Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR IMPROVEMENT OF LUNG FUNCTION USING
TGF-[3 INHIBITORS
Background of the Invention
Field~of the Invention
The present invention concerns methods of treatment using transforming growth
factor /3
(TGF-(3) inlubitors. More specifically, the invention concerns methods of
improving lung
function by adrriinistering TGF-(3 inhibitors that inhibit biological
activities mediated by the type
I TGF-[3 receptor (TGF(3-Rl).
Description~of the Related Art
Transforming growth factor-beta (TGF-(3) denotes a family of proteins, TGF-
(31, TGF-(32,
and TGF-(33, which are pleiotropic modulators of cell growth and
differentiation, embryonic and
bone development; extracellular matrix formation, hematopoiesis, irmnune and
inflammatory
responses (Roberts and Sporn Handbook of Experimental Pharmacology (1990)
95:419-58;
Massague et al. Ann Rev Cell Biol (1990) 6:597-646). Other members of this
superfamily
include activin, inhibin, bone morphogenic,protein, and Mullerian inhibiting
substance. TGF-(3
initiates intracellular signaling pathways leading ultimately to the'
expression of genes that
regulate the cell cycle, control proliferative responses, or relate to
extracellular matrix proteins
that mediate outside-in cell signaling, cell adhesion, migration a~zd
intercellular communication.
TGF-(3 exerts its biological activities through a receptor system including
the type I and
type II single transmembrane TGF-[3 receptors (also referred to as receptor
subunits) with
intracellular serine-threonine kinase domains, that signal through the Smad
.family of
transcriptional regulators. Binding of TGF-(3 to the extracellular domain of
the type II receptor
induces phosphorylation and activation of the type I receptor (TGF(3-Rl) by
the type II receptor
(TGF(3-R2). The activated TGF(3-Rl phosphorylates a receptor-associated co-
transcription factor
Smad2/Smad3, thereby releasing it into the cytoplasm, where it binds to Smad4.
The Smad
complex translocates into the nucleus, associates with a DNA-binding cofactor,
such as Fast-1,
binds to enhancer regions of specific genes, and activates transcription. The
expression of these
genes leads to the synthesis of cell cycle regulators that control
proliferative responses or
extracellular matrix proteins that mediate outside-in cell signaling, cell
adhesion, migration, and
intracellular communication. Other signaling pathways Iike the MAP kinase-ERK
cascade are
also activated by TGF-(3 signaling. For review, see, e.g. Whitman, Genes Dev.
12:2445-62
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2.
(1998); and Miyazono et al., Adv. Immufzol. 75:111-57 (2000), which are
expressly incorporated
herein by reference.
Susnmarx of the Invention
The invention concerns a method for the improvement of lung function,
comprising the
administration, to a mammalian subject diagnosed with a disease or condition
benefiting from
the improvement of lung function, an effective amount of a molecule capable of
inhibiting a
biological activity mediated by a TGF(3-Rl kinase receptor.
The invention further concerns a method for the treatment of a mammalian
subject
having impaired lung function, comprising administering to such subject an
effective amount of
a molecule capable of inhibiting a biological activity mediated by a TGF(3-Rl
lcinase receptor.
The subject preferably is human.
In a particular embodiment, the molecule is a TGF-(3 inlubitor specifically
binding to a
TGF(3-Rl kinase, receptor. In another particular embodiment, the molecule is a
non-peptide
small molecule, e.g. a small organic molecule.
The disease or condition benefiting from the improvement of lung function may,
for
example, be selected from the group consisting of emphysema, chronic
bronchitis, chronic
obstructive pulmonary disease (OOPD), , pulmonary edema, cystic fibrosis,
occlusive lung
disease, acute respiratory deficiency syndrome CARDS), asthma, radiation-
induced injury of the
lung, lung injuries resulting from infectious causes, inhaled toxins, or
circulating exogenous
toxins, aging and genetic predisposition to impaired lung function.
In a further embodiment, the small molecule inhibitor additionally inhibits a
biological
activity mediated by p38 kinase.
In another embodiment, the small molecule inhibitor preferentially inhibits a
biological
activity mediated by TGF-[3-RI kinase relative to a biological activity
mediated by p38 kinase.
In a further embodiment, the small molecule inhibitor is other than an
imidazole
derivative.
In a still further embodiment, the small molecule inhibitor is a compound of
formula (1)
(L)n Ar'
Zs ~ Z ~ Zs
A B (~)
Z7
\ Z$ N R3
or the pharmaceutically acceptable salts thereof
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wherein R3 is a noninterfering substitueni;
each Z is CR2 or N, wherein no more than two Z positions in ring A are N, and
wherein
two adjacent Z positions in ring A cannot be N;
each R2 is independently a noninterfering substituent;
L is a linker;
n is 0 or 1; and
Ar' is the residue of a cyclic aliphatic, cyclic heteroaliphatic, aromatic or
heteroaromatic
moiety optionally substituted with 1-3 noninterfering substituents.
In a preferred embodiment, the compound of formula (1) is a quinazoline
derivative.
In another preferred group of compounds of formula (1) Z3 is N; and ZS-Zg are
CR2.
In a different group, Z3 is N; and at least one of ZS-Z8 is nitrogen.
Compounds in which
R3 is an optionally substituted phenyl moiety are specifically included.
Another group of compounds fox use in the methods of the present invention is
represented by the following formula (2)
Ar
Z (2)
R3
(R2)n
and the pharmaceutically acceptable salts and prodrug forms thereof;.wherein .
Ar represents an optionally substituted aromatic or optionally substituted
heteroaromatic
moiety containing 5-12 ring members wherein said heteroaromatic moiety
contains one or
more O, S, andlor N;
X is NRI, O, or S;
Rl is H, alkyl (1-8C), alkenyl (2-8C), or alkynyl (2-8C);
Z represents N or CR4;
each of R3 and R4 is independently H, or a non-interfering substituent;
each R2 is independently a non-interfering substituent; and
n is 0, l, 2, 3, 4, or 5. In one embodiment, if n>2, and the R2's are
adjacent, they can be
joined together to form a 5 to 7 membered non-aromatic, heteroaromatic, or
aromatic ring
containing 1 to 3 heteroatoms..where each heteroatom can independently be O,
N, or S.
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Another group of the compounds of the nvention is represented by formula (3)
Y
X1
.
Y~ .'
~2
Yq
Y6
wherein Y1 is phenyl or naphthyl optionally substituted with one or more
substituents selected from halo, alkoxy(1-6 C), alkyltluo(1-6 C), alkyl(1-6
C), haloalkyl (1-6C), -
O-(CH2)m Ph, -S-(CH2)m-Ph, cyano, phenyl, and C02R, wherein R is hydrogen or
alkyl(1-6 C),
and m is 0-3; or phenyl fused with a 5- or 7-membered aromatic or non-aromatic
ring wherein
said ring contains up to three heteroatoms, independently selected from N, O,
and S:
Y2, Y3, Y4, and Ys independently represent hydrogen, alkyl(1-6C), alkoxy(1-6
C), haloalkyl(1-6 .C), halo, NH2, NH-alkyl(1-6C), or NH(CHZ)n Ph wherein n is
0-3; or an
adjacent pair of Y2, Y3, Y4, and Ys form a fused 6-membered aromatic ring
optionally containing
up to 2 nitrogen atoms, said ring being optionally substituted by one o more
substituents
independently selected from alkyl(1-6 C), alkoxy(a-6 C), haloalkyl(1-6 C),
halo, NH2, NH-
alkyl(1-6 C), or NH(CH2)~ Ph, 'wherein n is 0-3, and the .remainder of Y2, Y3,
Y4, and Ys
represent hydrogen, alkyl(1-6 C), alkoxy(1-6C), haloalkyl(1-6 C), halo, NH2,
NH-alkyl(1-6 C),
or NH(CH2)"-Ph wherein n is 0-3; and
one of XI and X2 is N and the other is NR6, wherein R6 is hydrogen or
alkyl(1-6 C).
As used in formula (3), the double bonds indicated by the dotted lined
represent possible
tautomeric ring forms of the compounds. Further information about compounds of
formula (3)
and their preparation is disclosed in WO 02/40468, published May 23, 2002, the
entire
disclosure of which is hereby expressly incorporated by reference.
Yet another group of compounds for use in the methods of the invention is
represented by
the following formula (4)
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Y1
X1
Y3
~~ N
Y~
wherein Y1 is naphthyl, anthracenyl, or phenyl optionally substituted with one
or
more substituents selected from the group consisting of halo, alkoxy(1-6 C),
alkylthio(1-6 C),
alkyl(1-6 C), -O-(CH2)-Ph, -S-(CH2)ri Ph, cyano, phenyl, and. C02R, wherein R
is hydrogen or
alkyl(1-6 C), and n is 0, 1, 2, or 3; or Yl represents phenyl fused with an
aromatic or non-
aromatic cyclic ring of 5-7 members wherein said cyclic ring optionally
contains up to two
heteroatoms, independently selected from N, O, and S; '
Y2 is H, NH(CH2)n Ph or NH-alkyl(1-6 C), wherein n is 0, 1, 2, or 3;
Y3 is C02H; CONH2, CN, N02, alkylthio(1-6 C), -SO2-alkyl(C1-6), alkoxy(C1-
6), SONH2, CONHOH, NH2, CHO, CH2NHz, or C02R, wherein R is hydrogen or alkyl(I-
6 C);
one of X~ aazd X2 is N or CR', and other is NR' or CHR' wherein R' is
hydrogen,
OH, alkyl(C-16), or cycloalkyl(C3-7); or when one ofXl and X2 is N or CR' then
the other may
beSorO.
Pharmaceutically acceptable salts of all compounds within the scope of the
invention are
specifically included.
Brief Description of the Drawings
Figure 1 shows the effect of a representative compound of formula (1) on the
respiratory
rate in a 5-day bleomycin rat lung injury model.
Figure 2 shows the effect of a representative compound of formula (1) on the
tidal
volume in a 5-day bleomycin rat lung injury model.
Figure 3 shows the effect of a representative compound of formula (1) on the
total BALF
IL-6 in a 5-day bleomycin rat lung injury model.
Figure 4 shows the effect of a representative compound of formula (1) on total
lung
capacity in a 5-day bleomycin rat lung injury model.
Figure 5 shows the efFect of a representative compound of formula (1) on
permeability in
a 5-day bleomycin rat lung injury model.
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' . Figure 6 illustrates that treatment with a representative compound of
formula (1) reduces
lung permeability as measured by fluorescence following RITC-Dextran
administration to rats
with bleomycin-induced lung injury.
Figure 7 shows that treatment with a representative compound of formula (1)
reduces
tissue damage in bleomycin 5 day rat lung injury model.
Figure 8 shows the effect of a representative compound of formula (1) on lung
hydroxyproline content following bleomycin-induced lung fibrosis.
Figure 9 shows the effect of a, representative compound of fornula (1) on
total lung
capacity following bleomycin-induced lung fibrosis.
Figure 10 shows that a representative compound of formula (1) significantly
reduces lung
fibrosis induced by bleomycin.
Figures 11 and 12 are histology pictures showing that treatment with a
representative
compound of formula (1) reduces fibrosis in the 14-day bleomycin rat lung
injury model.
Detailed Description of the Preferred Embodiment
A. Definitions
The terms "improvement of lung function," and "improvement of pulmonary
function"
are used interchangeably, and refer to an improvement in any parameter
,suitable to measure
lung performance. Thus, improvement of pulmonary function can be measured, for
example, in
muriile bleomycin-induced lung injury models, such as .the bleomycin rat lung
injury model
described in the Examples below, which monitors improvements in respiratory
rate and tidal
volume. Parameters that are typically monitored in human patients as a measure
of lung function
include, but are not limited to, inspiratory and expiratory flow rates, lung
volume (also referred
to as lung capacity), and diffusing capacity for carbon monoxide, ability to
forcibly exhale,
respiratory rate, and the like. ~ Methods of quantitatively determining
pulmonary function in
patients are well known in the art, and include timed measurement of
inspiratory and expiratory
maneuvers to measure specific parameters. For example, forced vital capacity
(FVC) measures
the total volume in liters exhaled by a patient forcefully from a deep initial
inspiration. This
parameter, when evaluated in conjunction with the forced expired volume in one
second (FEVI),
allows bronchoconstriction to be quantitatively evaluated. In addition to
measuring volumes of
exhaled air as indices of pulmonary function, the flow in liters per minute
measured over
differing portions of the expiratory cycle can be useful in determining the
status of a patient's
pulmonary fiuiction. In particular, the peak expiratory flow, taken as the
highest air flow rate in
liters per minute during a forced maximal exhalation, is well correlated with
overall pulmonary
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function in a patient with respiratory diseases. Metliods and tools for
measuring these and
similar parameters are well known in the art, and routinely used in everyday
clinical practice.
The term "tidal volume" refers to the volume of air inspired or expired with
each normal
breath.
A "biological activity mediated by the TGF(3-Rl kinase receptor" can be any
activity
associated with the activation of TGF(3-Rl and downsteam intracellular
signaling events, such as
the phosphorylation of Smad2/Smad3.
The term "treatment" refers to both therapeutic treatment and prophylactic or
preventative measures, wherein the object is to prevent or slow down (lessen)
the targeted
pathologic condition or disorder. Those in need of treatment include those
already with the
disorder as well as those prone to have the disorder or those in whom the
disorder is to be
prevented. Thus, in the context of improving lung function, treatment includes
prevention and
treatment of a disease or condition negatively impacting lung function or
otherwise benefiting
from the improvement of lung function, relieving one or more symptoms of such'
disease, .
prevention and treatment of complications resulting from such disease,
improving exercise
tolerance of patients with compromised lung function, and reduction in
mortality.
The "pathology" of a disease or condition negatively impacting lung function
includes
all phenomena that compromise the well-being of the patient.
A "disease or condition benefiting from the improvement of lung function"
includes all
diseases, disorders and conditions which involve a negative change in at least
one parameter
suitable for measurement of lung performance. Such diseases and conditions
include, without
limitation, emphysema, chronic bronchitis, chronic obstructive pulmonary
disease (COPD),
pulmonary edema, cystic fibrosis, occlusive lung disease, acute respiratory
deficiency syndrome
CARDS), asthma, radiation-induced injury of the lung, and lung injuries
resulting from other
factors, such as, infectious causes, inhaled toxins, or circulating exogenous
toxins, aging and
genetic predisposition to impaired lung function.
The term "inhibitor" as .used herein refers to a molecule, e.g. a nonpeptide
small
molecule, having the ability to inhibit the biological function of a native
TGF-(3 molecule
mediated by the TGF[3-Rl receptor. Accordingly, the term "inhibitor" is
defined in the context
of the biological role of TGF-(3 and its receptors. Preferred inhibitors
within the scope of the
invention specifically bind a TGF(3-Rl receptor. Other preferred inhibitors
preferentially inhibit
the function of a TGF(3-Rl receptor through specific binding to that receptor
or otherwise.
The teens "specifically binding," "binds specifically," "specific binding,"
and
grammatical equivalents thereof, are used to refer to binding to a unique
epitope within the type I
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TGF-[3 receptor (TGF(3-Rl). The binding must occur with an affinity to
effectively inhibit TGF-
(3 signaling through TGF(3-Rl.
The term "preferentially inhibit" as used herein means that the inhibitory
effect on the
target that is "preferentially inhibited" is significantly greater than on any
other target. Thus, in
the context of preferential inhibition of TGF-(3-Rl kinase relative to the p38
kinase, the term
means that the inhibitor inhibits biological activities, e.g. profibrotic
activities, mediated by the
TGF-[3-Rl kinase significantly more than biological activities mediated by the
p38 kinase. The
difference in the degree of inhibition, in favor of the preferentially
inhibited receptor, generally is
at least about two-fold, more preferably at least about f ve-fold, even more
preferably at least
about ten-fold.
The term "mammal" for purposes of treatment refers to any animal classified as
a
mammal, including humans, domestic and farm animals, and zoo, sports, or pet
animals, such as
dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the
mammal is human.
Administration "in combination with" one or more further therapeutic agents
includes
simultaneous (concurrent) and consecutive administration in any order.
A "therapeutically effective amount", in reference to the treatment of a
disease, e.g. when
inhibitors of the present invention are used, refers to an amount capable of
invoking one or more
of the following effects: (1) inlubition (i.e., reduction, slowing down or
complete stopping) of
the development or progression of a disease or condition negatively affecting
lung function; (2)
inhibition (i.e., reduction, slowing down or complete stopping) of
consequences of or
complications resulting from such disease or condition; and (3) relief, to
some extent, of one or
more symptoms associated with such disease or condition, or symptoms of
consequences of or
complications resulting from such disease and/or condition.
As 'used herein, a "noninterfering substituent" is a substituent wluch leaves
the ability of
the compound of formula (1) to inhibit TGF-(3 activity qualitatively intact.
Thus, the substituent
may alter the degree of inhibition. However, as long as the compound of
formula (1) retains the
ability to inhibit TGF-(3 activity, the substituent will be classified as
"noninterfering."
As used herein, "hydrocarbyl residue" ,refers to a residue which contains only
carbon and
hydrogen. The residue may be aliphatic or aromatic, straight-chain, cyclic,
branched, saturated
or unsaturated. The hydrocarbyl residue, when indicated, may contain
heteroatoms over and
above the carbon and hydrogen members of the substituent residue. Thus, when
specifically
noted as containing such heteroatoms, the hydrocarbyl residue may also contain
carbonyl groups,
amino groups, hydroxyl groups and the like, or contain heteroatoms within the
"backbone" of the
hydrocarbyl residue.
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As used herein, the term "alkyl," "alkenyl" and "alkynyl" include straight-
and branched-
chain and cyclic monovalent substituents. Examples include methyl, ethyl,
isobutyl, cyclohexyl,
cyclopentylethyl, 2-propenyl, 3-butynyl, and .the like. Typically, the alkyl,
alkenyl and alkynyl
substituents contain .l-lOC (alkyl) or 2-lOC (alkenyl or alkynyl). Preferably
they contain 1-6C
(alkyl) or 2-6C (alkenyl or alkynyl). Heteroalkyl, heteroalkenyl and
heteroalkynyl are similarly
defined but may contain 1-2 O, S or N heteroatoms or combinations thereof
within the backbone
residue.
As used herein, "acyl" encompasses the definitions of alkyl, allcenyl, alkynyl
and the
related hetero-forms which are coupled to an additional residue through a
carbonyl group.
"Aromatic" moiety refers to a monocyclic or fused bicyclic moiety such as
phenyl or
naphthyl; "heteroaromatic" also refers to monocyclic or fused bicyclic ring
systems containing
one ore more heteroatoms selected from O, S and N. The inclusion of a
heteroatom permits
inclusion of 5-membered rings as well as 6-membered rings. Thus, typical
aromatic systems
include pyridyl, pyrimidyl, indolyl, benzimidazolyl, benzotriazolyl,
isoquinolyl, quinolyl,
benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl,
imidazolyl and the
like. Any nionocyclic or fused ring bicyclic system which has the
characteristics of aromaticity
in terms of electron distribution throughout the ring system is included in
this definition.
Typically, the ring systems contain 5-12 ring member atoms.
Similarly, "arylalkyl" and "heteroalkyl" refer to aromatic and heteroaromatic
systems
which are coupled to another residue through a carbon chain, including
substituted or
unsubstituted, saturated or unsaturated, carbon chains, typically of 1-6C.
These carbon chains
may also include a carbonyl group, thus making them able to provide
substituents as an acyl
moiety.
B. Modes of Carr in out the Invention
As discussed before, the biological activities of TGF-(3 are mediated by two
distinct types
of receptors designated type I' and type ~II (Derynck and Feng, Biochifn.
Bio~ahys. Acta
1333:F105-F150 (1997); Massague, Annu. Rev. Biochem., 67:753-9I (1998)). Both
receptors are
serine-threonine kinases. Upon binding of TGF-(3 to the type II receptor, the
type II receptor
phosphorylates the type 1 receptor, which is activated and is, in turn,
responsible for intracellular
signaling. 'In addition, TGF-(3 has a non-serine-theronine kinase receptor,
termed type III
receptor, which is believed to facilitate or modulate signaling through the
type I/II receptor pair
(Lopez-Casillas et al., Cell 73:996-1005 (1993)).
The present invention is based on the surprising finding that certain
quinazoline and
imidazole derivatives specifically inlubiting TGF-(3 signaling through the
type I TGF-(3 receptor
(TGF(3-Rl), e.g. by specifically binding TGF(3-Rl, can improve lung function.
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In a preferred embodiment, the inhibitors of the present invention selectively
inhibit
biological responses mediated by the type I receptor, without affecting the
type II receptor-
mediated cell proliferation.
In another preferred embodiment, the compounds of the present invention
preferentially
inhibit TGF(3-Rl kinase relative to p38 kinase.
Compounds of the Invention .
The inhibitors of the present invention typically are small organic molecules
(non-peptide
small molecules), generally less than about 1,000 daltons in size. Preferred
non-peptide small
molecules have molecular weights of less than about 750, daltons, more
preferably less than
about SOO daltons, and even more preferably less than about 300 daltons.
In a preferred embodiment, the compounds of the invention are of the formula
5
z6 /. z ~ Z3
A B
7
Z \ Z8 N R3
or the pharmaceutically acceptable salts thereof .
wherein R3 is a noninterfering substituent;
each Z is CR2 or N, wherein no more than two Z positions in ring A are N,
and~wherein two adjacent Z positions in ring A cannot be N;
each R2 is independently a noninterfering substituent;
L is a linker;
nis0orl;and
Ar' is the residue ~ of a cyclic aliphatic, cyclic . heteroaliphatic, aromatic
or
heteroaromatic moiety optionally substituted with 1-3 noninterfering
substituents.
In a preferred embodiment, the small organic molecules herein are .
derivatives of
quinazoline and related compounds containing mandatory substituents at
positions corresponding
to the 2- and 4-positions of quinazoline. In general, a quinazoline nucleus is
preferred, although
alternatives within the scope of the invention are also illustrated below.
Preferred embodiments
for Z3 are N and CH; preferred embodiments for ZS-Z8 are CR2. However, each of
ZS-Z8 can
also be N, with the proviso noted above. Thus, with respect to the basic
quinazoline type ring
system, preferred embodiments include quinazoline pen se, and embodiments
wherein all of
ZS-Zs as well as Z3 are either N or CH.. Also preferred are those embodiments
wherein Z3 is N,
and either ZS ox Z8 or both ZS and Z8 are N and Z6 and Z' are CH or CR2. Where
R2 is other than
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H, it is preferred that CR2 occur at positions 6land/or 7. Thus, by way of
example, quinazoline
derivatives within the scope of the invention include compounds comprising a
quinazoline
nucleus, having an aromatic ring attached in position 2 as a non-interfering
substituent (R3),
which may be further substituted.
With respect to the substituent at the positions corresponding to the 4-
position of
quinazoline, LAr', L is present or absent and is a linker which spaces the
substituent Ar' from
ring B at a distance of 2-81~r, preferably 2-6~, more preferably 2-4~. The
distance is measured
from the ring carbon in ring B to which one valence of L is attached to the
atom of the Ar' cyclic
moiety to wluch the other valence of the linker is attached. The Ar' moiety
may also be coupled
directly to ring B (i.e., when n is 0). Typical, but nonlimiting, embodiments
of L are of the
formula S(CR22)m, -NR1S02(CR22)I, NRl(CR22)m, NR1C0(CRZZ)i, O(CR22)m,
OCO(CR22)u and
-N Z
(CRa
wherein Z is N or CH and wherein m is 0-4 and I is 0-3, preferably I-3 and
1-2, respectively. L preferably provides -NRl- coupled directly to ring B. A
preferred
embodiment of Rl is H, but Rl may also be aryl, alkyl, arylacyl or arylalkyl
where the aryl
moiety may be substituted by 1-3 groups such as alkyl, alkenyl, alkynyl, acyl,
aryl, alkylaryl,
aroyl, N-aryl, NH=alkylaryl, NH-aroyl, halo, OR, NR2, SR, -SOR, -NRSOR, -
NRSOZR, -S02R,
-OCOR, -NRCOR, -NRCONR2, -NRCOOR, -OCONR2, -RCO, -COOR, -S03R, -CONR2,
S02NR2; CN, CF3, and N02, wherein each R is independently H or alkyl (1-4C),
preferably the
substituents are alkyl (1-6C), OR, SR or NR2 wherein R is H or lower alkyl (I-
4C). More
preferably, Rl is H or alkyl (1-6C). Any aryl groups contained in the
substituents may further be
substituted by for example alkyl, alkenyl, alkynyl, halo, OR, NR2, SR, -SOR, -
SOZR, -OCOR,
-NRCOR, =NRCONR2, -NRCOOR, -OCONR2, -RCO, -COOR, S02R, NRSOR, NRS02R,
-S03R, -CONR2, S02NR2, CN, CF3, or N02, wherein each R is independently H or
alkyl (1-4C).
Ar' is aryl, heteroaryl, including 6-5 fused heteroaryl, cycloaliphatic or
cycloheteroaliphatic. Preferably Ar' is phenyl, 2-, 3- or 4-pyridyl, indolyl,
2- or 4-pyrimidyl,
benzimidazolyl, indolyl, preferably each optionally substituted with a group
selected from the
group consisting of optionally substituted alkyl, alkenyl, alkynyl, aryl, N-
aryl; NH-aroyl, halo,
OR, NR2, SR, -OOCR, -NROCR, RCO, -COOR, -CONR2, SO~NR2, CN, CF3, and N02,
wherein
each R is independently H or alkyl (1-4C).
Ar' is more preferably indolyl, 6-pyrimidyl, 3- or 4-pyridyl, or optionally
substituted
phenyl.
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For embodiments wherein Ar' is optionally substituted phenyl, substituents
include,
without limitation, alkyl, alkenyl, alkynyl, aryl, alkylaryl, amyl, N-aryl, NH-
alkylaryl, NH-aroyl,
halo, OR, NR2, SR, -SOR, -S02R, -OCOR, -NRCOR, -NRCONR2, -NRCOOR, -OCONR2,
RCO, -COOR, -S03R, -CONR2, S02NR2, CN, CF3, and N02, wherein each R is
independently
H or alkyl (1-4C). Preferred substituents include halo, OR, SR, and NRZ
wherein R is H or
methyl or ethyl. These substituents may occupy all five positions of the
phenyl ring, preferably
1-2 pbsitions, preferably one position. Embodiments of,Ar' include substituted
or unsubstituted
phenyl, 2-, 3-, or 4-pyridyl, 2-, 4- or 6-pyrimidyl, indolyl, isoquinolyl,
quinolyl,.benzimidazolyl,
benzotriazolyl, benzothiazolyl, benzofuranyl, pyridyl,,thienyl, fiuyl,
pyrrolyl, thiazolyl, oxazolyl,
imidazolyl, and morpholinyl. Particularly preferred as an embodiment of Ar' is
3- or 4-pyridyl,
especially 4-pyridyl in unsubstituted form.
Any of the aryl moieties, especially the phenyl moieties, may also comprise
two
substituents which, when taken together, form a 5-7 membered carbocyclic or
heterocyclic
aliphatic ring.
Thus, preferred embodiments of the substituents at the position of ring B
corresponding
to , 4-position of the quinazoline include 2-(4-pyridyl)ethylamino; 4-
pyridylamino; 3-
pyridylamino; 2-pyridylamino; 4-indolylamino; .5-indolylamino; 3-
methoxyanilinyl; 2-(2,5-
difluorophenyl)ethylamino-, and the like.
R3 is generally a hydrocarbyl residue (1-20C) containing 0-5 heteroatoms
selected from
O, S and N. Preferably R3 is alkyl, aryl, arylalkyl, hetexoalkyl, heteroaryl,
or heteroarylalkyl,
each unsubstituted or substituted with 1-3 substituents. The substituents are
independently
selected from a group that includes halo, OR, NR2, SR, -SOR, -S02R, -OCOR,
NRCOR,
-NRCONRZ, -NRCOOR, -OCONR2, RCO, -COOR, -S03R, NRSOR, NRSOZR, -CONR2,
SOZNR2, CN, CF3, and NOZ, wherein each R is independently H or alkyl (1-4C)
and with respect
to any aryl or heteroaryl moiety, said group further including alkyl (1-6C) or
alkenyl or alkynyl.
Preferred embodiments of R3 (the substituent at position corresponding to the
2,-position of the
quinazoline) comprise a phenyl moiety optionally substituted with 1-2
substituents preferably
halo, alkyl (1-6C), OR; NR2, and SR wherein R is as defined above. Thus,
preferred substituents
at he 2-position .of the quinazoline include phenyl, 2-halophenyl, e.g., 2-
bromophenyl,
2-chlorophenyl, 2-fluorophenyl; 2-alkyl-phenyl, e.g., 2-methylphenyl, 2-
ethylphenyl; 4-
halophenyl, e.g., 4-bromophenyl, 4-chlorophenyl, 4-fluorophenyl; 5-halophenyl,
e.g. 5-
bromophenyl, 5-chlorophenyl, 5-fluorophenyl; 2,4- or 2,5-halophenyl, wherein
the halo
substituents at different positions may be identical or different, e.g. 2-
fluoro-4-chlorophenyl; 2-
bromo-4-chlorophenyl; 2-fluoro-5-chlorophenyl; 2-chloro-5-fluorophenyl, and
the like. Other
preferred embodiments of R3 comprise a cyclopentyl or cyclohexyl moiety.
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As noted above, RZ is a noninterfering substituent. As set forth above, a
"noninterfering
substituent" is one whose ,presence does not substantially destroy the TGF-(3
inhibiting ability of
the compound of formula (1).
Each R2 is also independently a hydrocaxbyl residue (1-20C) containing 0-5
heteroatoms
selected from O, S and N. Preferably, R2 is independently H, alkyl, alkenyl,
alkynyl, acyl or
hetero-forms thereof or is aryl, arylalkyl, heteroalkyl, heteroaryl, or
heteroarylalkyl, each
unsubstituted or substituted with 1-3 substituents selected independently
from. the group
consisting of alkyl, alkenyl, allcynyl, aryl, alkylaryl, amyl, N=aryl, NH-
alkylaryl, NH-aroyl, halo,
OR, NR2, SR, -SOR, -S02R, -OCOR, -NRCOR, -NRGONR2, -NRCOOR, NRSOR, NRS02R,
-OCONR2, RCO, -COOR, -SO3R, NRSOR, NRS02R, -CONR2, S02NR2, CN, CF3, and N02,
wherein each R is independently H or alkyl (1-4C). The aryl or aroyl groups on
said substituents
may be further substituted by, for example, alkyl, alkenyl, alkynyl, halo, OR,
NR2, SR, -SOR,
-S02R, -OCOR, -NRCOR, -NRCONR2, -NRCOOR, -OCONR2, RCO, -COOR, -S03R, -CONR2,
SO2NR2, CN, CF3, and N02, wherein each R is independently H or alkyl (l-4C).
More
preferably the substituents on R2 are selected from R4, halo, OR4, NR42, SR4, -
OOCR4,
-NROCR4, -COOR4, R4C0, -CONR42, -S02NR42, CN, CF3, and N02, wherein each R4 is
independently H, or optionally substituted alkyl (1-6C), or optionally
substituted arylalkyl
(7-12C) and wherein two R4 or two substituents on said alkyl or arylalkyl
taken together may
form a fused aliphatic ring of 5-7 members.,
R2 may also, itself, be selected from the group consisting of halo, OR, NRZ,
SR, -SOR,
-S02R, -OCOR, -NRCOR, -NRCONR2, -NRCOOR, NRSOR, NRS02R, -OCONR2, RCO,
-COOR, -S03R, NRSOR, NRSOZR, -CONRa, S02NR2, CN, CF3, and N02, wherein each R
is
independently H or alkyl (1-4C).
More preferred substituents represented by R2 are those as set forth with
regard to the
phenyl moieties contained in Ar' or R3 as set forth above. Two adjacent CR2
taken together may
form a carbocyclic or heterocyclic fused aliphatic ring of 5-7 atoms.
Preferred R2 substituents
are of the formula R4, -OR4, SR4 or R4NH-, especially R4NH-, wherein R4 is
defined as above.
Particularly preferred are instances wherein R4 is substituted arylalkyl.
Specific representatives
of the compounds of formula (1) are shown in Tables 1-3 below. All compounds
listed in Table
1 have a quinazoline ring system (Z3 is N), where the A ring is unsubstituted
(ZS-Z8 represent
CH). The substituents of the B ring are listed in the following Table 1.
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_ Table
1
Compound L Ar' R3
No.
1 NH -pyridyl -chlorophenyl
2 ~ NH -pyridyl 2,6-dichlorophenyl
3 NH -pyridyl -methylphenyl
4 NH -pyridyl -bromophenyl
NH -pyridyl -fluorophenyl
6 . NH -pyridyl 2,6-difluorophenyl
7 NH -pyridyl phenyl
8 NH -pyridyl -fluorophenyl
9 NH -pyridyi -methoxyphenyl
NH -pyridyl 3-fluorophenyl
11 * N* -pyridyl phenyl
12 N -pyridyl phenyl
Z 3 NHCH~ -pyridyl phenyl
14 ~ NHCHZ -pyridyl , , -chlorophenyl
NH 3-pyridyl phenyl.
16 NHCH2 -pyridyl phenyl .
17 NHCH~ 3-pyridyl phenyl
18 NHCHZ 2-pyridyl phenyl
19 NHCHZCHz 2-pyridyl phenyl
NH 6-pyrimidinyl phenyl
21 NH' -pyrimidinyl phenyl
~ .
22 NH phenyl phenyl
23 NHCHZ phenyl 3-chlorophenyl
24 NH 3-hydroxyphenylphenyl
NH -hydroxyphenyl phenyl
26 NH -hydroxyphenyl phenyl
27 NH -indolyl phenyl
28 NH 5-indolyl phenyl
29 NH -methoxyphenyl phenyl
NH 3-methoxyphenylphenyl
31 NH -methoxyphenyl phenyl
32 NH -(2- phenyl
hydroxyethyl)phenyl
33 NH 3-cyanophenyl phenyl
34 NHCHZ 2,5-difluorophenylphenyl
NH -(2-butyl)phenylphenyl
36 NHCHz -dimethylaminophenylphenyl
37 NH -pyridyl cyclopentyl
38 NH 2-pyridyl phenyl
39 NHCHZ 3-pyridyl phenyl
NH -pyrimidyl phenyl
41~ N -pyridyl phenyl
42 NH p-aminomethylphenylphenyl
43 NHCH2 -aminophenyl phenyl
44 NH -pyridyl ~ 3-chlorophenyl
NH phenyl -pyridyl
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46 NH N\ phenyl
// ,NH
-
-
-
~~~'
47 NH -pyr -butyl
i
yl
d
48 NH -benzylamino-3-phenyl
pyridyl
49 NH -benzylamino-4-phenyl
pyridyl
50 ~ NH 3-benzyloxyphenyiphenyl
51 NH -pyridyl 3-aminophenyl
52 NH -pyridyl -pyridyl
53 NH -pyridyl 2-naphthyl
54 /~ -pyridyl phenyl
-N, r--CHI-
~
55 ~ phenyl phenyl
_/~_
56 ~---~ -pyridyl phenyl
-N N-
57 NHCH2CH2 - Vo phenyl .
58 not present_N~CONI~ phenyl
59 not present-N NH phenyl
60 NH -pyridyl cyclopropyl
61 NH -pyridyl -trifluoromethyl
phenyl
62 NH -aminophenyl phenyl
63 NH -pyridyl cyclohexyI
64 NH 3-methoxyphenyl-fluorophenyl
65 NH -methoxyphenyl -fluorophenyl
66 NH ~ -pyrimidinyl -fluorophenyl
67 NH 3-amino-4-pyridylphenyl
68 NH -pyridyl
benzylaminophenyl
69 NH 2-benzylaminophenylphenyl
70 NH -benzylaminophenyl-cyanophenyl
71 NH 3'-cyano-2- phenyl
benzylaminophenyl
*R'=2-propyl
tR'=4-methoxyphenyl
$R' = 4-methoxybenzyl
The compounds in Table 2 contain modifications of the quinazoline nucleus as
shown.
All of the compounds in Table 2 are embodiments of formula (1) wherein~Z3 is N
and Z6 and Z'
represent CH. In all cases the linker, L, is present and is NH.
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Table 2
_
Compound Z$ Z$ Ar' R3
No.
72 CH N -pyridyl -fluorophenyl
73 CH N -pyridyl -chlorophenyl
74 CH N -pyridyl 5-chloro-2-
luorphenyl
75 CH N -(3-methyl)-pyridyl5-chloro-2-
luorphenyl
.
76 CH N -pyridyl Phenyl
77 N N -pyridyl phenyl
78 N CH -pyridyl Phenyl
79 N N -pyridyl 5-chloro-2-
luorphenyl
80 N N -(3-methyl)-pyridyl5-chloro-2-
luorphenyl
Additional compounds were prepared wherein ring A contains CRZ at Z6 or Z'
where RZ
is not H. These compounds, which are all quinazoline derivatives, wherein L is
NH and Ar' is
4-pyridyl, are shown in Table 3.
Tabl e 3
Compound
No. R3 CRZ as noted
81 2-chlorophenyl6,7-dimethoxy
82 2-fluorophenyl-nitro
83 2-fluorophenyl6-amino
84 2-fluorophenyl7-amino
85 2-fluorophenyl6-(3-methoxybenzylamino)
86 2-fluorophenyl6-(4-methoxybenzylamino)
87 2-fluorophenyl6-(2-isobutylamino)
88 ~ 2-fluorophenyl6-(4-
methylmercaptobenzylamino)
89 2-fluorophenyl-(4-methoxybenzoyl
amino)
90 4-fluorophenyl7-amino
91 4-fluorophenyl7-(3-methoxybenzylamino)
Structures representative of quinazoline derivatives are shown below in Table
4.
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Table 4
~N
~JI
HN
~N~ ~ N
IN N
~N
HN
N ~N F
w
~N
HN
N ~N
CN N ~ CI
N~
~NH
N \N
N~' ~ OCH3
/
N'
~NH
N ~ N OCH3
N N~
N'
~NH
N~ ~ N CI
~N N
/
.J
HN N
N~ ~ N
CN N~ ~ CI
/
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~N
H3C, N ~~JL
N
I ~ N
CN Nr ~ CI
I~
HzN ~ N
HN' J
N ~N
I
Nr ~ CI
~N
HN
N ~N F
CN Nr
I \ ,
F
~N
HN
N ~ N Cl
CN Nr
~N
HN
N ~N F
CN N ~ CI
I~
~N
HN
r
CN I rN F F
N N ~F
~N
HN
r
N ~N
CN I N ~ F
I
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19
~N
HN~~'' \ II
CN I ~ N F
~N N~
CI
~N
'''' w~JI
HN
N ~N
CN I N ~ Br
I /
~N:
HN
N ~N F
I
N N~ W
I/
Br
~N
HN
N \N
C N N.~ \
I
I / F
~N
'~JI
HN
N ~N F
CN N \
I
/
F F
F
~N
HN
N \N
w
~N I N
I /
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HsC~N
\ I
HN
~N F
N N' \
CI
OH
N ~N F
N N' \
CI
CH3
/ ~N
\ I
HN
N ~N F
CN N \
CI
Br / N
\ I
HN
N wN C P
CN N~ \
/
CI
/ N
\ !
HN
N~ ~ N
~ /
N
/ N
~\ I ,
HN' J
~N
N V 'N' \
~N
HN
N \ ~N
/ ' \
N
~' /
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~N
\ I
HN
I \ wN
N~N \ CI
I~
i N
HN' J
\ ~N
I.N N I \ O~CHs
~N
\ I
HN
~N
I
~N N I w
CHs
~N
'\ II
HN
~N
IN N l \
H3C.0 /
~N
\ I
HN
~N
I N~N O
0
~N
HN!\ II
~N
I ~ r ~ O~CH3
N N
I ~ CHs
~N
'''' \ II
HN
\ ~N
I r ~ O~CHs
N N
. ~N
HN
\ ~N I
I r r O \
N N \
I~
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22
~N
HN
~sN
N N ~ CI
~N
HN
~N
N- _N ~ CI
HsC.O ~ /
~N
~/ ~JJI
HN
~N
CI
~/
CI
~N
HN
~N
N~N . W
CI
~N
HN
~N
CI
~N
HN
~N
N- -N
H3C.0
Although the invention is illustrated with reference to certain quinazoline
derivatives, it is
not so limited. Inhibitors of the present invention include compounds having a
non-quinazoline,
such as, a pyridine, pyrimidine nucleus carrying substituents like those
discussed above with
respect to the quinazoline derivatives.
Another group of compounds for use in the methods of the present invention is
represented by the following formula (2)
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23
Ar
Z (2)
R3
~R2)n
and the pharmaceutically acceptable salts and prodrug forms thereof; wherein
Ar represents an optionally substituted aromatic or optionally substituted
heteroaromatic
moiety containing 5-12 ring members wherein said heteroaromatic moiety
contains one or
more O, S, and/or N;
X is NRI, O, or S;
Rl is H, alkyl (1-8C); alkenyl (2-8C), or alkynyl (2-8C);
Z represents N or CR4;
each of R3 and R4 is independently H, or a non-interfering substituent;
each RZ is independently a non-interfering substituent; and
n is 0, 1, 2, 3, 4, or 5. In one embodiment, if n>2, and the R2's are
adjacent, they can be
joined together to forma 5 to 7 membered non-aromatic, heteroaromatic, or
aromatic ring
containing 1 to 3 heteroatoms where each heteroatom can independently be O, N,
or S.
In preferred embodiments, Ar represents an optionally substituted aromatic or
optionally
substituted heteroaromatic moiety containing 5-9 ring members wherein said
heteroaxomatic
moiety contains one or more N; or
Rl is H, alkyl (1-8C), alkenyl (2-8C), or alkynyl (2-8C); or
Z represents N or CR4; wherein
R4 is H, alkyl (1-lOC), alkenyl (2-lOC), or alkynyl (2-lOC), acyl (1-lOC),
aryl, alkylaryl,
aroyl, O-aryl, O-alkylaryl, O-aroyl, NR-aryl, NR-alkylaryl, NR-aroyl, or the
hetero forms of any
of the foregoing, halo, OR, NR2, SR, -SOR, -NRSOR, -NRS02R, -S02R, -OCOR, -
NRCOR,
-NRCONR2, -NRCOOR, -OCONRZ, -COOR, -S03R, -CONR2, -S02NR2, -CN, -CF3, or -N02,
wherein each R is independently H or alkyl (1-lOC) or a halo or heteroatom-
containing form of.
said alkyl, each of which may optionally be substituted. Preferably R4 is H,
alkyl (1-lOC), OR,
SR or NR2 wherein R is H or alkyl (1-l OC) or is O-aryl; or
R3 is defined in the same manner as R4 and preferred forms are similar, but R3
is
independently embodied; or
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each R2 is independently alkyl (1-8C), alkenyl (2-8C), alkynyl (2-8C), acyl (1-
8C), aryl,
alkylaryl, aroyl, O-aryl, O-alkylaryl, O-aroyl, NR-aryl, NR-alkylaryl, NR-
amyl, or the hetero
forms of any of the foregoing, halo, OR, NRa, SR, -SOR, -NRSOR, -NRS02R, -
NRS02R2,
-S02R, -OCOR, -OS03R, -NRGOR, -NRCONR2, -NRCOOR, -OCONRa, -COOR, -S03R,
-CONRZ, S02NR2, -CN, -CF3, or -N02, wherein each R is independently H or lower
alkyl
(1-4G). Preferably R2 is halo, alkyl (1-6C), OR, SR or NR2 wherein R is H or
lower alkyl
(1-4C), more preferably halo; or
n is 0-3.
The optional substituents on the aromatic or heteroaromatic moiety represented
by Ar
include alkyl (1-lOC), alkenyl (2-lOC), alkynyl (2-lOC), acyl (1-lOG), aryl,
alkylaryl, aroyl,
O-aryl, O-alkylaryl, O-aroyl, NR-aryl, NR-alkylaryl, NR-amyl, or the hetero
forms of any of the
foregoing, halo, OR, NR2, SR, -SOR, -NRSOR, -NRS02R, -S02R, -OCOR, -NRCOR,
-NRCONR2, -NRCOOR, -OCONR2, -COOR, -S03R, -CONR2, -SOZNR2, -CN, -CF3, .and/or
N02; wherein each R is independently H or lower. alkyl (1-4C). Preferred
substituents include
alkyl, OR, NRZ, O-alkylaryl and NH-alkylaryl.
Because tautomers are theoretically possible, phthalimido is also considered
aromatic,
and phthalimido-substituted alkyl and phthalimido-substituted alkoxy are
preferred embodiments
of R3' and R4.
In general, any alkyl, alkenyl, alkynyl, acyl, or aryl group contained in a
substituent may
itself optionally be substituted by additional substituents. The nature of
these substituents is
similar to those recited with regard to the primary substituents themselves.
Thus, where an
embodiment of, for example, R4 is alkyl, this alkyl may optionally be
substituted by the
remaining substituents listed as embodiments for R4 where this makes chemical
sense, and where
this does not undermine the size limit of alkyl pe~° se; e.g., alkyl
substituted by alkyl or by
alkenyl would simply extend the upper limit of carbon atoms for these
embodiments. However,
alkyl substituted by aryl, amino, alkoxy, and the like would be included
within the scope of the
invention. The features of the compounds are defined by formula (2) and the
nature of the
substituents is less important as long as the substituents do not interfere
with the stated biological
activity of this basic structure.
Nori-interfering substituents embodied by RZ, R3 and R4, include, but are not
limited to,
alkyl, alkenyl, alkynyl, halo, OR, NR2, SR, -SOR, -SOZR, -OCOR, -NRCOR, -
NRCONR2,
-NRCOOR, -OCONR2, -RCO, -COOR, S02R, NRSOR, NRS02R, -S03R, -CONR2, SOaNRz,
wherein each R is independently H or alkyl (1-8C), -CN, -CF3, and N02, and
like substituents.
R3 and R4 can also be H. Preferred embodiments for R3 and R4 are H, alkyl (1-
lOC) or a
heteroatom-containing form thereof, each optionally substituted, especially (1-
4C) alkyl; alkoxy
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(1-8C), acylamido, aryloxy, arylalkyloxy, especially wherein the aryl group is
a phthalimido
group, and alkyl or arylalkyl amine. Preferred embodiments of R2 include lower
alkyl, alkoxy,
and halo, preferably halo. Halo, as defined herein includes fluoro, chloro,
bromo and iodo.
Fluoro acid chloro are preferred.
Preferably, Ri is H or lower alkyl (1-4C), more preferably H.
Preferably Ar is optionally substituted phenyl, 2-, 3- or 4-pyridyl, indolyl,
2- or
4-pyrimidyl, pyridazinyl, benzotriazol or benzimidazolyl. More preferably Ar
is phenyl, pyridyl,
or pyrimidyl. Each of these embodiments may optionally be substituted with a
group such as
alkyl, alkenyl, alkynyl, aryl, O-aryl, O-alkylaryl, O-aroyl, NR-aryl, N-
alkylaryl, NR-aroyl, halo,
OR, NR2, SR, -OOCR, -NROCR, RCO, -COOR, -CONR2, and/or SO2NR2, wherein each R
is
independently H or alkyl ~ ( 1-8C), and/or by -CN, -CF3, and/or NO2. Alkyl,
allcenyl, alkynyl and
aryl portions of these may be further substituted by similar substituents.
Preferred substituents on Ar include alkyl, alkenyl, alkynyl, halo, OR, SR,
NR2 wherein
R is H or alkyl (1-4C); and/or arylamino, arylalkylamino, including alkylamino
which is
substituted by more than one aryl. As stated above, any aryl or alkyl group
included within a
substituent may itself be substituted similarly. These substituents may occupy
alI available
positions of the ring, preferably 1-2 positions, or more preferably only one
position.
Any of the aryl moieties, including those depicted in formula (2) especially
the phenyl
moieties, may also comprise two substituents wluch, when taken together, form
a 5-7 membered
carbocyclic or heterocyclic aliphatic ring. Similarly, R4 may be bridged to R3
to obtain a 5-7
membered carbocyclic or heterocyclic ring.
Structures representative of pyrimidine derivatives are shown below in Table
5.
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Table 5
/~N
HN
NC w N F
MeS N
I.
%~N
HN
Me02C ~ N F
MeS N
II.
~N
~ I
HN
NC w N F
Me2N N
III.
~N
I
HN
/ N
wN I ~ CI
/
IV.
/~N
~ I
HN
/ N F
I
N
V.
~N
HN
/ ~N F
I
N
CI
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/~N
\ II
HN
/ N
I
AcHN ~N I \ CI
VII.
N
'~\ I
HN
Me0 / N
wN I ~ CI
I /
VIII.
~~N
HN
Me0 / N F
~ I \
N
~. CI
~N
/\ II
HN
Et0 / N F
N
X.
/~N
\ II
HN
/ ~N
H N ~N~ I . ~ CI
2 I
XI. /
_ / N
\ I
HN
Et0 / N
wN I ~ CI
I/
XII.
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/~N
HN
/ N
I
Me0 ~N I ~ CI
XIII.
(' N
HN
/ ~N F
I
Me0 N
XIV. CI
~/~N
HN
Me0 / N F
I
N I /
XV. CI
J~N
HN
Me0 / N ~ F
I
Me0 \N,
XVI. CI
MeS / N
HN
Me0 ~ N F
N I /
XVII. CI
Me0 / N
I
HN
Me0 ~ N F
N
XVIII. CI
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~~N.
HN
Et0 ~ N
I
N
XIX. /
/~N
I
HN
~N
I
N I W
XX.
/~N
I
HN
~N F
I
N I /
XXI. CI
./' ~N
I
HN
~N F
I
N I /
XXII. CI
/~N
HN
~N F
~ y
N I /
XXIII. CI
~/~N
HN
C I ~N F
. N I /
XXIV. CI
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I ~N
NH
HN
Me0 ~ N O F
N
XXV. CI
HO / N
~ I
HN
Me0 ~ N F
I
N I/
XXVI. CI
~J
HN N
Me0 ~ ~ N F
I
N
XXVII. CI
~J
HN N
\N
I N ~ CI
XXVIII.
\J
HN N
I ~N F
i
N
XXIX.
/ I ~N
H N /~\~
~N
~N ~ ~ CI
XXX.
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31
/ N
~ I
NN
HO w N F
I / \
N.
~:XXI. CI
~~~N
HN
O \N F
I
Et N ~ ~ \
N I
CI
~/~N
HN
I ~N F
N I \.
~;XXIII. CI
\/~N
\ I
HN
O ~O I ~ N F
N N~ \
I/
'O
~;XXIV. CI
~/~N
HN
I
\N F
Me0 N
~:XXV. Cl
~~~N
HN
/0 ~ N F
I
GN N I \
CI
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32
~O / N
I
HN
Me0 -w N F
r
N
~X_XVII. CI
O / N
I
HN
Me0 w N F
r
N
/
X:XXVIII. CI
O
/~O~O / N
HN
MeO w N F
r
. N
XXXIX.
~/~N
I
HN
~O ~ N F
H2N N
CI
r~N
HN
~N F
r
N I /
XLI. CI
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33
~N
\ I
HN
I.~N F
r
N
XLII.
/ ~N
J('\~I
HN
I ~N
N ~ CI
I
XLIII.
~N
\ I
HN
~N
I r \
N
XLIV.
~N
\ I
HN
I wN
\ OMe
I ~
XLV.
~N
/\ I
HN
~N
N ~I /~
'OMe
XLVI.
~N
\ II
HN
~N
I r \
N
XLVII.
~N
I
HN
I ~N
Nr \ F
I
XLVIII.
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34
/~N
I
HN
~N
N ~'~~
F
XLIX.
/~N
\ I
HN
N CI
\
N
L.
/~N
\ I
HN
~N
N
v 'CI
LI.
/~N
\ II
HN
\N
N
LII.
/~N
\ I
HN
~N F
N \ F
LIII.
~ I
~~N
HN
~N F
N
F
LIV.
CA 02494367 2005-02-O1
WO 2004/010929 PCT/US2003/023240
/~N
.\ II
HN
~N F
N I,/
LV. F
/~N -
''\\\ II
HN
~N F
i
N
F
LVI.
~/~N
HN
~N F
N y
N
H CI
LVII.
I / N
HN
Me0 ~ N F
N
LVIII. CI
N
\ I
HN
F ~N F
N
LIX. CI
~N
HN
I
~N F
N ~ /
LX. CI
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36
O
Me0 , Y/ 'N
HN \ II
Me0 ~ N F
N
LXI. ~ CI
~/~N
HN
~~N F
N I
LXII. CI
~/~N
O HN
~N F
I
N I / ..
LXIII. CI
O
MeHN Y/ 'N
HN
Me0 w N F
I
N I /
LXIV. CI
N~N
HN
Me0 ~ N F
. N I /
LXV. CI
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37
~/~N
I
HN
~N F
y OMe I N \
i
LXVI. C)
Me0~0, ~ N
HN
Me0 ~ N F
~ \
N
LXVII. CI
\%~N
\ I
NO ~ HN
~N F
N
LXVIII. CI
O
H2N ~N .
HN \
Me0 w N F
N I \
LXIX. CI
J
HN N
Me0 ~ N F
\
N
LXX. CI
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38
MeO~ N
H
H
...__~ ~N F
N
LXXI. CI
O
NON / N
H
HN
Me0 ~ N F
N
LXXII. CI
(~N~O / N .
OJ ~ I
HN
Me0 w N F
N
LXXIII. CI
Another group of compounds for use in the methods of the present invention is
represented by the formula (3)
Y,
X1
,
,
3 s
Y
~2
Y4
Y6
wherein Y1 is phenyl or naphthyl optionally substituted with one or more
substituents selected from halo, alkoxy(1-6 C), alkylthio(1-6 C), alkyl(1-6
C), haloallcyl (1-6C),
O-(CH2)m Ph, -S-(CH2)m-Ph, cyano, phenyl, and C02R, wherein R is hydrogen or
alkyl(1-6 C),
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39
and m is 0-3; or phenyl fused with a 5- or 7-membered aromatic or non-aromatic
ring wherein
said ring contains up to three heteroatoms, independently selected from N, O,
and S:
Y2, Y3, Y4, and Ys independently represent hydrogen, alkyl(1-6C), alkoxy(1-6
C), haloalkyl(1-6 C), halo, NH2, NH-alkyl(1-6C), or NH(CH2)p Ph wherein n is 0-
3; or an
adjacent pair of YZ, Y3, Y4, and Ys form a fused 6-membered aromatic ring
optionally containing
up to 2 nitrogen atoms, said ring being optionally substituted by one o more
substituents
independently selected from alkyl(1-6 C), alkoxy(a-6 C), haloalkyl(1-6 C),
halo, NH2, NH-
alkyl(1-6 C), or NH(CH2)n Ph, wherein n is 0-3, and the remainder of Y2, Y3,
Y4, and Ys
represent hydrogen, alkyl(1-6 C), alkoxy(1-6C), haloalkyl(1-6 C), halo, NH2,
NH-alkyl(1-6 C),
or NH(CH2)"Ph wherein n is 0-3; and ~ .
one of Xl and X2 is N and the other is NR6, wherein R6 is hydrogen or alkyl(1-
6 C).
As used in formula (3), the double bonds indicated by the dotted lined
represent possible
tautomeric ring forms of the compounds. Further information about compounds of
formula (3)
and their preparation is disclosed in WO 02/40468, published May 23, 2002, the
entire
disclosure of which is hereby expressly incorporated by reference.
Yet another group of compounds for use in the methods of the invention is
represented by
Y1
X1
' ~' ~ Y
' ~ 3
'
>.
\ xz
Y~
the following formula (4)
wherein Yl is naphthyl, anthracenyl, or phenyl optionally substituted with one
or more substituents selected from the group consisting of halo, alkoxy(1-6
C), allcylthio(1-6 C),
alkyl(1-6 C), -O-(CH2)-Ph, -S-(CH2)"Ph, cyano, phenyl, and COZR, wherein R is
hydrogen or
alkyl(1- .6 C), and n is 0, l, 2, or 3; or Yl represents phenyl fused with an
aromatic or non-
aromatic cyclic ring of 5-7 members wherein said cyclic ring optionally
contains up to two
heteroatoms, independently selected from N, O, and S;
YZ is H, NH(CH2)"-Ph or NH-alkyl(1-6 C), wherein n is 0, 1, 2,
or 3;
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Y3 is CO2H, CONH2, CN, N02, alkylthio(1-6 C), -S02-alkyl(C1-6),
alkoxy(Cl-6), SONH2, CONHOH, NH2, CHO, CH2NH2, or C02R, wherein R is hydrogen
or
alkyl(1-6 C);
one of Xl and X2 is N or CR', and other is NR' or CHR' wherein R' is
hydrogen, OH, alkyl(C-16), or cycloalkyl(C3-7); or when one of Xl and XZ is N.
or CR' then the
other may be S or O.
Further details of the compounds of formula (4) and their modes of preparation
are
disclosed in WO 00/61576 published October 19, 2000, the entire disclosure of
which is hereby
expressly incorporated by reference.
The compounds of the formulas (1) - (4), may be supplied in the form of their
pharmaceutically acceptable acid-addition salts including salts of inorganic
acids such as
hydrochloric, sulfuric, hydrobromic, or phosphoric acid or salts of organic
acids such as acetic,
tartaric, succinic, benzoic, salicylic, and the like. If a carboxyl moiety is
present on the
compound of formula (1) - (4), the compound may also be supplied as a salt
with a
pharmaceutically acceptable cation.
The compounds of formulas (1) - (4) may also be supplied in the form of a
"prodrug"
which is designed to release the compound of formulas (1) - (4) when
administered to a subject.
Prodrug formed designs are well known in the art, and depend on the
substituents contained in
the compounds of formulas (1) - (4). For example, a substituent containing
sulflzydryl could be
coupled to a carrier which renders the compound biologically inactive until
removed by
endogenous enzymes or, for example, by enzymes targeted to a particular
receptor or location in
the subject.
In the event that any of the substituents in the above formulas contain chiral
centers, as
some, indeed, do, the compounds include all stereoisomeric forms thereof, both
as isolated
stereoisomers and mixtures of these stereoisomeric forms. .
Synthesis of the Compounds of the Invention
The compounds of formula (1) of the invention may be synthesized from the
corresponding 4-halo-2-phenyl quinazoline as described in Reaction Scheme 1;
which may be
obtained from the corresponding 4-hydroxyquinazoline as shown in Reaction
Scheme 2.
Alternatively, the compounds can be prepared using anthranylamide as a
starting material and
benzoylating the amino group followed by cyclization to obtain the
intermediate 2-phenyl-
4-hydroxy quinazoline as shown in Reaction Scheme 3. Reaction Schemes 4-6 are
similar to
Reaction Scheme 3 except that an appropriate pyridine or 1,4-pyrimidine
nucleus, substituted
with a carboxamide residue and an adjacent amino residue, is substituted for
the anthranylimide.
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41
The compounds of the invention Wherein R1 is H can be further derivatized to
comprise other
embodiments of Rl as shown in Reaction Scheme 7.
Reaction Scheme 1
I N / I
\ N\ \ 4-Aminopyridine/K ZC03/Reflux \ \ \
N
/N
CI ~ HN\
Reaction Scheme 1 is illustrative of the simple conversion of a halogenated
quinazoline
to compounds of the invention. Of course, the phenyl of the illustration at
position 2 may be
generalized as R3 and the 4-pyridylamino at position 2 can be generalized to
Ar'-L or Ar'-.
Reaction Scheme 2
/ cl , / cl
\ N\ \ I \ N\ \
SOCIZ/CHCI3/DMF/Reflux
/ /N / . /N
OH CI
/ CI / CI
\ N\ \ I \ N\ \
4-Aminopyridine/K ZC03/Reflux I
/ '/N / /N
CI , HN
/ N
Reaction Scheme 2 can, of course, be generalized in the same manner as set
forth for
Reaction Scheme 1.
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42
Reaction Scheme 3
0 0 0
cl
NHz F ~ i ~ NHZ
CHCI3 / Pyridine
NHZ NH
/ Ethanol / Reflux
HCI
F
F
~ CHCI3 / DMA
F
F
F F
4-Aminopyridine i KzC03 / Reflux
or
4.aminopyridine / TEA / DMF ! reflu~
Again, Reaction Scheme 3 can be generalized by substituting the corresponding
acyl
halide, R3COCl for the parafluorobenzoyl chloride. Further, Ar' or Ar'-L may
be substituted for
4-aminopyridine in the' last step.
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43
Reaction Scheme 4
o off
NHz ~ 2 ~ ~ \N
->
N"NHz N' -N- _R3
CI L-Ar'
~N 4 ~ ~ \N
N/ N' -R3 N/ N~R3
1. Acid chloride / Chloroform / Pyridine
2. Sodium Hydroxide (aqueous) / Ethanol / Reflux
3. Thionyl chloride l Chloroform / DMF
4. Nucleophile (Amine, Alcohol), TEA, DMF / Reflux
Reaction Scheme 5
O OH
N\ NHz Z ~ N\ \ N
NHz ~ ~ Rs
CI L-Ar'
3 N\ \ N 4 ~ N\ \ N
N/ R3 / Ra
1. Acid chloride I Chloroform / Pyridine
2. Sodium Hydroxide (aqueous) / Ethanol / Reflux
3. Thionyl chloride / Chloroform / DMF
4. Nucleophile (Amine, Alcohol), TEA, DMF / Reflux
Reaction Scheme 6
' O O OH
N\ NHz ~ ~ N\ NHz 2 ~ N\ \ N
N/ NHz N NH N/ N"R3
O R
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44
Ci L-Ar'
N . N\ \
\ \N 4 I ~N
N/ N% \Rs N/ N% \Rs
1. Acid chloride / Chloroform / Pyridine
2. Sodium Hydroxide (aqueous) / Ethanol / Reflux
3. Thionyl chloride / Chloroform / DMF
4. Nucleophile (Amine, Alcohol), TEA, DMF l Reflux
It is seen that Reaction Scheme 1 represents the last step of Reaction Schemes
2-6 and
that Reaction Scheme 2 represents the last two steps of Reaction Scheme 3-6.
Reaction Scheme 7 provides conditions wherein compounds of formula (1) are
obtained
wherein Rl is other than H.
Reaction Scheme 7
' N I
N\ ~ I ~. \
4-Methoxybenzylchloride/KOH/ / N
/ N Acetone/Reflux
HN ~ N ~
I 1 / I /~
I
~o
Reaction Scheme ~ is a modification of Reaction Scheme 3 which .simply
demonstrates
that substituents on ring A are carried through the synthesis process. The
principles of the
behavior of the substituents apply as well to Reactions Schemes 4-6.
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0 0
OOH ~ OOH
z I z I
H / NHz _
Reaction Scheme 8 shows a modified form of Reaction Scheme 3 which includes
substituents R2 in the quinazoline ring of formula (1). The substituents are
carried throughout
the reaction scheme. In step a, the starting material is treated with thionyl
chloride in the
presence of methanol and refluxed for 12 hours. In step b, the appropriate
substituted benzoyl
chloride is reacted with the product of step a by treating with the
appropriately substituted
benzoyl chloride in pyridine for 24 hours. In embodiments wherein X (shown
illustratively in
the ortho-position) is fluoro, 2-fluorobenzoyl chloride is used as a reagent;
where X is (for
illustration ortho-chloro), 2-chlorobenzoyl chloride is used.
In step c, the ester is converted to the amide by treating in ammonium
hydroxide in an
aprotic solvent such as dimethyl formamide (DMF) for 24 hours. The product is
then cyclized in
step d by treatment with 10 N NaOH in ethanol and refluxed for 3 hours.
The resulting cyclized form is then converted to the chloride in step a by
treating with
thionyl chloride in chloroform in the presence of a catalytic amount of DMF
under reflex for 4
hours. Finally, the illustrated 4-pyridylamino compound is obtained in step f
by treating with 4-
amino pyridine in the presence of potassium carbonate and DMF and refluxed for
2 hours.
In illustrative embodiments of Reaction Scheme 8, R~' may, for example,
provide two
methoxy substituents so that the starting material is 2-amino-4,5-dimethoxy
benzoic acid and the
product is, for example, 2-(2-chloxophenyl)-4-(4-pyridylamino)-6,7-
dimethoxyquinazoline.
Reaction Scheme 8
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46
In another illustrative embodiment, RZ provides a single nitro; the starting
material is
thus, for example, 2-amino-5-nitrobenzoic acid and the resulting compound is,
for example, 2-
(2-fluorophenyl)-4-(4-pyridylamino)-5-nitroquinazoline.
Reaction Schemes 4-6 can be carried out in a manner similar to that set forth
in Reaction
Scheme 8, thus carrying along R2 substituents through the steps of the
process.
In compounds of the. invention wherein R2 is nitro, the nitro group may be
reduced to
amino and further derivatized as indicated in Reaction Scheme 9.
Reaction Scheme 9
g
HN \
HN
~N F
N
In Reaction Scheme 9, the illustrative product of Reaction Scheme 8 is first
reduced in
step g by treating with hydrogen and palladium on carbon (10%) in the presence
of acetic acid
and methanol at atmospheric pressure for 12 hours to obtain the amino
compound. The resulting
amino compound is either converted to the acyl form (R=acyl) using the
appropriate acid
chloride in the presence of chloroform and pyridine for four hours, or is
converted to the
corresponding alkylated amine (R=alkyl) by treating the amine intermediate
with the appropriate
aldehyde in the presence of ethanol, acetic acid, and sodium
triacetoxyborohydride for 4 hours.
While the foregoing exemplary Reaction Schemes are set forth to illustrate the
synthetic
methods of the invention, it is understood that the substituents shown on the
quinazoline ring of
the products are generically of the formula (1) as described herein and that
the reactants may be
substituted accordingly. Variations to accommodate various substituents which
represent
embodiments of R3 other thaai the moieties shown in these illustrative
examples or as Ar' in these
illustrative examples may also be used. Similarly, embodiments wherein the
substituent at
\~
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47
position 4 contains an arylalkyl can be used in these schemes. Methods to
synthesize the
compounds of the invention are, in general, known in the art.
Thus, a number of synthetic routes may be employed to produce the compounds of
formula (2). In general, they may be synthesized using reactions lcnovim in
the art. One useful
method, especially with regard to embodiments which contain nitrile
substitutions (which also,
of course, can be hydrolyzed to the corresponding carboxylic acids or reduced
to the amines) is
shown in Reaction Scheme 10, shown below. In Reaction Scheme 10, an
intermediate wherein
the pyrimidine ring is halogenated is obtained; the halide is then displaced
by an aryl amine. In
this method, the pyrimidine ring is generated in the synthetic scheme,
resulting in the compound
formed in reactions labeled a.
Reaction Scheme 10
O ~ CI
NC CN NC CN NC I NH F NC i ~ N F
Me N"SMe Me N N ~ Me2N N
MeS SMe z 2
b
/ N
p CI ~ I
HN
NC NH F NC ~ N F a
NC
MeS I N ~ ~ I ~ N F
MeS N I / ~ / R N
R = SMe, NMe2
In Reaction Scheme 11, the pyrimidine ring is obtained by cyclizing an amido
moiety
and, again, a halo group on the pyrimidine ring is displaced by an aryl amide
to obtain the
compounds of the invention in step b. Further substitution on the resulting
invention compound
can then also be performed as shown in subsequent steps bl, b2, and b3.
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48
Reaction Scheme 11
OH CI
F F NH
Me0 / N F MeO / N F
CN I ~ NH2 \N I
~ w N ~ w
i
/
CI CI
CI CI
b
N ~ N ~~ N
HN HN
b3 O ~ N F bz HO ~ N F b1 M
I ~ I
PhthN \N ~ ~N
CI CI
Reaction Schemes 12, 13, 14 and 15, shown below, provide alternative routes to
the
pyrimidine nucleus, and further substitution thereof.
Reaction Scheme 12
OH CI CI
F NH / N F / N F
~ ~N F
. NH2 HO \N I
/ '' I ~ ~ CI N I ~ -"~ Me0 N I w
/ / /
CI
CI CI CI
Reaction Scheme 13
OH OH
F NH
/~N F / ~N F
NH2
H2N N I ~ -' AcHN N I
CI
CI CI
Reaction Scheme 14
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49
N~ N~ Nw
Me0 I / RHN
HN O O HN O O HNUO
o
0
N
Me0 I / R = H, Me,
~ N~
O NH2 ~'~,.,~~OMe RHN I /
.TFA ~O O NH2
~-~NJ
.TFA
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Reaction Scheme 15
N\ I N~ I Nw
/ HO ~ R~ /
N O ~ HN~O HN~O
1
p ,
N\ R = Me, O
OMe
RO / ~~'~ ~~r'
NH2
.TFA . NFmoc ~NFmoc
~J N
. '''w'~ . 'fir
Small organic molecules other than quinazoline derivatives can be synthesized
by well
known methods of organic chemistry as described in standard textbooks.
Methods of treatment
There are numerous conditions and diseases that require or may benefit from
the
improvement of lung function, including, without limitation, emphysema,
chronic bronchitis,
chronic obstructive pulmonary disease (COPD), pulmonary edema, cystic
fibrosis, occlusive
lung disease, acute respiratory deficiency syndrome CARDS), asthma, radiation-
induced injury of
the lung, and lung injuries resulting from other factors, such as, infectious
causes, inhaled toxins,
or circulating exogenous toxins, aging and genetic predisposition to impaired
lung function.
Chronic bronchitis, emphysema and COPD are typically associated with cigarette
smoking, and often coexist, causing abnormalities in lung structure and
function, and obstruction
of air flow, negatively impacting the quality of life of patients. COPD is
commonly used to
describe a spectrum of conditions, diseases and symptoms that may occur
individually or in
combination, including, for example, chronic obstructive bronchitis,
emphysema, and chronic
airway obstruction. Over the time, as the diseases progress, gradually more
serious symptoms
can develop. COPD is currently the fourth leading cause of death in the United
States.
Current treatments of COPD, and related conditions that require or benefit
from the
improvement of lung function, include the administration of bronchodilators,
such as (3-
adrenergic agonists, anticholinergic agents, and theophylline, and
corticosteroid therapy,
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51
although the. benefits of these and similar treatments vary from patient to
patient, and long term
benefits. have not been clearly demonstrated.
According to the present inventions, the foregoing diseases and other lung
conditions that
require or benefit from the improvement of lung function are treated by
administration of small
molecules specifically binding to the type I TGF-(3 receptor (TGF(3-Rl).
The manner of administration and formulation of the compounds useful in the
invention
and their related compounds will depend on the nature of the condition, the
severity of the
condition, the particular subject to be treated, and the judgement of the
practitioner; formulation
will depend on mode of administration. The small molecule compounds of the
invention are
conveniently. administered by oral administration by compounding them with
suitable
pharmaceutical excipients so as to provide tablets, capsules, syrups, and the
like. Suitable
formulations for oral administration may also include minor components such as
buffers,
flavoring agents and the like. Typically, the amount of active ingredient in
the formulations will
be in the range of about 5%-95% of the total formulation, but wide variation
is permitted
depending on the carrier. Suitable carriers include sucrose, pectin, magnesium
.stearate, lactose,
peanut oil, olive oil, water, and the like.
The compounds useful in the invention may also be administered through
suppositories
or other transmucosal vehicles. Typically, such formulations will include
excipients that
facilitate the passage of the compound through the mucosa such as
pharmaceutically acceptable
detergents.
The compounds may also be administered topically, for topical conditions such
as
psoriasis or ophthalmic treatments, or in formulation intended to penetrate
the skin or eye. These
include lotions, creams, ointments, drops and the like which can be formulated
by known
methods.
The compounds may also be administered by injection, including intravenous,
intramuscular, subcutaneous, intrarticular or intraperitoneal injection.
Typical formulations for
such use are liquid formulations in isotonic vehicles such as Hank's solution
or Ringer's
solution.
Alternative formulations include aerosol inhalants, nasal sprays, liposomal
formulations,
slow-release formulations, and the like, as are known in the art.
A preferred route of administration for the treatment of a disease or
condition that
requires or benefits from the improvement of lung function is aerosol
delivery. Aerosol delivery
to various parts of the respiratory tract, including the lungs has been
extensively used for
delivery of various pharmaceutical agents. Pharmaceutical agents, including
small molecule
drugs, are generally delivered to the respiratory tract in the form of a fine
mist or aerosol which
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52
is breathed into the lungs through the nose or mouth of the patient.
Typically, a nebulizer is used
to convert a liquid into a fine aerosol, and the aerosol is introduced into
the lungs by means of a
mouthpiece which delivers the aerosol through the mouth only, or by means of a
face mask
which delivers the aerosol through both the mouth and nose of the patient. The
first commercial
inhaleable systems developed were developed in the early 1950s, and dispensed
drugs for
treating asthma or COPD. Various aerosol inhalation devices have been
developed and are
disclosed, for example, in U.S. Patent Nos. 4,823,784; 4,106,503; 4,677,975;
and 5,479,920.
Inhalation devices suitable for the purposes of the present invention
specifically include metered
dose inhalers (MDIs), nebulisers, and dry powder inhalers (DPIs). A
particularly preferred route
of administration is intrapulmonary delivery directly to the lungs. The deep
lung epithelium,
composed of a thin, nonciliated, mucus-free cell layer, offers a very
efficient port of entry for the
direct delivery of pharmaceuticals, such as small molecule drugs, directly
into the patient's blood
stream.
The pharmaceutical compositions of the present invention can be prepared by
art-known
methods, such as those disclosed in Remin~ton's Pharmaceutical Sciences,
latest edition, Mack
Publishing Company, Easton, PA. Reference to this manual is routine in the
art.
The dosages of the compounds of the invention will depend on a number of
factors which
will vary from patient to patient. However, it is believed that generally, the
daily oral dosage
will utilize 0.001-100 mg/kg total body weight, preferably from 0.01-50 mglkg
and more
preferably about 0.01 mg/kg-10 mg/kg. The dose regimen will vary, however,
depending on the
conditions being treated and the judgment of the practitioner.
It should be noted that the compounds of formula (1) can be administered as
individual
active ingredients, or as mixtures of several embodiments of this formula. In
addition, the
TGF-(3 inhibitors can be used as single therapeutic agents or in combination
with other
therapeutic agents. Drugs that could be usefully combined with these compounds
include natural
or synthetic corticosteroids, particularly prednisone and its derivatives,
bronchodilators,
monoclonal antibodies targeting cells of the immune system or genes associated
with the
development or progression of pulmonary diseases, and small molecule
inhibitors of cell
division, protein synthesis, or mRNA transcription or translation, or
inhibitors of immune cell
differentiation or activation.
As implicated above, although the compounds of the invention may be used in
humans,
they are also available for veterinary use in treating non-human mammalian
subjects.
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Animal Models
Prior to administration to human or veterinary patients, the safety and
efficacy of small
molecule drug candidates is typically tested in ifz vita°o and in vivo
assays, including animal
models of the target disease.
In view of the similarities in lung development and lung structure between
humans and
other mammals, animal models can provide valuable insights into the
pathogenesis of diseases
and conditions characterized by reduced or compromised lung function, and may
be developed
for testing drug candidates. In particular, the mice have been considered as
prefeiTed for
developing animal models because the mouse genome has been extensively studied
and
sequences, and close similarities exist with the human genome. Since many of
the lung
conditions benefiting from the improvement of lung function are, associated
with smoking, and
have complex etiologies that are not clearly understood, traditionally
meaningful animal models
have been scarce. However, in recent times several groups have made
significant progress to
remedy this situation.
Animal models of COPD have been discussed at the First International
Conference on
Animal Models of Chronic Obstructive Pulmonary Disease, Certosa di Pontignano,
University of
Siena, Italy, September 30-October 2, 2001. A meeting report authored by David
Hele has been
published in Respir Res 3:12 (2002).
Emphysema-induced changes in lung function can be demonstrated in the rat,
using
elastase to generate an emphysematous pathology.
Smoking models have been developed by several laboratories. Cigarette smoke-
induced
lesions in animal models have been shown to be similar to those observed in
humans. Mice,
such as B6C3F1 mice, were demonstrated to show an inflammatory and
emphysematous
response to chronic exposure to cigarette smoke. After long term exposure to
cigarette smoke,
A/J mice showed a faster development of emphysema than C57BI/6J mice used for
comparison.
Other researchers have suggested that it is important to check the
antiprotease and antioxidant
status of an animal strain before establishing an animal model of COPD. It has
been shown that
G57BI/6J and DBA/2J mice (reduced antielastase and increased sensitivity to
antioxidants) were
more responsive to cigarette smoke exposure than were ICR mice, which have a
normal level of
antielastase and lack sensitivity to antioxidants.
In ,non-cigarette smoke driven models ozone, lipopylsaccharides, sulphur
dioxide,
nitrogen dioxide, diesel particles, and the like have been used to produce
aspects of COPD, such
as cough, inflammation, and mucus hypersecretion.
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Transgenic animal, e.g. mouse models are also known in the art. For example,
the
development of spontaneous emphysema has been described in the pallid mouse,
an animal that
has reduced elastase inhibitory capacity. Emphysema development can be
accelerated by
treatment with formyl-methionyl-leucyl phenylalanine or exposure to cigarette
smoke.
For further discussion of animal models of COPD see, also Dawkins and
Stockley,
Thorax 56:973-977 (2001).
Further details of the invention will be apparent from the following non-
limiting
examples.
Example 1
Effect of a Representative Compound of Formula (1) on Resbiratory Rate, Tidal
Volume and
Total BALF IL-6 in a 5-Day Bleomycin-Induced Lung Iniury Model
Material and Methods
Ani~rzal Model
Male Sprague-Dawley rats weighing 225 to 250 were purchased from Charles River
Laboratories, Inc. Rats were housed in groups of two in an animal facility
provided with filtered
air and constant temperature and hwnidity. All animal maintenance was in
accordance with
Scios' guidelines for animal welfare. The rats were allowed to acclimate to
the new environment
for one week before treatment. A 12:12 hour light-dark cycle was maintained,
and the animals
had free access to ad libitum food and water.
P~~otocol
Group n Rxl Rx2 (1 Day after
Rx 1 )
1 24 Saline 1% MC
2 24 Bleomycin1 % MC
3 6 Bleomycin10 ,mg/kg
Com ound A
4 12 Bleomycin30 mg/kg
Compound A
24 Bleomycin60 mg/kg
Compound A
Ti°eatment Pf~otocol
Day 0: To induce pulmonary injury, rats were intubated with 0.5 ml of saline
or O.SmI of
2.0 mit/ml of bleomycin by intratracheal injection under anesthesia. The
anesthetic,solution
used was a mixture of 0.4 ml of ketamine (100 mg/ml) and 0.25 ml of xylazine
(20 mg/ml) at a
dose of 1.3 ml/kg.
Day 1 to Day 5:
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Group 1 & 2: Rats were weighed and orally dosed with 5 ml/kg of 1 % methyl
cellulose (MC) two times a day.
Group 3: Rats were weighed and orally dosed with 5 ml/kg of 2.0 mg/ml of
Compound A. two times a day.
Group 4: Rats were weighed and orally dosed with 5 mI/kg of 6.0 mg/mI of
Compound A two times a day.
Group 5: Rat were weighed and orally dosed injected with 5 ml/kg of 12.0
mg/ml of Compound A two times a day.
Day 4: After dosing, rats were placed in the Buxco system to measure lung
functions.
Day 5: After dosing, rats were sacrificed, BALF was collected and stored at -
80°G
for IL-6 analysis.
Lun~Functions
To measure other lung functions, the Buxco whole body plethysmograph system
was
used (Buxco Electronics, Inc., Sharon, CT), to measure respiratory rate, and
tidal volume.
Briefly, the Buxco system was first calibrated, then rats were placed into the
whole body
unrestrained plethysmographs for . l hour to be acclimatized, and then lung
functions were
continuously collected for 30 minutes by the BioSystem XA for Windows
Software.
B~°ofzclzoalveolaf° lava~e fluid BALF) Collections
Rats were sacrificed by overdose of ketamine/xylazine cocktail, and then
trachea, heart
and lung were removed en bloc. BALF was collected from the lungs slowing
injecting 4 ml of
1X PBS into the lungs and slowly withdrawing the 1X PBS out of the lungs. This
process is
repeated for three times. BALF was then centrifuged at 4°C for 15
minutes at 3000 rpm. The
supernatant was saved and stored at -80°C for measurement of IL-6, and
protein.
Detefrfzirzation AIL-6
Total BALF IL-6 was measured by the R & D System ELISA Kit (cat #: R6000).
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Statistical analysis
The data were analyzed using a one-way analysis of variance (ANOVA) with a
Bonferroni's multiple comparisons post tests. . A value of p <_ 0.05 was
considered statistically
significant. Values are reported as mean ~ SD.
Results:
The results are illustrated on Figures 1-3.
Figure 1 shows the respiratory rate 'measured in control (MC-treated) and
bleomycin-
treated animals as well as animals treated with 10 mg/kg, 30 mg/kg, and 60
mg/kg doses of
Compound A following bleomycin treatment as described above. In Figure 1 * * *
indicates
p<0.001, and * indicates p<0.05. The first and second graphs show that
bleomycin significantly
increases the respiratory rate in rats (Saline+1 % MC versus Bleo+1 % MC)
relative to saline-
treated control animals. The Figure further shows that Compound A
significantly reduces
respiratory rate induced by bleomycin (Bleo+1% MC versus Bleo-Compound A).
Figure 2 shows the tidal volume measured in control (MC-treated) and bleomycin-
treated
animals as well as animals treated with 10 mg/kg, 30 mg/kg, and 60 mg/kg doses
of Compound
A following bleomycin treatment as described above. In Figure 2 * * *
indicates p<0.001, and *
indicates p<0.01. The first and second graphs show that bleomycin
significantly decreases tidal
volume in rats (Saline+1% MC.versus Bleo+1% MC) compared to saline-treated
control. The
Figure further shows that treatment with Compound A significantly increases
tidal volume
induced by bleomycin (Bleo+1% MC versus Bleo-Compound A).
Figure 3 shows the effect of Compound A on total BALF IL-6 induced by
bleomycin. In
Figure 3 * indicates p<0.05; ** indicates p<0.01; and ***indicates p<0.001.
The first and
second graphs show that bleomycin significantly increases total BALF IL-6 in
rats (Saline+1%
MC versus Bleo+1% MC). The Figure further shows that treatment with Compound A
significantly decreases total BALF IL-6 induced by bleomycin (Bleo+1 % MC
versus Bleo-
Compound A (10), bleo+1% MC versus Bleo-Compound A (30), bleo+1% MC versus
Bleo-
Compound A (60)).
Conclusion:
Bleomycin-treated rats that dosed with Compound A show a significant
improvement in
lung functions, and a significant decrease in total BALF IL-6 compared to
bleomycin-treated rats
orally dosed with the 1 % MC. Since these data were obtained in a 5-day
bleomycin study, they
> are indicative of the ability of Compound A to improve lung function
following acute lung
injury, before the development of fibrosis.
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Example 2
Effect of a Representative Compound of Formula (11 on Total Lung Capacity and
Lung
Permeability in a 5-Day Bleomycin-Induced Lung Iniury Model
Material and Methods
Animal Model
Male Sprague-Dawley rats weighing 225 to 250 were purchased from Charles River
Laboratories, Inc. Rats were housed in groups of two in the animal facility
provided with
filtered air and constant temperature and humidity. All animal maintenance was
in accordance
with Scios' guidelines for aumal welfare. The rats were allowed to acclimate
to the new
environment for one week before all treatment. A 12:12 hour light-dark cycle
was maintained,
and the animals had free access to ad libitum food and water.
Protocol
Group n Rxl Rx2 (1 Day after
Rx 1
1 4 Saline 1% MC
2 4 Bleomycin 1 % MC
3 4 Bleomycin 60 mg/kg
Compound A
Ti°eatment P~°otocol
Day 0: To induce pulmonary injury, rats were intubated with 0.5 ml of saline
or O.SmI of
2.0 unit/ml of bleomycin by intratracheal injection under anesthesia. The
anesthetic solution
used is a mixture of 0.4 ml of ketamine (100 mg/ml) and 0.25 mI of xylazine
(20 mg/ml) at a
dose of 1.3 ml/kg.
Day 1 to Day 5:
Group 1 & 2: Rats were weighed and orally dosed with 5 mllkg of 1% methyl
cellulose (MC) two times a day.
Group 3: Rats were weighed and orally dosed with 5 ml/kg of 12.0 mg/ml of
Compound A two times a day.
Day 5: After dosing, rats were injected intravenously with 3ml/kg of 10 mg/ml
of
rhodamine labeled dextran. Two hours after inj ection of rhodamine labeled
dextran, rats were
sacrificed, and lungs were inflated and fixed for histological analysis
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Lung Functions
To estimate total lung capacity, lungs were inflated with 4% formalin .at a
constant
pressure of 15 cm of water. Total lung capacity is equal to the volume of 4%
formalin used to
inflate the lung. The maximum volume to inflate the lung is 10 ml.
HistoloQ-v
Lungs were first inflated with 4% formalin at a constant pressure of 15 cm of
water and
the maximum volume to be inflated is 10 ml. After inflation, the inflated
lungs were then fixed
in 10% fonnalin for 48 hours. Each lung was cut into seven segments and each
segment was
embedded in O.C.T. Two six micrometer frozen sections were cut from each
segment. One for
H & E stain and one unstained for rhodamine labeled dextran analysis.
Tissues analyses were totally blinded and randomized using the NikonE600
microscope
equipped with spot digital camera aided by Image-Pro-Plus 4.5 software. To
examine the
vascular permeability in the alveolar wall, tissues were analyzed for the
presence of the positive
rhodamine-~i-isothiocyanate (RITC)-labeled dextran under the NikonE600
fluorescence ,
microscope using rhodamine filter at magnification of 600X. Ten fields from
each seven
sections (70 fields . per lung) were evenly chosen and the positive
fluorescent signals were
measured by Image-Pro-Plus-Mirco.
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Statistical analysis
The data were analyzed using a one-way analysis of variance (ANOVA) with a
Bonferroni's multiple comparisons post tests. A value of p <_ 0.05 was
considered statistically
significant. Values are reported as mean ~ SD.
Results:
The effect of Compound A on total lung capacity following bleomycin-induced
lung
injury is shov~m in Figure 4. In Figure 4, ** indicates p<O.Ol;.and ***
indicates p<0.001. The
first two graphs show that bleomycin significantly decreases total lung
capacity in rats
(Saline+1 % MC versus Bleo+1 % MC). The third graph shows that treatment with
Compound A
as described above significantly increases total lung capacity induced by
bleomycin (Bleo+1
MC versus Bleo-Compound A (60)).
The effect of Compound A on lung permeability following bleomycin-induced lung
injury is shown in Figure 5. In Figure 5, * * * represents p<0.0001. The first
two graphs show
that bleomycin significantly increases lung permeability in rats (Saline+1% MC
versus Bleo+1%
MC). The third graph shows that treatment with Compound A as described above
significantly
decreases lung permeability induced by bleomycin (Bleo+1 % MC versus Bleo-
Compound A
(60)).
Figure 6 shows the effect of Compound A on lung permeability following
bleomycin-
induced lmg injury, as measured by fluorescence following RITC-dextran
administration to rats
as described above.
Figure 7 shows H & E stained tissue sections after bleomycin treatment and
subsequent
treatment with Compound A. The tissue sections clearly show that treatment
with Compound A
reduces tissue damage in bleomycin 5-day rat lung injury model.
Conclusion:
Bleomycin-treated rats orally dosed with Compound A show a significant
improvement
in lung function, and a significant decrease in lung permeability compared to
bleomycin-treated
rats orally dosed with the 1%' MC. Since these data were obtained in a 5-day
bleomycin study,
they are indicative of the ability of Compound A to improve lung function
following acute lung
injury, before the development of fibrosis.
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Example 3
Effect of a Representative Compound of Formula (11 on Lung Hyroxyproline
Content Following
Bleomycin-Induced Lung Fibrosis
Material and Methods
Anin2al Model
Male Sprague-Dawley rats weighing 225 to 250 were purchased from Charles River
Laboratories, Inc. Rats were housed in groups of two in the animal facility
provided with
filtered air and constant temperature and humidity. All animal maintenance was
in accordance
with Scios' guidelines for animal welfare. The rats were allowed to acclimate
to the new
environment for one week before all treatment. A 12:12 hour light-dark cycle
was maintained,
and the animals had free access to ad libitum food and water.
Protocol
Group n Rxl Rx2 (1 Day after
Rx 1 )
1 6 No Rx No Rx
2 8 Saline Saline
3 8 BleomycinSaline
4 7 Bleomycin30 mg/kg
Compound B
5 10 Bleomycin8 mg/kg
Triamcinolone
Ti°eatment Protocol
Day 0: To induce pulmonary fibrosis, rats were intubated with 0.5 ml of saline
or O.SmI
of 1.0 unit/ml of bleomycin by intratracheal injection under anesthesia. The
anesthetic solution
used is a mixture of 0.4 ml of ketamine (100 mg/ml) and 0.25 ml of xylazine
(20 mg/ml) at a
dose of 1.3 ml/kg.
DayltoDayl4:
Group 1: Rats were weighed daily
Group 2 & 3: Rats were weighed and orally dosed with 5 ml/kg of saline
three times a day
Group 4: Rats were weighed and orally dosed with 5 ml/kg of 6.0 mg/ml
of Compound B three times a day
Group 5: Rats were weighed and intraperitoneally injected with 1 ml/kg of
8 mg/ml of Triamcinolone every other day
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Day 14: After dosing, rats were sacrificed by overdose of the
ketamine/xylazine cocktail,
and then trachea, heart and lung were removed en bloc. All lung lobes were
dissected and
collected and stored in -80°C for hydroxyproline assays.
Detennzination ofHydroxyy°oline
To estimate the total amount of collagen in fibrotic lungs, the hydroxyproline
content of
the whole Lung was measured in each group according to the method described by
Woessner
Biochim Biophys Acta. 1967 Jun 27;140(2):329-38.
Briefly, hulgs were harvested and homogenized in 15 ml of 1X PBS with a
Polytron
homogenizer. Each sample (1 ml) was digested in 2 ml of 6 N HCl for 18 hours
at 110C. The
samples were neutralized with 3 N NaOH. The hydroxyproline content was the
measured using
the method of Woessner.
Statistical analysis
The data were analyzed using a one-way analysis of variance (ANOVA) with a
Bonferroni's multiple comparisons post tests. A value of p <_ 0.05 was
considered statistically
significant. Values are reported as mean ~ SD.
Results:
Figure 8 shows that both Triamcinolone and Compound B attenuated bleomycin-
induced
lung fibrosis in rats by significantly reducing lung hydroxyproline content.
In Figure 8, **
represents p<0.01, and * * * represents p<0.001. The first three graphs
demonstrate that
bleomycin significantly increased the amount of hydroxyproline in rats
(Saline+Saline versus
Bleo+Saline). The third and fourth graphs show that both Triamcinolone and
Compound B
attenuated the effect of bleomycin on the amount of hydroxyproline as the
amount of
hydroxyproline in the bleo+093 and Bleo+Triam groups were significantly less
than bleo+saline.
Conclusion:
Compound B attenuated bleomycin-induced lung fibrosis in rats by significantly
reducing
Iung hydroxyproline content.
Example 4
Effect of a Representative Compound of Formula (1) on Total Lung Capacity and
Lung Fibrosis
in a 14-Da~Bleomycin-Induced Lung Injury Model
Material and Methods
> Anin2al Model
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Male Sprague-Dawley rats weighing 225 to 250 were purchased from Charles River
Laboratories, Inc. Rats were housed in groups of two in the animal facility
provided with
filtered air and constant temperature and humidity. All animal maintenance was
in accordance
with Scios' guidelines for animal welfare. The rats were allowed to acclimate
to the new
environment for one week before all treatment. A 12:12 hour light-dark cycle
was maintained,
and the animals had free access to ad libitum food and water.
Protocol
Group n Rxl Rx2 (1 Day after
Rx 1
1 4 Saline 1% MC
2 4 Bleomycin1% MC
3 3 Bleomycin60 mg/kg
Compound A
Ty~eatnzent P~°otocol
Day 0: To induce pulmonary fibrosis, rats were intubated with 0.5 ml of saline
or O.SmI
of 2.0 unithnl of bleomycin by intratracheal injection under anesthesia. The
anesthetic solution
used is a mixture of 0.4 ml of ketarnine (100 mg/ml) and 0.25 ml of xylazine
(20 mg/ml) at a
dose of 1.3 ml/kg.
Day 1 to Day 14:
Group 1 & 2: Rats were weighed and orally dosed with 5 ml/kg of 1%
methyl cellulose (MC) two times a day.
Group 3: Rats were weighed and orally dosed with 5 ml/kg of 12.0 mg/ml
of Compound A two times a day.
Day 14: After dosing, rats were sacrificed by overdose of the
ketamine/xylaziiie cocktail,
and lungs were inflated and fixed for histological analysis
LungFunctions
To estimate total lung capacity, lungs were inflated with 4% formalin at a
constant .
pressure of 15 cm of water. Total lung capacity is equal to the volume of 4%
formalin used to
inflate'the lung. The maximum volume to inflate the lung is 10 ml.
HistoloQy
Lungs were first inflated with 4% formalin at a constant pressure of 15 cm of
water and
then fixed in 10% formalin for 48 hours. Each lung was cut into seven segments
and each
segment was embedded in O.C.T. Six micrometer sections were cut from each
segment. The
slides were stained for H & E and trichrome for imaging analysis.
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Imaging analysis was totally bliizded and randomized using the NikonE600
microscope
equipped with spot digital camera aided by Image-Pro-Plus 4.5 software.
Statistical analysis
The data were analyzed using a one-way analysis of variance (ANOVA) with a
Bonferroni's multiple comparisons post tests. A value of p <_ 0.05 was
considered statistically
significant. Values are reported as mean ~ SD.
Results:
Figure 9 shows the effect of Compound A on total lung capacity following
bleomycin-
induced lung fibrosis. In the Figure, ** represents p<0.41. As shown in Figure
S, bleomycin
significantly decreases total lung capacity in rats (Saline+1% MC versus
Bleo+1% MC), and
Compound A significantly increases total lung capacity induced by bleomycin
(Bleo+1 % MC
versus Bleo-Compound A (60)).
Figure 10 shows that bleomycin induces lung fibrosis in rats (Saline+1% MC
versus
Bleo+1% MC), and Compound A significantly reduces lung fibrosis induced by
bleomycin
(Bleo+1 % MC versus Bleo-Compound A (60)).
Figures 11 and 12 are histology pictures showing that treatment with Compound
A
reduces fibrosis in this 14-day bleomycin rat lung injury model.
Example 5
Identif~n~ Compounds for Use in the Methods of the Invention
Compounds that are useful for the invention can be tested for their ability to
inhibit TGF-
~i by a TGF(3-Rl autophosphorylation protocol. This was conducted as follows:
Compound
dilutions and reagents were prepared fresh daily. Compounds were diluted from
DMSO stock
solutions to 2 times the desired assay concentration, keeping final DMSO
concentration in the
assay less than or equal to 1%. TGF~i-Rl was diluted to 4 times the desired
assay concentration
in buffer + DTT. . ATP was diluted into 4xreaction buffer, and gamma-33P-ATP
was added at
60uCihnL.
The assay was performed by adding 10,1 of the enzyme to 20,1 of the compound
solution. The reaction was initiated by the addition of l Op.l of ATP mix.
Final assay conditions
included lOuM ATP, 170nM TGF Rl, and 1M DTT in 20mM MOPS, pH7. The reactions
were
incubated at room temperature for 20 minutes. The reactions were stopped by
transferring 231
of reaction mixture onto a phosphocellulose 96-well filter plate, which had
been pre-wetted with
l5ul of 0.25M H3P04 per well. After 5 minutes, the wells were washed 4x with
75mM H3P04
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and once v~ith 95% ethanol. The plate was dried, scintillation cocktail was
added to each well,
and the wells were counted in a Packard TopCount microplate scintillation
counter.
All references cited throughout the specification are expressly incorporated
herein by
reference. While the present invention has been described with reference to
the specific
embodiments thereof, it should be understood by those skilled in the art that
various changes
may be made and equivalents may be substituted without departing from the true
spirit and scope
of the invention. In addition, many modifications may be made to adapt a
particular situation,
material, composition of matter, process, and the like. All such modifications
are within the
scope of the claims appended hereto.
