Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
Integrin Receptor Antagonists
FIELD OF THE INVENTION
This invention relates to pharmaceutically active compounds which bind to
integrins, such as the vitronectin receptor and fibrinogen receptor. Such
compounds
are useful for inhibiting platelet aggregation and osteoclast attachment to
bone.
BACKGROUND OF THE INVENTION
Integrins are a family of heterodimeric proteins which generally mediate cell
adhesion. Typical of such proteins are the vitronectin receptor (an av(33
heterodimer) and the fibrinogen receptor (an aIIb~3 heterodimer). The natural
ligands of these receptors (e.g., vitronectin and fibrinogen) have been found
to share
a common -Arg-Gly-Asp- amino acid sequence, which appears to be critical for
binding. In fact, many of the integrin receptors appear tv cross react with
ligands
which possess such an amino acid sequence. For instance, the allb[33 receptor
reacts
with fibronectin and vitronectin, thrombospondin and von Willebrand factor, as
well
as fibrinogen. Functionally fibrinogen, a dimer having two binding sites for
allb(~3~
reacts with activated receptors found on the surface of platelets. The binding
of
allb(33 receptors on adjacent platelets, by fibrinogen leads to crosslinking
and is
considered to be a major factor in platelet aggregation. Compounds which
inhibit
the binding of the aIIb~3 receptor to fibrinogen have been shown to inhibit
the
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platelet aggregation in vitro, and thrombus formation in vivo. See, for
instance, EP-
A 0 341 915.
The vitronectin receptor is found on a variety of cell types, such as on
osteoclasts and the endothelial cells lining blood vessels. Recent studies
have
indicated that the attachment of osteoclasts to the bone matrix is mediated
through
these cell surface adhesion receptors. For instance, Davies, et al., J. Cell
Biol., 1989,
109, 1817, disclose that the osteoclast functional antigen, which is
implicated in the
regulation of bone resorption, is biochemically related to the vitronectin
receptor.
The vitronectin receptor is known to bind to bone matrix proteins, such as
osteopontin, bone sialoprotein and thrombospondin, which contain the tri-
peptide
Arg-Gly-Asp (or RGD) motif. Thus, Horton, et al., Exp. Cell Res. 1991,195,
368,
disclose that RGD-containing peptides and an anti-vitronectin receptor
antibody
(23C6) inhibit dentine resorption and cell spreading by osteoclasts. Bertolini
et al.,
J. Bone Min. Res., 6, Sup. l, S 146, 252 have shown that cylco-S,S-Na-acetyl-
cysteinyl-Na-methyl-argininyl-glycyl-aspartyl-penicillamine amide inhibits
osteoclast attachment to bone. In addition, Sato, et al., J. Cell Biol. 1990,
lll, 1713
disclose that echistatin, a snake venom peptide which contains the RGD
sequence, is
a potent inhibitor of hone resorption in tissue culture, and inhibits
attachment of
osteoclasts to bone. Fisher, et aL, Endocrinology 1993, 132, 1411, has further
shown that echistatin inhibits bone resorption in vivo in the rat. EP 528 587
and 528
586 report substituted phenyl derivatives which inhibit osteoclast mediated
bone
resorption.
Bondinell, et al., in WO 93/00095 (PCT/US92/05463) and WO 94/14776
(PCT/LJS93/12436) disclose that certain compounds which have a substituted 6-7
bicyclic ring system are useful for inhibiting the fibrinogen (aIIb~3)
receptor. Other
6-7 bicyclic ring systems which inhibit the fibrinogen receptor are disclosed
by
Blackburn et al. in WO 93/08174 (PCT/LJS92/08788). Blackburn et al., WO
95/04057 {PCT/US94/07989) also disclose compounds which have a five- or six-
membered ring fused to such 6-7 bicyclic ring to form a tricyclic ring system,
which
are useful as antagonists of the fibrinogen receptor. Other compounds having 6-
7
bicyclic ring systems that selectively inhibit the vitronectin receptor are
disclosed in
WO 96/00730 (PCT/US95/08306) and WO 96/00574 (PCT/US95/08146). It has
now been discovered that certain new tricyclic ring systems are useful
templates for
preparing integrin receptor antagonists. It has also been discovered that such
a ring
system may be used as a template, which may be suitably substituted to prepare
compounds which are selective for either the fibrinogen receptor or the
vitronectin
receptor.
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SUMMARY OF THE INVENTION
It is an object of this invention to provide compounds of the formula (I), as
described hereinafter, which have pharmacological activity for the inhibition
of
integrin receptors. It is an object of this invention to provide a template
which may
be suitably substituted to provide selective binding for specific integrin
receptors,
especially the fibrinogen (aIIb~3) or the vitronectin (av(33) receptor
relative to each
other and other integrin receptors.
This invention is also a pharmaceutical composition comprising a compound
according to formula (I) and a pharmaceutically carrier.
This invention is also a method of treating diseases in which the pathology
may be modified by binding to an integrin receptor, especially the vitronectin
or the
fibrinogen receptor. In a particular aspect, the compounds of this invention
are
useful for treating osteoporosis, atherosclerosis, restenosis, cancer and
conditions in
which it is desirable to inhibit platelet aggregation, such as stroke,
transient ischemia
attacks, myocardial infarction and rethrombosis following thrombolytic
therapy.
DETAILED DESCRIPTION
This invention comprises compounds of formula (I):
R4
X=A E .
RB Rs
R~ ~ X~-N
\~CH2 ),n COR2
(I)
wherein:
AisCorN;
E is a five- or six-membered heteroaromatic or heterocyclic ring, or a
six-membered aromatic ring;
X1 is CHR', C(O) or C(S);
X2 is CRSRs~, NRs, S(O)u or O;
R1 is H, C~_6alkyl, C3_7cycloalkyl-C0.4alkyl or Ar-Cp~alkyl;
R2 is -OR', -NRIt", -NR'S02R"', -NR'OR', -OCR 2C(O)OR', -OCR 20C(O)-
R', -OCR 2C(O)NR'2, CF3 or -COCR 2R2';
R2' is -OR', -CN, -S(O)rR', S(O)2NR 2, -C(O)R' C(O)NR'2 or -C02R';
R' is H, C1_6alkyl, C3_7cycloalkyl-Cp~alkyl or Ar-Cp~alkyl;
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R" is R', -C(O)R' or -C(O)ORS;
R'~ is CI_6alkyl, C3_7cycloalkyl-Cp_4alkyl or Ar-Cp~alkyl;
RS and RS' are independently H, C1_6alkyl, C3_7cycioalkyl-Cp_4alkyl or
Ar_Cp_aalkYl;
R6 is W-(CR 2)q-Z-(CR'Rlp)r-U-(CR 2)s-V- or W'-(CR'2)q-U-(CR 2)s-;
R3, R4 and R7 are independently H, halo, -OR12, -SR12, -CN, -NR~tl2,
-N02, -CF3, CF3S(O)r-, -C02R', -CONR'2, R14_Cp_6alkyi-, R14-C1_6oxoalkyl-,
R14-C2-6alkenyl-, R14-C2-6alkYnyi-, R14-Cp_6alkyloxy-, R~4-Cp_6alkylamino- or
R 14-Cp_6alkyl-S(O)r-;
R8 is R', C(O)R', CN, N02, S02R' or C(O)ORS;
R9 is R', -CF3, -SR', or -OR';
Rlp is H, C1_4alkyl or -NR~t";
R12 is R', -C(O)R', -C(O)NR'2, -C(O)ORS, -S(O)mR' or S(O)2NR'2;
R14 is H, C3_6cycloalkyi, Het or Ar;
R'f is H, C 1 _ ~ palkyl, C3_~cycloalkyl-Cp_galkyl or Ar-Cp_galkyl;
U and V are absent or CO, CR'Z, C(=CR'S2), S(O)S, O, NR'S, CR's'OR'S,
CR'(OR")CR'2, CR 2CR'(OR"), C(O)CR'2, CR"2C(O), CONR", NR'SCO, OC(O),
C(O)O, C(S)O, OC(S), C(S)NR'S, NR'SC(S), S02NR'S, NR'sS02 , N=N, NR'sNR's,
NR"CR'S2, NR'SCR'sZ, CR'S20, OCR'S2, C=C , CR's=CR'S, Het, or Ar, provided
that U and V are not simultaneously absent;
W is R~t'N-, Rat"NRN-, Rat"NR~1C0-, R 2NR~TC(=NR~-,
R~2N R~2N NR" NR
R'ONR'C(=NR~-, R9~N', R~2N~ N' , R9~NR~ ~ R~~R'N~ y'
NR' NR a
RNR'N~ NR'-X-, R"R'N ~NR'- or O ;
R'
G~ a
R ~ J Rb Q N R~ ~ R
I ~ ~ N
a
W' is (~~W , Rc N , R ,
Rc
R'
Rb
N~ ~~ Ra
Ra Rc~N '_IVN.R
, , or ;
QisNR',OorS;
Ra is H, C ~ _6alkyl, Ar-Cp_6alkyl, Het-Cp_6alkyl, or C3_6cycloalkyl-
Cp_6alkyl,
halogen, OR1, SRS, COR1, OH, N02, N(R1)2, CO(NR1}2, CH2N(R1}2;
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Rb and R~ are independently selected from H, Ct_6alkyl, Ar-Co-6alkyl, Het-
Cp~,alkyl, or C3-6cycloalkyl-C~6alkyl, halogen, ORS, SRi, COR1, OH, N02,
N(R 1 )2, CO(NR ~ )2, CH2N(R 1 )2, or Rb and R~ are joined together to form a
five or
six membered aromatic or non-aromatic ring, optionally substituted by halogen,
C~_4alkyl, OR1, SRS, COR1, OH, N02, N(R1)2, CO(NR1)2, CH2N(R1)2, CN, or
R"R~iC(=NR~-;
X is N=CR', C(O) or O;
Y is absent, S or O;
Z is (CH2)i, Het, Ar or C3_~cycloalkyl;
m is 1 or 2;
nis0, l,2or3;
qis0, l,2or3;
r is 0, 1 or 2;
s is 0, 1 or 2;
t is 0, 1 or 2;
a is 0, 1 or 2;
v is 0, 1 or 2; and
wis0or l; or
a pharmaceutically acceptable salt thereof.
Also included in this invention are pharmaceutically acceptable addition
salts, complexes or prodrugs of the compounds of this invention. Prodrugs are
considered to be any covalently bonded carriers which release the active
parent drug
according to formula (I) in vivo. In cases wherein the compounds of this
invention
may have one or more chiral centers, unless specified, this invention includes
each
unique nonracemic compound which may be synthesized and resolved by
conventional techniques. In cases in which compounds have unsaturated carbon-
carbon double bonds, both the cis (Z) and traps (E) isomers are within the
scope of
this invention. In cases wherein compounds may exist in tautomeric forms, such
as
O OR'
keto-enol tautomers, such as ~ and ~ , and tautomers of guanidine-type
NR' NR'p
groups, such as R"R'N~ NR'-X- and R"R'N- _ N-X- , each tautomeric form is
contemplated as being included within this invention whether existing in
equilibrium
or locked in one form by appropriate substitution with R'. The meaning of any
substituent at any one occurrence is independent of its meaning, or any other
substituent's meaning, at any other occurrence, unless specified otherwise.
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In one embodiment, this invention is a carbocyclic formula (I) compound
according to formula (II):
R3
RB
X~-N
'-COR2 (u).
With regards to formula (I):
Suitably, A1 is C.
Preferably, X 1 is CH2 and X2 is O.
Suitably, R2 is -OH. Suitably, R3 and R4 are H.
Suitably, U is CONR15, NRi5C0, CH2CH2, or CH20, where R15 is
C1_~palkyl, optionally substituted by N02, CN, COZR', RI4-Co-6alkY1 or R~4-
Cp-6alkylamino.
Suitably, when U is Ar, it is a phenyl ring, preferably 1,3 disubstituted.
Suitably R~5 is R'. More suitably R15 is C1-6alkyl, most suitably H or
methyl.
Suitable substituents for R6 when fibrinogen antagonist acitivity is desired
are:
~G. (CHa~2-s-U (CH~rU R~° ~ (CH~2-U
R~~ N J R" N V NJ
U
R"-HN ~ / ~ ~ U R"-HN / ~ CH2 U
NR" R"-HN \ \
~G,CO
~(J
N
~ R"HNC =NH NH- CH CHR1~ -U and R"HN- CH -U
( ) ( 2)3( ) ~ ( 2)5
wherein G is N or CH, R2~ is hydrogen, amino, mono or di-C~~alkylamino,
hydroxy or C ~ alkyl, and U is NR'CO, CONR', (CH2)CO, CH=CH, C~ C , CH20,
OCH2 and (CH2)2.
Particularly good substituents for promoting selective fibrinogen antagonist
activity are:
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CONR' NR'CO
R"HN I / R"HN I / ~ ~ NR CO-
NH , NH ~ R"HN \
(CH~2NR'CO
\ R2° (CH~2NR'CO
R"N R"N N
. ,
N, CO
(CH ~NR'CO
. (CH~~C NJ
R"N ~ ~ N I
"
. . R NJ
N-CO
(CH~4- ~ N (CH~3-O
R"-N J R"N J R"N J
. . , .
~N~ (CH~ZNR'CO NR'
R"N R"HN ~ N~ CONR'
or R Ri° wherein R' are H or
C i _4alkyl. Preferably R' is methyl and R" is H.
Particularly preferred of such groups for R6 are:
~N-CO
(CH~2N(CH~CO
H-N H-N
and
Preferred substituents for W' when vitronectin binding activity is desired
are:
R'
Rb Rb
Ra! / ~~~ --- ~~ Ra r~~ ~N N
~ N J Rc N ~ Rc ~ ~v ' R' ~ ~d Ra
.
wherein Q is NH. Preferably, Rb and Rc are joined to form a cyclohexyl, phenyl
or
pyridyl ring. Suitably, Ra is C1_6alkyl, C1_6alkoxy, halogen or RNH.
Suitably, -(CR 2)g-U- is (CHZ)q-NR'CO, (CH2)q-CH20 or (CH2)g-CH2CH2.
Specific preferred R6 substituents for enhancing vitronectin activity are
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~N~ N ~ ,CO- N
Ra ~ R, Ra (i' ~ ~(CH2ls-O- ~ NR'CO
//
, Ra ,
Rb R'HN N~ ~1
-NHCO
CH2NR'CO / ~>-CHR'NR'CO
Rc N _ . ~ N
/ ~ H H
N~CH2NR'CO- ~-CHR'NR'CO N~--(CH~3-
N
N N , and /
By appropriate selection of the spacing of the substituent W and/or W' from
the phenyl ring of the 6-7 ring system, compounds having selective activity
for
either the vitronectin and fibrinogen receptor, or dual activity for both
receptors,
may be obtained. In general, fibrinogen antagonist activity will be favored by
an
intramolecular distance of about 16 angstroms between the oxygen of the
carbonyl
moiety attached to the seven-membered ring, and the basic nitrogen moiety of W
or
W'; while vitronectin antagonist activity will be favored by about 14
angstroms
between the respective acidic and basic centers.
A specific compound of this invention is 3-[3-(2-pyridyl)aminopropyloxy]
10,11-dihydrodibenzo[b,f][1,4]oxazepine-10-acetic acid or a pharmaceutically
acceptable salt thereof.
The compounds of formula (I) inhibit the binding of vitronectin and other
RGD-containing peptides to the vitronectin (ocv(33) receptor. Inhibition of
the
vitronectin receptor on osteoclasts inhibits osteoclastic bone resorption and
is useful
in the treatment of diseases wherein bone resorption is associated with
pathology,
such as osteoporosis and osteoarthritis. Additionally, since the compounds of
the
instant invention inhibit vitronectin receptors on a number of different types
of cells,
said compounds would be useful in the treatment of inflammatory disorders,
such as
rheumatoid arthritis and psoriasis, and cardiovascular diseases, such as
atherosclerosis and restenosis. The compounds of Formula (I) of the present
invention may be useful for the treatment or prevention of other diseases
including,
but not limited to, thromboembolic disorders, asthma, allergies, adult
respiratory
distress syndrome, graft versus host disease, organ transplant rejection,
septic shock,
eczema, contact dermatitis, inflammatory bowel disease, and other autoimmune
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diseases. The compounds of the present invention may also be useful for wound
healing.
In particular, the compounds of the present invention are useful for the
treatment, including prevention, of angiogenic disorders. The term "
angiogenic
disorders" as used herein includes conditions involving abnormal
neovascularization. Where the growth of new blood vessels is the cause of, or
contributes to, the pathology associated with a disease, inhibition of
angiogenisis
will reduce the deleterious effects of the disease. An example of such a
disease
target is diabetic retinopathy. Where the growth of new blood vessels is
required to
support growth of a deleterious tissue, inhibition of angiogenisis will reduce
the
blood supply to the tissue and thereby contribute to reduction in tissue mass
based
on blood supply requirements. Examples include growth of tumors where
neovascularization is a continual requirement in order that the tumor grow and
the
establishment of solid tumor metastases. Thus, the compounds of the present
invention inhibit tumor tissue angiogenesis, thereby preventing tumor
metastasis and
tumor growth.
Thus, according to the methods of the present invention, the inhibition of
angiogenesis using the compounds of the present invention can ameliorate the
symptoms of the disease, and, in some cases, can cure the disease.
A preferred therapeutic target for the compounds of the instant invention are
eye diseases chacterized by neovascularization. Such eye diseases include
corneal
neovascular disorders, such as corneal transplantation, herpetic keratitis,
luetic
keratitis, pterygium and neovascular pannus associated with contact lens use.
Additional eye diseases also include age-related macular degeneration,
presumed
ocular histoplasmosis, retinopathy of prematurity and neovascular glaucoma.
In another aspect of the invention is the use of the formula (I) compounds in
the inhibition of platelet aggregation and smooth muscle cell migration
following
vascular injury from percutaneous transluminal coronary angioplasty (PTCA).
The
instant compounds are useful in vascular remodeling.
Abbreviations and symbols commonly used in the peptide and chemical arts
are used herein to describe the compounds of this invention.
Cl~alkyl as applied herein is meant to include methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl and t-butyl. C~_6alkyl additionally includes
pentyl, n-
pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers
thereof. Any
C~_4alkyl or C1_6alkyl group may be optionally substituted by R~ unless
otherwise
indicated. C~4alkyl and CQ6alkyl additionally indicates that no alkyl group
need be
present (e.g., that a covalent bond is present).
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C2_6alkenyl as applied herein means an alkyl group of 2 to 6 carbons wherein
a carbon-carbon single bond is replaced by a carbon-carbon double bond. C2_
6alkenyl includes ethylene, 1-propene, 2-propene, 1-butene, 2-butene,
isobutene and
the several isomeric pentenes and hexenes. Both cis and trans isomers are
included.
Any C2_6alkenyl group may be optionally substituted by R~ unless otherwise
indicated.
C2_6alkynyl means an alkyl group of 2 to 6 carbons wherein one carbon-
carbon single bond is replaced by a carbon-carbon triple bond. C2_6 alkynyI
includes acetylene, 1-propyne, 2-propyne, 1-butyne, 2-butyne, 3-butyne and the
simple isomers of pentyne and hexyne. Any spa carbon atom in the C2_6alkynyl
group may be optionally substituted by R~.
C1_4oxoalkyl refers to an alkyl group of up to four carbons wherein a CH2
group is replaced by a C(O), or carbonyl, group. Substituted formyl, acetyl, 1-
propanal, 2-propanone, 3-propanal, 2-butanone, 3-butanone, 1- and 4-butanal
groups
are representative. Cl_6oxoalkyl includes additionally the higher analogues
and
isomers of five and six carbons substituted by a carbonyl group.
C3_6oxoalkenyl and
C3_6oxoalkynyl refers to a C3_6alkenyl or C3_6alkynyl group wherein a CH2
group is
replaced by C(O) group. C3_4oxoalkenyl includes 1-oxo-2-propenyl, 3-oxo-1-
propenyl, 2-oxo-3-butenyl and the like.
A substituent on a Cl_b alkyl, C2_6 alkenyl, C2_6 alkynyl or C~_6 oxoalkyl
group, such as R~, may be on any carbon atom which results in a stable
structure,
and is available by conventional synthetic techniques.
R t 4-C 1 _6 alkyl refers to a C 1 _6 alkyl group wherein in any position a
carbon-
hydrogen bond is replaced by a carbon-R14 bond. R~4-C2_6 alkenyl and R~4-CZ_6
alkynyl have a similar meaning with respect to C2_6 alkenyl and C2_6 alkynyl.
Ar, or aryl, as applied herein, means phenyl or naphthyl, or phenyl or
naphthyl substituted by one to three moieties R~. In particular, R~ may be
C~_4alkyl,
C1_4alkoxy, C~_4alkthio, trifluoroalkyl, OH, F, Cl, Br or I.
Het, or heterocycle, indicates an optionally substituted five or six membered
monocyclic ring, or a nine or ten-membered bicyclic ring containing one to
three
heteroatoms chosen from the group of nitrogen, oxygen and sulfur, which are
stable
and available by conventional chemical synthesis. Illustrative heterocycles
are
benzofuran, benzimidazole, benzopyran, benzothiophene, furan, imidazole,
indole,
indoline, morpholine, piperidine, piperazine, pyrrole, pyrrolidine,
tetrahydropyridine, pyridine, thiazole, thiophene, quinoline, isoquinoline,
and tetra-
and perhydro- quinoline and isoquinoline. A six membered ring heterocycle
containing one or two nitrogens, such as piperidine, piperazine,
tetrahydropyridine
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and pyridine, are preferred heterocycles for the moiety Z. Any accessible
combination of up to three subsdtuents, such as chosen from R~, on the Het
ring that
is available by chemical synthesis and is stable is within the scope of this
invention.
C3_?cycloalkyl refers to an optionally substituted carbocyclic system of three
to seven carbon atoms, which may contain up to two unsaturated carbon-carbon
bonds. Typical of C3_~cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl,
cyclopentenyl, cyclohexyl, cyclohexenyl and cycloheptyl. Any combination of up
to
three substituents, such as chosen from R7, on the cycloalkyl ring that is
available by
conventional chemical synthesis and is stable, is within the scope of this
invention.
N
~ as used herein indicates a nitrogen heterocycle, which may be a
saturated or unsaturated stable five-, six- or seven-membered monocyclic ring,
or a
seven- to ten-membered bicyclic ring containing up to three nitrogen atoms or
containing one nitrogen atom and a heteroatom chosen from oxygen and sulfur,
and
which may be substituted on any atom that results in a stable structure. The
nitrogen
atom in such ring may be substituted so as to result in a quaternary nitrogen.
The
nitrogen heterocycle may be substituted in any stable position by R2~, for
instance
H, C~-4alkoxy, F, Cl, Br, I, N02, NRi, OH, C02R', CONHR', CF3, R~4-C~4alkyl,
R~4-C~_4alkyl-S(O)S (e.g., where a is 0, 1 or 2) or C1_4alkyl substituted by
any of the
N
aforementioned sustituents. Representative of ~ are pyrroline, pyrrolidine,
imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine,
piperidine, piperazine, morpholine, pyridine, pyridinium, tetrahydropyridine,
tetrahydro- and hexahydro-azepine, quinuclidine, quinuclidinium, quinoline,
isoquinoline, and tetra- and perhydro- quinoline and isoquinoline. In
particular,
N
may be pyridyl, pyrolidinyl, piperidinyl, piperazinyl, azetidinyl,
quinuclidinyl
or tetrahydropyridinyl. ~ is preferably 4-pyridyl, 4-{2-amino-pyridyl), 4-
tetrahydropyridyl, 4-piperidinyl or 4-piperazinyl.
When Rb and R~ are joined together to form a five- or six-membered
aromatic or non-aromatic ring fused to the ring to which Rb and R~ are
attached, the
ring formed will generally be a five- or six-membered heterocycle selected
from
those listed above for Het, or will be a phenyl, cyclohexyl or cyclopentyl
ring.
Benzimidazolyl, 4-azabenzimidazolyl, 5-azabenzimidazolyl and substituted
derivatives thereof are preferred moieties for W'.
Certain radical groups are abbreviated herein. t-Bu refers to the tertiary
butyl
radical, Boc refers to the t-butyloxycarbonyl radical, Fmoc refers to the
fluorenylmethoxycarbonyl radical, Ph refers to the phenyl radical, Cbz refers
to the
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benzyloxycarbonyl radical, BrZ refers to the o-bromobenzyloxycarbonyl radical,
CIZ refers to the o-chlorobenzyloxycarbonyl radical, Bn refers to the benzyl
radical,
4-MBzI refers to the 4-methyl benzyl radical, Me refers to methyl, Et refers
to ethyl,
Ac refers to acetyl, Alk refers to Cl_4alkyl, Nph refers to 1- or 2-naphthyl
and cHex
refers to cyclohexyl. MeArg is Na-methyl arginine. Tet refers to S-tetrazolyl.
Certain reagents are abbreviated herein. DCC refers to
dicyclohexylcarbodiimide, DMAP refers to dimethylaminopyridine, DIEA refers to
diisopropylethylamine, EDC refers to N-ethyl-N'(dimethylaminopropyl)-
carbodiimide. HOBt refers to 1-hydroxybenzotriazole, THF refers to
tetrahydrofuran, DMF refers to dimethyl formamide, NBS refers to N-bromo-
succinimide, Pd/C refers to a palladium on carbon catalyst, DPPA refers to
diphenylphosphoryl azide, BOP refers to benzotriazol-1-yloxy-
tris(dimethylamino)phosphonium hexafluorophosphate, HF refers to hydrofluoric
acid, PPA refers to polyphosphoric acid, TEA refers to triethylamine, TFA
refers to
trifluoroacetic acid, PCC refers to pyridinium chlorochromate.
In certain cases, it may be desirable to further modify the group W or W' by
appropriate reactions to introduce a functional group, or remove a protecting
group,
as further illustrated herein. The coupling will generally result in the
formation of
the U or V group, and methods for such coupling reactions are well known in
the art.
WO 93/08174 (PCT/US92/08788; Genentech), WO 93/08174 (PCT/US92/08788;
Genentech}, WO 96/00730 (PCT/US95/08306; SmithKline Beecham), WO
96/00574 (PCT/LJS95/08146; SmithKline Beecham), WO 93/00095
(PCT/US92/05463; SmithKline Beecham) and WO 94/14776 (PCT/US93/12436;
SmithKline Beecham) generally disclose such reactions and are incorporated
herein
by reference.
Compounds of formula (I) are prepared by methods analogous to those
described in Schemes I-III and by methods analogous to those described in WO
97/01540 (PCT/LJS96/11108; SmithKline Beecham}, the entire disclosure of which
is incorporated herein by reference.
Compounds of the formula (I) wherein X2 is O, X 1 is C(O) or CH2 and m is
1 to 3 are prepared by methods analogous to those shown in Scheme I.
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WO 99/11626 PCT/US98/18379
Scheme I
CH30 ~ OH F ~ CH30 ~ O
/ i- ~/ '~--~ ~/ ~/
CO CH O N
2 3 Z (;p2(iH3 Np2
i 2
CH30 ~ O ~ CEO i O w
\ / ~ / ~ \ I 1 /
H
O
4 ø
i,
CH30 CH30
i O ~. ~.- O -~..
\ I ~ / \ /
O '-CO2CH3 ~C02Chi3
I9 I9
HO ~' H ~'O
i O ~ ~ O
\ / ~ / \ I 1 /
O ~COZCH~ ~CO2CH3
a) K2C03, EtOH; b) H2, 10% Pd/C, MeOH; c) toluene, reflux; d) LiAlH4, THF;
e) i - NaH, DMF, ii - methyl bromoacetate; f) methyl bromoacetate, Et3N, THF;
g)
BBr3, CH2C12.
Hydroxy esters, such as commercially available methyl 2-hydroxy-4
methoxybenzoate (I-1), is condensed with a fluoronitroarene, such as
commercially
available 2-fluoro-1-nitrobenzene (I-2), in the presence of a mild base, such
as
KZC03, in a suitable solvent, such as EtOH, to give the di-aryl ether I-3. If
neccessary, the reaction may be heated to reflux to effect this condensation.
The
reduction of the nitro group is accomplished by hydrogenation over a suitable
catalyst, such as 10% PdlC, in a suitable solvent, such as MeOH. Many
alternative
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WO 99/11626 PCT/US98/18379
methods of reducing the nitro group exist and can be found in reference
volumes
such as Larcock, "Comprehensive Organic Transformations" (published by VCH
Publishers).
The resulting aminoester is heated in a suitable solvent, such as toluene, to
give the ring closed procuct I-4. Alternatively, the ester of I-3 is first
saponified
with aqueous base, such as NaOH, in a polar solvent, such as MeOH. The
resulting
carboxylate is activated in situ by standard reagents, such as DCC, and
allowed to
react in an intramolecular fashion to give the cyclic amide, I-4.
Compound I-4 is then deprotonated with a suitable base, such as NaH, in a
suitable solvent, such as DMF, and allowed to react with a suitable halo-
ester, such
as methyl bromoacetate to give I-8. Alternatively, the carbonyl group of I-4
is
reduced by standard methods, such as treating with lithium aluminum hydride in
an
aprotic polar solvent, such as THF. Many alternative methods of reducing the
amide
carbonyl group exsist and can be found in reference volumes such as
"Compendium
of Organic Synthetic Methods", Vol. I-VI (published by Wiley Interscience).
The
resulting amine I-5 is reacted with a suitable halo-ester, such as methyl
bromoacetate, in the presence of a mild base, such as triethylamine, in a
polar
solvent, such as THF, to give I-6.
The methyl ether of the tricyclic compounds, such as I-6 or I-8, are
deprotected with a Lewis acid, such as BBr3, in a suitable solvent, such as
CH2C12
to give the corresponding phenols, such as I-7 and I-9. Alternatively, the
methyl
ethers can be removed by treatment with AlClg and a thiol, such as
ethanethiol, in a
suitable solvent , such as CH2C12. The resulting phenols, such as I-7 or I-9,
are then
reacted according to the procedures found in WO 97/01540 (PCT/LTS96/11108;
SmithKline Beecham).
Compounds of formula (I) wherein X2 is CH2, X 1 is CH2 or C(O) and m is
1 to 3 are prepared by methods analogous to those described in Schemes II and
III.
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WO 99/11626 PCT/US98/18379
Scheme II
coZH
~I -~ ~OH --~
CH O- v HzN ~ O
CH3O
1 2
o CEO y y ~ d
-~ I~ I~ --
COzli NHz
4
CH30 CH30
i w i w
\ I NI / ~ \ I NI / -
H H
Z
CH30 HO
i ~ i
\ I ~ ~ \ I 1 /
~COzC~"~3 ~CO2CH3
a) 2-amino-2-methyl-1-propanol; b) i- s-BuLi, Et20; ii - 2-methyl-4H-3,1-
benzoxazin-4-one; c) HCI; d) DCC; e) LiAlH4, THF; f) methyl bromoacetate,
Et3N, THF; g) BBr3, CHZC12
A suitable aryl carboxylic acid, such as 4-methoxybenzoic acid, is reacted
with a suitable aminoalcohol, such as 2-amino-2-methyl-1-propanol, according
to
the procedure of Meyers A I, et al. in J. Org. Chem. 1981, 46, 783 to give the
resulting oxazoline II-3. Deprotonation of the aromatic ring ortho to the
oxazoline
is accomplished with a strong base, such as s-butyl lithium, in an aprotic
polar
solvent, such as Et20. The resulting anion is the quenched with suitable
electrophile, such as 2-methyl-4H-3,1-benzoxazin-4-one, to give the
corresponding
keto-amide II-4. Hydrolysis of the oxazoline and acetamide is accomplished by
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WO 99/11626 PCTlUS98/18379
treatment with a strong acid, such as aqueous hydrochloric acid, to give the
amino
acid II-5. The carboxylic acid of II-5 is activated in situ by standard
methods, such
as DCC, and allowed to react in an intramolecular fashion to give the cyclic
amide
II-6. The ketone and carbonyl group of the amide are reduced simultaneously
with a
strong reducing agent, such as lithium aluminum hydride, in a suitable
solvent, such
as THF, to give the cyclic amine II-7. If neccessary, the reaction can be
heated to
reflux to affect the transformation.
Scheme III
CH~O \ MgB~ a CH30 \ \ b,c
I ~ + I \ O ~ I ~ I ~ -~.
O
1 ~ $
CH30 \ \ CEO ~ ~ CH30 w.
I ~ I ~ d,e-.'. \ I \ / ~ \ I 1
HzN -N H
4 ;z !!1
a) THF; b) Zn(Hg), HCI; c) HCI; d) HC02H; e) POCI3, PPA; f) H2, 10% Pd/C,
EtOH
Alternatively, II-7 can be prepared by a process analogous to that shown in
Scheme III. Treatment of III-2 with a suitable organometallic reagent, such as
3-
methoxyphenylmagnesium bromide, gives the corresponding ketoamide, such as III-
3. Clemmensen reduction of the ketone using the general method of Dauben, et
al.
J. Am. Chem. Soc. 19'54, 76, 3864, followed by hydrolysis of the acetamide
with a
strong acid, such as hydrochloric acid gives the amine III-4. Many alternative
methods exist for reducing the ketone to the corresponding methylene compound
and can be found in such reference volumes such as Larock, "Comprehensive
Organic Transformations" (published by VCH publishers). The cyclization of III-
4
is accomplished by a Pictet-Spengler reaction. Thus, formylation of IIL4 with
formic acid followed by an acid catalyzed cyclization with , for example,
polyphosphoric acid, in POC13 give the cyclic imine III-5. Standard methods,
such
as catalytic hydrogenation over Pd on carbon, are used to reduce the imine to
the
corresponding cyclic amine II-7.
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The resulting amine II-7 is reacted with a suitable halo-ester (Scheme In,
such as methyl bromoacetate, in the presence of a mild base, such as
triethylamine,
in a polar solvent, such as THF, to give II-8. The methyl ether of II-8 is
demethylated with a Lewis acid, such as BBr3, in a suitable solvent, such as
CH2C12, to give the corresponding phenol, such as II~9. Alternatively, the
methyl
ethers can be removed by treatment with AlCl3 and a thial, such as
ethanethiol, in a
suitable solvent , such as CH2C12. The resulting phenols, II-9 is then reacted
with
the previously described Arg-VNR according to the procedures found in WO
97/OI540 (PCT/US96/11108; SmithKline Beecham).
Acid addition salts of the compounds are prepared in a standard manner in a
suitable solvent from the parent compound and an excess of an acid, such as
hydrochloric, hydrobromic, hydrofluoric, sulfuric, phosphoric, acetic,
trifluoroacetic,
malefic, succinic or methanesulfonic. Certain of the compounds form inner
salts or
zwitterions which may be acceptable. Cationic salts are prepared by treating
the
IS parent compound with an excess of an alkaline reagent, such as a hydroxide,
carbonate or alkoxide, containing the appropriate cation; or with an
appropriate
organic amine. Cations such as Li+, Na~, K+, Ca++, Mg++ and NH4+ are specific
examples of cations present in pharmaceutically acceptable salts.
This invention also provides a pharmaceutical composition which comprises
a compound according to formula {I) and a pharmaceutically acceptable carrier.
Accordingly, the compounds of formula (I) may be used in the manufacture of a
medicament. Pharmaceutical compositions of the compounds of formula (I)
prepared as hereinbefore described may be formulated as solutions or
lyophilized
powders for parenteral administration. Powders may be reconstituted by
addition of
a suitable diluent or other pharmaceutically acceptable carrier prior to use.
The
liquid formulation may be a buffered, isotonic, aqueous solution. Examples of
suitable diluents are normal isotonic saline solution, standard 5% dextrose in
water
or buffered sodium or ammonium acetate solution. Such formulation is
especially
suitable for parenteral administration, but may also be used for oral
administration or
contained in a metered dose inhaler or nebulizer for insufflation. It may be
desirable
to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose,
acacia,
polyethylene glycol, mannitol, sodium chloride or sodium citrate.
Alternately, these compounds may be encapsulated, tableted or prepared in a
emulsion or syrup for oral administration. Pharmaceutically acceptable solid
or
liquid Garners may be added to enhance or stabilize the composition, or to
facilitate
preparation of the composition. Solid carriers include starch, lactose,
calcium
sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc,
pectin, acacia,
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agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline
and water.
The carrier may also include a sustained release material such as glyceryl
monostearate or glyceryl distearate, alone or with a wax. The amount of solid
carrier varies but, preferably, will be between about 20 mg to about 1 g per
dosage
unit. The pharmaceutical preparations are made following the conventional
techniques of pharmacy involving milling, mixing, granulating, and
compressing,
when necessary, for tablet forms; or milling, mixing and filling for hard
gelatin
capsule forms. When a liquid carrier is used, the preparation will be in the
fonm of a
syrup, elixir; emulsion or an aqueous or non-aqueous suspension. Such a liquid
formulation may be administered directly p.o. or filled into a soft gelatin
capsule.
For rectal administration, the compounds of this invention may also be
combined with excipients such as cocoa butter, glycerin, gelatin or
polyethylene
glycols and molded into a suppository.
The compounds described herein which are antagonists of the vitronectin
receptor, are useful for treating diseases wherein the underlying pathology is
attributable to ligand or cell which interacts with the vitronectin receptor.
For
instance, these compounds are useful for the treatment of diseases wherein
loss of
the bone matrix creates pathology. Thus, the instant compounds are useful for
the
treatment of ostoeporosis, hyperparathyroidism, Paget's disease, hypercalcemia
of
malignancy, osteolytic lesions produced by bone metastasis, bone loss due to
immobilization or sex hormone deficiency. The compounds of this invention are
also believed to have utility as antitumor, antiinflammatory, anti-angiogenic
and
anti-metastatic agents, and be useful in the treatment of cancer,
atherosclerosis and
restenosis. In particular, the compounds of this invention are useful for
inhibiting
restenosis following angioplasty.
The compounds of this invention which inhibit fibrinogen binding provide a
method of inhibiting platelet aggregation and clot formation in a mammal,
especially
a human, which comprises the internal administration of a compound of formula
(I)
and a pharmaceutically acceptable canrier. Indications for such therapy
include acute
myocardial infarction (AMI), deep vein thrombosis, pulmonary embolism,
dissecting anurysm, transient ischemia attack (TIA), stroke and other infarct-
related
disorders, and unstable angina. Chronic or acute states of hyper-
aggregability, such
as disseminated intravascular coagulation (DIC), septicemia, surgical or
infectious
shock, post-operative and post-partum trauma, cardiopulmonary bypass surgery,
incompatible blood transfusion, abruptio placenta, thrombotic thrombocytopenic
purpura (TTP), snake venom and immune diseases, are likely to be responsive to
such treatment. In addition, the compounds of this invention may be useful in
a
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WO 99/11626 PCT/US98/18379
method for the prevention of metastatic conditions, the prevention or
treatment of
fungal or bacterial infection, inducing immunostimulation, treatment of sickle
cell
disease, and the prevention or treatment of diseases in which bone resorption
is a
factor.
This invention further provides a method for inhibiting the reocclusion of an
artery or vein following fibrinolytic therapy, which comprises internal
administration of a compound of formula (I) and a fibrinolytic agent.
Administration of a compound of formula (I) in fibrinolytic therapy either
prevents
reocclusion completely or prolongs the time to reocclusion. When used in the
context of this invention the term fibrinolytic agent is intended to mean any
compound, whether a natural or synthetic product, which directly or indirectly
causes the lysis of a fibrin clot. Plasminogen activators are a well known
group of
fibrinolytic agents. Useful plasminogen activators include, for example,
anistreplase, urokinase (UK), pro-urokinase (pUK), streptokinase (SK}, tissue
plasminogen activator (tPA) and mutants, or variants, thereof.
The compounds of this invention may also be used in vitro to inhibit the
aggregation of platelets in blood and blood products, e.g., for storage, or
for ex vivo
manipulations such as in diagnostic or research use.
The compound is administered either orally or parenterally to the patient, in
a
manner such that the concentration of drug is sufficient to inhibit bone
resorption, or
inhibit platelet aggregation or other such indication. The pharmaceutical
composition containing the compound is administered at an oral dose of between
about 0.1 to about 50 mg/kg in a manner consistent with the condition of the
patient.
Preferably the oral dose would be about 0.5 to about 20 mg/kg. For acute
therapy,
parenteral administration is preferred. An intravenous infusion of the
compound in
5% dextrose in water or normal saline, or a similar formulation with suitable
excipients, is most effective, although an intramuscular bolus injection is
also useful.
Typically, the parenteral dose will be about 0.01 to about 100 mg/kg;
preferably
between 0.1 and 20 mg/kg. The compounds are administered one to four times
daily at a level to achieve a total daily dose of about 0.4 to about 400
mg/kg/day.
The precise level and method by which the compounds are administered is
readily
determined by one routinely skilled in the art by comparing the blood level of
the
agent to the concentration required to have a therapeutic effect.
The compounds may be tested in one of several biological assays to
determine the concentration of compound which is required to have a given
pharmacological effect.
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INHIBITION OF VITRONECTIN BINDING
Solid-Phase ~3HJ-SK&F-107260 Binding to a"~33: Human placenta or human
platelet a"~i3 (0.1-0.3 mg/mL) in buffer T (containing 2 mM CaCl2 and 1 %
octylglucoside) was diluted with buffer T containing 1 mM CaCl2, 1 mM MnCl2,
1 mM MgCl2 (buffer A) and 0.05% NaN3, and then immediately added to 96-well
ELISA plates (Corning, New York, NY) at 0.1 mL per well. 0.1 - 0.2 pg of a"(i3
was added per well. The plates were incubated overnight at 4°C. At the
time of the
experiment, the wells were washed once with buffer A and were incubated with
0.1 mL of 3.5% bovine serum albumin in the same buffer for 1 hr at room
temperature. Following incubation the wells were aspirated completely and
washed
twice with 0.2 mL buffer A.
Compounds were dissolved in 100% DMSO to give a 2 mM stock solution,
which was diluted with binding buffer ( 15 mM Tris-HCl (pH 7.4), 100 mM NaCI,
1 mM CaCl2, 1 mM MnCl2, 1 mM MgCl2) to a final compound concentration of
100 pM. This solution is then diluted to the required final compound
concentration.
Various concentrations of unlabeled antagonists (0.001 - 100 pNi) were added
to the
wells in triplicates, followed by the addition of 5.0 nM of [3H]-SK&F-107260
(65 -
86 Cilmmol).
The plates were incubated for 1 hr at room temperature. Following
incubation the wells were aspirated completely and washed once with 0.2 mL of
ice
cold buffer A in a well-to-well fashion. The receptors were solubilized with
0.1 mL
of 1% SDS and the bound [3H]-SK&F-107260 was determined by liquid
scintillation counting with the addition of 3 mL Ready Safe in a Beckman LS
Liquid
Scintillation Counter, with 40% efficiency. Nonspecific binding of [3H]-SK&F-
107260 was determined in the presence of 2 ~.iM SK&F-107260 and was
consistently
less than 1 % of total radioligand input. The ICsp (concentration of the
antagonist to
inhibit 50% binding of [3H]-SK&F-107260) was determined by a nonlinear, least
squares curve-fitting routine, which was modified from the LUNDON-2 program.
The K; (dissociation constant of the antagonist) was calculated according to
the
equation: K; = ICgp/( 1 + L/Kd), where L and Kd were the concentration and the
dissociation constant of [3H]-SK&F-107260, respectively.
Compounds of the present invention inhibit vitronectin binding to SK&F
107260 in the concentration of about 0.1 micromolar.
Compounds of this invention are also tested for in vitro and in vivo bone
resorption in assays standard in the art for evaluating inhibition of bone
formation,
such as the pit formation assay disclosed in EP 528 587, which may also be
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WO 99/11626 PCT/US98/18379
performed using human osteoclasts in place of rat osteoclasts, and the
ovarectomized
rat model, described by Wronski et al., Cells and Materials 1991, Sup. 1, 69-
74.
PARATHYROIDECTOMIZED RAT MODEL
Each experimental group consists of 5-6 male Sprague-Dawley rats. The rats are
parathyroidectomized (by the vendor, Taconic Farms) 7 days prior to use.
Twenty four
hours prior to use, circulating ionized calcium levels are measured in whole
blood
immediately after it has been withdrawn by tail venipuncture into heparinized
tubes. Rats
are included if ionized Ca level (measured with a Ciba-Corning model 634
calcium pH
analyzer) is ~ 1.2 mM/L. The rats are then put on a diet of calcium-free chow
and
deionized water. At the start of the experiment the rats weigh approximately
100g.
Baseline Ca levels are measured and the rats are administered control vehicle
(saline) or
compound (dissolved in saline) as a single intravenous (tail vein) bolus
injection followed
immediately by a single subcutaneous injection of either human parathyroid
hormone 1-
34 peptide (hPTHI-34, dose 0.2mg/kg in saline/0.1% bovine serum albumen,
Bachem,
Ca) or the PTH vehicle. The calcemic response to PTH (and any effect of
compound on
this response) is measured 2h after compound/PTH administration.
RAT ULNA DRIFT MODEL
Each experimental group consists of 8-10 male Sprague-Dawley or Wistar rats of
approximately 30-40g body weight at the start of the experiment. The agent
being tested
is administered by an appropriate route as single or multiple daily doses for
a period of
seven days. Prior to administration of the first dose, the rats are given a
single dose of a
fluorescent marker (tetracycline 25mg/kg, or calcein lOmg/kg) that labels the
position of
bone forming surfaces at that point in time. After dosing of compound has been
completed, the rats are killed and both forelimbs are removed at the elbow,
the foot is
removed at the ankle and the skin removed. The sample is frozen and mounted
vertically
on a microtome chuck. Cross sections of the midshaft region of the ulna are
cut in the
cryostat. The rate of bone resorption is measured morphometrically in the
medial-dorsal
portion of the cortical bone. The measurement is done as follows: the amount
of bone
resorbed at the periosteal surface is equal to the distance by which the
periosteal surface
has advanced towards the fluorescent label which had been incorporated at the
endosteal
bone formation surface on day zero; this distance is calculated by subtracting
the width of
bone between the label and the periosteal surface on day 7 from the width on
day zero; the
resorption rate in microns per day is calculated by dividing the result by 7.
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HUMAN OSTEOCLAST RESORPTION ASSAY ("PIT ASSAY")
~ Aliquots of osteoclastoma-derived cell suspensions are removed from liquid
nitrogen strorage, warmed rapidly at 37°C and washed xl in RPMI-1640
medium by
centrifugation ( 100orpm, 5 rains at 4°C).
~ Aspirate the medium and replace it with murine anti-HLA-DR antibody, diluted
1:3 in RPMI-1640 medium. Incubate for 30 rains on ice and mix the cell
suspension
frequently.
~ The cells are washed x2 with cold RPMI-1640 by centrifugation (1000rpm, 5
rains
at 4°C) and the cells are transferred to a sterile 15 ml centrifuge
tube. The number of
mononuclear cells are enumerated in an improved Neubauer counting chamber.
~ Sufficient magnetic beads (5 / mononuclear cell), coated with goat anti-
mouse IgG,
are removed from their stock bottle and placed into 5 ml of fresh medium (this
washes away the toxic azide preservative). The medium is removed by
immobilizing
the beads on a magnet and is replaced with fresh medium.
~ The beads are mixed with the cells and the suspension is incubated for 30
rains on
ice. The suspension is mixed frequently.
~ The bead-coated cells are immobilized on a magnet and the remaining cells
(osteoclast-rich fraction) are decanted into a sterile 50 ml centrifuge tube.
~ Fresh medium is added to the bead-coated cells to dislodge any trapped
osteoclasts.
This wash process is repeated x 10. The bead-coated cells are discarded.
~ The osteoclasts are enumerated in a counting chamber, using a large-bore
disposable plastic pasteur to charge the chamber with the sample.
~ The cells are pelleted by centrifugation and the density of osteoclasts
adjusted to
1.5x104/ml in EMEM medium, supplemented with 10% fetal calf serum and
1.7g/litre of sodium bicarbonate.
~ 3ml aliquots of the cell suspension ( per treatment) are decanted into l5ml
centrifuge tubes. The cells are pelleted by centrifugation.
~ To each tube 3m1 of the appropriate treatment are added (diluted to 50 uM in
the
EMEM medium). Also included are appropriate vehicle controls, a positive
control
(87MEM1 diluted to 100 ug/ml) and an isotype control (IgG2a diluted to 100
uglml). Incubate at 37°C for 30 rains.
~ 0.5m1 aliquots of the cells are seeded onto sterile dentine slices in a 48-
well plate
and incubated at 37°C for 2 hours. Each treatment is screened in
quadruplicate.
~ The slices are washed in six changes of warm PBS (10 ml / well in a 6-well
plate)
and then placed into fresh treatment or control. Incubate at 37°C for
48 hours.
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WO 99/11626 PCT/US98/18379
tartrate resistant acid phosphatase (trap) procedure (selective stain for
cells of
the osteoclast lineage).
~ The slices are washed in phosphate buffered saline and fixed in 2%
gluteraldehyde
(in 0.2M sodium cacodylate) for 5 rains.
~ They are washed in water and incubated in TRAP buffer for 5 rains at
37°C.
~ Following a wash in cold water they are incubated in cold acetate buffer /
fast red
garnet for 5 rains at 4°C.
~ Excess buffer is aspirated, and the slices are air dried following a wash in
water.
~ The TRAP positive osteoclasts are enumerated by bright-field microscopy and
are
then removed from the surface of the dentine by sonication.
~ Pit volumes are determined using the Nikon/Lasertec ILM21 W confocal
microscope.
INHIBITION OF RGD-MEDIATED OCIIb(~3 BINDING
Purification of lxl/b~33
Ten units of outdated, washed human platelets (obtained from Red Cross)
were lyzed by gentle stirnng in 3%a octylglucoside, 20 mM Tris-HCI, pH 7.4,
140
mM NaCI, 2 mM CaCl2 at 4°C for 2 h. The lysate was centrifuged at
100,000g for 1
h. The supernatant obtained was applied to a 5 mL lentil lectin sepharose 4B
column (E.Y. Labs) preequilibrated with 20 mM Tris-HCI, pH 7.4, 100 mM NaCI, 2
mM CaCl2, 1 % octylglucoside (buffer A). After 2 h incubation, the column was
washed with 50 mL cold buffer A. The lectin-retained ocIlb~3 w~ eluted with
buffer
A containing 10% dextrose. All procedures were performed at 4°C. The
atIb(33
obtained was >95% pure as shown by SDS polyacrylamide gel electrophoresis.
Incorporation of a/Ib/33 in Liposomes
A mixture of phosphatidylserine (70%} and phosphatidylcholine (30%)
(Avanti Polar Lipids) were dried to the walls of a glass tube under a stream
of
nitrogen. Purified a,IIbp3 w~ Muted to a final concentration of 0.5 mg/mL and
mixed with the phospholipids in a protein:phospholipid ratio of 1:3 (w:w). The
mixture was resuspended and sonicated in a bath sonicator for 5 min. The
mixture
was then dialyzed overnight using 12,000-14,000 molecular weight cutoff
dialysis
tubing against a 1000-fold excess of 50 mM Tris-HCI, pH 7.4, 100 mM NaCI, 2 mM
CaCl2 (with 2 changes). The aiib(~3-containing liposomes wee centrifuged at
12,DDOg for 15 min and resuspended in the dialysis buffer at a final protein
concentration of approximately 1 mg/mL. The liposomes were stored at -
70°C until
needed.
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Competitive Binding to attb~(33
The binding to the fibrinogen receptor (aIIb~3) was assayed by an indirect
competitive binding method using [3H]-SK&F-107260 as an RGD-type ligand. The
binding assay was performed in a 96-well filtration plate assembly (Millipore
Corporation, Bedford, MA) using 0.22 um hydrophilic durapore membranes. The
wells were precoated with 0.2 mL of 10 pg/mL polylysine (Sigma Chemical Co.,
St.
Louis, MO.) at room temperature for 1 h to block nonspecific binding. Various
concentrations of unlabeled benzadiazapines were added to the wells in
quadruplicate. [3H]-SK&F-107260 was applied to each well at a final
concentration
of 4.5 nM, followed by the addition of 1 llg of the purified platelet aIIb~3-
containing
liposomes. The mixtures were incubated for 1 h at room temperature. The aIIb~3-
bound [3H]-SK&F-107260 was seperated from the unbound by filtration using a
Millipore filtration manifold, followed by washing with ice-cold buffer (2
times,
each 0.2 mL). Bound radioactivity remaining on the filters was counted in 1.5
mL
Ready Solve (Beckman Instruments, Fullerton, CA) in a Beckman Liquid
Scintillation Counter (Model LS6800), with 40% efficiency. Nonspecific binding
was determined in the presence of 2 pM unlabeled SK&F-107260 and was
consistently less than 0.14% of the total radioactivity added to the samples.
All data
points are the mean of quadruplicate determinations.
Competition binding data were analyzed by a nonlinear least-squares curve
fitting procedure. This method provides the IC50 of the antagonists
(concentration
of the antagonist which inhibits specific binding of [3H]-SK&F-107260 by 50%
at
equilibrium). The IC50 is related to the equilibrium dissociation constant
(Ki) of the
antagonist based on the Cheng and Prusoff equation: Ki = IC50/( 1+L/Kd), where
L
is the concentration of [3H]-SK&F-107260 used in the competitive binding assay
(4.5 nM), and Kd is the dissociation constant of [3H]-SK&F-107260 which is 4.5
nM as determined by Scatchard analysis.
Inhibition of platelet aggregation may be measured by the method described
in WO 93/00095 (PCT/LTS/92/05463). In vivo thrombus formation is demonstrated
by recording the systemic and hemodynamic effects of infusion of the peptides
into
anesthetized dogs according to the methods described in Aiken et al.,
Prostaglandins, 19, 620 ( 1980).
Vascular smooth muscle cell migration assay
The compounds of the instant invention were tested for their ability to
inhibit
the migration and proliferation of smooth muscle tissue in an artery or vein
in order
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WO 99/11626 PCT/US98/18379
to assess their ability to prevent restenosis of an artery, such as that which
typically
occurs following angioplasty.
Rat or human aortic smooth muscle cells were used. The cell migration was
monitored in a Transwell cell culture chamber by using a polycarbonate
membrane
S with pores of 8 um (Costar). The lower surface of the filter was coated with
vitronectin. Cells were suspended in DMEM supplemented with 0.2% bovine serum
albumin at a concentration of 2.5 - 5.0 x 106 cells/mL, and were pretreated
with test
compound at various concentrations for 20 min at 20°C. The solvent
alone was used
as control. 0.2 mL of the cell suspension was placed in the upper compartment
of
the chamber. The lower compartment contained 0.6 mL of DMEM supplemented
with 0.2% bovine serum albumin. Incubation was carried out at 37°C in
an
atmosphere of 95% air/5% C02 for 24 hr. After incubation, the non-migrated
cells
on the upper surface of the filter were removed by gentle scraping. The filter
was
then fixed in methanol and stained with 10% Giemsa stain. Migration was
measured
I S either by a) counting the number of cells that had migrated to the lower
surface of
the filter or by b) extracting the stained cells with 10% acetic acid followed
by
determining the absorbance at 600 nM.
Examples
Nuclear magnetic resonance spectra were obtained using either a Bruker AM
250 or Bruker AC 400 spectrometer. Chemical shifts are reported in parts per
milliom (S) downfield from the internal standard tetramethylsilane. Mass
spectra
were taken on either VG 70 FE or VG ZAB HF instruments using fast atom
bombardment (FAB) or electrospray (ES) ionization techniques. Elemental
analyses
were performed by Quantitative Technologies Inc., Whitehouse, New 3ersey.
Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254 thin layer plates
were used for thin layer chromatography. Flash chromatography was carried out
on
E. Merck Kieselgel 60 (230-400 mesh) silica gel. Analytical and preparative
HPLC
were carried out on Bechman Chromatographs. PRP-10 is a polymeric (styrene
divinylbenzene) chromatographic support, and is a registered trademark of
Hamilton
Co., Reno, Nevada.
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xam le 1
Preparation of 3-f3-(2-pvridylamino)nropyloxyl-10.11-
dihydrodibenzofb.fif 1.41oxazepine-10-acetic acid
a) Methyl4-methoxy-2-(o-nitrophenoxy)-benzoate
2-Fluoronitrobenzene (6.00 mL, 56.9 mmol) and potassium carbonate (30.5
g., 22.1 mmol) were added to methyl 2-hydroxy-4-methoxybenzoate (10.1 g., 55.6
mmol) in DMF ( 100 mL). The reaction was heated in an oil bath at 1 I O
°C. After
20 h, the reaction was diluted with water and extracted with ethyl acetate
(3x). The
organic extracts were dried over anhydrous MgS04, filtered and concentrated
under
vacuum to give a dark residue. Flash chromatography (20°lo
EtOAc/hexanes, silica
gel) gave 16.1 g of the desired product as a yellow solid. ~H NMR (250 MHz,
CDC13). b 7.95-8.05 (m, 2H), 7.40-7.50 (m, 1H), 7.10-7.17 (m, 1H), 6.75-6.85
(m,
2H), 6.63 (t, 1H), 3.85 (s, 3H), 3.70
b) Methyl4-methoxy-2-(o-aminophenoxy)-benzoate
A reaction vessel was charged with methyl 4-methoxy-2-(o-nitrophenoxy)-
benzoate ( 16.1 g, 53.1 mmol) and 10°lo Pd/C ( 100 mg) in MeOH ( 100
mL). The
reaction vessel was flushed with hydrogen and then fitted with a hydrogen
filled
balloon. After 24 hours, the reaction was filtered through celite and the
residue was
evaporated under vacuum to yield 14.3 g of the desired product. MS (ES+) m/z
274.2 (M+H}+
c) 3-Methoxy-10,11-dihydrodibenzo[b,f][1,4]oxazepine-I1-one
To methyl 4-methoxy-2-(o-aminophenoxy)-benzoate (14.3 g, 52.3mmol) in
toluene (500 mL) was added 1N NaOH (150 mL, 150 mmol). The reaction was
heated to 70 °C for 3 days. The solvent was removed under reduced
pressure and
the product was isolated by flash chromatography (EtOAc, silica gel) to give
9.85 g
of the desired product. MS (ES+) m/z 242.2 (M+H+).
d) 3-Methoxy-10,11-dihydrodibenzo[b,fj[1,4]-oxazepine
To 3-methoxy-10,11-dihydrodibenzo[h,fj[1,4]oxazepine-11-one (9.84 g, 40.9
mmol) in THF ( 150 mL) at RT was added LiAIH4 (30 mL, 1.0 M in THF, 30
mmol). After 18 h, the reaction was diluted with toluene and cooled to 0
°C. The
reaction was quenched by adding water (1.6 mL.) and NaF (5.0 g.) and stirring
vigorously for 1 h. The resulting precipitate was removed by filtration and
the
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WO 99/llb2b PCT/US98/18379
eluent was concentrated under vacuum to give the crude product. Flash
chromatography (CHCl3, silica gel) gave 6.28 g of the desired material as a
pale
yellow solid. 1H NMR (250 MHz, CDC13). b 6.50-7.15(m, 7H), 4.42 (s, 2H), 3.78
(s, 3H), 3.28 (br. s., 1H).
e) Methyl3-methoxy-10,11-Dihydrodibenzo[b,f][1,4]-oxazepine-10-acetate
3-Methoxy-10,11-dihydrodibenzo[b,f][1,4]oxazepine (0.43 g, 1.90 mmol)
was dissolved in THF (5 mL). Methyl bromoacetate (0.25 mL, 2.64 mmol.) was
added followed by triethyIamine (0.25 mL, 1.80 mmol). After 24 h at reflux,
the
solvent was removed under vacuum and the residue was adsorbed onto silica gel.
Flash chromatography (CHC13 to 10% MeOH/CHCl3, silica gel) gave 0.40 g of the
desired material. MS(ES+) m/z 300.2 (M+H+).
f) Methyl 3-hydroxy-10,11-dihydrodibenzo[b,f] [ 1,4]-oxazepine-10-acetate
Methyl 3-methoxy-10,11-dihydrodiibenzo[b,f][I,4]oxazepine-acetate (0.40
g, 1.34 mmol) in CH2C12 (5 mL) at 0 oC was treated with BBr3 (6.70 mL, 1.0 M
in
CH2Cl2, 6.70 mmol). After 20 minutes, the reaction was quenched with methanol
and the solvent was removed under vacuum. Flash chromatography (CHC13, silica
gel) gave 0.25 g of the desired material. MS (ES+) m/z 286.3 (M+H+).
g) Methyl3-[3-(N-t-butoxycarbonyl-2-aminopyridyl)propyloxy]-10,11-
dihydrodibenzo [b,f J [ 1,4]-oxazepine-10-acetate-N-oxide
To N-(t-butoxycarbonyl)-3-(2-aminopyridyl)-propanol (0.49 g, 1.81 mmol}
in pyridine (5 mL) at 0 °C was added mesyl chloride (0.18 mL, 2.26
mmol). After 1
h at 0 °C, the reaction was extracted with EtOAc. The combined organic
extracts
were washed with 1N HCI, 1N, NaHC03 and dried over MgS04. The solvent was
removed under reduced pressure and the crude mesylate was used without further
purification in the next step.
To the methyl-3-hydroxy-10,11-dihydrodibenzo[b,f][1,4]oxazepine-1-acetate
(0.25 g, 0.88 mmol) in DMSO ( 15 mL) was added NaH (27 mg, 60% dispersion in
oil, 0.68 mmol). After the bubbling stopped, the crude mesylate (obtained
above)
was added and the reaction was allowed to proceed at RT. After 20 h, the
solvent
was removed under reduced pressure and the product isolated by flash
chromatography (EtOAc, silica gel) to give 0.19 g of the desired product.
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WO 99/11626 PCTNS98/18379
h) Methyl 3-[3-(2-aminopyridyl)pmpyloxy]-10,11-dihydrodibenzo[b,f][ 1,4]-
oxazepine-10-acetate
Methyl 3-[3-(N t-butoxycarbonyl-2-aminopyridyl)propyloxy]-10,11-
dihydrodibenzo[b,f][1,4]-oxazepine-10-acetate-N oxide (0.19 g, 0.36 mmol) was
treated with 4 N HCUdioxane (5 mL) at RT. After 1 h, the solvent was removed
under vacuum and the residue was azeotroped with toluene (2x). This material
was
redissolved in ethanol (5 mL) and triethylamine (0.10 mL, 0.72 mmol),
cyclohexene
(0.50 mL, 4.90 mmol) and 10% Pd/C were added. The reaction was heated to
reflux
for 20 h. After allowing the reaction to cool to RT, the catalyst was removed
by
filtration through celite and the filtrate was concentrated under vacuum to
give 0.17
g of the desired material. This was used without further purification. MS
(ES+) m/z
420.2 (M+H+).
i) 3-[3-(2-Pyridylamino)propyloxy]-10,11-dihydrodibenzo[b,f][1,4]oxazepine-10-
acetic acid
To methyl 3-[3-(2-aminopyridyl)propyloxy]-10,11-dihydrodibenzo[b,f][1,4]-
oxazepine-10-acetate {0.17 g, 0.42 mmol) in MeOH (2 mL) was added 1N NaOH (2
mL). The reaction was heated to 55 °C for 20 h. The reaction was
allowed to cool
to RT and then nuetralized with 1N HCI. Cooling the solution to 0 °C
resulted in the
product forming as a precipitate. This was collected and dried under vacuum to
give
17 mg of the desired material as a white solid. MS(ES+) m/z 406.1 (M+H+).
Anal.
~C23H23N304'0~75HC1) calcd: C, 63.83; H, 5.53; N, 9.71. Found: C, 63.67; H,
5.27; N, 9.49.
Example 2
Oral Dosage Unit Composition
A tablet for oral administration is prepared by mixing and granulating 20 mg
of sucrose, I50 mg of calcium sulfate dihydrate and 50 mg of the compound of
Example 1 with a 10% gelatin solution. The wet granules are screened, dried,
mixed
with 10 mg starch, 5 mg talc and 3 mg stearic acid; and compressed into a
tablet.
The above description fully discloses how to make and use the present
invention. However, the present invention is not limited to the particular
embodiments described hereinabove, but includes all modifications thereof
within
the scope of the following claims. The various references to journals, patents
and
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WO 99/11626 PCT/US98/183'79
other publications which are cited herein comprises the state of the art and
are
incorporated herein by reference as though fully set forth.
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