 Osm,
even more preferably between 280 and 300 mOsm, such as 290 mOsm.
In certain embodiments, the pH of the solution will be in the range of 3 to 6,
preferably 3.5 to 5, even more preferably between 4 and 4.5.
Thus, generally, an aqueous solution may comprise up to about 7% of a
carbohydrate or polyol such as mannitol, lactose, or dextrose, e.g., up to
about 6%,
or about 3% to about 6%, or about 4 to about 5%, up to about 50 mM of a salt
selected from sodium acetate and sodium citrate, e.g., up to about 20 mM, or
about 2
mM to about 20 mM, or about 5 mM to about 15 mM, and sufficient
pharmaceutically acceptable solutes, such as sodium chloride, to result in an
osmolarity between about 200 and 400 mOsm, preferably between 250 and 350
Osm, even more preferably between 280 and 300 mOsm. In certain embodiments,
the aqueous solution is substantially free of a carbohydrate or polyol,
substantially
free of sodium acetate and sodium citrate, or both, e.g., may consist
essentially of
physiological saline and a salt of a subject compound, or is substantially
free of salts
such as sodium chloride, sodium acetate, and sodium citrate and consists
essentially
of an aqueous solution of a carbohydrate or polyol and a salt of a subject
compound.
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Thus, for example, an aqueous solution of a subject compound, such as
compound D, may comprise 10 mM sodium acetate in physiological saline at a pH
of 4.2. Alternatively, an aqueous solution may comprise 10 mM sodium acetate
in a
5% dextrose solution, or simply a 5% dextrose solution.
Tjl. Synthetic Schemes aid Ide~tificatio~c ofActive Anta~o~ists
The subject antagonists, and congeners thereof, can be prepared readily by
employing the cross-coupling technologies of Suzulci, Stille, and the like.
These
coupling reactions are carried out under relatively mild conditions and
tolerate a
wide range of "spectator" functionality.
a. Conzbi~atorial Lib~a~ies
The compounds of the present invention, particularly libraries of variants
having various representative classes of substituents, are amenable to
combinatorial
chemistry and other parallel synthesis schemes (see, for example, PCT WO
94/08051). The result is that large libraries of related compounds, e.g. a
variegated
library of compounds represented above, can be screened rapidly in high
throughput
assays in order to identify potential hedgehog antagonist lead compounds, as
well as
to refine the specificity, toxicity, and/or cytotoxic-kinetic profile of a
lead
compound. For instance, ptc, hedgehog, or smoothe~zed bioactivity assays, such
as
may be developed using cells with either a ptc loss-of function, hedgehog gain-
of
function, or smoothened gain-of function, can be used to screen a library of
the
subject compounds for those having agonist activity toward ptc or antagonist
activity
towards hedgehog or smoothened.
Simply for illustration, a combinatorial library for the purposes of the
present
invention is a mixture of chemically related compounds which may be screened
together for a desired property. The preparation of many related compounds in
a
single reaction greatly reduces and simplifies the number of screening
processes
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which need to be carried out. Screening for the appropriate physical
properties can
be done by conventional methods.
Diversity in the library can be created at a variety of different levels. For
instance, the substrate aryl groups used in the combinatorial reactions can be
diverse
in terms of the core aryl moiety, e.g., a variegation in terms of the ring
structure,
and/or can be varied with respect to the other substituents.
A variety of techniques are available in the art for generating combinatorial
libraries of small organic molecules such as the subject hedgehog antagonists.
See,
for example, Blondelle et al. (1995) Trends Anal. Chem. 14:83; the Affymax
U.S.
Patents 5,359,115 and 5,362,899: the Ellman U.S. Patent 5,288,514: the Still
et al.
PCT publication WO 94/08051; the ArQule U.S. Patents 5,736,412 and 5,712,171;
Chen et al. (1994) JACS 116:2661: I~err et al. (1993) JACS 115:252; PCT
publications W092/10092, W093/09668 and WO91/07087; and the f,erner et al.
PCT publication W093/20242). Accordingly, a variety of libraries on the order
of
about 100 to 1,000,000 or more diversomers of the subject hedgehog antagonists
can
be synthesized and screened for particular activity or property.
In an exemplary embodiment, a library of candidate hedgehog antagonists
diversomers can be synthesized utilizing a scheme adapted to the techniques
described in the Still et al. PCT publication WO 94/08051, e.g., being linked
to a
polymer bead by a hydrolyzable or photolyzable group, optionally located at
one of
the positions of the candidate antagonists or a substituent of a synthetic
intermediate.
According to the Still et al. technique, the library is synthesized on a set
of beads,
each bead including a set of tags identifying the particular diversomer on
that bead.
The bead library can then be "plated" with ptc loss-of function, hedgehog gain-
of
function, or smoothened gain-of function cells for which an 7zedgehog
antagonist is
sought. The diversomers can be released from the bead, e.g. by hydrolysis.
The structures of the compounds useful in the present invention lend
themselves readily to efficient synthesis. The nature of the structures, as
generally
described by formulas I to VI, allows the assembly of such compounds using
some
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combination of Rl, R2, R3, and R4 moieties, as set forth above. For example,
these
subunits can be attached to the core ring through common acylation or
allcylation
reactions. The vast majority of such reactions, including those depicted in
Figures
11, 12, 15, and 16 are both extremely mild and extremely reliable, and are
thus
perfectly suited for combinatorial chemistry. The facile nature of such a
combinatorial approach towards the generation of a library of test compounds
is
apparent in the exemplary scheme below (P = protecting group), wherein the
various
groups of a compound according to the above formulae are linked
combinatorially
(e.g., using one of the methods described above). Even greater diversity may
be
attained by, for example, utilizing a range of reactive fimctionalities when
appending
a subunit, e.g., using a range of R-L-C(O)Cl, PO-Ar-L-NCO, PO-Ar-L-S02C1, etc.
when appending an Rl subunit.
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t2LCH0/NaBHq
or RZLCHZX RtLCOX
alkylate acylate
Q n o_
RqLXH
E
couple
Bead 0
N
X
0
~N~
RZL ~LRt
Many variations on the above and related pathways permit the synthesis of
widely diverse libraries of compounds which may be tested as inhibitors of
hedgel2og function.
Preparation of Exemplary Compounds of the Present Invention
A series of compounds conforming to the general structures disclosed herein
were prepared and tested for biological activity (vide ihft~a). A suitable
core structure
can be readily prepared from commercially available ti~ans-4-hydroxy-L-proline
as
sLUinnarized in the scheme below:
104
acylate or I ~ deprotect
alkylate 2. R3LCOX or
R3LCHZX
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HO~~~ MsO,~ '
OFi 1. MeOH, H+ OMe 1. NaN3 OMe
H~~ 2' (BOC)ZO ' Ni~ 2. H+
\'\' 3. MsCI, bas \\e
O O I O
BOC
Ti~ans-4-h, day-L-proline methyl ester hydrochloride:
Acetyl chloride (249 mL, 3.47 mol) was added dropwise to methanol (2090
mL) with stirring and cooling to maintain the temperature below 30 °C.
After
complete addition, stirring was continued for a further 60 min. before
addition of
tT°aTZS-4-hydroxy-L-proline (325 g, 2.48 mol) as a solid. The reaction
mixture was
heated to reflux for 24 h, cooled to 0 °C, and test-butyl methyl ether
(TBME, 5220
mL) was added slowly over 30 min. The precipitated solid was collected on a
filter
and washed with ice-cold TMBE (2 x 1 L). The product was dried at 40 °C
overnight
in a vacuum to yield 424 g of the desired ester.
Ty~ans-1-(test-butoxycarbon~)-4-h day-L-proline methyl ester:
The product ester of the previous reaction (423 g, 2.32 mol) was suspended
in dichloromethane (6.5 L). Under stirring and cooling, triethylamine (1019
mL,
7.32 mol) was added over 30 min., followed by di-test-butyl dicarbonate (588
g, 2.70
mol) over 30 min. to maintain the internal temperature below 15 °C.
After complete
addition, the mixture was stirred at room temperature for 3 hours, followed by
addition of 1 M aqueous citric acid solution (650 mL). The mixture was stirred
1
hour, and the organic layer was separated, washed with 1 M aqueous KHC03 (920
mL), water (2 x 1 L), and dried over MgS04 in the presence of activated
charcoal
(15 g). The solvent was removed in vacuo and the residue purified by flash
chromatography (2x1800 g silica gel, 3:1 to 2:1 hexane:EtOAc eluent) to give
the
desired carbamate (489 g).
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(4R)-1- test-butoxycarbon~)-4-[(methylsulfon~)oxy]-L-proline methyl ester:
The carbamate above (478 g, 1.95 mol), N-diisopropylethylamine (DIPEA,
373 mL, 2.15 mol), and 4-dimethylaminopyridine (DMAP, 23.8 g, 0.195 mol) were
dissolved in dichloromethane (7650 mL). Methanesulfonyl chloride (167 mL, 2.15
mol) in dichloromethane (950 mL) was added dropwise over 50 min. with cooling
to
maintain a temperature below 10 °C. The mixture was stirred at -6
°C for 2 h, water
(750 mL) was added, the mixture was stirred 15 min. more, and the layers were
separated. The organic layer was washed with 1 M aqueous KHC03 (950 mL), 1 M
aq. citric acid (2 x 950 mL), and water (750 mL) and dried over MgS04. The
solvent
was removed in vacuo and the residue crystallized with hexane (1.9 L). The
crystalline mesylate was collected on a filter, washed with hexane (2 x 500
mL), and
dried at 40 °C in vacuo to give 624 g of the product.
~4S)-1-(test-butoxycarbonyl)-4-azido-L-proline meth 1
A solution of the above mesylate (624 g, 1.93 mol) and sodium azide (716 g,
11.01 mol) in dimethylformamide (DMF, 3120 mL) was stirred for 22 h at 60
°C,
the solution was cooled to 0 °C, water (3 L) was added over 40 min. to
lceep the
temperature below 20 °C, and EtOAC (3 L) was added. The mixture was
stirred
vigorously 20 min, the layers were separated, and the aqueous phase extracted
with
EtOAc (3 L). The combined organic layers were washed with water (750 mL), 0.1
M
aq. HCl (400 mL), and water (750 mL), then dried over MgS04. The solvent was
removed in vacuo and the residue purified by flash chromatography (2x1800 g
silica
gel, 2:1 hexane:EtOAc) to give the desired azide (516 g).
(4S)-4-azido-L-nroline methyl ester hydrochloride:
A saturated solution of HCl in dioxane (1940 mL) was prepared at 10-16
°C,
and a solution of the azide (523 g, 1.94 mmol) in dioxane (480 mL) was added
dropwise with stirring and cooling over 30 min. to beep the temperature below
25
°C. After complete addition, the "reaction mixture was stirred at room
temperature for
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2 hours, TBME (2 L) was added, and the resulting mixture stirred at 0
°C for 1 hour.
The precipitated solid was collected on filter paper, washed with TBME (4x500
mL), and dried at 40 °C in vacuo to give the desired hydrochloride salt
(348 g).
Subject compounds can be prepared from the above core, or from related
compounds or derivatives, using solution-phase or solid-phase techniques, as
shown
in the schemes below:
Scheme 1: Solution-Phase Route 1
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BOC
H ~ ~N~
BOC-piperazine,
N
couple
FMOC
1. base, deprotect
2. allcylate
BOC
~BOC ~ N
H~ ~ N 1. mesylate
2. sodium azide
N 3. reduce
R
R
allrylate
BOC acyl
~N/ ~NH
1. acylate
2. deprotect
Scheme 2: Solution-Phase Route 2
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.acyl
1. reduce
2. allcylate
3. acylate
BOC BOC
1. saponify
NR2
2. amidation
R~
Scheme 3: Solid-Phase Route
109
I. deprotect
2. allcylate
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solid solid
support support
N ~ N
couple
NH
~C
reductive amination
acyl\
solid
1. acylate support
HN ~ E N
2. deprotect
N 3. allcylate N
4. cleave
p K~ 7C
These routes, together with the exemplary solid-phase route, provide access
to a wide range of compounds having different substituents and stereochemical
relationships. One of ordinary skill in the art will appreciate that the use
of
piperazine in the above schemes is exemplary ouy, and other amines caai be
employed to obtain an even more diverse array of subject compounds. Similarly,
the
use of BOC, FMOC, and other protecting groups is exemplary only, and one of
shill
in the art can select other protecting groups suitable for the functional
group and the
subsequent reaction conditions without departing from the scope or spirit of
the
present invention. Furthermore, although the above schemes typically begin
with the
trans-hydroxy-L-proline compound, all isomers of this compound are
commercially
available, including cis/trans and D/L compounds, providing access to a wide
range
of diastereomerically pure intermediates and subject compounds. A
t~°avrs-
aminoproline core can be obtained from a tr~ans-hydroxyproline starting
material by
forming an intermediate czs-bromoproline (by forming, for example, a triflate
or
mesylate of the hydroxyl acid displacing the sulfonate with bromide ion),
followed
by a second displacement with azide, to provide net retention of the t~a~cs
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stereochemical relationship, as is well known in the art. Alternatively,
diastereomeric mixtures may be prepared, as in the above Scheme 3, followed by
an
optional separation of the isomers.
b. Sc~ee~ihg Assays
There are a variety of assays available for determining the ability of a
compound to agonize ptc function or antagonize smoothened or hedgehog
function,
many of which can be disposed in high-tluoughput formats. In many drug
screening
programs which test libraries of compounds and natural extracts, high
throughput
assays are desirable in order to maximize the number of compounds surveyed in
a
given period of time. Thus, libraries of synthetic and natural products can be
sampled for other compounds which are hedgehog antagonists.
In addition to cell-free assays, test compounds can also be tested in cell-
based assays. In one embodiment, cell which have a ptc loss-of function,
hedgelzog
gain-of function, or smoothened gain-of function phenotype can be contacted
with a
test agent of interest, with the assay scoring for, e.g., inhibition of
proliferation of
the cell in the presence of the test agent.
A number of gene products have been implicated in patched mediated signal
transduction, including patched, transcription factors of the cubitus
ihtef°~uptus (ci)
family, the serine/threonine lcinase fused (fu) and the gene products of
costal-2,
smoothened and supp~~esso~ of fused.
The induction of cells by hedgehog proteins sets in motion a cascade
involving the activation and inhibition of downstream effectors, the ultimate
consequence of which is, in some instances, a detectable change in the
transcription
or translation of a gene. Potential transcriptional targets of hedgehog-
mediated
signaling are the patched gene (Hidalgo and Ingham, 1990 Development 110, 291-
301; Marigo et al., 1996 ) and the vertebrate homologs of the drosophila
cubitus
interruptus gene, the GLI genes (Hui et al. (1994) Dev Biol 162:402-413).
Patched
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gene expression has been shown to be induced in cells of the limb bud and the
neural plate that are responsive to Shh. (Maxigo et al. (1996) PNAS 93:9346-
51;
Marigo et al. (1996) Development 122:1225-1233). The Gli genes encode putative
transcription factors having zinc finger DNA binding domains (Orenic et al.
(1990)
Genes & Dev 4:1053-1067; Kinzler et al. (1990) Mol Cell Biol 10:634-642).
Transcription of the Gli gene has been reported to be upregulated in response
to
hedgehog in limb buds, while transcription of the Gli3 gene is domzregulated
in
response to hedgehog induction (Maxigo et al. (1996) Development 122:1225-
1233).
By selecting transcriptional regulatory sequences from such target genes,
e.g., from
hatched or Gli genes, that are responsible for the up- or dorm-regulation of
these
genes in response to hedgehog signalling, and operatively linl~ing such
promoters to
a reporter gene, one can derive a transcription based assay which is sensitive
to the
ability of a specific test compound to modify hedgehog-mediated signalling
pathways. Expression of the reporter gene, thus, provides a valuable screening
tool
for the development of compounds that act as antagonists of hedgehog.
Reporter gene based assays of this invention measure the end stage of the
above described cascade of events, e.g., transcriptional modulation.
Accordingly, in
practicing one embodiment of the assay, a reporter gene construct is inserted
into the
reagent cell in order to generate a detection signal dependent on ptc loss-of
function,
hedgehog gain-of function, snzoothe~ed gain-of fwction, or stimulation by SHH
itself. The amount of transcription from the reporter gene may be measured
using
any method known to those of shill in the art to be suitable. For example,
mRNA
expression from the reporter gene may be detected using RNAse protection or
RNA-
based PCR, or the protein product of the reporter gene may be identified by a
characteristic stain or an intrinsic biological activity. The amount of
expression from
the reporter gene is then compared to the amount of expression in either the
same
cell in the absence of the test compound or it may be compared with the amomt
of
transcription in a substantially identical cell that laclcs the target
receptor protein.
Any statistically or otherwise significant decrease in the amount of
transcription
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indicates that the test compound has in some mariner agonized the normal ptc
signal
(or antagonized the gain-of function hedgehog or smoothefzed signal), e.g.,
the test
compound is a potential hedgehog antagonist.
Exemplification
The invention now being generally described, it will be more readily
understood by reference to the following examples which are included merely
for
purposes of illustration of certain aspects and embodiments of the present
invention,
and are not intended to limit the invention.
Syhtlzesis of Exemplafw Ifihibitors
N 1-((3R,5.5~-1-(1,3-benzodioxol-5-ylmethyl)-5-(piperazinocarbonyl)tetrahydro-
1H 3-pyrrolyl] N 1-(4-methoxybenzyl)-3,3-dimethylbutanamide. "Ti°av~s-
aminoproline"
HO HO HO Br
'/~OH ~ ,I~~OMe - 'I~OMe --~ home
(1) O (2) OO boc 0 boc 0
OMe (3) (4)
OMe
. .~ 1
HN HZN~ NCI N
~N,,
O ~~OMe ~ '/home ~ ~~OMe ~ I~~OMe
Nboc ~O' boc rOI Nboc ~O Nboc ~O
i$) (~) (s> (5)
OMe
-r IVp
.OH
(11 )
(9a) TFA Salt
(9b) Free Base
113 Trans Amino Proline
(13a) TFA Salt
a (13b) Free. Base
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1-(tent-Butyl) 2-methyl (2S, 4S)-4-bromotetrahydro-1H 1, 2-
pyrroledicarboxylate (4)
1-(tey~t-Butyl) 2-methyl (2S, 4R)-4-hydroxytetrahydro-1H 1,2-
pyrroledicarboxylate (3) (2.0 g, 8.15 mmol) was weighed into an oven-dried
flaslc
and azeotropically dried using toluene. Dichloromethane (16 mL) and carbon
tetrabromide (10.81 g, 8.15 rninol) were added and the solution was stirred,
cooled
to 0 °C and treated with triphenylphosphine (8.5 g, 32.41 mmol). The
mixture was
stirred for 5 h at 0 °C, then methanol (1.8 mL) was added and stirring
was continued
overnight at room temperature. The mixture was diluted with diethyl ether (80
ml)
and the resulting suspension was filtered and washed with diethyl ether (30
ml). The
solvents were combined and evaporated under reduced pressure and the crude
product was purified by silica gel column chromatography eluting with
hexane/ethyl
acetate (19:1 to 4:1, v/v) to give the title bromide (4) (1.0 g, 40 %) as a
colourless
oil:
8H (360 MHz; CDC13) 1.41 and 1.46 (2xs, 9H, rotamers), 2.38-2.46 (m, 1H),
2.75-2.87 (m, 1H), 3.67-3.74 (m, 1H), 3.76 (s, 3H), 3.96-4.07 (m, 1H) and 4.24-
4.42
(m, 2H); LRMS (from LC-MS) (ES+) m/z 210 (100).
1-(tent-Butyl) 2-methyl (2S, 4R)-4-azidotetrahydro-1H l, 2-
pyrroledicarboxylate
A dispersion of sodium azide (0.90 g, 13.84 mmol) and 1-(test-butyl)-2-
methyl (2S, 4S)-4-bromotetrahydro-1111,2-pyrroledicarboxylate (4) (1.0 g, 3.24
mmol) in anhydrous dimethylformamide (32 mL) was heated for 64 h under an
atmosphere of nitrogen. The mixture was cooled to room temperature, poured
into
ice-cold water and extracted with ethyl acetate. The orgailic extracts were
combined,
washed with water and brine, dried (MgS04) and evaporated under reduced
pressure.
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The crude product was purified by silica gel column chromatography eluting
with
hexane-ethyl acetate (3:1 to 1:l, v/v) to give the title azide (5) (0.88 g, 93
%) as a
pale yellow oil:
~H (360 MHz; CDC13) 1.41 and 1.46 (2xs, 9H, rotamers), 2.13-2.20 (m, 1H),
2.27-2. 38 (m, 1H), 3.45-3.49 and 3.57-3.60 (2xm, 1H, rotamers), 3.68-3.73 (m,
1H),
3.74-3.75 (2xs, 3H, rotamers), 4.15-4.23 (m, 1H) and 4.30-4.35 and 4.39-4.43
(2xm,
1H, rotamers); LRMS (from LC-MS) (ES+) m/z 171 [(M+H)+ - CSH902] (100).
1-(tent-Sutyl) 2-methyl (2S,4R)-4-ammoniotetrahydro-1H 1,2-
pyrroledicarboxylate chloride (6)
Palladium on carbon (10%, 0.5 g) was added to a solution of 1-(tef°t-
butyl)-2-
methyl (2S, 4R)-4-azidotetrahydro-1H 1,2-pyrroledicarboxylate (5) (0.81 g, 3.0
mmol) in 2% v/v hydrochloric acid in ethanol (8 mL). The reaction mixture was
evacuated and purged with nitrogen (three times), then placed under an
atmosphere
of hydrogen and vigorously stirred at room temperature overnight. The mixture
was
filtered through a pad of CELITE'° and evaporated under reduced
pressuxe to give
the crude product. This was triturated with diethyl ether at 0 °C and
the resulting
slung was filtered, washed with ice-cold diethyl ether and dried under vacuum.
The
title salt (6) was obtained in quantitative yield:
bH (360 MHz; CD30D) 1.46 and 1.51 (2xs, 9H, rotamers), 2.35-2.47 (m,
2H), 3.50-3.55 (m, 1H), 3.74-3.86 [m, 4H, {containing at 3.79 and 3.80 (2xs,
3H,
rotamers)}], 3.89-3.95 (m, 1H) and 4.46-4.50 (m, 1H); LRMS (from LC-MS) (ES+)
m/z 210 (100).
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1-(tart-Butyl) 2-methyl (2S,4R)-4-[3-methoxybenzyl)amino]tetrahydro-1H 1,2-
pyrroledicarboxylate (7)
A solution of 1-(tent-butyl) 2-methyl (2S, 4R)-4-ammoniotetrahydro-1H 1,2-
pyrroledicarboxylate chloride (6) (0.83 g, 2.96 mmol) and 3-
methoxybenzaldehyde
(0.38 g, 2.8 mmol) in trimethyl orthoformate (8 mL) was stirred for 45 min at
room
temperature. The solution was treated slowly with sodium cyanoborohydride
(0.28 g,
4.46 mmol) and the course of the reaction was monitored by thin layer
chromatography (TLC) analysis. Once completed (~1.5 h), the reaction was
quenched with saturated aqueous potassium hydrogensulfate solution and
extracted
with dichloromethane. The pH value of the aqueous phase was adjusted to 9 and
baclc-extracted with dichloromethane. The combined organic extracts were dried
(MgS04) and evaporated under reduced pressure to give the title amine (7) in
quantitative yield:
8H (360 MHz; CDC13) 1.40 and 1.45 (2xs, 9H, rotamers), 2.07-2.19 (m, 2H),
3.18-3.23 and 3.32-3.36 (2xm, 1H), 3.43-3.53 (m, 1H), 3.70-3.74 [m, 4H,
{containing at 3.72 and 3.73 (2xs, 3H, rotamers)~] 3.81 (s, 3H), 4.32-4.36 and
4.40-
4.44 (2xm, 1H), 6.79-6.81 (m, 1H), 6.87-6.89 (m, 2H) and 7.24 (t, 1H); LRMS
(from
LC-MS) (ES+) mlz 265 [(M+H)+ - CSH902] (100).
1-(tent-Butyl) 2-Methyl (2S,4R)-4-[(3,3-dimethylbutanoyl)(3-
methoxybenzyl)amino]tetrahydro-1H 2-pyrrolecarboxylate (8)
A solution of 1-(test-butyl)-2-methyl (2S,4R)-4-[3-
methoxybenzyl)amino]tetrahydro-1H 1,2-pyrroledicarboxylate (7) (0.3 g, 0.82
mmol) and N,N diisopropylethylamine (0.106 g, 0.82 mmol) in anhydrous
dichloromethane (0.8 mL) was stirred at room temperature under an atmosphere
of
nitrogen. The solution was treated dropwise with test-butylacetyl chloride
(0.133 g,
0.99 mmol) and stirred overnight. The solvent was evaporated under reduced
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pressure and the residue was purified by silica gel column chromatography
(hexane-
ethyl acetate, 2:1, v/v) to give the title amide (8) (1.0 g, 40 %) as a
colourless oil:
8H (360 MHz; CDC13) 1.01 and 1.05 (2xs, 9H, rotamers), 1.37 and 1.41 (2xs,
9H, rotamers), 1.87-2.56 [m, 4H (containing at 2.16 (s, 2H)], 3.17-3.35 (m,
1H),
3.62-3.85 [m, 7H, containing at 3.70 and 3.79 (2 x s, 6H))] 4.21-4.24 and 4.28-
4.35
(2xm, 1H, rotamers), 4.40-4.58 (m, 2H), 4.73-4.95 and 5.03-5.21 (2xm, 1H,
rotamers), 6.54-6.88 (m, 3H) and 7.19-7.31 (m, 1H); LRMS (from LC-MS) (ES+)
m/z 363 (100).
(2S, 4R)-4-[(3,3-dimethylbutanoyl)(3-methoxyanilino)]-2-
(methoxycarbonyl)tetrahydro-1H 2-pyrrolium 2,2,2-trifluoroacetate (9a)
1-(test-Butyl) 2-methyl(2S,4R)-4-[(3,3-dimethylbutanoyl)(3-
methoxybenzyl)amino]-tetrahydro-1H 2-pyrrolecarboxylate (8) (0.01 g, 21.6
~mol)
was added to a 30% solution of trifluoroacetic acid in dichloromethane(0.5 mL)
at
room temperature and stirred for 30 min. The solution was evaporated to
dryness
under reduced pressure to give the title pyrrolimn salt (9a) in quantitative
yield:
8H (360 MHz; CDC13) 1.05 (s, 9H), 2.38-2.57 (m, 4H), 3.59-3.68 (m, 2H),
3.75 (s, 3H), 3.79 (s, 3H), 4.09-4.15 (m, 1H), 4.52-4.63 (m, 2H), 4.78-4.94
(m, 1H),
6.66 (s, 1H), 6.70 (d, 1H), 6.86-6.88 (dd, 1H) and 7.31 (t, 1H).
Methyl (2S,4R)-1-(1,3-benzodioxol-5-ylmethyl)-4-[(3,3-dimethylbutanoyl)(3-
methoxybenzyl)amino]tetrahydro-1H 2-pyrrolecarboxylate (10)
1-(teT~t-Butyl) 2-methyl(2S,4R)-4-[(3,3-dimethylbutanoyl)(3-
methoxybenzyl)amino]-tetrahydro-1H 2-pyrrolecarboxylate (8) (0.15 g, 0.32
mmol)
was added to a solution of 30% v/v trifluoroacetic acid in dichloromethane (3
mL) at
room temperature. The mixture was stirred for 30 min and evaporated to dryness
under vacuum. The residue was partitioned between dichloromethane and
saturated
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aqueous potassium carbonate and shaken vigorously for 5 mins. The organic
layer
was separated, dried (MgS04) and evaporated under reduced pressuxe to give 140
mg of crude methyl (2S,4R)-4-[(3,3-dimethylbutanoyl)-3-
methoxyanilino]tetrahydro
1H 2-pyrrolecarboxylate (9b) which was used in the following reaction without
further purification.
A solution of the crude amine (9b) ( 140 mg) prepared above, piperonal (74
mg, 0.49 mmol) and glacial acetic acid (2 drops) in 1,2-dichloroethane (0.5
mL) was
stirred for 30 min at room temperature. 95% Sodium cyanoborohydride (32 mg,
0.48
mmol) was added in small portions and stirring was continued for 1 h. The
reaction
was quenched with saturated aqueous sodium bicarbonate solution (2 mL),
extracted
with dichloromethane, dried (MgS04) and evaporated under reduced pressure. The
residue was purified by silica gel column chromatography eluting with
dichloromethane-ethyl acetate (90:10-75:25) to give the title pynole (10) (115
mg,
71.4 %) as a pale yellow oil:
8H (360 MHz; CDC13) 0.98-1.08 (m, 9H), 2.09-2.59 [m, 4H, {containing at
2.13 (s, 2H)~], 2.96-3.07 (m. 1H), 3.47-3.85 (m, 11H), 4.46-4.63 (m, 1H), 4.83-
4.94
(m, 1H), 5.92-5.95 (m, 2H), 6.63-6.89 (m, 6H) and 7.15-7.34 (m, 1H); LRMS
(from
LC-MS) (ES+) f~2~z 497 [(M+H)+] (100).
(2S,4R)-1-(1,3-benzodioxol-5-ylmethyl)-4-[(3,3-dimethylbutanoyl)(3-
methoxybenzyl)amino]tetrahydro-1H 2-pyrrolecarboxylic acid (11)
Lithium hydroxide monohydrate (17 mg, 0.405 mmol) was added to a
solution of methyl (2S,4R)-1-(1,3-benzodioxol-5-ylmethyl)-4-[(3,3-
dimethylbutanoyl)(3-methoxybenzyl)amino]tetrahydro-1H 2-pyrrolecarboxylate
(10)
(100 mg, 0.20 mmol) in 66 % v/v methanol in water (1.0 mL). The mixture was
stirred overnight at room temperature, then the solvent was removed under
reduced
pressure and the residue partitioned between dichloromethane (1.0 mL) and
water
(1.0 ml). The aqueous phase was acidified with 1.0 M aqueous citric acid and
the
two layers were vigorously stirred for 10 min at room temperature. The layers
were
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separated and the aqueous layer was bacle-extracted with dichloromethane. The
combined dichloromethane extracts were dried (MgSO4) and evaporated under
reduced pressure to give the title acid (11) (70 mg, 72%) as an off white
solid:
8I~ (360 MHz; CDC13) 1.00 and 1.03 (2xs, 9H, rotamers), 2.17-2.39 (m, 3H),
2.62-2.71 (m, 1H), 3.28-3.34 (m. 1H), 3.47-3.56 (m, 1H), 3.76 (m, 3H), 3.96-
4.13
(m, 1H), 4.21-4.26 (m, 2H), 4.36-4:58 (m, 3H), 5.93 (d, 2H), 6.62-6.90 (m, 6H)
and
7.21-7.25 (m, 1H); LRMS (from LC-MS) (ES+) nz/z 483 [(M+H)+] (100).
tent-Butyl 4-({(2S,4R)-1-(1,3-benzodioxol-5-ylmethyl)-4-[(3,3-
dimethylbutanoyl)-3-methoxyanilino]tetrahydro-1H 2-pyrrolyl}carbonyl)-1-
piperazinecarboxylate (12)
A mixture of (2S,4R)-1-(1,3-benzodioxol-5-ylmethyl)-4-[(3,3-
dimethylbutanoyl)(3-methoxybenzyl)amino]tetrahydro-1H 2-pyrrolecarboxylic acid
(11) (60 mg, 0.12 mmol), O-benzotriazol-1-yl-N,N,N;N'-tetramethyluronium
tetrafluoroborate (48 mg, 0.15 mmol) and N,N diisopropylethylamine (54 ~,L,
0.31
m~nol) in dimethylformamide (1 mL) was stirred at room temperature for 1.5 h.
The
mixture was diluted with water and extracted with ethyl acetate. The aqueous
phase
was back-extracted with ethyl acetate and the combined extracts were dried
(MgS04) and evaporated to dryness under reduced pressure. The residue was
partially purified by silica gel colurml chromatography eluting with 100%
dichloromethane, dichloromethane/ethyl acetate (4:1, v/v) and 100% ethyl
acetate to
give the crude product, contaminated with N,N dimethylformamide.
Dichloromethane was added and the resulting solution was washed with water.
The
aqueous layer was baclc extracted with dichloromethane and the combined
organic
extracts were dried (MgS04) and evaporated under reduced pressure to give the
title
piperazine (12) (33.1 mg, 41%):
LRMS (from LC-MS) (ES+) ~z/z 651 [(M+H)+] (100).
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Nl-[(3R,5S~-1-(1,3-benzodioxol-5-ylmethyl)-5-(piperazinocarbonyl)tetrahydro-
1H 3-pyrroliumyl] Nl-(3-methoxybenzyl)-3,3-dimethylbutanamide 2,2,2-
trifluoroacetate (13a).
A solution of tart-butyl 4-({(2S,4R)-1-(1,3-benzodioxol-5-ylmethyl)-4-[(3,3-
dimethylbutanoyl)-3-methoxyanilino]tetrahydro-1H 2-pyrrolyl J carbonyl)-1-
piperazinecarboxylate (12) (24 mg, 36.9 ~.mol) in dichloromethane (0.8 mL) was
treated with trifluoroacetic acid (0.1 mL, 1.3 mmol). The mixture was stirred
at room
temperature and the course of the reaction was monitored by TLC analysis. Once
completed, the solvent was evaporated under reduced pressure to give the title
trifluoroacetate salt (13a) in quantitative yield. This salt was used in the
following
experiment without further purification:
LRMS (from LC-MS) (ES+) Jz~/z 551 [(M+H)+] (100).
Nl-[(3R,5,S~-1-(1,3-benzodioxol-5-ylmethyl)-5-(piperazinocarbonyl)tetrahydro-
1H 3-pyrrolyl] Nl-(4-methoxybenzyl)-3,3-dimethylbutanamide (13b)
A biphasic mixture of dichloromethane (0.8 mL) and water (0.8 mL)
containing 26 mg of crude Nl-[(3R,SS)-1-(1,3-benzodioxol-5-ylmethyl)-5
(piperazinocarbonyl)-tetrahydro-1H 3-pyrroliumyl]-Nl-(3-methoxybenzyl)-3,3
dimethylbutanamide 2,2,2-trifluoroacetate (13a) was vigorously stirred and
treated
dropwise with 2.0 M aqueous sodium hydroxide solution until the pH value of
the
aqueous phase was adjusted to 12. The layers were separated and the aqueous
layer
was extracted with dichloromethane (2x1 mL). The organic extracts were
combined,
dried (MgS04) and evaporated under reduced pressure to give the title
piperazine
(13b) (12.7 mg, 59%):
LRMS (from LC-MS) (ES+) ~z/z 551 [(M+H)+] (100).
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Nl-[(3S,5S)-1-(1,3-benzodioxol-5-ylmethyl)-5-(piperazinocarbonyl)tetrahydro-
1H 3-pyrrolyl] Nl-(4-methoxybenzyl)-3,3-dimethylbutanamide. "Cis-
aminopr oline"
1-(tent-Butyl) 2-methyl (2S,4S)-4-ammoniotetrahydro-1H 1,2-
pyrroledicarboxylate chloride (16)
HO HO HO Ms0
'I~OH ~ '~~~OMe ---~ 'I~ N OMe ---~ '~ N OMe
(1) 0 2 0 boc 0 boc 0
()
OMe (3) (14)
1
HN~ HZN .HCI N
home ~ N OMe ~ N OMe
~I
boc O boc 0 hoc O
(18) (1~) (1g)
(15)
w
(19a) TFA Salt
(19b) Free Base Cis Amino Proline
(23a) TFA Salt
(23b) Free Base
A suspension of palladium on carbon (10%, 0.25 g) and 1-(tee°t-
butyl)-2-
methyl (ZS, 4S)-4-azidotetrahydro-1H 1,2-pyrroledicarboxylate (15) (1.00 g,
3.7
mmol) in a degassed solution of 2% v/v hydrochloric acid in ethanol (10 mL)
was
vigorously stirred at room temperature under an atmosphere of hydrogen (1
atin).
After stirring overnight, the mixture was filtered tluough a pad of CELITE"
and
washed thoroughly with ethanol. The filtrate was evaporated umder reduced
pressure
and the residue was triturated with test-butyl methyl ether at 0 °C.
The resulting
slurry was filtered, washed with ice-cold tee°t-butyl methyl ether and
dried under
vacuum to give the title hydrochloride salt (I6) (0.74 g, 7I%) as a white
solid:
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~H (360 MHz; Da0) 1.21 and 1.26 (2xs, 9H, rotaaners), 1.85-2.03 (m, 1H),
2.52-2.65 (m, 1H), 3.29-3.48 (m, 1H), 3.58-3.83 9(m, SH) and 4.14-4.34 (m,
1H);
LRMS (from LC-MS) (ES+) m/z 189 (100).
1-(tent-Sutyl) 2-methyl (2S,4S)-4-[3-methoxybenzyl)amino]tetrahydro-1H 1,2-
pyrroledicarboxylate (17)
A solution of 1-(test-butyl) 2-methyl (2S, 4S)-4-ammoniotetrahydro-1H 1,2-
pyrroledicarboxylate chloride (16) (3.00 g, 10.70 mmol) and 3-
methoxybenzaldehyde (1.30 mL, 10.7 mmol) in trimethyl orthoformate (8 mL) was
stirred for 45 min at room temperature. Sodium triacetoxyborohydride (2.26 g,
10.70
mmol) was added to the solution in small portions over 30 rains and the course
of
the reaction was monitored by TLC analysis. Once completed (about 30 min), the
reaction was quenched with saturated aqueous sodium hydrogencaxbonate solution
(15 mL) and extracted with ethyl acetate (15 mL). The organic extract was
washed
with saturated aqueous sodium hydrogencarbonate solution (2x15 mL), dried
(MgSO~) and evaporated under reduced pressure. The residue was purified by
flash
colurm chromatography on silica gel using 100% dichloromethane and then 100%
ethyl acetate as eluents to give the title amine (17) (2.48 g, 64%) as a
yellow oil:
1-(tart-Butyl) 2-Methyl (2S,4S)-4-[(3,3-dimethylbutanoyl)(3-
methoxybenzyl)amino]tetrahydro-1H 2-pyrrolecarboxylate (18)
A stirred solution of 1-(test-butyl) 2-methyl (2S,4S)-4-[3-methoxybenzyl)-
amino]tetrahydro-1H 1,2-pyrroledicarboxylate (17) (1.37 g, 3.76 mmol) and
triethylamine (0.63 mL, 4.52 mmol) in anhydrous dichloromethane (14 mL) was
treated dropwise with tey~t-butylacetyl chloride (0.53 mL, 3.82 mmol). After
stirring
overnight at room temperature, the mixture was diluted with dichloromethane
(50
mL) and washed with 1.0 M aqueous citric acid solution (2x50 mL). The layers
were
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separated and the aqueous layers were baclc-extracted with dichloromethane (25
mL). The combined organic layers were washed with saturated aqueous sodium
hydrogencarbonate solution, dried (MgS04) and evaporated under reduced
pressure.
The residue was purified by silica gel column chromatography (hexane/ethyl
acetate,
2:1, v/v) to give the title amide (18) (1.5 g, 86 %) as a pale yellow oil:
8H (360 MHz; CDCl3) 1.00 and 1.06 (2xs, 9H, rotamers), 1.38 and 1.42 (2xs, 9H,
rotamers), 1.81-1.93 (m, 1H), 2.15 (s, 2H) 2.30-2.51 (m, 1H), 3.18-3.25 (m,
1H),
3.62-3.85 [m, 4H, f containing at 3.69 (s, 3H)}], 3.78 (m, 3H), 4.15-4.25 (m,
1H),
4.45-4.61 (rim, 2H), 5.10-5.23 (m, 1H), 6.64-6.82 (m, 3H) and 7.13-7.31 (m,
1H);
LRMS (from LC-MS) (ES+) m/z 363 (100).
(2S, 4S)-4-[(3,3-dimethylbutanoyl)(3-methoxyanilino)]-2-
(methoxycarbonyl)tetrahydro-1H 2-pyrrolium 2,2,2-trifluoroacetate (19a)
1-(tent-Butyl) 2-methyl (2S,4S)-4-[(3,3-dimethylbutanoyl)(3
methoxybenzyl)amino]-tetrahydro-1H 2-pyrrolecarboxylate (18) (524 mg, 1.13
rmnol) was added to a 21% v/v solution of trifluoroacetic acid in
dichloromethane
(6.6 mL) at room temperature. The mixture was stirred for 50 min and then
evaporated to dryness under reduced pressure to give 0.98 g of a mixture of
the title
pyrrolium salt (19a) and trifluoroacetic acid:
8H (360 MHz; CDC13) 1.08 (s, 9H), 2.34-2.42 (m, 1H), 2.47 (s, 3H), 2.63-
2.72 (m, 1H), 3.61- 3.71 (m, 2H), 3.82 (s, 3H), 3.83 (s, 3H), 4.07-4.14 (m,
1H), 4.43-
4.54 (m, 1H), 4.57-4.67 (m, 2H), 6.68-6.74 (m, 2H), 6.90-6.93 (dd, 1H) and
7.34 (t,
1 H).
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Methyl (2S,4S)-1-(1,3-benzodioxol-5-ylmethyl)-4-[(3,3-dimethylbutanoyl)(3-
methoxybenzyl)amino]tetrahydro-1H 2-pyrrolecarboxylate (20)
1-(tet°t-Butyl) 2-methyl (2S,4S)-4-[(3,3-dimethylbutanoyl)(3-
methoxybenzyl)amino]-tetrahydro-1H 2-pymolecarboxylate (18) (138 mg, 0.38
mmol) was added to a solution of 30% v/v trifluoroacetic acid in
dichloromethane (3
mL) at room temperature. The mixture was stirred for 30 min and evaporated to
dryness under reduced pressure. The residue was partitioned between
dichloromethane and saturated aqueous potassium carbonate and shalcen
vigorously
for 5 mins. The organic layer was separated, dried (MgS04) and evaporated
under
reduced pressure to give 140 mg of crude methyl (2S,4R)-4-[(3,3-
dimethylbutanoyl)-
3-methoxyanilino]tetrahydro-1H 2-pyrrolecarboxylate (19b), which was used in
the
following reaction without further purification.
A solution of methyl (2S,4S)-4-[(3,3-dimethylbutanoyl)(3
methoxybenzyl)amino]-tetrahydro-1H 2-pyrrolecarboxylate (19b) (138 mg, 0.38
mmol), piperonal (58 mg, 0.39 mmol) and glacial acetic acid (225 ~L, 3.93
mmol) in
tetrahydrofuran (2.8 mL) was stirred for 30 min at room temperature. 95%
Sodium
cyanoborohydride (125 mg, 1.88 mmol) was added in small portions and stirring
was
continued for 45 min at the same temperature. After dilution with ethyl
acetate (5
mL), the reaction mixture was washed with saturated aqueous sodium
hydrogencarbonate solution (2x5 mL) and brine (5 mL), dried (MgS04) and
evaporated under reduced pressure. The residue was purified by silica gel
column
chromatography eluting with 100% dichloromethane and dichloromethane-
ethylacetate (4:1, v/v) to give the title pyrrole (20):
~H (360 MHz; CDC13) 0.89 and 0.99 (2xs, 9H, rotamers), 1.65-1.79 (m, 1H,),
1.87-2.11 [m, 3H, {contaiung at 1.94 (s, 2H)}], 2.21-2.66 (m. 2H), 3.04-3.15
(m,
2H), 3.56 (s, 3H), 3.67-3.74 [m, 4H, {containing at 3.67 (s, 3H); ], 4.43-4.64
(m,
2H), 4.74-4.92 (m, 1H), 5.10-5.14 (m, 1H), 5.74-5.88 (m, 2H), 6.49-6.78 (m,
6H)
and 7.02-7.10 (m, 1H); LRMS (from LG-MS) (ES+) m/z 497 [(M+H)+] (100).
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(2S,4S)-1-(1,3-benzodioxol-5-ylmethyl)-4-[(3,3-dimethylbutanoyl)(3-
methoxybenzyl)amino]tetrahydro-1H 2-pyrrolecarboxylic acid (21)
Lithium hydroxide monohydrate (17 mg, 0.405 mmol) was added to a
solution of methyl (2S,4S)-1-(1,3-benzodioxol-5-ylmethyl)-4-[(3,3-
dimethylbutanoyl)(3-methoxybenzyl)amino]tetrahydro-1H 2-pyTOlecarboxylate (20)
(100 mg, 0.20 mmol) in 66 % v/v methanol in water (1.0 mL). The mixture was
stirred overnight at room temperature, then the solvent was removed under
reduced
pressure and the residue partitioned between dichloromethane (1.0 mL) and
water
(1.0 ml). The aqueous phase was acidified with 1.0 M aqueous citric acid
solution
and the two layers were vigorously stirred for 10 min at room temperature. The
layers were separated and the aqueous layer was back-extracted with
dichloromethane. The combined dichloromethane extracts were dried (MgS04) and
evaporated under reduced pressure. The residue was purified by flash column
chromatography on silica gel using dichloromethane/ethyl acetate (1:1, v/v)
and then
dichloromethane/methanol (9:1, v/v) as eluents to give the title acid (21) (88
mg,
91 %) as an off white solid:
8H (360 MHz; CDC13) 0.95 (s, 9H), 2.18 [m, 3H, f containing at 2.18 (s,
2H)}], 2.62-2.87 (m, 1H), 3.17-3.28 (m, 1H), 3.42-3.47 (m, 1H), 3.73 (m, 3H),
3.82-
3.93 (m, 1H), 3.94-4.60 (m, 1H), 4.41-4.65 (m, 4H), 5.90-5.94 (m, 2H), 6.63-
6.93
(m, 6H) and 7.21-7.25 (m, 1H); LRMS (from LC-MS) (ES+) m/z 483 [(M+H)+]
(100).
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tart-Butyl 4-(~(2S,4S)-1-(1,3-benzodioxol-5-ylmethyl)-4-[(3,3-
dimethylbutanoyl)-
3-methoxyanilino]tetrahydro-1H 2-pyrrolyl)carbonyl)-1-piperazinecarboxylate
(22)
A mixture of (2S,4S)-1-(1,3-benzodioxol-5-ylmethyl)-4-[(3,3-
dimethylbutanoyl)(3-
methoxybenzyl)amino]tetrahydro-1H 2-pyrrolecarboxylic acid (21) (96.5 mg, 0.20
mmol), O-benzotriazol-1-yl-N,N,N;N'-tetramethyluronium tetrafluoroborate (77
mg,
- 0.24 mmol) and N,N diisopropylethylamine (87 ~.L, 0.50 mmol) in
dimethylformamide (1 mL) was stirred for 1.5 h at room temperature. The
mixture
was diluted with water and extracted with ethyl acetate. The aqueous phase was
back-extracted with ethyl acetate and the combined organic extracts were dried
(MgS04) and evaporated to dryness under reduced pressure. The residue was
purified by silica gel column chromatography using hexane-ethyl acetate (1:l,
v/v)
and then 100% ethyl acetate as eluents to give the title piperazine (22) (89
mg, 68%):
8H (360 MHz; CDC13) 0.95 and 0.97 (2xs, 9H, rotamers), 1.46 (s, 9H), 1.74
(s, 2H), 2.01-2.24 and 2.38-2.44 (2xm, 2H, rotamers), 2.55-2.59 and 2.70-2.81
(2xm,
2H, rotamers), 3.09-3.58 (m, 9H), 3.74-3.85 [m, 4H, containing at 3.76 and
3.79 (2
x s, 3H, rotamers))J, 3.94-4.05 and 4.06-4.19 (2xm, 1H, rotamers), 4.24-4.41
and
4.61-4.69 (2xm, 2H, rotamers), 4.86-4.96 and 5.11-5.21 (2xm, 1H, rotamers),
5.89-
6.01 (m, 2H, rotamers), 6.58-6.98 (m, 6H), and 7.13-7.18 and 7.25-7.27 (2xm,
1H,
rotamers); LRMS (from LC-MS) (ES+) m/z 651 [(M+H)+J (100).
Nl-[(3S;SSA-1-(1,3-benzodioxol-5-ylmethyl)-5-(piperazinocarbonyl)tetrahydro-
1H 3-pyrroliumyl] Nl-(3-methoxybenzyl)-3,3-dimethylbutanamide 2,2,2-
trifluoroacetate (23a)
A solution of test-butyl 4-({(2S,4S)-1-(1,3-benzodioxol-5-yhnethyl)-4-[(3,3-
dimethylbutanoyl)-3-methoxyanilino]tetrahydro-1H 2-pyrrolyl J carbonyl)-1-
piperazinecarboxylate (22) (21.7 mg, 33.3 ~.mol) in dichloromethane (0.5 mL)
was
treated with a 95 % v/v solution of trifluoroacetic acid in dichloromethane
(0.1 mL,
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1.2 mmol). The mixture was stirred at room temperature and the course of the
reaction was monitored by TLC analysis. Once completed (1 h), the solvent was
evaporated udder reduced pressure to give 22.8 mg of a mixture of the title
trifluoroacetate salt (23a), ethyl acetate and trifluoroacetic acid. This salt
was used in
the following experiment without further purification:
SH (360 MHz; CDC13) 0.98 (s, 9H), 2.08-2.18 (m, 1H), 2.32 (d, 1H), 2.43 (d,
1H), 2.73-2.82 (m, 1H), 3.30-3.73 (m, 8H), 3.77 (s, 3H), 3.90-3.96 (m, 1H),
4.06-
4.19 (m, 1H), 4.36-4.46 (m, 2H), 4.57 (d, 1H), 4.65-4.73 (m, 1H), 5.57-6.01
(m, 2H),
6.61-6.66 (m, 2H), 6.76-6.91 (m, 4H), and 7.29 (t, 1H); LRMS (fiom LC-MS)
(ES+)
r~2/z 551 [(M+H)+] (100).
N 1-[(3S,5.S~-1-(1,3-benzodioxol-5-ylmethyl)-5-(piperazinocarbonyl)tetrahydro-
1H 3-pyrrolyl] Nl-(4-methoxybenzyl)-3,3-dimethylbutanamide (23b)
A biphasic mixture of dichloromethane (0.5 mL) and water (0.5 mL)
containing 22.8 mg of crude N 1-[(3S,SSA-1-(1,3-benzodioxol-5-ylmethyl)-5-
(piperazinocarbonyl)-tetrahydro-1H 3-pyrroliumyl]-N 1-(3-methoxybenzyl)-3,3-
dimethylbutanamide 2,2,2-trifluoroacetate (23a) was treated with 2.0 M aqueous
sodium hydroxide solution until the pH value of the aqueous layer was adjusted
to
12. The mixture was vigorously stirred for 5 min at room temperature and the
layers
were separated. The aqueous layer was extracted with dichloromethane (2x0.5
mL)
and the combined organic extracts were dried (MgS04) and evaporated under
reduced pressure to give the title piperazine (23b) (14.5 mg, 79%):
8H (360 MHz; CDC13) 0.97 and 1.09 (2xs, 9H, rotamers), 1.66-1.79 [m, 3H,
{containing at 1.79 (s, 2H)~], 2.03 (d, 1H), 2.13 (d, 1H), 2.32-2.47 (m, 1H),
2.54-
2.88 (m, SH), 3.05-3.12 (m, 1H), 3.29-3.67 (m, SH), 3.76 (s, 3H), 3.86 (d,
1H), 4.66
(d, 1H), 4.97 (d, 1H), 5.08-5.22 (m, 1H), 5.89-5.92 (m, 2H), 6.59-6.6.81 (m,
6H) and
7.09-7.18 (m, 1H); LRMS (from LC-MS) (ES+) ~rz/z 551 [(M+H)''-] (100).
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Variations of the protecting group scheme can increase the efficiency and
speed with wluch compounds of the subject invention may be prepared. The
schemes below, which can be readily executed by one of skill in the art based
on the
disclosure above together with known methods in the art, provide rapid,
efficient
routes to compounds which may inhibit hedgehog activity. As will be
understood,
the particular moieties, groups, and reactions (e.g., electrophilic or
reductive
alkylation of the amine) may be varied to produce a wide range of compounds
having a structure according to any of Formula I-VI, for example. See also
J.W.
Miclcelson, K.L. Belongs and E. J. Jacobsen, J. Org. Chem., 1995, 60, 4177-
4183.
Scheme 1
HZN
1. protec
OMe
~ 2. deprot
N' 11 3, alkylal
1,0
Boc
~NH
1, alkylation
2.isocyanate
3. deprotect
Scheme 2
128
I. saponify
2. couple
~ r~Pnrr,tP~r
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1. protect
Y
2. saponify
7. amidation
2. deprotection
N
1. alkylation O OMe
--.-
2. deprotectio ~1n
N
H
N
N
Bac
scheme 3
CN
HZN
1. alkylate
OMe N
~ 2. couple
N' 11 3. deprotect
11 0
O OMe
Boc
N
H
O
deprotect
i
129
1. allylate
2. saponify
3. couple
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Scheme 4
HzN
1. allcylate Aliyl-o
OMe -a
2. protect
N 3, deprotect
0
O
Boc
Solid phase route
Allyl-O
1, deprotect
2, couple
3. deprotect
The synthetic route used to carry out the production on this template is
described in Scheme 5.
130
1. allcylate
2. saponify
3. couple
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O
HO~ H
O fmoc' ~R1-~ o (N] ~-R1-~ N'R2
--R1-NHz "
B
A
fmoc fmoc
R3 R3
CapCr [H] ~-R1-~~N'R2 ~apD ~H] ~ ~-R1-~ N'R2
O/~/ J\ N J O
fmoc R4
R3
S
95% TFA R1_p~N'R2
E //~ J' JN
O R4
Scheme 5
Washing Protocols
Method 1: water (3x), acetone (2x), N,N dimethylformamide (3x), water
(2x), acetone (lx), N,N dimethylformamide (3x), water (2x), acetone (3x),
methanol
(3x), acetone (3x) and methanol (3x);
Method 2: dichloromethane, hexane, N,N dimethylformamide,
dichloromethane, hexane, dichloromethane and hexane;
Method 3: water, N,N dimethylformamide, water, 1.0 M aqueous sodium
hydroxide solution, water, N,N dimethylformamide, water, 1.0 M aqueous sodium
hydroxide solution, water, N,N dimethylformamide, dichloromethane, methanol,
dichloromethane and methanol
Method 4: N,N dimethylformamide, dichloromethane, N,N
dimethylformamide, dichloromethane, methanol, dichloromethane , methanol (2x)
and ether (2x).
Method 5: N,N dimethylformamide, dichloromethane, N,N
dimethylformamide, dichloromethane, methanol, dichloromethane and methanol
(2x).
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Resin swelling in solvents was based on a standard of 10 mL of solvent per
gram of
resin.
Step A: The p~eparatiosz of (Nit~ophen-4 =yloxycarboxy)behz-4-
yloxymethyl polystys~ene-(Wahg PNP carbonate polystys ene)
15
Hydroxybenz-4-yloxymethyl polystyrene (Wang resin)
Sodium methoxide (233 g, 4.31 mol) was added slowly to a stirred mixture of
chloromethyl polystyrene (2.4 lcg, 3.6 mol functionalised loading) and 4-
hydroxybenzyl alcohol (581 g, 4.68 mol) in N,N dimethylacetamide (10 L) under
nitrogen. After dilution with N,N dimethylacetamide (13 L), the mixture was
heated
at 50 °C for 5 h and then filtered via cannula through a P-ETFE mesh
(70 ~,m). The
crude product was washed extensively using the sequence listed in method l,
then
dried under vacuum at 60 °C to give 2630 g of the title resin.
(Nitrophen-4'-yloxycarboxy)benz-4-yloxymethyl polystyrene-(Wang PNP
carbonate polystyrene)
4-Methylmorpholine (660 mL, 6.0 mol) was added dropwise over 2 h to a
stirred mixture of hydroxybenz-4-yloxymethyl polystyrene (2000 g, 2.5 mol
functionalised loading) and 4-nitrophenol chloroformate (1209 g, 6.0 mol) in
dichloromethane (22 L) at 0 °C under nitrogen. The mixture was warmed
gradually
to room temperature, stirred overnight and filtered via cannula through a P-
ETFE
mesh (70 ~,m). The crude resin was washed extensively using the sequence
listed in
method 2, then dried underwacuum at room temperature to give 2728 g of a
mixture
of the title resin and 4-methylmorpholine hydrochloride.
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Step B: The prepafatioh of T~ahg resin-bound diamines
~ General Method (for ~berazine, homo~perazine a~.id ty°aj~s-1,4-
diaminocyclohexane):
Crude (nitrophen-4'-yloxycarboxy)benz-4-yloxymethyl polystyrene (1002.5
g, ~0.9 mol functionalised loading) was swollen over 15 min in a mixture of
anhydrous dichloromethane and N,N dimethylformamide (1:l, v/v, 9 L) under
nitrogen. N,N diisopropylamine (626 mL, 5 mol equivalents) and the appropriate
diamine (5 mol equivalents) were added and the mixture was stirred vigorously
overnight at room temperature. The mixture was filtered through a P-ETFE mesh
(70
Vim), washed extensively using the sequence listed in method 3 and dried under
vacuum at 60 °C to give the resin-bound diamine.
~ Ethylenediamine bound to Wan resin
Crude (nitrophen-4'-yloxycarboxy)benz-4-yloxymethyl polystyrene (1002.5 g,
~0.9 mol functionalised loading) was swollen over 15 min in dichloromethane (7
L)
under nitrogen and treated with ethylenediamine (181 ,mL, 2.7 mol). The
resulting
thick, yellow suspension was diluted with dichloromethane (2 L) and vigorously
stirred overnight at room temperature. The mixture was filtered tluough a P-
ETFE
mesh (70 Vim), washed extensively using the sequence listed in method 3 and
dried
under vacuum at 60 °C to give the title resin-bound diamine.
~ m-~ylylenediamine bound to Wang resin
Crude (nitrophen-4'-yloxycarboxy)benz-4-yloxymethyl polystyrene (1002.5 g,
~0.9 mol functionalised loading) was swollen in tetrahydroftvran (7 L) over 15
min
under nitrogen and treated with a solution of m-xylylenediamine (828 mL, 6.27
mol)
in tetrahydrofuran (1 L). The resulting thick, yellow suspension was diluted
with
dichloromethane (2 L) and vigorously stirred overnight at room temperature.
The
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mixture was filtered through a P-ETFE mesh (70 ~.m), washed extensively using
the
sequence listed in method 3 and dried under vacuum at 60 °C to give the
title resin-
bound diamine.
Step C: Building Block loadi~zg onto Waug DiamiiZe:
The appropriate resin was swollen in N,N dimethylformamide over 15 min,
then gently agitated and treated with 1-[9H 9-fluorenylmethoxycarbonyl]-4-oxo-
2(S)-pyrrolidinecarboxylic acid (2 equivalents). After 30 min, 1-
hydroxybenzotriazole hydrate (2 equivalents) and N,N°-
diisopropylcarbodiimide (2
equivalents) were added and the resin suspension was agitated gently overnight
at
room temperature. After filtration, the resin was washed extensively using the
sequence listed in method 4 and dried under vacuum at 40 °C.
Step D: Reductive Amir~atiou at C 4
The appropriate resin was swollen in a 50% v/v mixture of aWydrous
tetrahydrofuran and methanol over 15 min, gently agitated and treated with
glacial
acetic acid (10 equivalents). The appropriate amine (5 equivalents) and sodium
cyanoborohydride (5 equivalents) were added and the resin suspension was
agitated
gently overnight at room temperature. After filtration, the resin was washed
extensively using the sequence listed in method 4 and dried under vacuum at 40
°C.
Step E: Reductive Alkylatiosa os Capping
~ Reductive All~lation
The appropriate resin was swollen in anhydrous N,N dimethylformamide,
then gently agitated and treated with glacial acetic acid (10 equivalents).
The
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appropriate aldehyde (5 equivalents) and sodium triacetoxyborohydride (5
equivalents) were added and the resin suspension was agitated cautiously for 1
h at
room temperature. The pressure that developed in the reaction vessel over this
period
was then released and gentle agitation of the suspension was continued
overnight at
room temperature. The resin was then filtered, washed extensively using the
sequence listed in method 4 and dried under vacuum at 40 °C.
~ Acid Chlorides Capping
The appropriate acid chloride (5 equivalents) and N,N diisopropylethylamine
(10 equivalents) were added to a gently agitated suspension of the appropriate
resin
in a 50% v/v mixture of anhydrous tetrahydrofuran and chloroform. After gentle
agitation at room temperature overnight, the resin was filtered, washed
extensively
using the sequence listed in method 4 and dried under vacuum at 40 °C.
Step F.' N Fmoc deprotectiofz
Resin analogues were suspended in a 20% v/v solution of piperidine in N,N
dimethylformamide and gently agitated for 30 min at room temperature. The
resin
suspension was subsequently filtered and washed with N,N dimethylformamide.
This treatment of the resin with piperidine in N,N dimethylformamide was
repeated
once more to ensure complete N Fmoc-deprotection. After standing for 30 min,
the
resin was filtered, washed using the sequence listed in method 4 and dried
under
vacuum at 40 °C.
Step G:Reductive Alkylatiou or Cappi~zg at 1V1
~ Reductive Alkylation
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' The appropriate resin (~60 mg per well in a 2 ml filter bloclc) was swollen
in
anhydrous N,N dimethylformamide (1 mL), then gently agitated and treated with
glacial , acetic acid (~50 ~,L, 10 equivalents), The appropriate aldehyde (5
equivalents) and sodium triacetoxyborohydride (~85 mg, 5 equivalents) were
added
and the filter blocks were then gently agitated at room temperature overnight.
Each
resin was subsequently filtered and washed using the sequence listed in method
5.
~ Acid Chlorides Capping
The appropriate resin (~60mg per well in a 2 ml filter block) was swollen in
a 50% vlv mixture of anhydrous tetrahydrofuran and chloroform (1 mL), then
gently
agitated and treated with the appropriate acid chloride (5 equivalents) and
N,N
diisopropylethylamine (~150~.1, 10 equivalents). After gentle agitation of the
filter
blocks overnight at room temperature, the resins were filtered and washed
using the
sequence listed in method 5.
Step H: Cleavage of Final P~ocluct fi om YYafZg tesiu using TFA:
The appropriate resin was swollen in DCM and the final product cleaved by
addition of 95% v/v TFA in dichloromethane. Four separate aliquots of TFA (2 x
300 ~,L, 75 ~L, and 500 ~.L) were added and the filtrates obtained from these
were
collected in plates containing 96 wells. Filtrates obtained from addition of
aliquots
1,2 and 4 were collected using the same 96 well plate. The filtrate obtained
after
addition of aliquot 3 (75 ~,L) was collected separately using an analytical 96
well
plate. All fractions were subsequently evaporated under reduced pressure using
a
Genevac apparatus to give the final product.
Biological Assays
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Lead Compound Discover~yl High-thr~oughput Screening Assay
Compounds to be tested are dissolved in DMSO to a concentration of IO
mM, and stored at -20 °C. To activate the Hedgehog pathway in the assay
cells, an
octylated (lipid-modified) form of the N-terminal fragment of the So>zic
Hedgehog
protein (OCT-SHH) is used. This N-terminal SHH fragment is produced
bacterially.
Compounds may be tested in the "Gli-Luc" assay below, using the cell line
lOT(sl2), wherein the cells contain a Hedgehog-responsive reporter construct
utilizing Luciferase as the reporter gene. In this way, Hedgehog pathway
signaling
activity can be measured via the Gli-Luc response.
lOtl/2(s12) cells are plated in a 96-well micro-titer plate (MTP) at 20,000
cells/well in full medium [DMEM with 10% FBS]. Then plates are placed in the
incubator for incubation overnight (0/N), at 37 °C and 5% CO2. After 24
h, the
medium is replaced with Luciferase-assay medium (DMEM with 0.5% FBS).
Compounds are thawed and diluted in assay medium at 3:1000 (about 300-fold)
resulting in a starting concentration of about 30 p,M.
Subsequently, 150 ~.l of each 30 ~.M sample is added to the first wells (in
triplicate). The MTP samples are then diluted at 3-fold dilutions to a total
of seven
wells, ultimately resulting in a regiment of seven dilutions in triplicate,
for each
compound. Next, the protein ligand OCT-SHH is diluted in Luciferase-assay
medium and added to each well at a final concentration of 0.3 ~.g/ml. Plates
are then
returned to the incubator for further incubation O/N, at 37 °C and 5%
CO2. After
about 24 h, plates are removed from the incubator and the medium is
aspirated/discarded. Wells are washed once with assay buffer [PBS + 1 mM Mg2+
and 1 mM Caa+]. Then 50 ~,l of assay buffer is added to each well. The
Luciferase
assay reagent is prepared as described by the vendor (LucLite lcit from
Pacl~ard), and
50 ~.l is added to each well. Plates are incubated at room temperature (RT)
for about
minutes after which the signals aa-e read, again at RT, on a Topcount
(Paclcard).
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Compounds identified in this assay are depicted in Figure 32. Testing of
individual diastereomers of the depicted compowds in the above assay has
demonstrated that cis isomers tend to exhibit greater activity, sometimes by
more
than 100-fold, than their tans isomer counterparts. Furthermore, ammonium salt
derivatives, such as TFA salts, of the subject compounds have been shown to
show
similar or greater activity in the above assay.
Activities of particular compounds are presented 'below in Table 1:
Table 1
Compound ICSO (~.M) Compound ICSO (wM)
A <1 S <1
C <1 D <1
E <0.1 F <1
G <1 H <0.1
I <10 J <0.1
K <0.1 L <10
M <1 N <1
O <O.1 P <0.1
Q <1 R <1
S <1 T <1
U <1 V <1
W <1 X <10
Y <10 Z <1
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A' <10 B' <10
C' <10 D' <10
E' <10 F' <10
G' <10 H' <10
I' <10 J' <10
K' <10 L' <10
M' <0.1 N' <1
O' <0.1 P' <10
Q' <10 R' <1
S' <10 T' <1
U' <1 V' <1
W' <1 X' <0.1
Y' <1 Z' <1
A" <1 B" <1
C" <1 D" <10
E" <10 F" <10
G" <1 H" <10
I" <0.1 J" <0.1
K" <1 L" <1
M" <1 N" <1
O" <1 P" <10
Q" <10 R" <1
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S" <1 T" <1
U" <10 V" <1
W" <10 X" <10
Y" <10 Z" <1
A"' <1 B"' <1
C"' <10 D"' <10
<1 O F"' <1
G"' <10 H"' <10
I"' <10 J"' <10
K"' <10 L"' <10
Ptc-yzull Assay
Methods
Ptc-null cells were cultured for 3 days in the presence of vehicle; jervine, a
known Patched pathway antagonist (i) used here as a positive control; or 1 ~,M
of
compound D. Total ribonucleic acid (RNA) was isolated from the cells and used
for
reverse transcriptase-polymerase chain reaction (RT-PCR). Specific primers for
the
detection of mouse gli-1 mRNA were used in the PCR, and the actin gene was
used
to demonstrate that equivalent amounts of mRNA samples were compared in the
experiment. The gli-l and actin mRNA samples were then loaded on 1.5% agarose
gel and were detected by staining with ethidium bromide. The same samples were
analyzed by the quantitative real-time polymerase chain reaction method to
quantify
the levels of gli-1 mRNA.
Results
Figure 33A shows the results of a representative experiment. It shows gli-1
mRNA expression in cells treated with a vehicle control (Lane 1); 5 ~,M
jervine, the
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positive control compound (Lane 2); and 1 ~,M D (Lane 3). Compared with
vehicle,
D and jervine significantly decreased the expression of gli-1 mRNA inptc-null
cells.
The levels of actin mRNA were equivalent in all conditions, indicating that
equal
quantities of RNA were analyzed in the experiment. This qualitative result was
confirmed by the quantitative real-time PCR analysis (Figure 33B), which shows
that D and jervine downregulated the gli-1 mRNA levels.
Together, these experiments confirm that exposure ofptc-null cells to D for 3
days downregulates the Patched pathway, as demonstrated by the inhibition of
the
expression of gli-1 mRNA transcripts.
ErrZbr yor~ic Mouse Skin PurZCIZ Assay: Effect of Py~olohged Exposure to D
Methods
A novel cell culture assay was established to determine the effects of D on
activation of the Patched pathway in shin. In tlv.s system, activation of the
Patched
pathway results in increased expression of the ptc gene.
To monitor the activity of the Patched pathway in embryonic skin, we
cultured pieces of slcin from transgenic Patched pathway reporter mice. These
mice
were genetically engineered to harbor a foreign gene (lacZ). The lacZ gene
encodes a
bacterial beta-galactosidase. The gene was inserted in the ptc locus but
allowed for
normal ptc function. Ptc activation in response to Shh-induced Patched pathway
activation can then be monitored by the production of the ZacZ gene product,
beta-
galactosidase, which is detectable by the enzymatic conversion of the
substrate X-gal
into a blue-colored reaction product.
Day 17.5 embryonic skin was explanted as 2 mm circular punches from these
transgenic reporter mice and cultured for 5 to 7 days in the presence of Shh
protein
(Figure 34). Shh protein should upregulate the expression of the ptc gene,
hence
increase the amount of X-gal staining in these cultures. To test the effect of
D, slcin
punches were cultured for 6 days in the presence of both Shh protein and D
(Figure
35).
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Results
As expected, adding Shh protein to cultured slcin explants resulted in ptc
activation as indicated by the blue X-gal staining of these cultvares (Figure
34A - X-
gal).
Hematoxylin and eosin (H&E) staining of sectioned slcin punches revealed
intensely stained cells with basophilic nuclei and a high nucleus to cytoplasm
ratio
(Figure 34A - H&E [10x] and H&E [40x]). These structures resemble BCCs in that
they were arranged in clusters throughout the dermal layer and were separated
by
palisades of normal appearing dermal cells.
X-gal staining demonstrated that the Patched pathway was active in cells
within these BCC-like structures (Figure 34A-Eosin+X-gal). Consistent with
published results and similar to human BCCs, the BCC-lilce clusters in the
mouse
skin punch expressed lceratin-14, a marlcer of undifferentiated lceratinocytes
(Figure
34B).
Shin punches were cultured for 6 days in the presence of both Shh protein
and D to test the effect of D. Figure 35 demonstrates the dose-dependent
effect of D
on the level of Patched pathway activity in Shh-treated skin punches.
Increasing
concentrations of D (from 0.01 to 1 ~,M) led to a dose-dependent decrease in
the
amount of pathway activity, as monitored by the amount of lacZ reporter enzyme
activity (Figure 35A). Reporter enzyme staining of D treated explants
demonstrated
that 0.2 ~M D decreased X-gal staining compared with the intense X-gal
staining of
skin pmiches treated with Shh protein alone (Figure 35B). This indicates that
D
blocked the activation of the Patched pathway and downregulated the expression
of
the ptc gene.
The next experiment demonstrated that inhibiting the Patched pathway with
D would prevent the formation of BCC-lilce structures. Figure 35C shows that D
completely blocked the formation of BCC-like structures without affecting the
integrity of normal slcin cells. This confirms that D can prevent the
appearance of
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BCC-lilce structures produced by activating the Patched pathway, the same
pathway
that underlies the human disease.
Emb~yohic Mouse Skih Puuch Assay: Effect of Sho~~t-te~~z Pret~eat~raerzt with
D
Methods
Transgenic mouse-derived skin punches were treated with vehicle or D for 5
hours in the absence of Shh. After the pretreatment, the vehicle or D was
removed.
The skin punches were washed twice and then cultured in the presence of S1W
for
6 days. At the end of the experiment, the slcin punches were fixed and stained
with
X-gal to determine Patched pathway activity.
Results
Slcin punches treated for 6 days with exogenous Shh protein alone showed
intense X-gal staining (i.e., activation) compared with those treated with
vehicle
alone (Figure 36, top row). Slcin punches pretreated with D at 10, 20 and 50
~,M for
5 hours before being exposed to exogenous Shh protein demonstrated complete
inhibition of Shh protein-induced upregulation of the Patched pathway, as
indicated
by the absence of X-gal staining (Figure 36, bottom row-3 slides on the
right).
Intense X-gal staining indicative of upregulation of the Patched pathway was
seen in
slcin punches pretreated with vehicle before exposure to S1W protein (Figure
36, bottom row, left). The short period of pretreatment was essentially
equivalent to
6-day exposure to D in terms of the level of ptc inhibition (compare top and
bottom
rows in Figure 36).
This result suggests that D binds tightly to its target and that the
l~irietics of
dissociation are slow or irreversible. The data also suggest that D might have
the
capacity to prevent the development of BCC.
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Ef~ab~ yo~cic Mouse Skin Punch Assay: Lorzg-te~~nz Ti~eaty~zefzt of P~~e-
existifzg BCC-
Like Sty~uctm~es with D
Methods
Day 17.5 embryonic skin punches from transgenic Patched pathway reporter
mice were cultured in the presence of Shh protein for 7 days to allow for the
development of BCC-like structures. The Shh protein was removed at the end of
the
7 days. The cultures were then exposed to Shh protein plus either vehicle or D
for 3
days. The cultures were analyzed histologically after 10 days to assess the
formation
of BCC-lilce structures that are indicative of activation of the Patched
pathway.
Results
Histological analysis showed that D, at either 1 or 5 ~,M, significantly
reduced the size and number of Shh-induced BCC-like stuuctures in treated skin
punches, as compared with vehicle treated explants (Figure 37A). Thus, it
appears
that exposing existing BCC-lilce structures to D for 3 days induced the
regression of
these structures. Furthermore, D did not appear to have general cytotoxic
effects on
slcin cells, as determined by their normal histology.
One possible mechanism for D-induced regression of BGC-like structures
rnay be apoptosis of the activated cells. To investigate this possibility,
parallel
explants were exposed to 5 ~M D for 2 days and were then stained by the
terminal
deoxynucleotidyltransferase mediated d-UTP nick end-labeling (TUNEL) method,
which is used to detect apoptotic nuclei. After 2 days of exposure to 5 ~.M D,
the
number of apoptotic nuclei (indicated by the brown color in the slides on the
right)
within the BCC-like structures was signif candy higher than in the vehicle
control on
the left (Figure 37B). Taken together, these results suggest that D-induced
regression
of BCC-life structures results, at least in part, from stimulating the
cellular suicide
pathway of cells in which the Patched pathway is activated.
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Embf youic Mouse Slri~ Punch Assay: Short-teem Ti~eatmeht of P~~e-existing BCC-
like Sts~uctu~~es with D
Methods
Day 17.5 embryonic slcin punches fiom transgenic Patched pathway reporter
mice were cultured in the presence of Shh protein for 7 days. The Shh protein
was
removed at the end of the 7 days. The slcin punches were then exposed to
vehicle or
1 or 5 ~M D for 5 hours on days 7 and 9. After each expostue to vehicle or D,
the
vehicle or D was washed off and the skin punches were cultured again in the
presence of Shh protein. Cultures were analyzed by X-gal staining after 10
days in
vitro to assess the'activity of the Patched pathway.
Results
Short-term treatment with D reduced the amount of X-gal staining associated
with exposure to Shh protein (Figure 38A), suggesting a downregulation of
pathway
activity in skin explants. Histological analysis showed that even at a
concentration of
1 ~M, D induced the regression of X-gal-positive BCC-like structures (Figure
38B).
Quantification of the gli-1 mRNA levels in D-treated punches demonstrated that
short-term treatment with D completely downregulated gli-1 transcription
(Figure
38C, left side). This effect appeared to be specific to the Patched pathway
and not
due to general cytotoxicity, as shown by the relatively constant mRNA levels
of a
houselceeping enzyme, glyceraldehyde-3-phosphate dehydrogenase or GAPDH
(Figure 38C, right side).
These results demonstrate that under certain conditions of short-term
exposure, D
has the capacity to inhibit the activity of the Patched pathway in cultured
embryonic
skin explants. Furthermore, D at concentrations of both l and 5 ~.M caused the
regressiomof existing Shh-induced BCC-like structures.
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Adult BCC Mouse Skiyz Punch Assay
Methods
Ptc heterozygous transgenic mice were irradiated 3 times weekly for
6 months, during which time many small, and often microscopic, BCC tumors
developed. Four-mm diameter skin punch explants, presmnably containing BCC
structures, were cultured for 6 days in the presence of vehicle, the positive
control
(jervine), or 5 ~.M D. At the end of the experiment, the explants were
analyzed by X-
gal staining to detect the level of Patched pathway activity, by histology to
determine
the effect of treatment on the morphology of ultraviolet radiation-induced
BCCs, and
by quantifying the level of gli-I mRNA expression to characterize the extent
of
pathway inhibition.
Results
X-gal staining of the treated explants shows that slcin punches cultured in
the
presence of vehicle alone developed intensely stained blue foci indicative of
a focal
upregulation of the Patched pathway and BCC structures (blue spots in Figure
39A).
Compared with vehicle, 5 ~,M D, lilce the positive control, decreased the
number and
size of established BCC structures. Histological analysis of sectioned
explants
demonstrated that D induced the regression of ultraviolet radiation-induced
BCC
tumors, as compared with the vehicle control (Figure 39B). In skin punches
from
these heterozygous transgenic mice, the levels of gli-1 mRNA were high because
of
the activation ofPtc target genes. D at concentrations of 1 and 5 ~.M also
significantly inhibited the level of gli-1 mRNA levels compared with vehicle
alone.
Quantification of gli-1 mRNA levels shows the almost complete inhibition of
target
gene activation by D (Figure 39C). This inhibition did not appear to be caused
by
non-specific cytotoxicity, as statistical comparison of the levels of the
housekeeping
GAPDH enzyme between treated and vehicle conditions shows no significant
difference among groups in general cellular metabolic activity. Thus, these
results
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demonstrate that D inhibits the Patched pathway and induces the regression of
ultraviolet radiation-induced, BCNS-like, BCC tumors in cultured skin
explants.
These data confirm the results of previous experiments and suggest that D
might be effective in treating BCC.
Hmna~ BCC Explaut Culture
Methods
Specimens obtained from surgical procedures (such as Mohs surgery or
curettage) were cultured on fresh, living, day 17 embryonic mouse dermis from
which all epidermal cells have been removed by digestion using disease. Since
disease treatment digests basement membrane components, Matrigel, a
commercially available basement membrane preparation, was applied between the
dermis and BCC. Cultures were assembled on top of a plastic grid and incubated
for
3 days (with or without D at a concentration of 10 ~,M) in a medium suitable
for the
long-term culture of human slcin. After culture, the samples were processed
for
routine histology and subjected to quantitative in situ hybridization.
Briefly, 7~.m
sections of paraformaldehyde-fixed, paraffin-embedded tissue containing laxge
basal
cell islands were cleared, re-hydrated, digested with proteinase K, acetylated
and
hybridized with [33P]- labeled RNA probes overnight. After high stringency
post-
hybridization washes, slides were exposed to a PhosphorImager screen in the
dark at
room temperature for 4-7 days. After developing, the [33P]-signal was scanned
using
a Storm Scanner (Molecular Dynamics). Individual basal cell islands were
selected
and the signal quantified and expressed in average counts/pixel using
ImageQuant
1.0 software.
Results
The morphological features characteristic of BCCs, such as islands of
undifferentiated basal cells, and in some cases, palisading of peripheral
cells and
stromal clefting (Figure 40A) were maintained when BCCs were cultured in this
system. Likewise, the differentiation markers that were expressed are
identical in
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WO 02/30421 PCT/USO1/32054
pattern to those of the pre-culture controls, as determined by
immunohistochemical
staining (data not shown). The GLI 1 gene, a pivotal indicator of Patched
signaling,
remained active at high levels in untreated cultures, as determined from
sections
exposed to 33P-labeled RNA probes (Figure 40B). Quantitative in situ
hybridization
showed that the level of GLI 1 expression was greatly reduced in the D-treated
samples as compaxed to vehicle-treated controls (Figure 41).
P~epa~atioh ~compouuds of the present ihveution
a. Illustrative synthetic schemes
Exemplary synthesis schemes for generating hedgehog antagonists useful in
the methods and compositions of the present invention are shown in Figures 1-
31.
The reaction conditions in the illustrated schemes of Figure 1-31 are as
follows:
1) R1CH2CN, NaNH2, toluene
(Arzneim-Forsch, 1990, 40, 11, 1242)
2) H~S04, H20, reflux
(Arzneim-Forsch, 1990, 40, 1 l, 1242)
3) H2S04, EtOH, reflux
(Arzneim-Forsch, 1990, 40, 11, 1242)
4) NaOH, EtOH, reflux
5) (Boc)20, 2M NaOH, THF
6) LiHDMS, R1X, THF
(Merclc Patent Applic # WO 96/06609)
7) Pd-C, H2, MeOH
8) t-BuONO, CuBr, HBr, H20
(J. Org. Chem. 1977, 42, 2426)
9) ArB(OH)2, Pd(PPh3)4, Dioxane
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WO 02/30421 PCT/USO1/32054
(J. Med. Chem. 1996, 39, 217-223)
10) R12(H)C=CR13R14, Pd(OAc)2, Et3N, DMF
(Org. React. 1982, 27, 345)
11 ) Tf20, THF
(J. Am. Chem. Soc. 1987, 109, 5478-5486)
12) ArSnBu3, Pd(PPh3)4, Dioxane
(J. Am. Chem. Soc. 1987, 109, 5478-5486)
13) KMn04, Py, H20
(J. Med. Chem. 1996, 39, 217-223)
14) NaORl, THF
15) NaSRI, THF
16) HNR1R13, THF
17) HONO, NaBF4
(Adv. Fluorine Chem. 1965, 4, 1-3 0)
18) Pd(OAc)2, NaH, DPPF, PhCH3~ R10H
(J. Org. Chem. 1997, 62, 5413-5418)
19) i. R1X, Et3N, CH2Cl2, ii. R13X
20) SOCl2, cat DMF
21 ) CH2N2, Et20
22) Ag20, Na2C03, Na2S2O3, H20
(Tetrahedron Lett. 1979, '2667)
23) Ag02CPh, Et3N, MeOH
(Org. Syn., 1970, 50, 77; J. Am. Chem. Soc. 1987, 109, 5432)
24) LiOH, THF-MeOH
25) (Et0)2P(O)CH2C02R, BuLi, THF
26) Me02CCH(Br)=P(Ph)3, benzene
27) KOH or I~OtBu
28) Base, X(CH2)nC02R
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WO 02/30421 PCT/USO1/32054
29) DPPA, Et3N, toluene
(Synthesis 1985, 220)
30) HONO, H20
31) 502, CuCI, HCI, H20
(Synthesis 1969, 1-10, 6)
32) Lawesson's reagent, toluene
(Tetrahedron Asym. 1996, 7, 1.2, 3553)
33) R2M, solvent
34) 30% H2O2, glacial CH3C02H
(Helv. Clum. Acta. 1968, 349, 323)
35) triphosgene, CH2Cl2
(Tetrahedron Lett., 1996, 37, 8589)
36) i. (Et0)2P(O)CHLiS02Oi-Pr, THF, ii. NaI
37) Ph3PCH3I, NaCH2S(O)CH3, DMSO
(Synthesis 1987, 498)
38) Br2, CHCl3 or other solvent
(Synthesis 1987, 498)
3 9) BuLi, Bu3 SnCI
40) C1SO20TMS, CC14
CChem. Ber. 1995, 128, 575-580)
41) MeOH-HCI, reflux
42) LAH, Et20 or LiBH4, EtOH or BH3-THF
(Tetrahedron Lett., 1996, 37, 8589)
43) MsCI, Et3N, CH2Cl2
(Tetrahedron Lett., 1996, 37, 8589)
44) Na2S03, H20
(Tetrahedron Lett., 1996, 37, 8589)
45) R2R4NH, Et3N, CH2C12
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WO 02/30421 PCT/USO1/32054
46) R2M, solvent
47) CH3NH(OCH3), EDC, HOBt, DIEA, CH2Cl2 or DMF
(Tetrahedron Lett, 1981, 22, 3815)
48) MeLi, THF
49) mCPBA, CH2Cl2
50) HONG, Cu20, Cu(N03)2, H20
(J. Org. Chem. 1977, 42, 2053)
51) R1M, solvent
52) HONO, NaS(S)COEt, H20
(Org. Synth. 1947, 27, 81)
53) HSR2 or HSR4, CH2Cl2
54) i-BuOC(O)Cl, Et3N, NH3, THF
55) R2R4NH, CH2Cl2, NaBH(OAc)3
56) R2R4NH, MeOH/CH3C02H, NaBH3CN
57) R2OH, EDC, HOBt, DIEA, CH2Cl2 or DMF
58) R20H, HBTU, HOBt, DIEA, CH2Cl2 or DMF
59) R2R4NH, EDC, HOBt, DIEA, CH2C12 or DMF
60) R2R4NH, HBTU, HOBt, DIEA, CH2Cl2 or DMF
61) POCl3, Py, CH2Cl2
62) R2R4NC0, solvent
63) R2OC(O)Cl, Et3N, solvent
64) R2C02H, EDC or HBTU, HOBt, DIEA, CH2Cl2 or DMF
65) R2X, Et3N, solvent
66) (CH3S)2C-N(CN),°DMF, EtOH
(J. Med. Chem. 1994, 37, 57-66)
67) R2S02C1, Et3N, CH2Cl2
68) R2- or R3- or R4CH0, MeOH/CH3C02H, NaBH3CN
(Synthesis 1975, 135-146)
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69) Boc(Tr)-D or L-CysOH, HBTU, HOBt, DIEA, CH2Cl2 or DMF
70) Boc(Tr)-D or L-CysH, NaBH3CN, MeOH/CH3C02H
(Synthesis 1975, 135-146)
71) S-Tr-N-Boc cysteinal, C1CH2CH2Cl or THF, NaBH(OAc)3
(J. Org. Chem. 1996, 61, 3849-3862)
72) TFA, CH2Cl2, Et3SiH or (3:1:1) thioanisole/ethanedithiol/DMS
73) TFA, CH2Cl2
74) DPPA, Et3N, toluene, HOCH2CH2SiCH3
(Tetrahedron Lett. 1984, 25, 3515)
75) TBAF, THF
76) Base, TrSH or BnSH
77) Base, R2X or Rq.X
78) R3NH2, MeOH/CH3C02H, NaBH3CN
79) N2H4, KOH
80) Pd2(dba)3, P(o-tol)3, RNH2, NaOtBu, Dioxane, R1NH2
(Tetrahedron Lett. 1996, 37, 7181-7184).
81 ) Cyanamide.
82) Fmoc-Gl, sodium bicarbonate.
83) BnCOCI, sodium carbonate.
84) AllyIOCOCI, pyridine.
85) Benzyl bromide, base.
86) Oxalyl chloride, DMSO.
87) RCONH2.
88) Carbonyldiimidazole, neutral solvents (e.g., DCM, DMF, THF, toluene).
89) Thiocarbonyldiimidazole, neutral solvents (e.g., DCM, DMF, THF, toluene).
90) Cyanogen bromide, neutral solvents (e.g., DCM, DMF, THF, toluene).
91) RCOCI, Triethylamine
92) RNHNH2, EDC.
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93) R02CCOCl, Et3N, DCM.
94) MsOH, Pyridine (J. Het. Chem., 1980, 607.)
95) Base, neutral solvents (e.g., DCM, toluene, THF)
96) H2NOR, EDC.
S 97) RCSNH2.
98) RCOCHBrR, neutral solvents (e.g., DCM, DMF, THF, toluene), (Org. Proc.
Prep. Intl., 1992, 24, 127).
99) CH2N2, HCI. (Synthesis, 1993, 197).
100) NH2NHR, neutral solvents (e.g., DCM, DMF, THF, toluene).
101) RS02C1, DMAP. (Tetrahedron Lett., 1993, 34, 2749).
102) Et3N, RX. (J. Org. Chem., 1990, SS, 6037).
103) NOCI or Cl2 (J. Org. Chem., 1990, 55, 3916).
104) H2NOH, neutral solvents (e.g., DCM, DMF, THF, toluene).
105) RCCR, neutral solvents (DCM, THF, Toluene).
106) RCHCHR, neutral solvents (DCM, THF, Toluene).
107) H2NOH, HCI.
108) Thiocarbonyldiimidazole, Si02 or BF30Et2. (J. Med. Chem., 1996, 39,
5228).
109) Thiocarbonyldiimidazole, DBU or DBN. (J. Med. Chem., 1996, 39, 5228).
110) HNO2, HCI.
111) C1CH2CO2Et (Org. Reactions, 1959,10,143).
112) Morpholine enamine (Eur. J. Med. Chem., 1982, 17, 27).
113) RCOCHR'CN
114) RCOCHR'C02Et
115) Na2S03
116) H2NCHRC02Et
117) Et02CCHRNCO
118) RCNHNH2.
119) RCOC02H, (J. Med. Chem., 1995, 38, 3741).
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120) RCHO, KOAc.
121) 2-Fluoronitrobenzene.
122) SnCl2, EtOH, DMF.
123) RCHO, NaBH3CN, HOAc.
124) NH3, MeOH.
125) 2,4,6-Me3PhS02NH2.
126) Et2NH, CH2Cl2
127) MeOC(O)Cl, Et3N, CH2C12
12~) R2NH2, EDC, HOBT, Et3N, CH2C12
129) DBU, PhCH3
130) BocNHCH(CH2STr)CH2NH2, EDC, HOBT, Et3N, CH2C12
131) R2NHCH2CO2Me, HBTU, HOBT, Et3N, CH2C12
132) BocNHCH(CH2STr)CH20Ms, LiHMDS, THF
133) R2NHCH2C02Me, NaBH(OAc)3, C1CH2CH2Cl or THF
134) R2NHCH2CH(OEt)2, HBTU, HOBT, Et3N, CH2C12
135) NaBH(OAc)3, C1CH2CH2C1 or THF, AcOH.
136) Piperidine, DMF.
137) Pd(Ph3P)4, Bu3SnH.
138) RC02H, EDC, HOBT, Et3N, DCM.
139) RNH2, neutral solvents.
140) RCHO, NaBH3CN, HOAc.
141) RNCO, solvent.
142) RCO2H, EDC or HBTU, HOBt, DIEA, CH2C12 or DMF.
143) RCOCI, Triethylamine
144) RS02C1, Et3N, CH2C12.
145) SnCl2, EtOH, DMF.
146) RNH2, EDC, HOBt, DIEA, CH2C12 or DMF.
147) Dibromoethane, Et3N, CH2Cla
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148) Oxalyl chloride, neutral solvents.
149) LiOH, THF-MeOH.
150) Carbonyldiimidazole, neutral solvents (e.g., DCM, DMF, THF, toluene).
151 ) RNH2, Et3N, CH2C12.
152) Base, RX.
153) DBU, PhCH3
154) DPPA, Et3N, toluene (Synthesis 1985, 220)
155) SOC12, cat DMF.
156) ArH, Lewis Acid (A1C13, SnCl4, TiCl4), CH2Cl2.
157) H2NCHRC02Et, neutral solvents.
158) BocHNCHRC02H, EDC OR HBTU, HOBt, DIEA, CHZC12 or DMF.
159) TFA, CH2Cl2.
All of the, references cited above are hereby incorporated by reference
herein.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than routine experimentation, many equivalents to the specific embodiments of
the
invention described herein. Such equivalents are intended to be encompassed by
the
following claims.
155
