Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
Vitronectin Receptor Antagonist
FIELD OF THE INVENTION
This invention relates to a pharmaceutically active compound which inhibits
the
vitronectin receptor and is useful for the treatment of inflammation, cancer
and
cardiovascular disorders, such as atherosclerosis and restenosis, and diseases
wherein bone
resorption is a factor, such as osteoporosis.
BACKGROUND OF THE INVENTION
Integrins are a superfamily of cell adhesion receptors, which are
transmembrane
glycoproteins expressed on a variety of cells. These cell surface adhesion
receptors include
gpIIb /IIIa (the fibrinogen receptor) and av133 (the vitronectin receptor).
The fibrinogen
receptor gpIIb /IIIa is expressed on the platelet surface, and mediates
platelet aggregation
and the formation of a hemostatic clot at the site of a bleeding wound.
Philips, et al.,
Blood., 1988, 71, 831. The vitronectin receptor avf3g is expressed on a number
of cells,
including endothelial, smooth muscle, osteoclast, and tumor cells, and, thus,
it has a variety
of functions. The a~f33 receptor expressed on the membrane of osteoclast cells
mediates the
adhesion of osteoclasts to the bone matrix, a key step in the bone resorption
process. Ross,
et al., J. Biol. Chem., 1987, 262, 7703. A disease characterized by excessive
bone
resorption is osteoporosis. The ava3 receptor expressed on human aortic smooth
muscle
cells mediates their migration into neointima, a process which can lead to
restenosis after
percutaneous coronary angioplasty. Brown, et al., Cardiovascular Res., 1994,
28, 1815.
Additionally, Brooks, et al., Cell, 1994, 79, 1157 has shown that an avB3
antagonist is able
to promote tumor regression by inducing apoptosis of angiogenic blood vessels.
Thus,
agents that block the vitronectin receptor would be useful in treating
diseases, such as
osteoporosis, restenosis and cancer.
The vitronectin receptor is now known to refer to three different integrins,
designated av131, av(33 and av135. Horton, et al., Int. J. Exp. Pathol., 1990,
71, 741. av131
binds fibronectin and vitronectin. a~,l3g binds a large variety of ligands,
including fibrin,
fibrinogen, laminin, thrombospondin, vitronectin, von Willebrand's factor,
osteopontin and
bone sialoprotein I. avf35 binds vitronectin. The vitronectin receptor avl3g
has been shown
to be involved in cell adhesion of a variety of cell types, including
microvascular
endothelial cells, (Davis, et al., J. Cell. Biol.,1993, 51, 206), and its role
in angiogenesis
has been confirmed. Brooks, et al., Science, 1994, 264, 569. This integrin is
expressed on
blood vessels in human wound granulation tissue, but not in normal skin.
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The vitronectin receptor is known to bind to bone matrix proteins which
contain the
tri-peptide Arg-Gly-Asp (or RGD) motif. Thus, Horton, et al., E.xp. 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. In
addition, Sato, et al.,
J. Cell Biol. 1990, Ill, 1713 discloses 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.
It has now been discovered that a certain compound is a potent inhibitor of
the
avf33 and avf35 receptors. In particular, it has been discovered that such a
compound is a
more potent inhibitor of the vitronectin receptor than the fibrinogen
receptor.
SUMMARY OF THE INVENTION
This invention comprises a compound of the formula (I) as described
hereinafter,
which has pharmacological activity for the inhibition of the vitronection
receptor and is
useful in the treatment of inflammation, cancer and cardiovascular disorders,
such as
atherosclerosis and restenosis, and diseases wherein bone resorption is a
factor, such as
osteoporosis.
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 which are mediated by the
vitronectin receptor. In a particular aspect, the compound of this invention
is useful for
treating atherosclerosis, restenosis, inflammation, cancer and diseases
wherein bone
resorption is a factor, such as osteoporosis.
DETAILED DESCRIPTION
This invention comprises a novel compound which is a more potent inhibitor of
the
vitronectin receptor than the fibrinogen receptor. The novel compound
comprises a
benzazepine core in which a nitrogen-containing substituent is present on the
aromatic six-
membered ring of the benzazepine and an aliphatic substituent containing an
acidic moiety
is present on the seven-membered ring of the benzazepine. The benzazepine ring
system is
believed to interact favorably with the vitronectin receptor and to orient the
substituent
sidechains on the six and seven membered rings so that they may also interact
favorably
with the receptor. It is preferred that about twelve to fourteen intervening
covalent bonds
via the shortest intramolecular path will exist between the acidic group on
the aliphatic
substituent of the seven-membered ring of the benzazepine and the nitrogen of
the nitrogen-
containing substituent on the aromatic six-membered ring of the benzazepine.
This invention comprises a compound of formula (I):
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O
CF3
O
,
CH3 '',--- C02H
(I)
or a pharmaceutically acceptable salt thereof. This compound is (S)-8-[3-(4-
methylpyridin-
2-ylamino)-1-propyloxy]-3-oxo-2-[4-(trifluoromethyl)benzyl]-2,3,4,5-tetrahydro-
1H-2-
benzazepine-4-acetic acid.
The compound of formula (I) inhibits the binding of vitronectin and other RGD-
containing peptides to the vitronectin 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.
In another aspect, this invention is a method for stimulating bone formation
which
comprises administering a compound of formula (I) which causes an increase in
osteocalcin
release. Increased bone production is a clear benefit in disease states
wherein there is a
deficiency of mineralized bone mass or remodeling of bone is desired, such as
fracture
healing and the prevention of bone fractures. Diseases and metabolic disorders
which
result in loss of bone structure would also benefit from such treatment. For
instance,
hyperparathyroidism, Paget's disease, hypercalcemia of malignancy, osteolytic
lesions
produced by bone metastasis, bone loss due to immobilization or sex hormone
deficiency,
Beh~et's disease, osteomalacia, hyperostosis and osteopetrosis, could benefit
from
administering a compound of this invention.
Additionally, since the compound of the instant invention inhibits vitronectin
receptors on a number of different types of cells, said compound would be
useful in the
treatment of inflammatory disorders, such as rheumatoid arthritis and
psoriasis, and
cardiovascular diseases, such as atherosclerosis and restenosis. The compound
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
diseases.
The compound of the present invention may also be useful for wound healing.
The compound of the present invention is also 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
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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 compound 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 compound of the present invention can ameliorate the
symptoms of
the disease, and, in some cases, can cure the disease.
Another therapeutic target for the compound 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.
This invention further provides a method of inhibiting tumor growth which
comprises administering stepwise or in physical combination a compound of
formula {I)
and an antineoplastic agent, such as topotecan and cisplatin.
Also included in this invention are 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. Thus, in another aspect of this
invention are
novel prodrugs, which are also intermediates in the preparation of the formula
(I)
compound, of formula (II):
O / N ~ ~ CF3
O
CH3 '~'~-- COZC~_saikyl
(II)
or a pharmaceutically acceptable salt thereof.
In yet another aspect of this invention are novel intermediates of formula
{IIi):
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O- _
CF
~N+ NCO /, N ~ / s
0
CH3 ~'~--- C02C1~alkyl
(III)
or a pharmaceutically acceptable salt thereof.
Abbreviations and symbols commonly used in the peptide and chemical arts are
used herein to describe the compounds of this invention. In general, the amino
acid
abbreviations follow the IUPAC-IUB Joint Commission on Biochemical
Nomenclature as
described in Eur. J. Biochem., 158, 9 (1984).
C1_6alkyl as applied herein means an optionally substituted alkyl group of 1
to 6
carbon atoms, and includes methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, t-butyl,
n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers
thereof.
Certain reagents are abbreviated herein. DCC refers to
dicyclohexylcarbodiimide,
DMAP refers to dimethylaminopyridine, DIEA refers to diisopropylethyl amine,
EDC
refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride. HOBt
refers to
1-hydroxybenzotriazole, THF refers to tetrahydrofuran, DIEA refers to
diisopropylethylamine, DEAD refers to diethyl azodicarboxylate, PPh3 refers to
triphenylphosphine, DIAD refers to diisopropyl azodicarboxylate, DME refers to
dimethoxyethane, DMF refers to dimethylformamide, NBS refers to N-
bromosuccinimide,
Pd/C refers to a palladium on carbon catalyst, PPA refers to polyphosphoric
acid, DPPA
refers to diphenylphosphoryl azide, BOP refers to benzotriazol-1-yloxy-
tris(dimethyl-
amino)phosphonium hexafluorophosphate, HF refers to hydrofluoric acid, TEA
refers to
triethylamine, TFA refers to trifluoroacetic acid, PCC refers to pyridinium
chlorochromate.
Compounds of the formula (I) are generally prepared by the methods described
in
Bondinell, et al., PCT application WO 93100095, published January 7, 1993 and
Bondinell,
et al., PCT application WO 94/14776, the entire disclosures of which are
incorporated
herein by reference.
Additionally, the compound of formula (I) is prepared by the methods detailed
in
the scheme hereinbelow.
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Scheme I
H FsC / -_.
HO / N ~ ~ O / N ~ ~ CF3
\ ~ O \ ~ O
,1 ~'~-C02CH3 2 ~'~--C02CH3
b HO CF3 c
O - "'
,N+ N~/'~O / ~ ~ CF3
O
CH3 4 ~~~.--CO2CH3
CFa
i NCO / N
\ ~ \ ~ O e,f
..
~~,-.-COZCH3
CH3 5
H ~ CF3
N N~/O /
\ ( O
~',--C02H
CH3 6
a) NaH, 4-(trifluoromethyl)benzyl bromide, DMF; b) H2, Pd(OH)2/C, MeOH; c) 2-
[(3-
hydroxy-1-propyl)amino]-4-methylpyridine-N-oxide, DEAD, (Ph)gP, CH2C12; d)
cyclohexene, 10% Pd/C, MeOH; e) 1.0 N NaOH, EtOH; t7 HCI, H20.
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Compound I-1, prepared by the general procedures described in Bondinell, et
al.,
PCT application WO 93/00095, published January 7, 1993 and Bondinell, et al.,
PCT
application WO 94/14776, is reacted with 4-(trifluoromethyl)benzyl bromide in
the
presence of a suitable base, generally sodium hydride or lithium
bis(trimethylsilyl)amide,
in an aprotic solvent, preferably DMF, THF, or mixtures thereof, to afford the
bis-alkylated
product I-2. The 4-(trifluoromethyl)benzyl ether of I-2 can be conveniently
removed by
hydrogenolysis to provide the phenol I-3. Methods for hydrogenolysis of benzyl
ethers are
well-known to those of skill in the art, and are described in appropriate
reference volumes,
for instance in Greene, "Protective Groups in Organic Synthesis" (published by
Wiley-
Interscience). Compound I-3, is reacted with 2-[(3-hydroxy-1-propyl)amino]-4-
methylpyridine-N-oxide in a Mitsunobu-type coupling reaction (Organic
Reactions 1992,
42, 335-656; Synthesis 1981, 1-28) to afford I-4. The reaction is mediated by
the complex
formed between diethyl azodicarboxylate and triphenylphosphine, and is
conducted in an
aprotic solvent, for instance THF, CH2Cl2, or DMF. The pyridine-N-oxide moiety
of I-4 is
reduced to the corresponding pyridine I-5 under transfer hydrogenation
conditions using a
palladium catalyst, preferably palladium metal on activated carbon, in an
inert solvent, for
instance methanol, ethanol, or 2-propanol. Cyclohexene, 1,4-cyclohexadiene,
formic acid,
and salts of formic acid, such as potassium formate or ammonium formate, are
commonly
used as the hydrogen transfer reagent in this type of reaction. The methyl
ester of I-5 is
hydrolyzed using aqueous base, for example, LiOH in aqueous THF or NaOH in
aqueous
methanol or ethanol, and the intermediate carboxylate salt is acidified with a
suitable acid,
for instance TFA or HCI, to afford the carboxylic acid I-6. Alternatively, the
intermediate
carboxylate salt can be isolated, if desired, or a carboxylate salt of the
free carboxylic acid
can be prepared by methods well-known to those of skill in the art.
Acid addition salts of the compound 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. Cationic salts are prepared by treating the 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 compound of formula (I) may be used in the manufacture of a
medicament. Pharmaceutical compositions of the compound of formula (I)
prepared as
hereinbefore described may be formulated as solutions or lyophilized powders
for
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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°!o 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, the compound may be encapsulated, tableted or prepared in a
emulsion
or syrup for oral administration. Pharmaceutically acceptable solid or liquid
carriers may
be added to enhance or stabilize the composition, or to facilitate preparation
of the
composition. Solid carriers include starch, lactose, calcium sulfate
dehydrate, terra alba,
magnesium stearate or stearic acid, talc, pectin, acacia, 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
far hard
gelatin capsule forms. When a liquid carrier is used, the preparation will be
in the form 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 compound of this invention may also be combined
with excipients such as cocoa butter, glycerin, gelatin or polyethylene
glycols and molded
mto a suppository.
The compound described herein is an antagonist of the vitronectin receptor,
and is
useful for treating diseases wherein the underlying pathology is attributable
to ligand or cell
which interacts with the vitronectin receptor. For instance, this compound is
useful for the
treatment of diseases wherein loss of the bone matrix creates pathology. Thus,
the instant
compound is 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 compound of this
invention is also
believed to have utility as an antitumor, anti-angiogenic, antiinflammatory
and anti-
metastatic agent, and be useful in the treatment of atherosclerosis and
restenosis.
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 other
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such indication. The pharmaceutical composition containing the compound is
administered
at an oral dose of between about 0. I to about 50 mglkg in a manner consistent
with the
condition of the patient. Preferably the oral dose would be about 0.5 to about
20 mglkg.
For acute therapy, parenteral administration is preferred. An intravenous
infusion of the
peptide 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 compound is 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 compound is 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.
This invention further provides a method for treating osteoporosis or
inhibiting
bone loss which comprises administering stepwise or in physical combination a
compound
of formula (1) and other inhibitors of bone resorption, such as
bisphosphonates (i.e.,
allendronate), hormone replacement therapy, anti-estrogens, or calcitonin. In
addition, this
invention provides a method of treatment using a compound of this invention
and an
anabolic agent, such as the bone morphogenic protein, iproflavone, useful in
the prevention
of bone loss andlor to increase bone mass.
Additionally, this invention provides a method of inhibiting tumor growth
which
comprises administering stepwise or in physical combination a compound of
formula (I)
and an antineoplastic agent. Compounds of the camptothecin analog class, such
as
topotecan, irinotecan and 9-aminocamptothecin, and platinum coordination
compiexes,
such as cisplatin, ormaplatin and tetraplatin, are well known groups of
antineoplastic
agents. Compounds of the camptothecin analog class are described in U.S.
Patent Nos.
5,004,758, 4,604,463, 4,473,692, 4,545,880 4,342,776, 4,513,138, 4,399,276, EP
Patent
Application Publication Nos . 0 418 099 and 0 088 642, Wani, et al., J. Med.
Chem., 1986,
29, 2358, Wani, et al., J. Med. Chem., 1980, 23, 554, Wani, et al., J. Med.
Chem., 1987, 30,
1774, and Nitta, et al., Proc. 14th International Congr. Chemotherapy., 1985,
Anticancer
Section l, 28, the entire disclosure of each which is hereby incorporated by
reference. The
platinum coordination complex, cisplatin, is available under the name
Platinol~ from
Bristol Myers-Squibb Corporation. Useful formulations for cisplatin are
described in U.S.
Patent Nos. 5,562,925 and 4,310,515, the entire disclosure of each which is
hereby
incorporated by reference.
In the method of inhibiting tumor growth which comprises administering
stepwise
or in physical combination a compound of formula (I) and an antineopiastic
agent, the
platinum coordination compound, for example cisplatin, can be administered
using slow
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intravenous infusion. The preferred carrier is a dextrose/saline solution
containing
mannitol. The dose schedule of the platinum coordination compound may be on
the basis
of from about 1 to about 500 mg per square meter (mg/m2} of body surface area
per course
of treatment. Infusions of the platinum coordiation compound may be given one
to two
times weekly, and the weekly treatments may be repeated several times. Using a
compound of the camptothecin analog class in a parenteral administration, the
course of
therapy generally employed is from about 0.1 to about 300.0 mg/m2 of body
surface area
per day for about five consecutive days. Most preferably, the course of
therapy employed
for topotecan is from about 1.0 to about 2.0 mglm2 of body surface area per
day for about
five consecutive days. Preferably, the course of therapy is repeated at least
once at about a
seven day to about a twenty-eight day interval.
The pharmaceutical composition may be formulated with both the compound of
formula (I) and the antineoplastic agent in the same container, but
formualtion in different
containers is preferred. When both agents are provided in solution form, they
can be
contained in an infusion/injection system for simultaneous administration or
in a tandem
arrangement.
For convenient administration of the compound of formula (I) and the
antineoplastic agent at the same or different times, a kit is prepared,
comprising, in a single
container, such as a box, carton or other container, individual bottles, bags,
vials or other
containers each having an effective amount of the compound of formula (I) for
parenteral
administration, as described above, and an effective amount of the
antineoplastic agent for
parenteral administration, as described above. Such kit can comprise, for
example, both
pharmaceutical agents in separate containers or the same container, optionally
as
lyophilized plugs, and containers of solutions for reconstitution. A variation
of this is to
include the solution for reconstitution and the lyophilized plug in two
chambers of a single
container, which can be caused to admix prior to use. With such an
arrangement, the
antineoplastic agent and the compound of this invention may be packaged
separately, as in
two containers, or lyophilized together as a powder and provided in a single
container.
When both agents are provided in solution form, they can be contained in an
infusionlinjection system for simultaneous administration or in a tandem
arrangement. For
example, the compound of formula (I) may be in an i.v. injectable form, or
infusion bag
linked in series, via tubing, to the antineoplastic agent in a second infusion
bag. Using such
a system, a patient can receive an initial bolus-type injection or infusion of
the compound
of formula (I) followed by an infusion of the antineoplastic agent.
The compound 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-1072b0 Binding to ay~33: Human placenta or human
platelet
avJi3 (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 av(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-HCI (pH 7.4), 100 mM NaCI, 1 mM
CaCl2, 1
mM MnCl2, 1 mM MgCl2) to a final compound concentration of 100 NM. This
solution is
then diluted to the required final compound concentration. Various
concentrations of
unlabeled antagonists (0.001 - 100 ~.iM) were added to the wells in
triplicates, followed by
the addition of 5.0 nM of [3H]-SK&F-107260 (65 - 86 Ci/mmol).
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 L.~M 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 Ki (dissociation constant of the antagonist) was
calculated
according to the equation: K; = ICSp/( 1 + L/Kd), where L and Kd were the
concentration
and the dissociation constant of [3H]-SK&F-107260, respectively.
The compound of the present invention inhibits vitronectin binding to SK&F
107260 at a concentration of about 0.003 micomolar.
The Compound of this invention is 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 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.
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Vascular smooth muscle cell migration assay
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 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 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.
Thyroparathyroidectomized rat model
Each experimental group consists of 5-6 adult male Sprague-Dawley rats (250-
400g body
weight). The rats are thyroparathyroidectomized (by the vendor, Taconic Farms)
7 days prior to
use. All rats receive a replacement dose of thyroxine every 3 days. On receipt
of the rats,
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
the ionized Ca level
(measured with a Ciba-Corning model 634 calcium pH analyzer) is <1.2 mM/L.
Each rat is fitted
with an indwelling venous and arterial catheter for the delivery of test
material and for blood
sampling respectively. The rats are then put on a diet of calcium-free chow
and deionized water.
Baseline Ca levels are measured and each rat is administered either control
vehicle or human
parathyroid hormone 1-34 peptide (hl'TH1-34, dose 1.25 ug/kg/h in saline/0.1%
bovine serum
albumin, Bachem, Ca) or a mixture of hPTHI-34 and test material, by continuous
intravenous
infusion via the venous catheter using an external syringe pump. The calcemic
response of each
rat is measured at two-hourly intervals during the infusion period of 6-8
hours.
Human osteoclast resorption and adhesion assays
Pit resorption and adhesion assays have been developed and standardized using
normal human osteoclasts derived from osteoclastoma tissue. Assay 1 was
developed for
the measurement of osteoclast pit volumes by laser confocal microscopy. Assay
2 was
developed as a higher throughput screen in which collagen fragments (released
during
resorption) are measured by competitve ELISA.
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Assay 1 (using laser confocal microscopy)
~ Aliquots of human osteoclastoma-derived cell suspensions are removed from
liquid
nitrogen strorage, warmed rapidly at 37oC and washed xl in RPMI-1640 medium by
centrifugation ( 1000rpm, 5 mins at 4oC).
The medium is aspirated and replaced with murine anti-HLA-DR antibody then
diluted 1:3 in RPMI-1640 medium. The suspension is incubated for 30 mins on
ice and
mixed frequently.
~ The cells are washed x2 with cold RPMI-1640 followed by centrifugation (1000
rpm, 5 mins at 4oC) and the cells are then 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
(Dynal, Great Neck, NY) 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
mins 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 viable osteoclasts are enumerated in a counting chamber, using
fluorescein
diacetate to label live cells. A large-bore disposable plastic pasteur pipet
is used to add
the sample to the chamber.
~ The osteoclasts are pelleted by centrifugation and the density adjusted to
the
appropriate number in EMEM medium (the number of osteoclasts is variable from
tumor to tumor), supplemented with 10% fetal calf serum and 1.7g/liter of
sodium
bicarbonate.
~ 3ml aliquots of the cell suspension (per compound treatment) are decanted
into
15m1 centrifuge tubes. The cells are pelleted by centrifugation.
~ To each tube, 3ml of the appropriate compound treatment are added (diluted
to 50
uM in the EMEM medium). Also included are appropriate vehicle controls, a
positive
control (anti-vitronectin receptor murine monoclonal antibody [87MEM1] diluted
to
100 ug/ml) and an isotype control (IgG~ diluted to 100 ug/ml). The samples are
incubated at 37oC for 30 mins.
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~ O.SmI 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 medium containing the compound treatment or control
samples. The samples are incubated at 37°C for 48 hours.
Tartrate resistant acid phosphatase (TRAP) procedure (selective stain for
cells of the
osteoclast lineage)
~ The bone slices containing the attached osteoclasts are washed in phosphate
buffered saline and fixed in 2% gluteraldehyde (in 0.2M sodium cacodylate) for
5 mins.
~ They are then washed in water and are incubated for 4 minutes in TRAP buffer
at
3?oC (0.5 mg/ml naphthol AS-BI phosphate dissolved in N,N-dimethylformamide
and
mixed with 0.25 M citrate buffer (pH 4.5), containing 10 mM sodium tartrate.
~ Following a wash in cold water the slices are immersed in cold acetate
buffer (0.1
M, pH 6.2) containing 1 mglml fast red garnet and incubated at 4oC for 4
minutes.
~ Excess buffer is aspirated, and the slices are air dried following a wash in
water.
~ The TRAP positive osteoclasts (brick redl purple precipitate) are enumerated
by
bright-field microscopy and are then removed from the surface of the dentine
by
sonication.
~ Pit volumes are determined using the NikonILasertec ILM21 W confocal
microscope.
Assay 2 (using an ELISA readout)
The human osteoclasts are enriched and prepared for compound screening as
described in the initial 9 steps of Assay 1. For clarity, these steps are
repeated hereinbelow.
~ Aliquots of human osteoclastoma-derived cell suspensions are removed from
liquid
nitrogen strorage, warmed rapidly at 37oC and washed xl in RPMI-1640 medium by
centrifugation (1000rpm, 5 mins at 4oC).
~ The medium is aspirated and replaced with murine anti-HLA-DR antibody then
diluted 1:3 in RPMI-1640 medium. The suspension is incubated for 30 mins on
ice and
mixed frequently.
~ The cells are washed x2 with cold RPMI-1640 followed by centrifugation (1000
rpm, 5 mins at 4oC) and the cells are then 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 I mononuclear cell), coated with goat anti-
mouse IgG
(Dynal, Great Neck, NY) are removed from their stock bottle and placed into 5
ml of
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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
mins 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 viable osteoclasts are enumerated in a counting chamber, using fluorescein
diacetate to label live cells. A large-bore disposable plastic pasteur pipet
is used to add
the sampie to the chamber.
~ The osteoclasts are pelleted by centrifugation and the density adjusted to
the
appropriate number in EMEM medium (the number of osteoclasts is variable from
tumor to tumor), supplemented with 10% fetal calf serum and 1.7g/liter of
sodium
bicarbonate.
In contrast to the method desribed above in Assay l, the compounds are
screened at
4 doses to obtain an IC5", as outlined below:
~ The osteoclast preparations are preincubated for 30 minutes at 37"C with
test
compound (4 doses) or controls.
~ They are then seeded onto bovine cortical bone slices in wells of a 48-well
tissue
culture plate and are incubated for a further 2 hours at 37°C.
~ The bone slices are washed in six changes of warm phosphate buffered saline
(PBS), to remove non-adherent cells, and are then returned to wells of a 48
well plate
containing fresh compound or controls.
~ The tissue culture plate is then incubated for 48 hours at 37°C.
~ The supernatants from each well are aspirated into individual tubes and are
screened in a competitive ELISA that detects the c-telopeptide of type I
collagen which
is released during the resorption process. This is a commercially available
ELISA
(Osteometer, Denmark) that contains a rabbit antibody that specifically reacts
with an 8-
amino acid sequence (Glu-Lys-Ala-His- Asp-Gly-Gly-Arg) that is present in the
carboxy-terminal telopeptide of the al-chain of type I collagen. The results
are
expressed as % inhibition of resorption compared to a vehicle control.
Human osteoclast adhesion assay
The human osteoclasts are enriched and prepared for compound screening as
described above in the inital 9 steps of Assay 1. For clarity, these steps are
repeated
hereinbelow.
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~ Aliquots of human osteoclastoma-derived cell suspensions are removed from
liquid
nitrogen strorage, warmed rapidly at 37oC and washed xl in RPMI-1640 medium by
centrifugation (1000rpm, S mins at 4oC).
~ The medium is aspirated and replaced with murine anti-HLA-DR antibody then
diluted 1:3 in RPMI-1640 medium. The suspension is incubated for 30 mins on
ice and
mixed frequently.
~ The cells are washed x2 with cold RPMI-1640 followed by centrifugation (1000
rpm, 5 mins at 4°C) and the cells are then 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
(Dynal, Great Neck, NY) 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
mins 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 viable osteoclasts are enumerated in a counting chamber, using
fluorescein
diacetate to label live cells. A large-bore disposable plastic pasteur pipet
is used to add
the sample to the chamber.
~ The osteoclasts are pelleted by centrifugation and the density adjusted to
the
appropriate number in EMEM medium (the number of osteoclasts is variable from
tumor to tumor), supplemented with 10% fetal calf serum and 1.7g/liter of
sodium
bicarbonate.
~ Osteoclastoma-derived osteoclasts are preincubated with compound (4 doses)
or
controls at 37"C for 30 minutes.
~ The cells are then seeded onto osteopontin-coated slides (human or rat
osteopontin,
2.Sug/ml) and incubated for 2 hours at 37°C.
Non adherent cells are removed by washing the slides vigorously in phosphate
buffered saline and the cells remaining on the slides are fixed in acetone.
~ The osteoclasts are stained for tartrate-resistant acid phosphatase (TRAP),
a
selective marker for cells of this phenotype (see steps 15 -17), and are
enumerated by
light microscopy. The results are expressed as % inhibition of adhesion
compared to a
vehicle control.
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Cell Adhesion Assay
Cells and Cell Culture
Human embryonic kidney cells ( HEK293 cells) were obtained from ATCC
(Catalog No. CRL 1573). Cells were grown in Earl's minimal essential medium
(EMEM)
medium containing Earl's salts, 10% fetal bovine serum, 1 % glutamine and 1 %
Penicillin-
Steptomycin.
Constructs and Transfections
A 3.2 kb EcoRI-KpnI fragment of the av subunit and a 2.4 kb XbaI- XhoI
fragment
of the (33 subunit were inserted into the EcoRI - EcoRV cloning sites of the
pCDN vector
(Aiyar et al., 1994 ) which contains a CMV promoter and a 6418 selectable
marker by
blunt end ligation. For stable expression, 80 x 10 6 HEK 293 cells were
electrotransformed
with av+~i3 constructs (20 pg DNA of each subunit) using a Gene Pulser
(Hensley et al.,
1994 ) and plated in 100 mm plates (5x105 cells/plate). After 48 hr, the
growth medium
was supplemented with 450 ~g/mL Geneticin (G418 Sulfate, GIBCO-BRL, Bethesda,
MD).
The cells were maintained in selection medium until the colonies were large
enough to be
assayed.
Immunocytochemical analysis of transfected cells
To determine whether the HEK 293 transfectants expressed the vitronectin
receptor, the cells were immobilized on glass microscope slides by
centrifugation, fixed in
acetone for 2 min at room temperature and air dried. Specific reactivity with
23C6, a
monoclonal antibody specific for the av(33 complex was demonstrated using a
standard
indirect immunofluorescence method.
Cell Adhesion Studies
Corning 96-well ELISA plates were precoated overnight at 4oC with 0.1 mL of
human vitronectin (0.2 ~g/mL in RPMI medium). At the time of the experiment,
the plates
were washed once with RPMI medium and blocked with 3.5% BSA in RPMI medium for
1
hr at room temperature. Transfected 293 cells were resuspended in RPMI medium,
supplemented with 20 mM Hepes, pH 7.4 and 0.1 % BSA at a density of 0.5 x 106
cells/mL.
0.1 mL of cell suspension was added to each well and incubated for 1 hr at
37°C, in the
presence or absence of various av~33 antagonists. Following incubation, 0.025
mL of a
10% formaldehyde solution, pH 7.4, was added and the cells were fixed at room
temperature for 10 min. The plates were washed 3 times with 0.2 mL of RPMI
medium
and the adherent cells were stained with 0.1 mL of 0.5% toluidine blue for 20
min at room
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temperature. Excess stain was removed by extensive washing with deionized
water. The
toluidine blue incorporated into cells was eluted by the addition of 0.1 mL of
50% ethanol
containing 50 mM HCI. Cell adhesion was quantitated at an optical density of
600 nm on a
microtiter plate reader (Titertek Multiskan MC, Sterling, VA).
Solid-Phase a.~,(35 Binding Assay:
The vitronectin receptor a~~3g was purified from human placenta. Receptor
preparation was diluted with 50 mM Tris-HCI, pH 7.5, 100 mM NaCI, 1 mM CaCl2,
1 mM
MnCl2, 1 mM MgCl2 (buffer A) and was immediately added to 96-well ELISA plates
at 0.1
ml per well. 0.1-0.2 Ng of a,,(33 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.
In a [3H)-SK&F-107260 competition assay, various concentrations of unlabeled
antagonists (0.001-100 pM) were added to the wells, followed by the addition
of 5.0 nM of
[3H]-SK&F-107260. 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 6800 Liquid Scintillation Counter,
with 40%
efficiency. Nonspecific binding of [3H]-SK8cF-107260 was determined in the
presence of 2
NM SK&F-107260 and was consistently less than 1 % of total radioligand input.
The ICgo
(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 Cheng and Prusoff equation: K; = ICgpl ( 1 + L/K~), where L and
Kd were the
concentration and the dissociation constant of [3H]-SK&F-10?260, respectively.
Inhibition of RGD-mediated GPIIb-IIIa binding
Purification of GPIIb-IIIa
Ten units of outdated, washed human platelets (obtained from Red Cross) were
lyzed by gentle stirring in 3% octylglucoside, 20 mM Tris-HCI, pH 7.4, I40 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%
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octylglucoside (buffer A). After 2 h incubation, the column was washed with 50
mL cold
buffer A. The lectin-retained GPIIb-IIIa was eluted with buffer A containing
10% dextrose.
All procedures were performed at 4°C. The GPIIb-IIIa obtained was >95%
pure as shown
by SDS polyacrylamide gel electrophoresis.
Incorporation of GPIIb-IIIa 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
GPIIb-IIIa was diluted 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 I()DO-fold
excess of 50
mM Tris-HCI, pH 7.4, 100 mM NaCI, 2 mM CaCl2 (with 2 changes). The GPIIb-IIIa-
containing liposomes wee centrifuged at 12,OOOg for IS min and resuspended in
the dialysis
buffer at a final protein concentration of approximately 1 mg/mL. The
liposames were
stored at -70C until needed.
Competitive Binding to GPIIb-IIIa
The binding to the fibrinogen receptor (GPIIb-IIIa) was assayed by an indirect
competitive binding method using [3H]-SK&F-107260 as an RGD-type iigand. 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 ug/mL polylysine (Sigma Chemical Co., St. Louis, MO.) at
room
temperature for i h to block nonspecific binding. Various concentrations of
unlabeled
benzazepines 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 pg
of the purified
platelet GPIIb-IIIa-containing liposomes. The mixtures were incubated for 1 h
at room
temperature. The GPIIb-IIIa-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
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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.
The compound of this invention has an affinity for the vitronectin receptor
relative
to the fibrinogen receptor of greater than 10:1. This compound has a ratio of
activity of
greater than 100:1.
The efficacy of the compound of formula (I) alone or in combination with an
antineoplastic agent may be determined using several transplantable mouse
tumor models.
See U. S. Patent Nos. 5,004,758 and 5,633,016 for details of these models
The examples which follow are intended in no way to limit the scope of this
invention, but are provided to illustrate how to make and use the compound of
this
invention. Many other embodiments will be readily apparent to those skilled in
the art.
EXAMPLES
General
1H nuclear magnetic resonance (NMR) spectra were recorded at either 250 or 400
MHz. Chemical shifts are reported in parts per million (b) downfield from the
internal
standard tetramethylsilane (TMS). Abbreviations for NMR data are as follows:
s=singlet,
d=doublet, t=triplet, q=quartet, m=multiplet, dd=doublet of doublets,
dt=doublet of triplets,
app=apparent, br=broad. J indicates the NMR coupling constant measured in
Hertz.
CDC13 is deuteriochloroform, DMSO-d6 is hexadeuteriodimethylsulfoxide, and
CD30D is
tetradeuteriomethanol. Infrared (IR) spectra were recorded in transmission
mode, and band
positions are reported in inverse wavenumbers (cm-1). Mass spectra were
obtained using
eiectrospray (ES) ionization techniques. Elemental analyses were performed by
Quantitative Technologies Inc., Whitehouse, NJ. Melting points were taken on a
Thomas-
Hoover melting point apparatus and are uncorrected. All temperatures are
reported in
degrees Celsius. Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254 thin
layer plates
were used for thin layer chromatography. Both flash and gravity chromatography
were
carried out on E. Merck Kieselgel 60 {230-400 mesh) silica gel. Analytical and
preparative
HPLC were carried out on Rainin or Beckman chromatographs. ODS refers to an
octadecylsilyl derivatized silica gel chromatographic support. 5 lr Apex-ODS
indicates an
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SU8ST1TUTE SHEET {RULE 26)
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octadecylsilyl derivatized silica gel chromatographic support having a nominal
particle size
of 5 N, made by Jones Chromatography, Littleton, Colorado. YMC ODS-AQ~ is an
ODS
chromatographic support and is a registered trademark of YMC Co. Ltd., Kyoto,
Japan.
PRP-1~ is a polymeric (styrene-divinylbenzene) chromatographic support, and is
a
registered trademark of Hamilton Co., Reno, Nevada. Celite~ is a filter aid
composed of
acid-washed diatomaceous silica, and is a registered trademark of Manville
Corp., Denver,
Colorado.
Example 1
Preparation of (S_)-8-1~3-(4-meth~pyridin-2-ylaminoZ 1-pro~yloxyl-3-oxo-2-f4-
(trifluoromethyl)benzyll-2.3,4,5-tetrahydro-1H-2-benzazenine-4-acetic acid
Preparation 1
Preparation of methyl (~)-8-h d~oxy-3-oxo-2.3,4,5-tetrahydro-1H-2-benzazepine-
4-acetate
a) 4-Bromo-3-bromomethylanisole
A mixture of 2-bromo-5-methoxytoluene (20 g, 0.10 mol), N-bromosuccinimide
(19.6 g, 0.11 mol), benzoyl peroxide (1 g, 4 mmol), and methylene chloride
(200 mL) was
irradiated for 18 hr with a flood lamp to effect gentle reflux. The mixture
was then cooled
to -10°C for several hours and the solution was decanted away from the
precipitated
succinimide. The solution was concentrated and the residue was crystallized
from
chloroform/hexane to give the title compound (19.7 g, 70%) as pale yellow
prisms: 1H
NMR (CDCl3) b 7.45 (d, J = 8.9 Hz, 1 H), 6.99 (d, J = 3 Hz, 1 H), 6.74 (dd, J
= 8.9, 3 Hz, 1
H), 4.55 (s, 2 H) 3.80 (s, 3 H).
b) 3-Bis(tent-butoxycarbonyl)aminomethyl-4-bromoanisole
A mixture of 4-bromo-3-bromomethylanisole (24 g, 86 mmol) and potassium di-
tert-butyl iminodicarboxylate (24 g, 94 mmol) in dimethylformamide (200 mL)
was stirred
under argon at room temperature for 18 hr. The reaction was then concentrated
under
vacuum and the residue was partitioned between ethyl acetate and water. The
organic
phase was washed with water and brine, dried(MgS04), and concentrated. The
residue was
recrystallized from hexane to give the title compound (15 g, 42%) as a white
solid: 1H
NMR (CDCl3) b 7.40 (d, J = 8.6 Hz, 1 H)), 6.68 (m, 2 H), 4.81(s, 2 H), 3.74
(s, 3 H), 1.44
(s, 18 H).
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c) Methyl {~)-3-carbomethoxy-4-[2-bis(tent-butoxycarbonyl)aminomethyl-4-
methoxyphenyl]-3-butenoate
A 500 mL flask was charged with 3-bis(tert-butoxycarbonyl)aminornethyl-4-
bromoanisole(15 g, 36 mmol), dimethyl itaconate (7.5 g, 47 mmol), tri-o-
tolylphosghine (I
g, 3 mol), palladium acetate (0.4 g, 2 mmol), diisopropylethylamine (12.8 mL,
72 mmol),
and propionitrile (150 mL). The mixture was purged with argon (several
evacuation/argon
flush cycles), then was heated to reflex under argon for 1 hr. The reaction
was allowed to
cool to RT, then was poured into ice-cold ethyl ether (500 mL). The resulting
precipitate
was removed by filtration and the filtrate was concentrated. The residue was
purified by
ID chromatography on silica gel (10% - 20% ethyl acetate in hexane) to give
the title
compound (11.8 g, 66%) as a pale yellow oil: IH NMR (CDC13) 8 7.94 (s, I H),
7.15 (d, J
= 8.1 Hz, I H)), 6.77 (d, J = 8.1 Hz, 1 H), 6.76 (s, 1 H), 4.73 (s, 2 H), 3.81
(s, 3 H), 3.79 (s,
3 H), 3.71 {s, 3 H), 3.38 (s, 2 H}, 1.45 (s, 18 H).
d) Methyl (f)-3-carbomethoxy-4-[2-bis(tent-butoxycarbonyl)aminomethyl-4-
methoxyphenyl]butanoate
A pressure vessel charged with methyl (~)-3-carbomethoxy-4-[2-bis(tert-
butoxycarbonyl}aminomethyl-4-methoxyphenyl]-3-butenoate (11.8 g), ethyl
acetate (120
mL}, and 10% palladium on charcoal ( I g) was shaken under 45 psi of hydrogen
for 18 hr.
The mixture was then filtered and the filtrate was concentrated to give the
title compound
( 12 g, I00%) as a colorless oil: 1 H NMR {CDC13) 8 7.00 (d, J = 8.2 Hz, 1 H),
6.71 (m, 2
H), 4.81 (s, 2 H), 3.75 (s, 3 H), 3.66 (s,3 H), 3.63 (s, 3 H), 3.05 (m, 2 H),
2.73 (m, 2 H),
2.42 (dd, J = 16.0, 4.8 Hz, 1 H), I .44 (s, 18 H).
e) Methyl (~)-3-carbomethoxy-4-[2-(aminomethyl)-4-methoxyphenyl]butanoate
A solution of methyl (~)-3-carbomethoxy-4-[2-bis(tert-
butoxycarbonyl)aminomethyl-4-methoxyphenyl]butanoate (12 g) in chloroform {100
mL)
and trifluoroacetic acid (50 mL) was stirred under argon at room temperature
for 4 hr. The
solution was then concentrated under vacuum to give the title compound ( 10 g,
100%) as a
viscous oil: MS (ES) m/e 296.2 (M + H)+.
f) Methyl (~)-8-methoxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate
A solution of methyl (~)-3-carbomethoxy-4-[2-(aminomethyl)-4
methoxyphenyl]butanoate {10 g, 24 mmol) and triethylamine (17 mL, 120 mmol) in
toluene
(100 mL) was heated at reflex for 18 hr. The reaction was then concentrated
and the
residue was partitioned between ethyl acetate and water. The aqueous layer was
extracted
twice with ethyl acetate and the combined organic extracts were washed with
brine, dried
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SUBSTITUTE SHEET (RULE 26)
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WO 99!15170 PCTIUS98/19949
(MgS04), and concentrated to afford the title compound (4.8 g, 76%) as tan
solid: MS
(ES) m/e 264.2 (M + H)+.
g) Methyl (t)-8-hydroxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate
Anhydrous aluminum chloride (7.6 g, 57 mmol) was added portionwise to a
stirred
solution of methyl (~)-8-methoxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-
acetate
(3.0 g, 11 mmol) and ethanethiol (4.2 mL, 57 mmol) in methylene chloride (100
mL) at 0°C
under argon. The resulting mixture was allowed to warm to room temperature and
stir
overnight, then was concentrated. The residue was triturated with ice-water,
and the
resulting solid was collected by filtration and dried to give the title
compound (2.64 g,
91%) as an off-white solid: MS (ES) m/e 250.2 (M + H)+.
Preparation 2
HPLC separation of the enantiomers of methyl C~)-8-h~rdroxy-3-oxo-2.3,4,5-
tetrahydro-1H-
2-benzazepine-4-acetate
a) Methyl (R)-(+)-8-hydroxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-
acetate and
methyl (S)-(-)-8-hydroxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate
Methyl (~)-8-hydroxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate was
resolved into its enantiomers by chiral HPLC using the following conditions:
Diacel
Chiralpak ASS column (21.2 x 250 mm), EtOH mobile phase, 7 mL/min flowrate, uv
detection at 254 nm, 70 mg injection; tR for methyl (R)-(+)-8-hydroxy-3-oxo-
2,3,4,5-
tetrahydro-1H-2-benzazepine-4-acetate = 21.5 min; tR for methyl (S)-(-)-8-
hydroxy-3-oxo-
2,3,4,5-tetrahydro-1 H-2-benzazepine-4-acetate = 39.1 min.
Preparation 3
HPLC separation of the enantiomers of methy~~)-8-methoxy-3-oxo-2.3.4,5-
tetrah,Ydro-
1 H-2-benzaze,.pine-4-acetate
a) Methyl {R)-(+)-8-methoxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-
acetate and
methyl (S)-(-)-8-methoxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate
Methyl (-~)-8-methoxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate was
resolved into its enantiomers by chiral HPLC using the following conditions:
Diacel
Chiralpak ASS column (21.2 x 250 mm), CH3CN mobile phase, 15 mL/min flowrate,
uv
detection at 254 nm, 500 mg injection; tR for methyl (R)-(+)-8-methoxy-3-oxo-
2,3,4,5-
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tetrahydro-1H-2-benzazepine-4-acetate = 10.2 min; tR for methyl (S)-(-)-8-
rnethoxy-3-oxo-
2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate = 19.0 min.
Preparation 4
Demethyiation of methy~S)-8-methoxy-3-oxo-2.3.4.5-tetrahydro-1H-2-benzazepine-
4-
acetate
a) Methyl (S)-8-hydroxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate
A solution of methyl (S)-8-methoxy-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-
acetate (15.0 g, 0.057 mole) in CHCI3 (160 mL) was added dropwise over 30 min
to a
solution of boron tribromide (20.53 mL, 0.217 mole) in CHCI3 (160 mL) at -8
°C under
argon, maintaining the temperature between -5 °C and 0 °C. The
reaction mixture was
stirred at ca. -8 °C for 30 min and then MeOH (200 mL) was added,
dropwise initially,
maintaining the temperature at ca. 0 °C. The reaction mixture was
concentrated to give a
viscous oil which was reconcentrated from MeOH (100 mL). The oil was dissolved
in
H20/MeOH and a small amount of dark solid was removed by filtration. The
filtrate was
neutralized (to pH 7) with 50 % sodium hydroxide, depositing a white solid.
The
suspension pH was adjusted to 4.5 by the addition of a small amount of acetic
acid and the
solid was collected and dried in vacuum to give afford the title compound (9.7
g, 68 %).
The product was assayed for chiral purity by HPLC: Chiralpak AS~ column (4.6 x
50
mm), 100% EtOH mobile phase, 0.5 mL/min flow rate, uv detection at 215 nm; tR
= 7.5
min (S-enantiomer, 99 %); tR = 4.4 min (R-enantiomer, 1 %).
Preparation 5
Preparation of methyl (S)-8-hydroxy-3-oxo-2-f4-(trifluorometh r~l)benz,~]-
2,3.4.5-
tetrahydro-1H-2-benzazepine-4-acetate via alkylation of methyl (S)-8-hydrox~r-
3-oxo-
2.3.4,5-tetrahydro-I H-2-benzazepine-4-acetate
a) Methyl (S)-3-oxo-8-[4-(trifluoromethyl)benzyloxy]-2-[4-
(trifluoromethyl)benzyl]-
2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate
To a solution of methyl (S)-8-hydroxy-3-oxo-2,3,4,5-tetrahydro-2-1H-
benzazepine-
4-acetate (0.31 g, 1.24 mmol) and 4-(trifluoromethyl)benzyl bromide (0.89 g,
3.72 mmol}
in DMF (10 mL) was added NaH (60% suspension in oil, 0.11 g, 2.75 mmol}. After
stirring
at RT for 4h, the bulk of the DMF was removed under vacuum. The residue was
partitioned between sat. NaHC03 and EtOAc. The aqueous phase was extracted
with
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SUBSTITUTE SHEET (RULE 26)
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EtOAc and the combined organic extracts were washed with sat. NaCI, dried over
Na2S04
and concentrated to give a clear oil (0.90 g). Radial chromatography (5%
acetone/CH2Ci2,
silica gel, 6 m plate) gave the title compound (0.53 g) as a white foam. MS
(ES) m/e 566.1
(M + H)+.
b) Methyl (S)-8-hydroxy-3-oxo-2-[4-{trifluoromethyl)benzyl]-2,3,4,5-tetrahydro-
1H-2-
benzazepine-4-acetate
A Parr hydrogenation flask was charged with methyl (S)-3-oxo-8-[4
(trifluoromethyl)benzyloxy]-2-[4-(trifluoromethyl)benzyl]-2,3,4,5-tetrahydro-
1H-2
IO benzazepine-4-acetate (0.78 g, 1.38 mmol) and Pearlman's catalyst (20 mg)
in MeOH (20
mL). After hydrogenating at 50 psi for 24 h, the reaction vessel was vented
and the catalyst
was removed by filtration. Removal of solvent gave a white foam (0.60 g).
Radial
chromatography {5% acetone/CH2C12, silica gel, 6 m plate) gave the title
compound (0.42
g) as a white foam. 1 H NMR {250 MHz, CDC13} d 7.50 {d, J = 8.5 Hz, 2H), 7.23
(d, J =
8.5 Hz, 2H), 6.90 (d, J = 7.5 Hz, 1H), 6.67 (dd, J = 7.5, 3.4 Hz, 1H), 6.39
(d, J = 3.4 Hz,
1 H), 5.05 (m, 2 H), 4.35 (d, J = 15.4 Hz, 1 H), 3.85 (m, 1 H), 3.70 (s, 3H),
3.60 (m, 1 H), 2.95
(m, 4H), 2.45 (dd, J = 17.1, 5.1 Hz, IH).
Preparation 6
Preparation of methyl (S)-8-hydroxy-3-oxo-2-(4-(trifluoromethyl)benz~rll-
2.3.4s5-
tetrahydro-1H-2-benzazepine-4-acetate via enantioselective synthesis
a) 4-Bromo-3-bromomethylanisole
To a stirred solution of 4-bromo-3-methylanisole (100 g, 497 mmol) in dry
dichloromethane (500 mL) was added N-bromosuccinimide (97 g, 545 mmol)
followed by
benzoyl peroxide (6 g, 25 mmol). The reaction was gently refluxed with a 150
watt flood
lamp with reflector placed approximately 12 inches from the reaction flask.
After 24 h the
reaction was concentrated by rotary evaporation to half its volume and allowed
to sit for 4
h. The white precipitate which formed was filtered off and rinsed with a small
volume of
dichloromethane. The filtrate was concentrated to dryness and the remaining
solid was
triturated with hexanes and filtered. Drying under vacuum gave the title
compound (100.25
g, 72%) as white needles: GC tR = 6.56 min (HP 530 p,m x 20 m methylsilicone
column,
He carrier flow 20 mLlmin, 100 °C initial temp., 1 min initial time, 10
°C/min rate, 200 °C
famal temp., 1 min final time); 1H NMR (400 MHz, CDCl3) 8 7.44 (d, J = 10 Hz,
1 H), 6.99
(d, J = 3 Hz, 1 H), 6.73 (dd, 1H), 4.55 (s, 2H), 3.80 (s, 3H).
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SUBSTITUTE S~IEET (RULE 26)
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b) 3-[N-(4-Trifluoromethylbenzyl)aminomethyl]-4-bromoanisole
To a stirred solution of 4-bromo-3-bromomethylanisole (35 g, 125 mmol) in
anhydrous DMSO {50 mL} and dry THF (50 mL) was added 4-
trifluoromethylbenzylamine
(30 g, 171 mmol) followed by triethylamine (18 mL, 129 mmol). After stirring
for 18 h at
RT the reaction was concentrated, diluted with aqueous I N NaOH (250 mL) and
extracted
with Et20 (2 x 250 mL). The combined organic layers were washed with brine,
dried
(Na2S04), and concentrated to dryness. The residue which remained was purified
by flash
chromatography on silica gel (10 to 20% EtOAc/CHCl3) to give the title
compound {34.17
g, 73%): TLC (20% EtOAc/CHCI3) Rf 0.63; 1H NMR (400 MHz, CDCl3) 8 7.59 (d, J =
8.2 Hz, 2H), 7.49 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.6 Hz, 1H), 6.96 (d, J =
3.1 Hz, 1H),
6.70 (dd, IH), 3.86 (s, 2H), 3.84 (s, 2H), 3.79 (s, 3H), 1.75 (br s, 1H).
c) 3-[N-(tert-Butoxycarbonyl)-N-(4-trifluoromethylbenzyl)aminomethyl]-4-
bromoanisole
To a stirred solution of 3-[N-(4-trifluoromethylbenzyl)aminomethyl]-4-
bromoanisole (34.17 g, 91 mmol) in dry THF (100 mL) was added di-tert-butyl
Bicarbonate
(22 g, 101 mmol). The reaction was stirred under argon for 18 h (vigorous gas
evolution
was observed). Concentration and silica gel chromatography (5 to 10%
EtOAclhexane)
gave the title compound (41.09 g, 95%) as a clear oil: TLC (silica, 20%
EtOAclhexane) Rf
0.44; 1 H NMR (400 MHz, CDCl3) 8 7.57 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 8.3
Hz, 2H),
7.39-7.33 (m, 2H), 6.83 and 6.72 (2 s, 1H), 6.71 (dd, 1H}, 4.54 and 4.50 (2 s,
2H), 4.43 (s,
2H), 3.75 (s, 3H), 1.47 (s, 9H).
d) Methyl 2-[N-(tert-butoxycarbonyl)-N-(4-trifluoromethylbenzyl)aminomethyl]-4-
methoxycinnamate
A solution of 3-[N-(tert-butoxycarbonyl)-N-(4-
trifluoromethylbenzyl)aminomethyl]-4-bromoanisole (37.08 g, 78 mmol), methyl
acrylate
(35 mL, 390 mmol), palladium acetate (0.88g, 3.9 mmol), tri-o-tolylphosphine
{2.38 g, 7.8
mol), and diisopropylethylamine (31 mL, 178 mmol) in acetonitrile (200 mL) was
deoxygenated (3 evacuation/argon purge cycles), then was heated to reflux
under argon (oil
bath set at 80 °C). After 6 hr additional palladium acetate (0.88 g,
3.9 mmol) and tri-o-
tolylphosphine ((2.38 g, 7.8 mmol) were added and the reaction was stirred
under reflux for
an additional 18 h. The reaction was concentrated to dryness, and the residue
was taken up
in 1:1 Et20/petroleum ether {300 mL) and allowed to stand for 4 h. A gray-
colored
precipitate was filtered off and washed with a small volume of 1:1
Et20/petroleum ether
(100 mL). The orangish-red filtrate was concentrated and purified by flash
chromatography on silica gel (15% ethyl acetate/hexanes). The resulting
residue was taken
up in hexane, and the mixture was allowed to stand for several hr, then was
filtered to
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SUBSTITUTE SHEET {RULE 26~
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WO 99/15170 PCTlUS98/19949
remove a yellow precipitate. Concentration of the filtrate left the title
compound (34.52 g,
92%) as a thick yellow oil: TLC (silica, 20% EtOAclhexanes) Rf0.45; 1H NMR
(400
MHz, CDCl3) 8 7.80 (br s, 1H), 7.57 (d, J = 8.1 Hz, 2H), 7.53 (d, J = 8.6 Hz,
1H), 7.29 (br
s, 2H), 6.83 (dd, 1 H), 6.72 (br s, 1 H), 6.23 {d, J = 15.7 Hz, 1 H}, 4.58 and
4.53 (2 br s, 2H),
4.46 and 4.37 (2 br s, 2H), 3.80 (s, 3H), 3.77 (s, 3H), 1.49 (s, 9H).
e) Methyl2-[N-(tent-butoxycarbonyl)-N-(4-trifluoromethylbenzyl)aminomethyl]-4-
methoxydihydrocinnamate
To 10% Pd/C (5 g, 4.7 mmol, prewetted with DMF) was added a solution of methyl
i0 2-[N-(tert-butoxycarbonyl)-N-(4-trifluoromethylbenzyl)aminomethyl]-4-
methoxycinnamate {34.52 g, 72 mmol) in methanol (100 mL). The mixture was
shaken
under hydrogen (50 psi) in a Parr apparatus for 7 hr, then was filtered
through a pad of
celiteOO to remove the catalyst. The filtrate was concentrated to afford the
title compound
(34.15 g, 98%) as a colorless oil: 1H NMR (400 MHz, CDC13) 8 7.58 (d, J = 8.1
Hz, 2H),
7.31 (br s, 2H), 7.09 (d, J = 8.4 Hz, 1H), 6.76 (dd, 1H), 6.66 (s, 1H), 4.47
(br s, 2H), 4.40
(br s, 2H), 3.76 (s, 3H), 3.63 (s, 3H), 2.79 (br s, 2H), 2.47 (t, 2H), 1.48
(s, 9H).
f) 2-[N-(tent-Butoxycarbonyl)-N-(4-trifluoromethylbenzyl)aminomethyl]-4-
methoxydihydrocinnamic acid
To a stirred solution of 2-[N-(tert-butoxycarbonyl)-N-(4-
trifluorobenzyl)aminomethyl]-4-methoxydihydrocinnamic acid (34.15 g, 71 mmol)
in
dioxane (150 mL) was added aqueous 1 N NaOH (85 mL, 85 mmol). The cloudy
reaction
was stirred at RT for 4 h. The resulting homogeneous solution was neutralized
with
aqueous 1 N HCl (85 mL, 85 mmol) and extracted with ethyl acetate (2 x 250
mL). The
combined organic layers were washed with brine (250 mL), dried (MgS04) and
concentrated to give the title compound (34.60 g, 100%) as a thick clear oil:
TLC (95:4:1
CHCI3/MeOH/HOAc) R f 0.49; 1 H NMR (400 MHz, CDC13) b 7.58 (d, J = 8.1 Hz,
2H),
7.30 (br s, 2H), 7.09 (d, J = 8.4 Hz, 1 H), 6.78 (dd, 1 H), 6.65 (d, J = 2.6
Hz, 1 H), 4.47 (br s,
2H), 4.42 (br s, 2H), 3.76 (s, 3H), 2.81 (br s, 2H), 2.53 (t, 2H), 1.47 {s,
9H).
g) (R)-4-Benzyl-2-oxazolidinonyl2-[N-(tert-butoxycarbonyl)-N-(4
trifluoromethylbenzyl)aminomethyl]-4-methoxydihydrocinnamide
To a stirred solution of 2-[N-(tent-butoxycarbonyl)-N-(4-
trifluoromethylbenzyl)aminomethyl]-4-methoxydihydrocinnamic acid (34.60 g, 71
mmol)
and pyridine (6.9 mL, 85 mmol) in dry dichloromethane (200 mL) under Argon was
added
cyanuric fluoride (4.4 mL, 48 mmol} via syringe. The reaction was stirred for
4 h at RT.
The resulting thick suspension was filtered through a pad of celite~ and
rinsed with a small
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SUBSTITUTE SHEET IF~cULE 26)
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volume of dry dichloromethane (50 mL). The clear filtrate was poured into a
separatory
funnel and washed with ice-cold water (500 mL). Drying (MgS04) and
concentration left
the crude acid fluoride (34.70 g, 100%) which was used without further
purification.
To a stirred solution of (R)-4-benzyl-2-oxazolidinone (I3.8 g, 78 mmol) in dry
THF (300 mL) under argon at -78 °C was added via syringe a solution of
n-BuLi in hexanes
(2.5 M, 30 mL, 75 mmol). The reaction was stirred at -78 °C for 15 min,
then a solution of
the above acid fluoride (34.70 g, 7 i mmol) in dry THF { 100 mL) was added via
syringe.
The reaction was stirred for 1 h at -78 °C then was quenched with
saturated NH4Cl and
extracted with ethyl acetate (2 x 200 mL). The combined organic layers were
washed with
brine (400 mL); dried (MgS04), and concentrated to dryness. Purification by
flash
chromatography on silica gel (20% ethyl acetate/hexanes) gave the title
compound (40.34
g, 90%) as a thick clear oil: TLC (20% EtOAc/hexane) R f 0.21; I H NMR (400
MHz,
CDCI3) 8 7.58 {d, J = 8.1 Hz, 2H}, 7.33-7.26 (m, 5H), 7.16 (m, 3H), 6.77 (dd,
1H), 6.67 (d,
J = 2.5 Hz, 1H), 4.62 (m, 1H), 4.60-4.40 (m, 4H}, 4.16 (m, 2H), 3.76 (s, 3H)>
3.27 (dd, IH),
I5 3.21-3.10 (m, 2H), 2.88 (br s, 2H), 2.72 (dd, IH), 1.48 (s, 9H).
h) (R)-4-Benzyl-2-oxazolidinonyl3-[2-[N-(tert-butoxycarbonyl)-N-(4-
trifluoromethylbenzyl)aminomethyl]-4-methoxyphenyl]-2(S)-methoxycarbonylmethyl-
propionamide
To a stirred solution of (R)-4-benzyl-2-oxazolidinonyl 2-[N-(tert-
butoxycarbonyl)-
N-(4-trifluormethylbenzyl)aminomethyl]-4-methoxydihydrocinnamide (40.30 g, 64
mmol}
in dry THF (300 mL) at -78 °C was added a solution of lithium
bis(trimethylsilyl)amide (70
mL, I M in THF, 70 mmol) via syringe. After 30 min, methyl bromoacetate (30
mL, 317
mmol) was added via syringe. After another 30 min at -78 °C the
reaction was allowed to
warm to -20 °C and stirred for an additional 6 h. The reaction was
quenched with saturated
NH4C1 (400 mL) and extracted with ethyl acetate (2 x 200 mL). The combined
organic
layers were washed with brine (300 mL), dried (MgS04), and concentrated to
dryness.
Purification by flash chromatography on silica gel (20% ethyl acetate/hexanes)
gave the
title compound (38.62 g, 86%} as a white solid: HPLC (Altex UltrasphereTM-Si
Su, 20%
EtOAc/hexane) showed approximately 20% unalkylated starting material was still
present.
HPLC of the crude reaction mixture gave a de of 90% for the reaction; 1H NMR
(400 MHz,
CDCl3) 8 7.57 (d, J = 8.1 Hz, 2H), 7.40-7.11 (m, 8H), 6.71 (dd, 1H), 6.63 (d,
J = 2.7 Hz,
1H), 4.57-4.34 (m, 6H), 4.03 (d, J = 8.6 Hz, 1H}, 3.85 (t, 1H), 3.72 (s, 3H),
3.61 (s, 3H),
3.28 {dd, 1 H), 2.90 (dd, 1 H), 2.86-2.71 (m, 2H), 2.70 (dd, 1 H), 2.44 (m, 1
H), 1.48 and 1.46
(2s, 9H).
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SUBSTITUTE SHEET (RULE 26)
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i) Methyl (S)-8-methoxy-3-oxo-2-(4-trifluoromethylbenzyl)-2,3,4,5-tetrahydro-
1H-2-
benzazepine-4-acetate
To a stirred solution of (R)-4-benzyl-2-oxazolidinonyl 3-[2-[N-(tert
butoxycarbonyl)-N-(4-trifluoromethylbenzyl}aminomethyl]-4-methoxyphenyl]-2(S)
methoxycarbonylmethyl-propionamide (38.0 g, 54 mmol) in THF (300 mL} and water
(100
mL) was added dropwise at 0 °C over 30 min a solution of 30% H202 (18.9
mL) and LiOH
H20 (2.3 g, 55 mmol) in water (62 mL). The cloudy solution was stirred for an
additional
1 h at 0 °C. The resulting homogeneous solution was treated slowly with
a solution of
sodium sulfite (34.3 g, 272 mmol) in water ( 17S mL) at 0 °C, then was
acidified with an
ice-cold solution of concentrated HCl (35 mL) in water ( 150 mL). The reaction
was
extracted with ethyl acetate (2x200 mL), and the combined organic layers were
washed
with brine (400 mL), dried (MgS04) and concentrated to dryness. The resulting
residue
was treated with 4.0 M HCl in dioxane (400 mL) with stirring at RT (slow gas
evolution
was observed). After 1 h, the reaction was concentrated and reconcentrated
from 1:1
i5 CHCI3/toluene (2 x), then the residue (37.65 g) was taken up in dry DMF
(400 mL). To
this solution with stirring under argon at 0 °C in a Dewar flask were
added triethylamine
(15.3 mL, 109 mmol) and NaHC03 (22.9 g, 273 mmol), followed by
diphenylphosphoryl
azide ( 13 mL, 60 mmol). After stirring for 24 h at 0 °C the reaction
was concentrated to
dryness. The residue was taken up in ethyl acetate (400 mL), and washed
sequentially with
water (300 mL) and brine (300 mL). Drying (MgS04), concentration, and flash
chromatography on silica gel {35% ethyl acetate/hexanes) gave the title
compound (16.87
g, 74%) as a clear thick oil: TLC (40% EtOAc/hexane) Rf 0.50; MS (ES) m/e
422.3 (M +
H)+; 1 H NMR (400 MHz, CDC13) 8 7.52 (d, J = 8.1, 2H), 7.29 (d, J = 8.1 Hz,
2H), 7.02 (d,
J = 8.5 Hz, 1 H), 7.75 (dd, 1 H}, 6.36 (d, J = 2.7 Hz, 1 H), 5.18 (d, J = 16.5
Hz, 1 H), 4.96 (d, J
= 15.4 Hz, 1H), 4.48 (d, J = 15.4 Hz, 1H), 3.87 (m, 1H), 3.74 (d, J = 16.5 Hz,
1H), 3.73 (s,
3H), 3.71 (s, 3H), 3.08 (dd, 1H), 3.02 (dd, 1H), 2.95 (dd, 1H), 2.48 (dd, 1H).
j) Methyl (S)-8-hydroxy-3-oxo-2-(4-trifluoromethylbenzyl)-2,3,4,5-tetrahydro-
1H-2-
benzazepine-4-acetate
A solution of boron tribromide in CH2C12 (1.0 M, 160 mL, 160 mmol) was added
dropwise over 30 min to a solution of methyl (S)-8-methoxy-3-oxo-2-(4-
trifluoromethylbenzyl)-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate (16.67 g,
39.6
mmol) in anhydrous CH2C12 ( 150 mL) at -20 °C under argon. After an
additional 1.5 hr at
-15 to -20 °C, the reaction was recooled to -20 °C and quenched
by careful dropwise
addition of MeOH (160 mL). The reaction was stirred at -10 to 0 °C for
1 hr, then was
concentrated on the rotavap. The residue was reconcentrated from MeOH (2 x).
Purification by flash chromatography on silica gel (50 to 100% ethyl
acetate/hexanes) gave
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the title compound (14.87 g, 92%) as a white solid: [a]D -81.8° (c,
1.0, MeOH); TLC
{silica, 50% EtOAc/hexane) Rf 0.54; MS (ES) m/e 408.2 (M + H)+; 1H NMR (400,
CDC13
+ 2% DMSO-d6) 8 7.53 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 8.1 Hz, 2H), 6.93 (d, J
= 8.4 Hz,
1 H), 6.70 (dd, I H), 6.41 (d, J = 2.3 Hz, 1 H), 5.16 (d, J = 16.4 Hz, 1 H),
5.01 (d, J = 15.6 Hz,
1H), 4.39 (d, J = 15.6 Hz, 1H), 3.84 (m, 1H), 3.73 (d, J = 16.4 Hz, 1H), 3.71
(s, 3H), 3.01
(dd, 1H), 2.98 (m, 1H), 2.90 (dd, 1H), 2.47 (dd, 1H).
Preparation 7
Preparation of ~(3-hydroxy-1-propel)aminol4-methylpyridine-N-oxide
a) 2-[(3-hydroxy-1-propyl)amino]-4-methylpyridine-N-oxide
A mixture of 2-chloro-4-methylpyridine-N-oxide (12.1 g, 0.068 mole) (Brown, E.
V. J. Amer. Chem. Soc. 1957, 79, 3565), 3-amino-I-propanol (10.33 mL, 0.14
mole),
NaHC03 (28 g, 0.34 mole), and tert-amyl alcohol (70 mL) was heated to reflux.
After 16
hr, the reaction was cooled, diluted with CH2C12 (300 mL), and suction
filtered to remove
insoluble materials. The filtrate was concentrated and reconcentrated from
toluene to leave
a yellow oil. Recrystallization from CH2C12IEt20 gave the title compound
(10.87 g, 88%)
as a yellow solid: TLC ( 15% MeOH/CH2CI2) R f 0.44; 1 H NMR (400, CDC13) 8
7.92 (d, J
= 6.7, 1 H), 7.28 (br t, 1 H), 6.43 (s, 1 H), 6.33 (dd, J = 6.6, 2.1 Hz, 1 H),
3.73 (t, J=5.7 Hz,
2 H), 3.47 (q, H=6.3 Hz, 2 H), 2.29 {s, 3 H), 1.82 - 1.88 (m, 2 H); MS (ES)
m/e 183 (M+
H)+.
Preparation 8
Preparation of~S)-8-f3-(4-methy~yridin-2=ylamino)-1-proQyloxyl-3-oxo-2-f4-
(trifluorometh,~l)benzyll-2.3,4.5-tetrah~rdro-1H-2-benzazepine-4-acetic acid
a) Methyl (S)-8-[3-(4-methyl-1-oxopyridin-2-ylamino)-1-propyloxy]-3-oxo-2-[4-
(trifluoromethyl)benzyl]-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate
To methyl (S)-8-hydroxy-3-oxo-2-[4-(trifluoromethyl)benzyI]-2,3,4,5-tetrahydro-
1H-2-benzazepine-4-acetate (0.42 g, 1.03 mmol) and Ph3P (0.41 g, 1.56 mmol) in
CH2C12
{6 mL) at 0 °C was added dropwise a solution of 2-[(3-hydroxy-1-
propyl)amino)-4-
methylpyridine-N-oxide (0.28 g, 1.54 mmol) and diethyl azodicarboxylate (0.24
mL, 1.52
mL). When the addition was complete, the ice bath was removed and the reaction
was
stirred at RT. After 20h, the solvent was removed and the product was isolated
by flash
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chromatography (100% CHCl3 to 10% MeOH/CHCi3, silica gel) to give the title
compound {0.57 g) as a clear oil. MS (ES) m/e 572.2 (M + H)+.
b) Methyl (S)-8-[3-(4-methylpyridin-2-ylamino)-1-propyloxy]-3-oxo-2-[4-
(trifluoromethyl)benzyl]-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate
To methyl (S)-8-[3-(4-methyl-1-oxa-pyridin-2-ylamino)-I-propyloxy]-3-oxo-2-[4-
(trifluoromethyl)benzyl]-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate (0.57
g, 1.00
mmol) and cyclohexene (1.00 mL, 9.87 mmol) in MeOH (10 mL) was added 10% Pd/C
(0.11 g). The reaction was heated to reflux for 20h. After cooling the
reaction to RT, the
catalyst was removed by filtration and the solvent was removed under vacuum to
give a
white foam (0.49 g). Radial chromatography (5% MeOH/CHCl3, silica gel, 6 mm
plate)
gave the title compound (0.42 g) as a white foam. MS (ES) m/e 556.1 (M + H)+.
c) (S)-8-[3-(4-Methylpyridin-2-ylamino)-1-propyloxy]-3-oxo-2-[4-
(trifluoromethyl)benzyl]-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid
To methyl {S)-8-[3-(4-methylpyridin-2-ylamino)-1-propyioxy]-3-oxo-2-[4-
(trifluoromethyl)benzyl]-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetate (0.42
g, 0.75
mmol) in EtOH (2 mL) was added 1 N NaOH ( 1.50 mL, 1.50 mmol). After stirring
at RT
for 3h, the bulk of the solvent was removed under vacuum. The residue was made
acidic
(pH = 3) with 1N HCI, frozen and lyophilized to dryness. Water was added to
the residue
and neutralized to pH = 7 with sat NaHC03. The aqueous layer was extracted
with CHCl3,
the combined organic extracts were dried over Na2S04 and concentrated to give
a white
foam (0.47 g). Radial chromatography (10% MeOH/CHC13, silica gel, 6 mm plate)
gave
the title compound (0.30 g) as a white solid. MS (ES) m/e 542.2 (M + H)+.
Anal. Calcd
for C29H30F3N304 ' 3.5 H20: C, 57.61; H, 6.17; N, 6.95. Found: C, 57.6; H,
5.30; N,
6.38.
Example 2
Parenteral Dosage Unit Composition
A preparation which contains 20 mg of the compound of Example 1 as a sterile
dry
powder is prepared as follows: 20 mg of the compound is dissolved in 15 mL of
distilled
water. The solution is filtered under sterile conditions into a 25 mL multi-
dose ampoule
and lyophilized. The powder is reconstituted by addition of 20 mL of 5%
dextrose in water
(D5W) for intravenous or intramuscular injection. The dosage is thereby
determined by the
injection volume. Subsequent dilution may be made by addition of a metered
volume of
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this dosage unit to another volume of DSW for injection, or a metered dose may
be added
to another mechanism for dispensing the drug, as in a bottle or bag for IV
drip infusion or
other injection=infusion system.
Example 3
Oral Dosage Unit Composition
A capsule for oral administration is prepared by mixing and milling 50 mg of
the
compound of Example 1 with 75 mg of lactose and 5 mg of magnesium stearate.
The
resulting powder is screened and filled into a hard gelatin capsule.
Example 4
Oral Dosage Unit Composition
A tablet for oral administration is prepared by mixing and granulating 20 mg
of
sucrose, 150 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 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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SUBSTITUTE SHEET (RULE 2fi)
