U.S. patent application number 09/952084 was filed with the patent office on 2002-04-25 for alpha v integrin receptor antagonists.
Invention is credited to Arison, Byron H., Cui, Donghui, Duggan, Mark E., Fang, Xiaojun, Halczenko, Wasyl, Hutchinson, John H., Prueksaritanont, Thomayant, Subramanian, Raju.
Application Number | 20020049224 09/952084 |
Document ID | / |
Family ID | 22872729 |
Filed Date | 2002-04-25 |
United States Patent
Application |
20020049224 |
Kind Code |
A1 |
Arison, Byron H. ; et
al. |
April 25, 2002 |
Alpha v integrin receptor antagonists
Abstract
The present invention relates to novel compounds formed by
metabolic conversion of compounds of structural formula (1),
pharmaceutical compositions containing such compounds, and their
use as .alpha.v.beta.3 integrin receptor antagonists. The compounds
of the present invention are useful for inhibiting bone resorption,
restenosis, angiogenesis, diabetic retinopathy, macular
degeneration, inflammatory arthritis, cancer, and metastatic tumor
growth. They are particularly useful for inhibiting bone resorption
and for the treatment and prevention of osteoporosis. 1
Inventors: |
Arison, Byron H.; (Watchung,
NJ) ; Cui, Donghui; (Newton, PA) ; Duggan,
Mark E.; (Schwenksville, PA) ; Halczenko, Wasyl;
(Lansdale, PA) ; Hutchinson, John H.;
(Philadelphia, PA) ; Prueksaritanont, Thomayant;
(Lansdale, PA) ; Subramanian, Raju; (Perkasie,
PA) ; Fang, Xiaojun; (Kalamazoo, MI) |
Correspondence
Address: |
MERCK AND CO INC
P O BOX 2000
RAHWAY
NJ
070650907
|
Family ID: |
22872729 |
Appl. No.: |
09/952084 |
Filed: |
September 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60232344 |
Sep 14, 2000 |
|
|
|
Current U.S.
Class: |
514/300 ;
546/122 |
Current CPC
Class: |
A61P 35/04 20180101;
C07D 471/04 20130101; A61P 27/02 20180101; A61P 19/08 20180101;
A61P 43/00 20180101; A61P 19/10 20180101; A61P 9/10 20180101; A61P
29/00 20180101; A61P 35/00 20180101; A61P 9/00 20180101; A61P 19/00
20180101; A61P 19/02 20180101 |
Class at
Publication: |
514/300 ;
546/122 |
International
Class: |
A61K 031/4745; C07D
471/02 |
Claims
What is claimed is:
1. A compound of structural formula (I): 24wherein at least one of
R.sup.1, R.sup.2, and R.sup.3 is hydroxy or oxo; and the individual
stereoisomers thereof, or a pharmaceutically acceptable salt
thereof.
2. The compound of claim 1 which is 25and the individual
stereoisomers thereof, or a pharmaceutically acceptable salt
thereof.
3. The compound of claim 2 which is 26and the individual
stereoisomers thereof, or a pharmaceutically acceptable salt
thereof.
4. The compound of claim 1 which is 27and the individual
stereoisomers thereof, or a pharmaceutically acceptable salt
thereof.
5. The compound of claim 4 which is 28and the individual
stereoisomers thereof, or a pharmaceutically acceptable salt
thereof.
6. A compound of structural formula 29or a pharmaceutically
acceptable salt thereof.
7. The compound of claim 1 which is 30and the individual
stereoisomers thereof, or a pharmaceutically acceptable salt
thereof.
8. The compound of claim 7 which is 31and the individual
stereoisomers thereof, or a pharmaceutically acceptable salt
thereof.
9. A compound of structural formula 32and the individual
stereoisomers thereof, or a pharmaceutically acceptable salt
thereof.
10. The compound of claim 9 which is 33or a pharmaceutically
acceptable salt thereof.
11. A pharmaceutical composition comprising a therapeutically
effective amount of a compound of claim 1 and a pharmaceutically
acceptable carrier.
12. A method of eliciting an .alpha.v.beta.3 integrin receptor
antagonizing effect in a mammal in need thereof, comprising
administering to said mammal a therapeutically effective amount of
a compound of claim 1.
13. The method of claim 12 wherein the .alpha.v.beta.3 integrin
receptor antagonizing effect is selected from the group consisting
of inhibition of bone resorption, restenosis, angiogenesis,
diabetic retinopathy, macular degeneration, inflammatory arthritis,
cancer, and metastatic tumor growth.
14. The method of claim 13 wherein the .alpha.v.beta.3 integrin
receptor antagonizing effect is inhibition of bone resorption.
15. A method of treating or preventing osteoporosis in a mammal in
need thereof, comprising administering to the mammal a
therapeutically effective amount of a compound of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present invention is related to U.S. provisional
application Serial No. 60/232,344, filed Sep. 14, 2000, the
contents of which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] U.S. Pat. No. 6,017,926 (issued Jan. 25, 2000) discloses
compounds of structural formula (1): 2
[0003] which include the two enantiomeric forms at the C-3 position
(marked with *) of the propionic acid side-chain.
[0004] These compounds are antagonists of the integrin receptor
.alpha.v.beta.3 and are therefore useful for inhibiting bone
resorption, restenosis, angiogenesis, diabetic retinopathy, macular
degeneration, inflammatory arthritis, cancer, and metastatic tumor
growth. They are particularly useful for inhibiting bone resorption
and for the treatment and prevention of osteoporosis.
SUMMARY OF THE INVENTION
[0005] The present invention relates to novel derivatives of
3-(6-methoxy-pyridin-3-yl)-3-{2-oxo-3-[3-(5,6,7,8-tetrahydro[1,8]naphthyr-
idin-2-yl)-propyl]-imidazolidin-1-yl}-propionic acid (1), methods
for their preparation, pharmaceutical compositions containing such
compounds, and methods for using these compounds as .alpha.v.beta.3
integrin receptor antagonists. These derivatives are formed by
metabolic conversion of the compounds of formula (1).
[0006] Because of their activity as .alpha.v.beta.3 integrin
receptor antagonists, the compounds of the present invention are
useful, inter alia, for inhibiting bone resorption and for the
treatment and prevention of osteoporosis.
DETAILED DESCRIPTION OF THE INVENTION
[0007] In one embodiment of the present invention, there are
provided compounds of structural formula (I): 3
[0008] wherein at least one of R.sup.1, R.sup.2, and R.sup.3 is
hydroxy or oxo; and the individual stereoisomers thereof, or a
pharmaceutically acceptable thereof.
[0009] One class of this embodiment of the present invention is
directed to compounds of structural formula (II): 4
[0010] and the individual stereoisomers thereof;
[0011] or a pharmaceutically acceptable salt thereof.
[0012] A subclass of this class is directed to compounds of
structural formula (III): 5
[0013] and the individual stereoisomers thereof at the hydroxylated
C-5 position (marked with **) of the tetrahydro-[1,8]naphthyridine
ring;
[0014] or a pharmaceutically acceptable salt thereof.
[0015] A second class of this embodiment of the present invention
is directed to compounds of structural formula (IV): 6
[0016] and the individual stereoisomers thereof;
[0017] or a pharmaceutically acceptable salt thereof.
[0018] A subclass of this class is directed to compounds of
structural formula 7
[0019] and the individual stereoisomers thereof at the hydroxylated
C-7 position (marked with **) of the tetrahydro-[1,8]naphthyridine
ring;
[0020] or a pharmaceutically acceptable salt thereof.
[0021] A third class of this embodiment of the present invention is
directed to compounds of structural formula (VI): 8
[0022] and the individual stereoisomers thereof; or a
pharmaceutically acceptable salt thereof.
[0023] A subclass of this class of this is directed to compounds of
structural formula (VII): 9
[0024] and the individual stereoisomers thereof at the hydroxylated
benzylic position (marked with **) of the
tetrahydro-[1,8]naphthyridine ring;
[0025] or a pharmaceutically acceptable salt thereof.
[0026] A fourth class of this embodiment of the present invention
is directed to compounds of structural formula (VIII): 10
[0027] and the individual stereoisomers thereof; or a
pharmaceutically acceptable salt thereof.
[0028] A subclass of this class is directed to the compound of
structural formula (IX): 11
[0029] or a pharmaceutically acceptable salt thereof.
[0030] A second embodiment of the present invention is directed to
compounds of structural formula (X): 12
[0031] and the individual stereoisomers thereof; or a
pharmaceutically acceptable salt thereof.
[0032] A class of this embodiment is directed to the compound of
structural formula (XI): 13
[0033] or a pharmaceutically acceptable salt thereof.
[0034] A third embodiment of the present invention is directed to
compounds of structural formula (XII): 14
[0035] and the individual stereoisomers thereof; or a
pharmaceutically acceptable salt thereof.
[0036] A class of this embodiment is directed to the compound of
structural formula (XIII): 15
[0037] or a pharmaceutically acceptable salt thereof.
[0038] In a further embodiment of the compounds of the present
invention, there are provided bis-hydroxylated derivatives of
structural formulae XIV-XVI: 16
[0039] and the individual stereoisomers thereof,
[0040] or a pharmaceutically acceptable salt thereof.
[0041] For use in medicine, the salts of the compounds of this
invention refer to non-toxic "pharmaceutically acceptable salts."
Other salts may, however, be useful in the preparation of the
compounds according to the invention or of their pharmaceutically
acceptable salts. Salts of basic compounds encompassed within the
term "pharmaceutically acceptable salts" refer to non-toxic salts
of the compounds of this invention which are generally prepared by
reacting the free base with a suitable organic or inorganic acid.
Representative salts of basic compounds of the present invention
include, but are not limited to, the following: acetate,
benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate,
borate, bromide, camsylate, carbonate, chloride, clavulanate,
citrate, dihydrochloride, edetate, edisylate, estolate, esylate,
fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,
hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,
hydroxynaphthoate, iodide, isothionate, lactate, lactobionate,
laurate, malate, maleate, mandelate, mesylate, methylbromide,
methylnitrate, methylsulfate, mucate, napsylate, nitrate,
N-methylglucamine ammonium salt, oleate, oxalate, pamoate
(embonate), palmitate, pantothenate, phosphate/diphosphate,
polygalacturonate, salicylate, stearate, sulfate, subacetate,
succinate, tannate, tartrate, teoclate, tosylate, triethiodide and
valerate. Furthermore, where the compounds of the invention carry
an acidic moiety, suitable pharmaceutically acceptable salts
thereof include, but are not limited to, salts derived from
inorganic bases including aluminum, ammonium, calcium, copper,
ferric, ferrous, lithium, magnesium, manganic, mangamous,
potassium, sodium, zinc, and the like. Particularly preferred are
the ammonium, calcium, magnesium, potassium, and sodium salts.
Salts derived from pharmaceutically acceptable organic non-toxic
bases include salts of primary, secondary, and tertiary amines,
cyclic amines, and basic ion-exchange resins, such as arginine,
betaine, caffeine, choline, N,N-dibenzylethylenediamine,
diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,
ethanolamine, ethylenediamine, N-ethylmorpholine,
N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine,
isopropylamine, lysine, methylglucamine, morpholine, piperazine,
piperidine, polyamine resins, procaine, purines, theobromine,
triethylamine, trimethylamine, tripropylamine, tromethamine, and
the like.
[0042] The compounds of the present invention can have chiral
centers and can thus occur as racemates, racemic mixtures, single
enantiomers, diastereomeric mixtures, and individual diastereomers,
with all isomeric forms being included in the present invention.
Therefore, where a compound is chiral, the separate enantiomers or
diastereomers, substantially free of the other, are included within
the scope of the invention; further included are all mixtures of
the two enantiomers.
[0043] Compounds of the present invention may be separated into
enantiomeric pairs of diastereoisomers by, for example, fractional
crystallization from a suitable solvent, for example, methanol or
ethyl acetate or a mixture thereof. The pair of enantiomers
(racemic mixture) thus obtained may be resolved into single
enantiomers by conventional means, for example, by the use of an
optically active acid as a resolving agent, or by HPLC using a
chiral stationary phase. Alternatively, any stereoisomer of a
compound of the present invention may be obtained by stereospecific
synthesis using optically pure starting materials or reagents of
known configuration.
[0044] Also included within the scope of the invention are
polymorphs and hydrates of the compounds of the instant
invention.
[0045] The term "therapeutically effective amount" shall mean that
amount of a drug or pharmaceutical agent that will elicit the
biological or medical response of a tissue, system, animal or human
that is being sought by a researcher or clinician.
[0046] The term "integrin receptor antagonist," as used herein,
refers to a compound which binds to and antagonizes the
.alpha.v.beta.3 receptor.
[0047] The term "bone resorption," as used herein, refers to the
process by which osteoclasts degrade bone.
[0048] Compounds of the present invention display an affinity for
the .alpha.v.beta.3 integrin receptor of less than 100 nanomolar.
Compounds of this invention are therefore useful for treating
mammals suffering from a bone condition caused or mediated by
increased bone resorption, who are in need of such therapy.
Pharmacologically effective amounts of the compounds, including
pharmaceutically acceptable salts thereof, are administered to the
mammal, to inhibit the activity of mammalian osteoclasts.
[0049] The compounds of the present invention are administered in
dosages effective to antagonize the .alpha.v.beta.3 receptor where
such treatment is needed, as, for example, in the prevention or
treatment of osteoporosis.
[0050] Illustrating the invention is the method for eliciting an
.alpha.v.beta.3 antagonizing effect. More particularly, the
.alpha.v.beta.3 antagonizing effect is selected from inhibition of:
bone resorption, restenosis, angiogenesis, diabetic retinopathy,
macular degeneration, inflammatory arthritis, cancer, and
metastatic tumor growth. In one embodiment of the method, the
.alpha.v.beta.3 antagonizing effect is the inhibition of bone
resorption.
[0051] More particularly illustrating the invention is a
pharmaceutical composition comprising any of the compounds
described above and a pharmaceutically acceptable carrier. Another
example of the invention is a pharmaceutical composition made by
combining any of the compounds described above and a
pharmaceutically acceptable carrier. Another illustration of the
invention is a process for making a pharmaceutical composition
comprising combining any of the compounds described above and a
pharmaceutically acceptable carrier.
[0052] Further illustrating the invention is a method of treating
and/or preventing a condition mediated by antagonism of the
.alpha.v.beta.3 integrin receptor in a mammal in need thereof,
comprising administering to the mammal a therapeutically effective
amount of any of the compounds described above. Preferably, the
condition is selected from bone resorption, osteoporosis,
restenosis, diabetic retinopathy, macular degeneration,
angiogenesis, atherosclerosis, inflammatory arthritis, cancer, and
metastatic tumor growth. More preferably, the condition is selected
from osteoporosis and cancer. Most preferably, the condition is
osteoporosis.
[0053] More specifically exemplifying the invention is a method of
eliciting an .alpha.v.beta.3 integrin antagonizing effect in a
mammal in need thereof, comprising administering to the mammal a
therapeutically effective amount of any of the compounds or any of
the pharmaceutical compositions described above. Preferably, the
.alpha.v.beta.3 antagonizing effect is selected from inhibition of
bone resorption, inhibition of restenosis, inhibition of
atherosclerosis, inhibition of angiogenesis, inhibition of diabetic
retinopathy, inhibition of macular degeneration, inhibition of
inflammatory arthritis, or inhibition of cancer or metastatic tumor
growth. More preferably, the .alpha.v.beta.3 antagonizing effect is
inhibition of bone resorption.
[0054] Additional examples of the invention are methods of
inhibiting bone resorption and of treating and/or preventing
osteoporosis in a mammal in need thereof, comprising administering
to the mammal a therapeutically effective amount of any of the
compounds or any of the pharmaceutical compositions decribed
above.
[0055] Additional illustrations of the invention are methods of
treating hypercalcemia of malignancy, osteopenia due to bone
metastases, periodontal disease, hyperparathyroidism, periarticular
erosions in rheumatoid arthritis, Paget's disease,
immobilization-induced osteopenia, and glucocorticoid treatment in
a mammal in need thereof, comprising administering to the mammal a
therapeutically effective amount of any of the compounds or any of
the pharmaceutical compositions described above.
[0056] More particularly exemplifying the invention is the use of
any of the compounds described above in the preparation of a
medicament for the treatment and/or prevention of osteoporosis in a
mammal in need thereof. Still further exemplifying the invention is
the use of any of the compounds described above in the preparation
of a medicament for the treatment and/or prevention of bone
resorption, cancer, metastatic tumor growth, restenosis,
atherosclerosis, diabetic retinopathy, macular degeneration,
inflammatory arthritis, and/or angiogenesis.
[0057] Also exemplifying the invention are compositions further
comprising an active ingredient selected from the group consisting
of
[0058] a) an organic bisphosphonate or a pharmaceutically
acceptable salt or ester thereof,
[0059] b) an estrogen receptor modulator,
[0060] c) an androgen receptor modulator,
[0061] d) a cytotoxic/antiproliferative agent,
[0062] e) a matrix metalloproteinase inhibitor,
[0063] f) an inhibitor of epidermal-derived, fibroblast-derived, or
platelet-derived growth factors,
[0064] g) an inhibitor of VEGF,
[0065] h) an antibody to a growth factor or to a growth factor
receptor,
[0066] i) an inhibitor of Flk-1/KDR, Flt-1, Tck/Tie-2, or
Tie-1,
[0067] j) a cathepsin K inhibitor,
[0068] k) a growth hormone secretagogue,
[0069] l) an inhibitor of osteoclast proton ATPase,
[0070] m) an inhibitor of urokinase plasminogen activator
(u-PA),
[0071] n) a tumor-specific antibody-interleukin-2 fusion
protein,
[0072] o) an inhibitor of HMG-CoA reductase, and
[0073] p) a prenylation inhibitor, such as a farnesyl transferase
inhibitor or a geranylgeranyl transferase inhibitor or a dual
farnesyl/geranylgeranyl transferase inhibitor; and mixtures
thereof.
[0074] (See, B. Millauer et al., "Dominant-Negative Inhibition of
Flk-1 Suppresses the Growth of Many Tumor Types in Vivo", Cancer
Research, 56, 1615-1620 (1996), which is incorporated by reference
herein in its entirety).
[0075] Preferably, the active ingredient is selected from the group
consisting of:
[0076] a) an organic bisphosphonate or a pharmaceutically
acceptable salt or ester thereof,
[0077] b) an estrogen receptor modulator,
[0078] c) an androgen receptor modulator,
[0079] d) an inhibitor of osteoclast proton ATPase,
[0080] e) an inhibitor of HMG-CoA reductase, and
[0081] f) a cathepsin K inhibitor; and mixtures thereof.
[0082] Nonlimiting examples of such bisphosphonates include
alendronate, etidronate, pamidronate, risedronate, ibandronate, and
pharmaceutically acceptable salts and esters thereof. A
particularly preferred bisphosphonate is alendronate, especially
alendronate monosodium trihydrate.
[0083] Nonlimiting examples of estrogen receptor modulators include
estrogen, progesterin, estradiol, droloxifene, raloxifene, and
tamoxifene.
[0084] Nonlimiting examples of cytotoxic/antiproliferative agents
are taxol, vincristine, vinblastine, and doxorubicin.
[0085] Cathepsin K, formerly known as cathepsin O2, is a cysteine
protease and is described in PCT International Application
Publication No. WO 96/13523, published May 9, 1996; U.S. Pat. No.
5,501,969, issued Mar. 3, 1996; and U.S. Pat. No. 5,736,357, issued
Apr. 7, 1998, all of which are incorporated by reference herein in
their entirety. Cysteine proteases, specifically cathepsins, are
linked to a number of disease conditions, such as tumor metastasis,
inflammation, arthritis, and bone remodeling. At acidic pH's,
cathepsins can degrade type-I collagen. Cathepsin protease
inhibitors can inhibit osteoclastic bone resorption by inhibiting
the degradation of collagen fibers and are thus useful in the
treatment of bone resorption diseases, such as osteoporosis.
[0086] Members of the class of HMG-CoA reductase inhibitors, known
as the "statins," have been found to trigger the growth of new
bone, replacing bone mass lost as a result of osteoporosis (see The
Wall Street Journal, Friday, Dec. 3, 1999, page B1). Therefore, the
statins hold promise for the treatment of bone resorption.
Nonlimiting examples of statins are lovastatin, simvastatin,
atorvastatin, and pravastatin.
[0087] Evidence for crucial role of the urokinase-urokinase
receptor (u-PA-u-PAR) in angiogenesis, tumor invasion,
inflammation, and matrix remodeling during wound healing and
development has been presented [see Y. Koshelnick et al.,
"Mechanisms of signaling through Urokinase Receptor and the
Cellular Response," Thrombosis and Haemostasis 82: 305-311 (1999)
and F. Blasi, "Proteolysis, Cell Adhesion, Chemotaxis, and
Invasiveness Are Regulated by the u-PA-u-PAR-PAI-1 System,"
Thrombosis and Haemostasis 82: 298-304 (1999)]. Thus, specific
antagonists of the binding of u-PA to u-PAR have been found to
inhibit cell-surface plasminogen activation, tumor growth, and
angiogenesis in both in vitro and in vivo models.
[0088] H. N. Lode and coworkers in PNAS USA 96: 1591-1596 (1999)
have observed synergistic effects between an antiangiogenic
.alpha.v integrin antagonist and a tumor-specific antibody-cytokine
(interleukin-2) fusion protein in the eradication of spontaneous
tumor metastases. Their results suggested this combination as
having potential for the treatment of cancer and metastatic tumor
growth.
[0089] The proton ATPase which is found on the apical membrane of
the osteoclast has been reported to play a significant role in the
bone resorption process. Therefore, this proton pump represents an
attractive target for the design of inhibitors of bone resorption
which are potentially useful for the treatment and prevention of
osteoporosis and related metabolic diseases (see C. Farina et al.,
"Selective inhibitors of the osteoclast vacuolar proton ATPase as
novel bone antiresorptive agents," DDT, 4: 163-172 (1999)).
[0090] Evidence has been presented that androgenic steroids play a
physiological role in the development of bone mass in men and women
and that androgens act directly on bone. Androgen receptors have
been demonstrated in human osteoblast-like cell lines and androgens
have been shown to directly stimulate bone cell proliferation and
differentiation. For a discussion, reference is made to S. R.
Davis, "The therapeutic use of androgens in women," J. Steroid
Biochem. Mol. Biol., 69: 177-184 (1999) and K. A. Hansen and S. P.
T. Tho, "Androgens and Bone Health," Seminars in Reproductive
Endocrinology," 16: 129-134 (1998). Thus, androgen receptor
modulators may have utility in the treatment and prevention of bone
loss in women.
[0091] Activators of the peroxisome proliferator-activated
receptor-.gamma. (PPAR.gamma.), such as the thiazolidinediones
(TZD's), inhibit osteoclast-like cell formation and bone resorption
in vitro. Results reported by R. Okazaki et al. in Endocrinology,
140: 5060-5065 (1999) point to a local mechanism on bone marrow
cells as well as a systemic one on glucose metabolism. Nonlimiting
examples of PPAR.gamma. activators include troglitazone,
pioglitazone, rosiglitazone, and BRL 49653.
[0092] The present invention is also directed to combinations of
the compounds of the present invention with one or more agents
useful in the prevention or treatment of osteoporosis. For example,
the compounds of the instant invention may be effectively
administered in combination with effective amounts of other agents
such as an organic bisphosphonate, an estrogen receptor modulator,
an androgen receptor modulator, a growth hormone secretagogue, a
cathepsin K inhibitor, an HMG-CoA reductase inhibitor, a
PPAR.gamma. activator, or an inhibitor of the osteoclast proton
ATPase.
[0093] Additional illustrations of the invention are methods of
treating tumor growth or metastasis in a mammal in need thereof,
comprising administering to the mammal a therapeutically effective
amount of a compound described above and one or more agents known
to be cytotoxic/antiproliferative. Also, the compounds of the
present invention can be administered in combination with radiation
therapy for treating cancer and metastatic tumor growth.
[0094] In addition, the integrin .alpha.v.beta.3 antagonist
compounds of the present invention may be effectively administered
in combination with a growth hormone secretagogue in the
therapeutic or prophylactic treatment of disorders in calcium or
phosphate metabolism and associated diseases. These diseases
include conditions which can benefit from a reduction in bone
resorption. A reduction in bone resorption should improve the
balance between resorption and formation, reduce bone loss or
result in bone augmentation. A reduction in bone resorption can
alleviate the pain associated with osteolytic lesions and reduce
the incidence and/or growth of those lesions. These diseases
include: osteoporosis (including estrogen deficiency,
immobilization, glucocorticoid-induced and senile), osteodystrophy,
Paget's disease, myositis ossificans, Bechterew's disease,
malignant hypercalcemia, metastatic bone disease, periodontal
disease, cholelithiasis, nephrolithiasis, urolithiasis, urinary
calculus, hardening of the arteries (sclerosis), arthritis,
bursitis, neuritis and tetany. Increased bone resorption can be
accompanied by pathologically high calcium and phosphate
concentrations in the plasma, which would be alleviated by this
treatment. Similarly, the present invention would be useful in
increasing bone mass in patients with growth hormone deficiency.
Thus, preferred combinations are simultaneous or alternating
treatments of an .alpha.v.beta.3 receptor antagonist of the present
invention and a growth hormone secretagogue, optionally including a
third component comprising an organic bisphosphonate, preferably
alendronate monosodium trihydrate.
[0095] In accordance with the method of the present invention, the
individual components of the combination can be administered
separately at different times during the course of therapy or
concurrently in divided or single combination forms. The instant
invention is therefore to be understood as embracing all such
regimes of simultaneous or alternating treatment, and the term
"administering" is to be interpreted accordingly. It will be
understood that the scope of combinations of the compounds of this
invention with other agents useful for treating integrin-mediated
conditions includes in principle any combination with any
pharmaceutical composition useful for treating osteoporosis.
[0096] As used herein, the term "composition" is intended to
encompass a product comprising the specified ingredients in the
specified amounts, as well as any product which results, directly
or indirectly, from combination of the specified ingredients in the
specified amounts.
[0097] The compounds of the present invention can be administered
in such oral dosage forms as tablets, capsules (each of which
includes sustained release or timed release formulations), pills,
powders, granules, elixirs, tinctures, suspensions, syrups and
emulsions. Likewise, they may also be administered in intravenous
(bolus or infusion), intraperitoneal, topical (e.g., ocular
eyedrop), subcutaneous, intramuscular or transdermal (e.g., patch)
form, all using forms well known to those of ordinary skill in the
pharmaceutical arts. An effective but non-toxic amount of the
compound desired can be employed as an .alpha.v.beta.3
antagonist.
[0098] The dosage regimen utilizing the compounds of the present
invention is selected in accordance with a variety of factors
including type, species, age, weight, sex and medical condition of
the patient; the severity of the condition to be treated; the route
of administration; the renal and hepatic function of the patient;
and the particular compound or salt thereof employed. An ordinarily
skilled physician, veterinarian or clinician can readily determine
and prescribe the effective amount of the drug required to prevent,
counter or arrest the progress of the condition.
[0099] Oral dosages of the present invention, when used for the
indicated effects, will range between about 0.01 mg per kg of body
weight per day (mg/kg/day) to about 100 mg/kg/day, preferably 0.01
to 10 mg/kg/day, and most preferably 0.1 to 5.0 mg/kg/day. For oral
administration, the compositions are preferably provided in the
form of tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0,
10.0, 15.0, 25.0, 50.0, 100 and 500 milligrams of the active
ingredient for the symptomatic adjustment of the dosage to the
patient to be treated. A medicament typically contains from about
0.01 mg to about 500 mg of the active ingredient, preferably, from
about 1 mg to about 100 mg of active ingredient. Intravenously, the
most preferred doses will range from about 0.1 to about 10
mg/kg/minute during a constant rate infusion. Advantageously,
compounds of the present invention may be administered in a single
daily dose, or the total daily dosage may be administered in
divided doses of two, three or four times daily. Furthermore,
preferred compounds for the present invention can be administered
in intranasal form via topical use of suitable intranasal vehicles,
or via transdermal routes, using those forms of transdermal skin
patches well known to those of ordinary skill in the art. To be
administered in the form of a transdermal delivery system, the
dosage administration will, of course, be continuous rather than
intermittent throughout the dosage regimen.
[0100] In the methods of the present invention, the compounds
herein described in detail can form the active ingredient, and are
typically administered in admixture with suitable pharmaceutical
diluents, excipients or carriers (collectively referred to herein
as `carrier` materials) suitably selected with respect to the
intended form of administration, that is, oral tablets, capsules,
elixirs, syrups and the like, and consistent with conventional
pharmaceutical practices.
[0101] For instance, for oral administration in the form of a
tablet or capsule, the active drug component can be combined with
an oral, non-toxic, pharmaceutically acceptable, inert carrier such
as lactose, starch, sucrose, glucose, methyl cellulose, magnesium
stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol
and the like; for oral administration in liquid form, the oral drug
components can be combined with any oral, non-toxic,
pharmaceutically acceptable inert carrier such as ethanol,
glycerol, water and the like. Moreover, when desired or necessary,
suitable binders, lubricants, disintegrating agents and coloring
agents can also be incorporated into the mixture. Suitable binders
include starch, gelatin, natural sugars such as glucose or
beta-lactose, corn sweeteners, natural and synthetic gums such as
acacia, tragacanth or sodium alginate, carboxymethylcellulose,
polyethylene glycol, waxes and the like. Lubricants used in these
dosage forms include sodium oleate, sodium stearate, magnesium
stearate, sodium benzoate, sodium acetate, sodium chloride and the
like. Disintegrators include, without limitation, starch, methyl
cellulose, agar, bentonite, xanthan gum and the like.
[0102] The compounds of the present invention can also be
administered in the form of liposome delivery systems, such as
small unilamellar vesicles, large unilamellar vesicles and
multilamellar vesicles. Liposomes can be formed from a variety of
phospholipids, such as cholesterol, stearylamine or
phosphatidylcholines.
[0103] Compounds of the present invention may also be delivered by
the use of monoclonal antibodies as individual carriers to which
the compound molecules are coupled. The compounds of the present
invention may also be coupled with soluble polymers as targetable
drug carriers. Such polymers can include polyvinylpyrrolidone,
pyran copolymer, polyhydroxypropylmethacrylamide-phenol,
polyhydroxy-ethylaspartamide-phen- ol, or
polyethyleneoxide-polylysine substituted with palmitoyl residues.
Furthermore, the compounds of the present invention may be coupled
to a class of biodegradable polymers useful in achieving controlled
release of a drug, for example, polylactic acid, polyglycolic acid,
copolymers of polylactic and polyglycolic acid, polyepsilon
caprolactone, polyhydroxy butyric acid, polyorthoesters,
polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked
or amphipathic block copolymers of hydrogels.
[0104] Methods of Preparation
[0105] Compounds of formulae I-XVI are biliary metabolites of a
compound of formula (1). They are obtained in vitro by suspension
in a cytochrome P-450-driven bioreactor; by incubation with rat,
dog, monkey, or human liver microsomes; or by incubation with rat,
dog, monkey, or human hepatocytes. They can also be isolated from
the bile of individuals who have ingested a compound of formula
(1), using methodologies that are well-known in the art, such as
reverse-phase high-performance liquid chromatography.
[0106] Illustratively, the preparation and characterization of the
metabolites of compound 2 are depicted in the Schemes and described
in the Examples below. 17
EXAMPLE 1
3(S)-(6-Methoxy-pyridin-3-yl)-3-{2-oxo-3-[3-(5(R)-hydroxy-5,6,7,8-tetrahyd-
ro[1,8]naphthyridin-2-yl)-propyl]-imidazolidin-1-yl}-propionic acid
and
3(S)-(6-methoxy-pyridin-3-yl)-3-{2-oxo-3-[3-(5(S)-hydroxy-5,6,7,8-tetrahy-
dro[1,8]naphthyridin-2-yl)-propyl]-imidazolidin-1-yl}-propionic
acid (3A, 3B)
[0107] The bioreactor system used for the production of
P450-derived metabolites has been described in "Bioreactor Systems
in Drug Metabolism: Synthesis of Cytochrome P450-Generated
Metabolites," T. H. Rushmore et al., Metabolic Engineering, 2
(2000) 1-11, which is incorporated by reference herein in its
entirety.
[0108]
3(S)-(6-Methoxy-pyridin-3-yl)-3-{2-oxo-3-[3-(5,6,7,8-tetrahydro[1,8-
]naphthyridin-2-yl)-propyl]-imidazolidin-1-yl}-propionic acid (2)
(for the preparation of 2, see U.S. Pat. No. 6,017,926) (100 .mu.M)
was incubated with cells (Sf21 insect cells infected with human
CYP450 2D6) in vitro at 27.degree. C. for 24 hours. Cells were
prepared by infecting with Baculovirus encoding human CYP450 2D6 or
oxidoreductase and grown at 27.degree. C. for 48 hours. The final
incubation volume was 1 liter, and the CYP450 2D6 concentration was
about 100 pmol/mL of cells. Incubates were centrifuged and the
supernatants were first purified using a solid phase extraction
column. The supernatants were loaded onto Varian Mega Bond Elut C18
(20 mL) columns, and the title compounds were eluted off the column
using acetonitrile-water (1:1), and the eluant was injected onto an
HPLC system for further isolation. The HPLC used in metabolite
isolation was a Waters 600 HPLC system. The title compounds were
separated using a Phenomenex Luna C18-2 preparative column (21.2
mm.times.150 mm, 5 micron). The mobile phase consisted of 0.1%
formic acid in water (solvent A) and 0.1% formic acid in
acetonitrile (solvent B). The eluting gradient started with an
isocratic condition of 15% B for 2 min followed by a linear
increase to 20% B in 10 min. The concentration of B was increased
to 80% B in the next minute and the column was washed for 2 min at
80% B before returning to 15% B over 1 min. The system was
equilibrated at 15% B for 10 min prior to the next injection. A
constant flow rate of 20 mL/min was used for all the analyses.
Under these HPLC conditions, isomer 3A eluted at about 5.5 min and
isomer 3B eluted at about 6.2 min. The NMR and mass spectra of 3A
and 3B were measured:
[0109] .sup.1H NMR data (400 MHz, d.sub.6-DMSO) for 3A and 3B:
.delta.8.10 (s, 1H), 7.75 (d, 1H, J=7.5 Hz), 7.70 (dd, 1H, J=8.7,
2.6 Hz); 6.82 (d, 1H, J=8.6 Hz); 6.67 (d, 1H, J=7.5 Hz), 5.20 (t,
1H, J=8.0 Hz), 4.62 (t, 1H, J=4.8 Hz), 3.82 (s, 3H), 3.41 (m, 1H),
3.29 (m, 1H), 3.20 (m, 1H), 2.92 (m, 1H), 2.97 (dd, 1H, J=15.5, 8.0
Hz), 2.87 (dd, 1H, J=15.5, 8.0 Hz), 2.59 (t, 1H, J=7.6 Hz), 1.80
(m, 1H), and 1.77 (m, 1H). Mass spectrum: found 456 (M+H).sup.+;
438 (M+H.sub.2O).
[0110] Compounds 4-9 whose structures are shown below were also
identified as metabolites of substrate 2 by means of comparison of
their mass and NMR spectra with those of authentic materials
prepared by unambiguous chemical synthesis as depicted in Schemes
2-5 and described in Examples 2-6 below. The preparation of
substrate 2 and its ethyl ester derivative 10 used as starting
materials in the synthetic transformations is described in U.S.
Pat. No. 6,017,926, which is incorporated by reference herein in
its entirety. 18 19
EXAMPLE 2
3(S)-(6-Methoxypyridin-3-yl)-3-{2-oxo-3-[3-(7-hydroxy-5,6,7,8-tetrahydro-[-
1,8]naphthyridin-2-yl)-propyl]-imidazolidin-1-yl}-propionic acid
(4)
[0111] To a solution of substrate 2 (0.88 g, 2 mmol) in water (20
mL) at room temperature were added 1N NaOH solution (2.0 mL, 2
mmol) and then KMnO.sub.4 (0.316 g, 2 mmol) and the mixture stirred
for 16 hr. After filtration, the solution was purified by HPLC
chromatography (DeltaPak C-18 column; 0.1% NH.sub.4HCO.sub.3 (aq)
and acetonitrile; gradient elution). Collection of the second
eluting peak followed by lyophilization afforded the title compound
4 as a white solid (1:1 mixture of 7-hydroxy epimers).
[0112] .sup.1H NMR (600 MHz, CD.sub.3OD): .delta.1.70 (1H, m), 1.86
(1H, m), 2.07 (1H, m), 2.12 (1H, m), 2.61 (1H, m), 2.71 (2H, m),
2.75-3.0 (6H, m), 3.17 (1H, q), 3.48 (1H, m), 3.62 (1H, m), 3.90
(3H, s), 4.73 (1H, m), 5.46 (1H, br d), 6.69 (1H, d), 6.80 (1H,
dd), 7.54 (1H, m), 7.67 (1H, dd), 8.09 (1H, d); Mass spectrum:
found 456.1 (M+H).sup.+.
EXAMPLE 3
3(S)-(6-Methoxypyridin-3-yl)-3-{2-oxo-3-[3-(7-oxo-5,6,7,8-tetrahydro-[1,8]-
naphthyridin-2-yl)-propyl]-imidazolidin-1-yl}-propionic acid
(5)
[0113] Following the procedure described for 4 but collecting the
first eluted product following HPLC chromatography of the crude
product, then concentration followed by lyophilization afforded 5
as a white solid.
[0114] .sup.1H NMR (500 MHz, CD.sub.3O): .delta.1.90 (2H, m), 2.57
(2H, m), 2.65 (2H, m), 2.87 (2H, m), 2.93 (3H, m), 3.13 (1H, m),
3.26 (3H, m), 3.40 (1H, q), 3.89 (3H, s), 5.37 (1H, t), 6.78 (1H,
d), 6.82 (1H, d), 7.39 (1H, d), 7.67 (1H, dd), 8.11 (1H, d).; Mass
spectrum: found 454.1 (M+H).sup.+. 20
EXAMPLE 4
3(S)-(6-Methoxypyridin-3-yl)-3-{2-oxo-3-[3-([1,8]naphthyridin-2-yl)-propyl-
]-imidazolidin-1-yl}-propionic acid (6)
[0115] To a solution of substrate 2 (0.44 g, 1 mmol) in pyridine (5
mL) at room temperature was added CrO.sub.3 (0.1 g, 1 mmol) and the
mixture stirred for 16 hr. The mixture was diluted with water (100
mL), filtered through celite and the solvent removed in vacuo to
give an oil. Purification by HPLC chromatography (DeltaPak C-18
column; 0.1% NH.sub.4HCO.sub.3 (aq) and MeOH; gradient elution)
afforded (after removal of the solvent in vacuo) a pale yellow
residue. The residue was taken up in water and lyophilized to give
6 as a solid.
[0116] .sup.1H NMR (300 MHz, CD.sub.3OD): .delta.2.12 (2H, m),
2.65-2.85 (4H, m), 3.03 (2H, t), 3.1-3.4 (4H, m), 3.75 (3H, s),
5.38 (1H, t), 6.75 (1H, d), 7.53 (1H, d), 7.57 (1H, dd), 7.64 (1H,
dd), 8.09 (1H, d), 8.22 (1H, d), 8.36 (1H, dd), 8.98 (1H, dd); Mass
spectrum: found 436.0 (M+H).sup.+. 21
EXAMPLE 5
3(S)-(6-Methoxypyridin-3-yl)-3-{2-oxo-3(R or
S)-[3-(5,6,7,8-tetrahydro-[1,-
8]naphthyridin-2-yl)-3-hydroxypropyl]-imidazolidin-1-yl}-propionic
acid trifluoroacetic acid salt and
3(S)-(6-methoxypyridin-3-yl)-3-{2-oxo-3(S or
R)-[3-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-3-hydroxypropyl]-imi-
dazolidin-1-yl}-propionic acid trifluoroacetic acid salt (7A and
7B)
Step A:
3(S)-(6-Methoxypyridin-3-yl)-3-{2-oxo-3-[3-(1N-oxide-5,6,7,8-tetra-
hydro-[1,8]naphthyridin-2-yl)-propyl]-imidazolidin-1-yl}-propionic
acid ethyl ester (11)
[0117] To a solution of the ethyl ester 10 (for preparation, see
U.S. Pat. No. 6,017,926) (1.51 g, 3.2 mmol) in CH.sub.2Cl.sub.2 (20
mL) was added m-chloroperbenzoic acid (70%; 0.96g, 3.9 mmol) and
the mixture stirred at room temperature for 4 hours. The mixture
was diluted with CH.sub.2Cl.sub.2, washed with NaHCO.sub.3
(.times.5), brine and dried over Na.sub.2SO.sub.4. The solvent was
removed and the residue purified by silica gel chromatography
(CHCl.sub.3/MeOH 97:3) to afford the title compound 11 as a viscous
oil.
[0118] Mass spectrum: found 484.1; (M+H) calculated: 484.3.
Step B:
3(S)-(6-Methoxypyridin-3-yl)-3-{2-oxo-3-[3-(8-acetyl-5,6,7,8-tetra-
hydro-[1,8]naphthyridin-2-yl)-3-acetoxypropyl]-imidazolidin-1-yl}-propioni-
c acid ethyl ester (12)
[0119] A solution of the N-oxide 11 (0.24 g, 0.5 mmol) in acetic
anhydride (2 mL) was heated to 90" C. for 7.5 hours then poured
onto ice and neutralized with NaHCO.sub.3. The mixture was
extracted with EtOAc (.times.3), washed with brine, dried
(Na.sub.2SO.sub.4) and the solvent removed. Purification of the
residue by column chromatography (silica gel; CHCl.sub.3/MeOH 97:3)
afforded the title compound 12 as a viscous oil.
[0120] Mass spectrum: found 568.2; (M+H) calculated: 568.3.
Step C: 3(S)-(6-Methoxypyridin-3-yl)-3-{2-oxo-3(R or
S)-[3-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-3-hydroxypropyl]-imidaz-
olidin-1-yl}-propionic acid trifluoroacetic acid salt and
3(S)-(6-Methoxypyridin-3-yl)-3-{2-oxo-3(S or
R)-[3-(5,6,7,8-tetrahydro-[1-
,8]naphthyridin-2-yl)-3-hydroxypropyl]-imidazolidin-1-yl}-propionic
acid trifluoroacetic acid salt (7A and 7B)
[0121] To a solution of the diester 12 (0.75 g, 1.32 mmol) in EtOH
(10 mL) was added 1N NaOH (6 mL, 6 mmol) and the solution heated to
reflux for 4 hours. The mixture was concentrated in vacuo and then
purified by reverse phase HPLC (C18 column; water/acetonitrile
+0.1% TFA; gradient) to give (after lyophilization) 7 as a TFA salt
and mixture of diastereomers.
[0122] Mass spectrum: found 456.1; (M+H) calculated: 456.2.
[0123] .sup.1H NMR (500 MHz, CD.sub.3OD): .delta.1.85-2.0 (4H, m),
2.82 (2H, t), 3.01 (2H, m), 3.07 (1H, m), 3.2-3-4 (4H, m), 3.44
(1H, m), 3.51 (2H, t), 3.9 (3H, s), 4.71 (1H, t), 5.37 (1H, t),
6.71 (1H, 2 overlapping d), 6.82 (1H, 2 overlapping d), 7.61 (1H, 2
overlapping d), 7.72 (1H, 2 overlapping dd), 8.11(1H, 2 overlapping
d).
[0124] The mixture of diastereomers 7 was separated by HPLC
chromatography using Chiralpak AD column eluting with hexane
+0.2%TFA/2-propanol/ethanol 70/25/5 to give (after lyophilization)
the faster eluting diastereomer 7A as a TFA salt.
[0125] .sup.1H NMR (500 MHz, CD.sub.3OD): .delta.1.88 (1H, septet),
1.95 (3H, m), 2.82 (2H, t), 3.01 (2H, m), 3.08 (1H, q), 3.3-3-4
(4H, m), 3.45 (1H, m), 3.51 (2H, t), 3.9 (3H, s), 4.71 (1H, dd),
5.37 (1H, t), 6.72 (1H, d), 6.82 (1H, d), 7.61 (1H, d), 7.72 (1H,
dd), 8.12 (1H, d). Continued elution afforded (after
lyophilization) the slower eluting diastereomer 7B as a TFA
salt.
[0126] .sup.1H NMR (500 MHz, CD.sub.3OD): .delta.1.89 (1H, septet),
1.95 (3H, m), 2.82 (2H, t), 3.01 (2H, d), 3.07 (1H, q), 3.24 (1H,
m), 3-34 (3H, m), 3.45 (1H, m), 3.51 (2H, t), 3.9 (3H, s), 4.71
(1H, dd), 5.37 (1H, t), 6.71 (1H, d), 6.82 (1H, d), 7.595 (1H, d),
7.72 (1H, dd), 8.12 (1H, d). 22
EXAMPLE 6
3(S)-(2(1H)-pyridone-5-yl)-3-{2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]naphthyr-
idin-2-yl)-propyl]-imidazolidin-1-yl}-propionic acid (8)
[0127] A mixture of the acid 2 (see U.S. Pat. No. 6,017,926; 100
mg, 0.23 mmol) and pyridine hydrochloride (300 mg) was heated in a
sealed vial at 125.degree. C. for 2.5 minutes then allowed to cool
to room temperature. The residue was dissolved in water and
purified by reverse phase HPLC (C-18 column; water/acetonitrile
+0.1 %TFA; gradient) to provide, after lyophilization, the title
compound 8 as a TFA salt. High resolution mass spectrum: found
426.2141; calculated (M+H)=426.2136.
[0128] .sup.1H NMR (300 MHz; CD.sub.3OD): .delta.1.86 (2H, m), 1.95
(1H, m), 2.66 (2H, t), 2.81 (2H, t), 2.96 (1H, m), 3.15 (1H, m),
3.23 (2H, t), 3.35-3.55 (5H, m), 5.23 (1H, t), 6.55 (1H, d), 6.64
(1H, d), 7.44 (1H, d), 7.58 (1H, d), 7.65 (1H, dd).
[0129] Assays for Determining Biological Activity 23
N-(4-Iodo-phenylsulfonylamino)-L-asparagine (A-2)
[0130] To a stirred solution of acid A-1 (4.39 g, 33.2 mmol), NaOH
(1.49 g, 37.2 mmol), dioxane (30 ml) and H.sub.2O (30 ml) at
0.degree. C. was added pipsyl chloride (10.34 g, 34.2 mmol). After
.about.5 minutes, NaOH (1.49, 37.2 mmol) dissolved in 15 ml
H.sub.2O, was added followed by the removal of the cooling bath.
After 2.0 h, the reaction mixture was concentrated. The residue was
dissolved in H.sub.2O (300 ml) and then washed with EtOAc. The
aqueous portion was cooled to 0.degree. C. and then acidified with
concentrated HCl. The solid was collected and then washed with
Et.sub.2O to provide acid A-2 as a white solid.
[0131] .sup.1H NMR (300 MHz, D.sub.2O) .delta.7.86 (d, 2H, J=8 Hz
), 7.48 (d, 2H, J=8 Hz) 3.70 (m, 1H), 2.39 (m, 2H).
2(S)-(4-Iodo-phenylsulfonylamino)-.beta.-alanine (A-3)
[0132] To a stirred solution of NaOH (7.14 g, 181.8 mmol) and
H.sub.2O (40 ml) at 0.degree. C. was added Br.sub.2 (1.30 ml, 24.9
mmol) dropwise over a ten minute period. After .about.5 minutes,
acid A-2 (9.9 g, 24.9 mmol), NaOH (2.00 g, 49.8 mmol) and H.sub.2O
(35 ml) were combined, cooled to 0.degree. C. and then added in a
single portion to the reaction. After stirring for 20 minutes at
0.degree. C., the reaction was heated to 90.degree. C. for 30
minutes and then recooled to 0.degree. C. The pH was adjusted to
.about.7 by dropwise addition of concentrated HCl. The solid was
collected, washed with EtOAc, and then dried in vacuo to provide
acid A-3 as a white solid.
[0133] .sup.1H NMR (300 MHz, D.sub.2O) .delta.8.02 (d, 2H, J=8 Hz),
7.63 (d, 2H, J=8 Hz), 4.36 (m, 1H), 3.51 (dd, 1H, J=5 Hz, 13 Hz)
3.21 (m, 1H).
Ethyl
2(S)-(4-iodo-phenylsulfonylamino)-.beta.-alanine-hydrochloride
(A-4)
[0134] HCl gas was rapidly bubbled through a suspension of acid A-3
(4.0 g, 10.81 mmol) in EtOH (50 ml) at 0.degree. C. for 10 minutes.
The cooling bath was removed and the reaction was heated to
60.degree. C. After 18 h, the reaction was concentrated to provide
ester A-4 as a white solid.
[0135] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta.7.98 (d, 2H, J=8
Hz), 7.63 (d, 2H, J=8 Hz), 4.25 (q, 1H, J=5 Hz), 3.92 (m, 2H), 3.33
(m, 1H), 3.06 (m, 1H), 1.01 (t, 3H, J=7 Hz).
Ethyl 4-[2-(2-Aminopyridin-6-yl)ethyl]benzoate (A-5a)
[0136] A mixture of ester A-5 (700 mg, 2.63 mmol), (for
preparation, see: Scheme 29 of PCT International Application
Publication No. WO 95/32710, published Dec. 7, 1995) 10% Pd/C (350
mg) and EtOH were stirred under 1 atm H.sub.2. After 20 h, the
reaction was filtered through a celite pad and then concentrated to
provide ester A-5a as a brown oil. TLC R.sub.f=0.23 (silica, 40%
EtOAc/hexanes)
[0137] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.7.95 (d, 2H, J=8
Hz), 7.26 (m, 3H), 6.43 (d, 1H, J=7 Hz), 6.35 (d, 1H, J=8 Hz), 4.37
(m, 4H), 3.05 (m, 2H), 2.91 (m, 2H), 1.39 (t, 3H, J=7 Hz).
4-[2-(2-Aminopyridin-6-yl)ethyl]benzoic acid hydrochloride
(A-6)
[0138] A suspension of ester A-5a (625 mg, 2.31 mmol) in 6N HCl (12
ml) was heated to 60.degree. C. After .about.20 h, the reaction was
concentrated to give acid A-6 as a tan solid.
[0139] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta.7.96 (d, 2H, J=8
Hz), 7.80 (m, 1H), 7.33 (d, 2H, J=8 Hz), 6.84 (d, 1H, J=9 Hz), 6.69
(d, 1H, J=7 Hz), 3.09 (m, 4H).
Ethyl
4-[2-(2-Aminopyridin-6-yl)ethyl]benzoyl-2(S)-(4-iodo-phenylsulfonyla-
mino)-.beta.-alanine (A-7)
[0140] A solution of acid 15-6 (400 mg, 1.43 mmol), amine A-4 (686
mg, 1.57 mmol), EDC (358 mg, 1.86 mmol), HOBT (252 mg, 1.86 mmol),
NMM (632 .mu.l, 5.72 mmol) in DMF (10 ml) was stirred for .about.20
h. The reaction was diluted with EtOAc and then washed with sat.
NaHCO.sub.3, brine, dried (MgSO.sub.4) and concentrated. Flash
chromatography (silica, EtOAc then 5% isopropanol/EtOAc) provided
amide A-7 as a white solid. TLC R.sub.f=0.4 (silica, 10%
isopropanol/EtOAc)
[0141] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta.7.79 (d, 2H, J=9
Hz) 7.61 (d, 2H, J=8 Hz), 7.52 (d 2H, J=9 Hz), 7.29 (m, 1H), 7.27
(d, 2H, J=8 Hz), 4.20 (m, 1H), 3.95 (q, 2H, J=7 Hz), 3.66 (dd, 1H,
J=6 Hz, 14 Hz), 3.49 (dd, 1H, J=8 Hz, 13 Hz), 3.01 (m, 2H), 2.86
(m, 2H), 1.08 (t, 3H, J=7 Hz).
4-[2-(2-Aminopyridin-6-yl)ethyl]benzoyl-2(S)-(4-iodophenyl-sulfonylamino)--
.beta.-alanine (A-8)
[0142] A solution of ester A-7 (200 mg, 0.3213 mmol) and 6N HCl (30
ml) was heated to 60.degree. C. After .about.20 h, the reaction
mixture was concentrated. Flash chromatography (silica, 20:20:1:1
EtOAc/EtOH/NH.sub.4OH/H.sub.2O) provided acid A-8 as a white
solid.
[0143] TLC R.sub.f=0.45 (silica, 20:20:1:1
EtOAc/EtOH/NH.sub.4OH/H.sub.2O)
[0144] .sup.1H NMR (400 MHz, DMSO) .delta.8.40 (m, 1H), 8.14 (Bs,
1H), 7.81 (d, 2H, J=8 Hz), 7.62 (d, 2H, J=8 Hz), 7.48 (d, 2H, J=8
Hz), 7.27 (m, 3H), 6.34 (d, 1H, J=7 Hz), 6.25 (d, 1H, J=8 Hz), 5.85
(bs, 2H), 3.89 (bs, 1H), 3.35 (m, 2H), 2.97 (m, 2H), 2.79 (m,
2H).
4-[2-(2-Aminopyridin-6-yl)ethyl)benzoyl-2(S)-(4-trimethylstannyl-phenylsul-
fonylamino-.beta.-alanine (A-9)
[0145] A solution of iodide A-8 (70 mg, 0.1178 mmol),
[(CH.sub.3).sub.3Sn].sub.2 (49 .mu.l, 0.2356 mmol),
Pd(PPh.sub.3).sub.4 (5 mg) and dioxane (7 ml) was heated to
90.degree. C. After 2 h, the reaction was concentrated and then
purified by preparative HPLC (Delta-Pak C.sub.18 15 .mu.M
100A.degree., 40.times.100 mm; 95:5 then 5:95 H.sub.2O/CH.sub.3CN)
to provide the trifluoroacetate salt. The salt was suspended in
H.sub.2O (10 ml), treated with NH.sub.4OH (5 drops) and then
lyophilized to provide amide A-9 as a white solid.
[0146] .sup.1H NMR (400 MHz, DMSO) .delta.8.40 (m, 1H), 8.18 (d,
1H, J=8 Hz), 7.67 (m, 5H), 7.56 (d, 2H, J=8 Hz), 7.29 (d, 2H, J=8
Hz), 6.95-7.52 (m, 2H), 6.45 (bs, 2H), 4.00 (m, 1H), 3.50 (m, 1H),
3.33 (m, 1H), 2.97 (m, 2H), 2.86 (m, 2H).
4-[2-(2-Aminopyridin-6-yl)ethyl]benzoyl-2(S)-4-.sup.125iodo-phenylsulfonyl-
amino-.beta.-alanine (A-10)
[0147] An iodobead (Pierce) was added to a shipping vial of 5 mCi
of Na.sup.125I (Amersham, IMS30) and stirred for five minutes at
room temperature. A solution of 0.1 mg of A-9 in 0.05 mL of 10%
H.sub.2SO.sub.4/MeOH was made and immediately added to the
Na.sup.125I/iodobead vial. After stirring for three minutes at room
temperature, approximately 0.04-0.05 mL of NH.sub.4OH was added so
the reaction mixture was at pH 6-7. The entire reaction mixture was
injected onto the HPLC for purification [Vydac peptide-protein C-18
column, 4.6.times.250 mm, linear gradient of 10% acetonitrile (0.1%
(TFA):H.sub.2O (0.1% TFA) to 90% acetonitrile (0.1% TFA):H.sub.2O
(0.1% TFA) over 30 minutes, 1 mL/min]. The retention time of A-10
is 17 minutes under these conditions. Fractions containing the
majority of the radioactivity were pooled, lyophilized and diluted
with ethanol to give approximately 1 mCi of A-10, which coeluted on
HPLC analysis with an authentic sample of A-8.
[0148] Instrumentation: Analytical and preparative HPLC was carried
out using a Waters 600E Powerline Multi Solvent Delivery System
with 0.1 mL heads with a Rheodyne 7125 injector and a Waters 990
Photodiode Array Detector with a Gilson FC203 Microfraction
collector. For analytical and preparative HPLC, a Vydac
peptide-protein C-18 column, 4.6.times.250 mm was used with a C-18
Brownlee modular guard column. The acetonitrile used for the HPLC
analyses was Fisher Optima grade. The HPLC radiodetector used was a
Beckman 170 Radioisotope detector. A Vydac C-18 protein and peptide
column, 3.9.times.250 mm was used for analytical and preparative
HPLC. Solutions of radioactivity were concentrated using a Speedvac
vacuum centrifuge. Calibration curves and chemical concentrations
were determined using a Hewlett Packard Model 8452A UV/Vis Diode
Array Spectrophotometer. Sample radioactivities were determined in
a Packard A5530 gamma counter.
[0149] The test procedures employed to measure .alpha.v.beta.3
binding and the bone resorption inhibiting activity of the
compounds of the present invention are described below.
[0150] Bone Resorption-Pit Assay
[0151] When osteoclasts engage in bone resorption, they can cause
the formation of pits in the surface of bone that they are acting
upon. Therefore, when testing compounds for their ability to
inhibit osteoclasts, it is useful to measure the ability of
osteoclasts to excavate these resorption pits when the inhibiting
compound is present.
[0152] Consecutive 200 micron thick cross sections from a 6 mm
cylinder of bovine femur diaphysis are cut with a low speed diamond
saw (Isomet, Beuler, Ltd., Lake Bluff, Ill.). Bone slices are
pooled, placed in a 10% ethanol solution and refrigerated until
further use.
[0153] Prior to experimentation, bovine bone slices are
ultrasonicated twice, 20 minutes each in H.sub.2O. Cleaned slices
are placed in 96 well plates such that two control lanes and one
lane for each drug dosage are available. Each lane represents
either triplicate or quadruplicate cultures. The bone slices in 96
well plates are sterilized by UV irradiation. Prior to incubation
with osteoclasts, the bone slices are hydrated by the addition of
0.1 ml .alpha.MEM, pH 6.9 containing 5% fetal bovine serum and 1%
penicillin/streptomycin.
[0154] Long bones from 7-14 day old rabbits (New Zealand White
Hare) are dissected, cleaned of soft tissue and placed in
.alpha.MEM containing 20 mM HEPES. The bones are minced using
scissors until the pieces are <1 mm and transferred to a 50 ml
tube in a volume of 25 ml. The tube is rocked gently by hand for 60
cycles, the tissue is sedimented for 1 min., and the supernatant is
removed. Another 25 ml of medium is added to the tissue and rocked
again. The second supernatant is combined with the first. The
number of cells is counted excluding erythrocytes (typically
.about.2.times.10.sup.7 cells/ml). A cell suspension consisting of
5.times.10.sup.6/ml in .alpha.MEM containing 5% fetal bovine serum,
10 nM 1,25(OH).sub.2D.sub.3, and pencillin-streptomycin is
prepared. 200 ml aliquots are added to bovine bone slices (200
mm.times.6 mm) and incubated for 2 hrs. at 37.degree. C. in a
humidified 5% CO.sub.2 atmosphere. The medium is removed gently
with a micropipettor and fresh medium containing test compounds is
added. The cultures are incubated for 48 hrs., and assayed for
c-telopeptide (fragments of the a1 chain of type I collagen) by
Crosslaps for culture media (Herlev, Denmark).
[0155] Bovine bone slices are exposed to osteoclasts for 20-24 hrs
and are processed for staining. Tissue culture media is removed
from each bone slice. Each well is washed with 200 ml of H.sub.2O,
and the bone slices are then fixed for 20 minutes in 2.5%
glutaraldehyde, 0.1 M cacodylate, pH 7.4. After fixation, any
remaining cellular debris is removed by 2 min. ultrasonication in
the presence of 0.25 M NH.sub.4OH followed by 2.times.15 min
ultrasonication in H.sub.2O. The bone slices are immediately
stained for 6-8 min with filtered 1% toluidine blue and 1%
borax.
[0156] After the bone slices have dried, resorption pits are
counted in test and control slices. Resorption pits are viewed in a
Microphot Fx (Nikon) fluorescence microscope using a polarizing
Nikon IGS filter cube. Test dosage results are compared with
controls and resulting IC.sub.50 values are determined for each
compound tested.
[0157] The appropriateness of extrapolating data from this assay to
mammalian (including human) disease states is supported by the
teaching found in Sato, M., et al., Journal of Bone and Mineral
Research, Vol. 5, No. 1, pp. 31-40, 1990, which is incorporated by
reference herein in its entirety. This article teaches that certain
bisphosphonates have been used clinically and appear to be
effective in the treatment of Paget's disease, hypercalcemia of
malignancy, osteolytic lesions produced by bone metastases, and
bone loss due to immobilization or sex hormone deficiency. These
same bisphosphonates are then tested in the resorption pit assay
described above to confirm a correlation between their known
utility and positive performance in the assay.
[0158] EIB Assay
[0159] Duong et al., J. Bone Miner. Res., 8: S378 (1993), describes
a system for expressing the human integrin .alpha.v.beta.3. It has
been suggested that the integrin stimulates attachment of
osteoclasts to bone matrix, since antibodies against the integrin,
or RGD-containing molecules, such as echistatin (European
Publication 382 451), can effectively block bone resorption.
[0160] Reaction Mixture
[0161] 1. 175 .mu.l TBS buffer (50 mM Tris.HCl pH 7.2, 150 mM NaCl,
1% BSA, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2).
[0162] 2. 25 ml cell extract (dilute with 100 mM octylglucoside
buffer to give 2000 cpm/25 .mu.l).
[0163] 3. .sup.125I-echistatin (25 .mu.l/50,000 cpm) (see EP 382
451).
[0164] 4. 25 .mu.l buffer (total binding) or unlabeled echistatin
(non-specific binding).
[0165] The reaction mixture was then incubated for 1 h at room
temp. The unbound and the bound .alpha.v.beta.3 were separated by
filtration using a Skatron Cell Harvester. The filters (prewet in
1.5% poly-ethyleneimine for 10 mins) were then washed with the wash
buffer (50 mM Tris HCl, 1 mM CaCl.sub.2/MgCl.sub.2, pH 7.2). The
filter was then counted in a gamma counter.
[0166] SPAV3 Assay
[0167] Materials
[0168] 1. Wheat germ agglutinin Scintillation Proximity Beads
(SPA): Amersham
[0169] 2. Octylglucopyranoside: Calbiochem
[0170] 3. HEPES: Calbiochem
[0171] 4. NaCl: Fisher
[0172] 5. CaCl.sub.2: Fisher
[0173] 6. MgCl.sub.2: SIGMA
[0174] 7. Phenylmethylsulfonylfluoride (PMSF): SIGMA
[0175] 8. Optiplate: PACKARD
[0176] 9. Compound A-10 (specific activity 500-1000 Ci/mmole)
[0177] 10. test compound
[0178] 11. Purified integrin receptor: .alpha.v.beta.3 was purified
from 293 cells overexpressing .alpha.v.beta.3 (Duong et al., J.
Bone Min. Res., 8:S378, 1993) according to Pytela (Methods in
Enzymology, 144:475, 1987)
[0179] 12. Binding buffer: 50 mM HEPES, pH 7.8, 100 mM NaCl, 1 mM
Ca.sup.2+/Mg.sup.2+, 0.5 mM PMSF
[0180] 13. 50 mM octylglucoside in binding buffer: 50-OG buffer
[0181] Procedure
[0182] 1. Pretreatment of SPA beads: 500 mg of lyophilized SPA
beads were first washed four times with 200 ml of 50-OG buffer and
once with 100 ml of binding buffer, and then resuspended in 12.5 ml
of binding buffer.
[0183] 2. Preparation of SPA beads and receptor mixture In each
assay tube, 2.5 .mu.l (40 mg/ml) of pretreated beads were suspended
in 97.5 .mu.l of binding buffer and 20 ml of 50-OG buffer. 5 ml
(.about.30 ng/.mu.l) of purified receptor was added to the beads in
suspension with stirring at room temperature for 30 minutes. The
mixture was then centrifuged at 2,500 rpm in a Beckman GPR Benchtop
centrifuge for 10 minutes at 4.degree. C. The pellets were then
resuspended in 50 .mu.l of binding buffer and 25 .mu.l of 50-OG
buffer.
[0184] 3. Reaction The following were sequentially added into
Optiplate in corresponding wells:
[0185] (i) Receptor/beads mixture (75 .mu.l)
[0186] (ii) 25 .mu.l of each of the following: compound to be
tested, binding buffer for total binding or A-8 for non-specific
binding (final concentration 1 .mu.M)
[0187] (iii) A-10 in binding buffer (25 .mu.l, final concentration
40 pM)
[0188] (iv) Binding buffer (125 .mu.l)
[0189] (v) Each plate was sealed with plate sealer from PACKARD and
incubated overnight with rocking at 4.degree. C.
[0190] 4. Plates were counted using PACKARD TOPCOUNT
[0191] 5. % inhibition was calculated as follows:
[0192] A=total counts
[0193] B=nonspecific counts
[0194] C=sample counts
[0195] % inhibition=[{(A-B)-(C-B)}/(A-B)]/(A-B).times.100
[0196] OCFORM Assay
[0197] Osteoblast-like cells (1.8 cells), originally derived from
mouse calvaria, were plated in CORNING 24 well tissue culture
plates in .alpha.MEM medium containing ribo- and
deoxyribonucleosides, 10% fetal bovine serum and
penicillin-streptomycin. Cells were seeded at 40,000/well in the
morning. In the afternoon, bone marrow cells were prepared from six
week old male Balb/C mice as follows:
[0198] Mice were sacrificed, tibiae removed and placed in the above
medium. The ends were cut off and the marrow was flushed out of the
cavity into a tube with a 1 mL syringe with a 27.5 gauge needle.
The marrow was suspended by pipetting up and down. The suspension
was passed through >100 mm nylon cell strainer. The resulting
suspension was centrifuged at 350.times.g for seven minutes. The
pellet was resuspended, and a sample was diluted in 2% acetic acid
to lyse the red cells. The remaining cells were counted in a
hemacytometer. The cells were pelleted and resuspended at
1.times.10.sup.6 cells/mL. 50 .mu.L was added to each well of 1.8
cells to yield 50,000 cells/well and 1,25-dihydroxy-vitamin D.sub.3
(D.sub.3) was added to each well to a final concentration of 10 nM.
The cultures were incubated at 37.degree. C. in a humidified, 5%
CO.sub.2 atmosphere. After 48 h, the medium was changed. 72 h after
the addition of bone marrow, test compounds were added with fresh
medium containing D.sub.3 to quadruplicate wells. Compounds were
added again after 48 h with fresh medium containing D.sub.3. After
an additional 48 h., the medium was removed, cells were fixed with
10% formaldehyde in phosphate buffered saline for 10 minutes at
room temperature, followed by a 1-2 minute treatment with
ethanol:acetone (1:1) and air dried. The cells were then stained
for tartrate resistant acid phosphatase as follows:
[0199] The cells were stained for 10-15 minutes at room temperature
with 50 mM acetate buffer, pH 5.0 containing 30 mM sodium tartrate,
0.3 mg/mL Fast Red Violet LB Salt and 0.1 mg/mL Naphthol AS-MX
phosphate. After staining, the plates were washed extensively with
deionized water and air dried. The number of multinucleated,
positive staining cells was counted in each well.
[0200] Compounds of structural formula (I) of the present invention
were tested and found to bind to human .alpha.v.beta.3 integrin.
These compounds were found to have IC.sub.50 values less than 100
nM in the SPAV3 assay.
Example of a Pharmaceutical Formulation
[0201] As a specific embodiment of an oral composition, 100 mgs of
Example 1 are formulated with sufficient finely divided lactose to
provide a total amount of 580 to 590 mg to fill a size 0 hard gel
capsule.
[0202] While the invention has been described and illustrated in
reference to certain preferred embodiments thereof, those skilled
in the art will appreciate that various changes, modifications and
substitutions can be made therein without departing from the spirit
and scope of the invention. For example, effective dosages other
than the preferred doses as set forth hereinabove may be applicable
as a consequence of variations in the responsiveness of the mammal
being treated for severity of bone disorders caused by resorption,
or for other indications for the compounds of the invention
indicated above. Likewise, the specific pharmacological responses
observed may vary according to and depending upon the particular
active compound selected or whether there are present
pharmaceutical carriers, as well as the type of formulation and
mode of administration employed, and such expected variations or
differences in the results are contemplated in accordance with the
objects and practices of the present invention. It is intended,
therefore, that the invention be limited only by the scope of the
claims which follow and that such claims be interpreted as broadly
as is reasonable.
* * * * *