WO1998046220A1 - Combination therapy for the prevention and treatment of osteoporosis - Google Patents

Combination therapy for the prevention and treatment of osteoporosis Download PDF

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Publication number
WO1998046220A1
WO1998046220A1 PCT/US1998/007065 US9807065W WO9846220A1 WO 1998046220 A1 WO1998046220 A1 WO 1998046220A1 US 9807065 W US9807065 W US 9807065W WO 9846220 A1 WO9846220 A1 WO 9846220A1
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alkyl
aryl
ethyl
amino
oxo
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PCT/US1998/007065
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French (fr)
Inventor
Arthur A. Patchett
Gideon A. Rodan
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Merck & Co., Inc.
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Priority claimed from GBGB9716883.5A external-priority patent/GB9716883D0/en
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to AU71054/98A priority Critical patent/AU7105498A/en
Publication of WO1998046220A1 publication Critical patent/WO1998046220A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention provides a combination therapy for the treatment and prevention of osteoporosis. More particularly, the combination of the present invention comprises an ⁇ v ⁇ 3 antagonist compound and a growth hormone secretogogue.
  • Osteoclasts are multinucleated cells of up to 400 mm in diameter that resorb mineralized tissue in vertebrates. They are actively motile cells that migrate along the surface of bone. They can bind to bone, secrete necessary acid and proteases and thereby cause the actual resorption of mineralized tissue from the bone.
  • osteoclasts are believed to exist in at least two physiological states.
  • the secretory state osteoclasts attach to the bone matrix via a tight attachment zone (sealing zone), become highly polarized, form a ruffled border, and secrete lysosomal enzymes and acid to resorb bone.
  • the adhesion of osteoclasts to bone surfaces is an important initial step in bone resorption.
  • the osteoclasts migrate across bone matrix and do not take part in resorption until they attach again to bone.
  • Integrins are transmembrane, heterodimeric, glycoproteins which interact with extracellular matrix and are involved in osteoclast attachment, activation and migration.
  • integrin receptor The most abundant integrin in osteoclasts (rat, chicken, mouse and human) is the vitronectin receptor, or ⁇ v ⁇ 3, thought to interact in bone with matrix proteins that contain the RGD sequence. Antibodies to ⁇ v ⁇ 3 block bone resorption in vitro and m vivo indicating that this integrin plays a key role in the resorptive process. There is increasing evidence to suggest that ⁇ v ⁇ 3 ligands can be used effectively to inhibit osteoclast mediated bone resoption in vivo in mammals.
  • osteoporosis hypercalcemia of malignancy
  • osteolysis due to bone metastases periodontal disease
  • hyperparathyroidism hyperparathyroidism
  • periarticular erosions in rheumatoid arthritis Paget's disease
  • immobilization- induced osteopenia loosening of bone prostheses and glucocorticoid induced osteoporosis.
  • All these conditions are characterized by bone loss, resulting from an imbalance between bone resorption (breakdown) and bone formation, which continues throughout life past about age 30 at the rate of about 0.5 - 1% per year on the average.
  • the rate of bone turnover differs from site to site, for example, it is higher in the trabecular bone of the vertebrae and the alveolar bone in the jaws than in the cortices of the long bones.
  • the potential for bone loss is directly related to turnover and can amount to over 5% per year in vertebrae immediately following menopause, a condition which leads to increased fracture risk.
  • ⁇ v ⁇ 3 antagonist compounds which are useful in the treatment and prevention of diseases involving bone resorption. Representative examples may be found in the following: PCT Patent Pub. Nos. WO 95/32710; WO 96/00574; WO 96/00730; WO 96/26190; WO 96/37492; EPO Patent Publication Nos. EP 0,578,083; EP 0,711,770; EP 0,727,425; EP 0,546,548.
  • the preparation of ⁇ v ⁇ 3 antagonists is well known in the art.
  • Certain non-peptidal growth hormone secretagogues are known to stimulate the pituitary gland to increase its secretion of growth hormone with utility in growth hormone deficient children and adults, in severe burn victims, in the treatment of Turners syndrome, for reversing the adverse effects of glucocorticoid treatment, for treating muscle and exercise tolerance deficiencies in growth hormone deficient adults, and for the treatment of osteoporosis.
  • Certain compounds have been developed which stimulate the release of endogenous growth hormone.
  • Peptides which are known to stimulate the release of endogenous growth hormone include growth hormone releasing hormone, the growth hormone releasing peptides GHRP-6 and GHRP-1 (described in U.S. Patent No. 4,411,890, PCT Patent Pub. No.
  • the present invention provides a combination which comprises an avb3 antagonist and a growth hormone secretagogue.
  • R2a is hydrogen, or Cl-C ⁇ alkyl
  • R3a and R3 are independently selected from the group consisting of: hydrogen, halogen, -C1-C6 alkyl, -OR2, cyano, -OCF3, methylenedioxy, nitro, -S(0)mR > -CF3 or -C(0)OR2 and when R3 a and R3b are in an ortho arrangement, they may be joined to form a C5 to C ⁇ aliphatic or aromatic ring optionally including 1 or 2 heteroatoms selected from oxygen, sulfur or nitrogen;
  • R4 and R5 are independently selected from the group consisting of: hydrogen, -C ⁇ -C6 alkyl, substituted C ⁇ -C6 alkyl wherein the substituents are selected from 1 to 5 halo, 1 to 3 hydroxy, 1 to 3
  • R4 and R5 can be taken together to form -(CH2) r L a (CH2)s- where L a is -C(R2)2-, -0-, -SCOW, or -N(R2)-, where r and s are independently 1 to 3 and R2 is as defined above;
  • R6 is hydrogen or Ci-C ⁇ alkyl
  • A is: (CH 2 ) -C — (CH 2 ) y -
  • R7 and R7a are independently selected from the group consisting of: hydrogen, -C ⁇ -CQ alkyl, -OR2, trifluoromethyl, phenyl, substituted Ci-C ⁇ alkyl where the substituents are selected from imidazolyl, phenyl, indolyl, p-hydroxyphenyl, -OR2, 1 to 3 fluoro, -S(0) m R2, -C(0)OR2, -C3- C7 cycloalkyl, -N(R2)(R2), -C(0)N(R2)(R2); or R7 and R7 a can independently be joined to one or both of R4 and R5 groups to form alkylene bridges between the terminal nitrogen and the alkyl portion of the R7 or R7 a groups, wherein the bridge contains 1 to 5 carbons atoms;
  • R8 and R ⁇ o are independently selected from the group consisting of:
  • R9 is selected from the group consisting of:
  • G, H, I and J are carbon, nitrogen, sulfur or oxygen atoms, such that at least one is a heteroatom and one of G, H, I or J may be optionally missing to afford a 5 or 6 membered heterocyclic aromatic ring; and pharmaceutically acceptable salts and individual diastereomers thereof.
  • Rl is selected from the group consisting of:
  • R3a is H, or fluoro
  • D is selected from the group consisting of:
  • N N '
  • aryl is phenyl or pyridyl and the phenyl may be substituted by 1-2 halogen;
  • R2 is H, or C1-C4 alkyl; m is 1 or 2; t is 0, 1, or 2; q is 1, 2, or 3; w is 2, 3, 4, 5, or 6; and the pharmaceutically acceptable salts and individual diastereomers thereof.
  • the growth hormone secretagogue is selected from the group consisting of:
  • the growth hormone secretagogue used in the combination is N-[l(R)-[(l,2-dihydro-l-methanesulfonyl-spiro[3H-indole- 3,4'-piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)-ethyl]-2-amino-2- methylpropanamide, or a pharmaceutically acceptable salt thereof.
  • the growth hormone secretagogue is N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3,4'-piperidin]-l'-yl)carbonyl]-2- (phenylmethyloxy)-ethyl]-2-amino-2-methylpropanamide methanesulfonate.
  • ⁇ v ⁇ 3 antagonist is a compound of the formula
  • X is a 9- to 10-membered polycyclic ring system, wherein one or more of the rings is aromatic, and wherein the polycyclic ring system contains 0, 1, 2, 3 or 4 heteroatoms selected from N, O or S, and wherein the polycyclic ring system is either unsubstituted or substituted on a carbon atom with Rl and R ⁇ ;
  • Y is selected from
  • Z is a 5-11 membered aromatic or nonaromatic mono- or polycyclic ring system containing 0 to 6 double bonds, and containing 0 to 6 heteroatoms chosen from N, O and S, and wherein the ring system is either unsubstituted or substituted on a carbon or nitrogen atom with one or more groups independently selected from R4, R5 ? R6 a nd R ⁇ ; provided that Z is not a 6-membered monocyclic aromatic ring system;
  • Rl, R2, R4, R5 ⁇ R13 and Rl4 are each independently selected from hydrogen, halogen, Ci-10 alkyl, C3-8 cycloalkyl, aryl, aryl Ci-8 alkyl, amino, amino Ci-8 alkyl, Cl-3 acylamino, Ci-3 acylamino Ci-8 alkyl, Cl-6 alkylamino, Cl-6 alkylamino- Ci-8 alkyl, Cl-6 dialkylamino, Cl-6 dialkylamino Ci-8 alkyl, Ci-4 alkoxy, Ci-4 alkoxy Cl-6 alkyl, hydroxycarbonyl, hydroxycarbonyl Cl-6 alkyl, Ci-3 alkoxycarbonyl,
  • R ⁇ is selected from hydrogen, aryl,
  • Ci-8 alkylaminocarbonyl amino sulfonyl, Ci-8 alkylaminosulfonyl, arylaminosulfonylamino, aryl Cl-8 alkylaminosulfonyl, Cl-6 alkylsulfonyl, aryl sulfonyl, aryl Cl-6 alkylsulfonyl, aryl Cl-6 alkylcarbonyl, Cl-6 alkylthiocarbonyl, arylthiocarbonyl, or aryl Cl-6 alkylthiocarbonyl, wherein any of the alkyl groups may be unsubstituted or substituted with
  • R6, R?, R8, R9, RlO and R 11 are each independently selected from hydrogen, aryl,
  • Ci-8 alkyl either unsubstituted or substituted, with one or more groups selected from: halogen, hydroxyl, Ci-5 alkylcarbonylamino, aryl Ci-5 alkoxy, Ci-5 alkoxycarbonyl, aminocarbonyl, Ci-5 alkylaminocarbonyl, Ci-5 alkylcarbonyloxy, C3-8 cycloalkyl, oxo, amino, Cl-3 alkylamino, amino Ci-3 alkyl, arylaminocarbonyl, aryl Ci-5 alkylaminocarbonyl, aminocarbonyl, aminocarbonyl Ci-4 alkyl, hydroxycarbonyl, or hydroxycarbonyl Ci-5 alkyl,
  • Ci-6 dialkylamino Ci-6 dialkylamino Cl-6 alkyl
  • l2 i s selected from hydrogen, Cl-8 alkyl, aryl, aryl Cl-8 alkyl, hydroxy, Cl-8 alkoxy, aryloxy, aryl Cl-6 alkoxy, Cl-8 alkylcarbonyl oxy Cl-4 alkoxy, aryl Cl-8 alkyl carbonyl oxy Cl-4 alkoxy, Cl-8 alkylaminocarbonylmethyleneoxy, or Cl-8 dialkylaminocarbonylmethyleneoxy;
  • n is an integer from 1 to 3
  • p is an integer from 1 to 4
  • q is an integer from 0 to 2
  • r is an integer from 0 to 6
  • s is an integer from 0 to 3; and the pharmaceutically acceptable salts thereof.
  • the ⁇ v ⁇ 3 antagonist is a compound wherein Z is selected from
  • ⁇ v ⁇ 3 antagonist is a compound of the formula
  • Y is selected from -(CH2) r - or -(CH2WNR 3 -;
  • R 3 is selected from hydrogen, -(CH 2 )p-aryl, Ci-5 alkoxycarbonyl, C3-8 cycloalkyl, arylaminocarbonyl, aryl Ci-5 alkylaminocarbonyl, Cl-8 alkyl, aryl Cl-6 alkyl, Cl-8 alkylsulfonyl, aryl sulfonyl, aryl Cl-6 alkylsulfonyl,
  • Exemplifying the invention is the combination wherein the ⁇ v ⁇ 3 antagonist is a compound of the formula
  • R ⁇ is selected from hydrogen
  • Rl2 is selected from hydrogen or Cl-8 alkyl; s is an integer from 0 to 3; wherein all other variables are as defined above; and the pharmaceutically acceptable salts thereof.
  • ⁇ v ⁇ 3 antagonist is selected from 2-Oxo-3-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]piperidin-l-yl- acetyl-3(S)-pyridin-3-yl- ⁇ -alanine ethyl ester;
  • the ⁇ v ⁇ 3 antagonist used in the combination is selected from
  • the growth hormone secretagogue is N-[1(R)-[(1,2- dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperidin]-l'- yl)carbonyl]-2-(phenylmethyloxy)-ethyl]-2-amino-2-methylpropanamide
  • the ⁇ v ⁇ 3 antagonist is a compound of the formula
  • Y is selected from -(CH2) r - or -(CH2 WNR 3 - R 3 is selected from hydrogen
  • the growth hormone secretagogue is N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)- ethyl]-2-amino-2-methylpropanamide methanesulfonate.
  • a pharmaceutical composition which comprises any of the combinations described above and a pharmaceutically acceptable carrier.
  • Another example of the invention is a pharmaceutical composition made by combining an ⁇ v ⁇ 3 ligand, a growth hormone secretagogue and a pharmaceutically acceptable carrier.
  • the disease is osteoporosis.
  • Another example of the invention is the use of a growth hormone secretagogue and an ⁇ v ⁇ 3 antagonist for the manufacture of a medicament for the treatment or prevention of a disease involving bone resorption which comprises an effective amount of a growth hormone secretagogue and an effective amount of an ⁇ v ⁇ 3 antagonist, together or separately.
  • Another illustration of the invention is a product containing a growth hormone secretagogue medicament and an ⁇ v ⁇ 3 antagonist medicament as a combined preparation for simultaneous, separate or sequential use in osteoporosis.
  • the present invention is concerned with the combination of an ⁇ v ⁇ 3 antagonist compound and a growth hormone secretagogue for the treatment and the prevention of disturbances of calcium and phosphate metabolism, in particular, the treatment and prevention of diseases involving bone resorption, especially, osteoporosis, Paget's disease, malignant hypercalcemia, and metastatic bone disease.
  • This particular combination produces unexpected results in the treatment and the prevention of such clinical disturbances.
  • compositions containing each of the compounds for use in the treatment of osteoporosis contains as a first element a growth hormone secretagogue.
  • Preferred growth hormone secretagogues for use in the present invention identified therein include:
  • Preferred growth hormone secretagogues for use in the present invention identified therein include:
  • Preferred growth hormone secretagogues for use in the present invention include:
  • Especially preferred growth hormone secretagogues for use in the present invention specifically include:
  • Preferred growth hormone secretagogues for use in the present invention identified therein include l-[2(R)-(2-Amino-2- methylpropionylamino)-3-(lH-indol-3-yl)propionyl]-3(S)-benzyl- piperidine-3-carboxylic acid ethyl ester:
  • Patent No. 5,310,737 U.S. Patent No. 5,317,017; U.S. Patent No. 5,374,721;
  • a compound as a "growth hormone secretagogue” and thus able to directly or indirectly stimulate or increase the endogenous release of growth hormone in an animal may be readily determined without undue experimentation by methodology well known in the art, such as the assay described by Smith, et al., Science, 260, 1640-1643 (1993) (see text of Figure 2 therein).
  • the assay described by Smith, et al., Science, 260, 1640-1643 (1993) see text of Figure 2 therein.
  • pituitary glands are aseptically removed from 150-200 g Wistar male rats and cultures of pituitary cells are prepared according to Cheng et al. Endocrinol, 124, 2791-2798 (1989).
  • the cells are treated with the subject compound and assayed for growth hormone secreting activity and intracellular cAMP levels as described by Chang et al.
  • the intrinsic growth horomone secretagogue activity of a compounds which may be used in the present invention may be determined by this assay
  • the second element is composed of an ⁇ v ⁇ 3 antagonist compound or a pharmaceutically acceptable salt thereof.
  • ⁇ v ⁇ 33 antagonists are well known in the literature.
  • Representative examples of various ⁇ v ⁇ 3 antagonist compounds and methods for the preparation may be found in the following: PCT Patent Pub. Nos. WO 95/32710; WO 96/00574; WO 96/00730; WO 96/26190; WO 96/37492; EPO Patent Publication Nos. EP 0,578,083; EP 0,711,770; EP 0,727,425; EP 0,546,548.
  • the utility of a compound as an avb3 antagonist may be demonstrated by the methodology known in the art, such as the assays described in WO 95/32710, published 7 December 1995. Additional ⁇ v ⁇ 3 antagonist compounds and assays for identifying ⁇ v ⁇ 3 antagonist compounds are described in detail herein.
  • ⁇ v ⁇ 3 antagonist compounds for use in the combinations and methods of the instant invention are compounds of the formula
  • X is a 9- to 10-membered polycyclic ring system, wherein one or more of the rings is aromatic, and wherein the polycyclic ring system contains 0, 1, 2, 3 or 4 heteroatoms selected from N, 0 or S, and wherein the polycyclic ring system is either unsubstituted or substituted on a carbon atom with Rl and R ⁇ ;
  • Y is selected from
  • Z is a 5-11 membered aromatic or nonaromatic mono- or polycyclic ring system containing 0 to 6 double bonds, and containing 0 to 6 heteroatoms chosen from N, 0 and S, and wherein the ring system is either unsubstituted or substituted on a carbon or nitrogen atom with one or more groups independently selected from R 4 , R5, R6 and R ⁇ ; provided that Z is not a 6-membered monocyclic aromatic ring system;
  • Rl, R2, R4 ; R5 ? R13 a nd R ⁇ 4 are each independently selected from hydrogen, halogen, Ci-io alkyl, C3-8 cycloalkyl, aryl, aryl Ci-8 alkyl, amino, amino Ci-8 alkyl, Ci-3 acylamino, Ci-3 acylamino Cl-8 alkyl, C ⁇ _6 alkylamino, Cl-6 alkylamino- Ci-8 alkyl, Ci-6 dialkylamino, Ci-6 dialkylamino Ci-8 alkyl, Ci-4 alkoxy, Cl-4 alkoxy Ci-6 alkyl, hydroxycarbonyl, hydroxycarbonyl Ci-6 alkyl, Ci-3 alkoxycarbonyl, Ci-3 alkoxycarbonyl Cl-6 alkyl, hydroxycarbonyl - Ci-6 alkyloxy, hydroxy, hydroxy Ci-6 alkyl, Ci-6 alk loxy- Ci-6 alkyl, nitro, cyano, trifluoromethyl, trifluoromethoxy
  • Ci- dialkylaminocarbonyl Ci-8 alkyl oxycarbonylamino, Cl-8 alkylaminocarbonyloxy or Ci-8 alkylsulfonylamino;
  • Ci-6 alkyl amino Ci-6 alkyl, arylaminocarbonyl, aryl Cl-5 alkylaminocarbonyl, hydroxycarbonyl Ci-6 alkyl,
  • Ci-6 alkylamino Ci-6 alkyl aryl Ci-6 alkylamino Ci-6 alkyl, Ci-6 dialkylamino Cl-6 alkyl,
  • R10 an( j R11 are eac h independently selected from hydrogen, aryl, -(CH 2 )p-aryl, halogen, hydroxyl, Cl-8 alkylcarbonylamino, aryl Cl-5 alkoxy, Cl-5 alkoxycarbonyl, aminocarbonyl, Cl-8 alkylaminocarbonyl , Ci-6 alkylcarbonyloxy,
  • Ci-8 alkyl either unsubstituted or substituted, with one or more groups selected from: halogen, hydroxyl, Cl-5 alkylcarbonylamino, aryl Ci-5 alkoxy,
  • Ci-8 alkylaminosulfonylamino Ci-6 alkyl, arylamino sulfonylamino Ci-6 alkyl, aryl Ci-8 alkylaminosulfonylamino, aryl Ci-8 alkylaminosulfonylamino Cl-6 alkyl, Ci-6 alkylsulfonyl,
  • Rl2 i s selected from hydrogen
  • Z is selected from
  • More preferred ⁇ v ⁇ 3 antagonists for use in the present invention are compounds of the formula
  • Y is selected from -(CH2) r - or -(CH2) m -NR 3 -;
  • R 3 is selected from hydrogen
  • the ⁇ v ⁇ 3 antagonist is a compound of the formula
  • R8 is selected from hydrogen
  • -(CH2)s CH CH Cl-6 alkyl aryl, -(CH2)s S02C 1-6 alkyl, or
  • ⁇ v ⁇ 3 antagonist compounds include:
  • the instant combination of an ⁇ v ⁇ 3 antagonist and a growth hormone secretagogue are useful 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.
  • osteoporosis including estrogen deficiency, immobilization, glucocorticoid induced and senile
  • osteogenesis imperfecta including 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.
  • the present invention would be useful in increasing bone mass in patients with growth hormone deficiency.
  • Combined therapy to inhibit bone resorption, prevent osteoporosis and enhance the healing of bone fractures may be illustrated by the combination of this invention of ⁇ v ⁇ 3 antagonists and growth hormone secretagogues.
  • an ⁇ v ⁇ 3 antagonist provides an unexpected effect in the treatment and prevention of diseases involving bone resorption when used in combination with a growth hormone secretagogue. While not being bound to any particular theory of operation, that is, an enhanced effect at reducing and reversing the rate of bone loss that occurs during the aging process, the process known as osteoporosis, is observed with the combination of drugs than would be expected from either drug alone.
  • combination therapy of a growth hormone secretagogue and an ⁇ v ⁇ 3 antagonist increase bone mass. This increase in bone mass is possibly a result of increased bone turnover or bone formation produced by elevated growth hormone/IGF- 1 levels resulting from the growth hormone secretagogue and decreased bone resorption produced by the ⁇ v ⁇ 3 antagonist.
  • bone formation and bone resorption generally respond to physiological stimuli and therapeutic intervention by changing in the same direction in a relationship referred to as "coupling".
  • Treatment with an ⁇ v ⁇ 3 antagonist alone is known to decrease bone turnover by decreasing bone resorption with a concomitant ("coupled") reduction in bone formation surface.
  • coupled concomitant
  • 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 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 include the following:
  • suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.
  • the compounds of the present invention may have chiral centers and occur as racemates, racemic mixtures and as individual diastereomers, or enantiomers with all isomeric forms being included in the present invention. Therefore, where a compound is chiral, the separate enantiomers, substantially free of the other, are included within the scope of the invention; further included are all mixtures of the two enantiomers. Also included within the scope of the invention are polymorphs and hydrates of the compounds of the instant invention.
  • the present invention includes within its scope prodrugs of the compounds of this invention.
  • prodrugs will be functional derivatives of the compounds of this invention which are readily convertible in vivo into the required compound.
  • the term “administering” shall encompass the treatment of the various conditions described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient.
  • Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs," ed. H. Bundgaard, Elsevier, 1985. Metabolites of these compounds include active species produced upon introduction of compounds of this invention into the biological milieu.
  • terapéuticaally 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.
  • bone resorption refers to the process by which osteoclasts degrade bone.
  • alkyl groups specified above are intended to include those alkyl groups of the designated length in either a straight or branched configuration which may optionally contain double or triple bonds.
  • alkyl groups are methyl, ethyl, propyl, ethinyl, isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl, isohexyl, allyl, propenyl, butenyl, butadienyl and the like.
  • alkoxy groups specified above are intended to include those alkoxy groups of the designated length in either a straight or branched configuration which may optionally contain double or triple bonds.
  • alkoxy groups are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy allyloxy, propinyloxy, isobutenyloxy, 2-hexenyloxy, and the like.
  • halogen is intended to include the halogen atom fluorine, chlorine, bromine and iodine.
  • aryl is intended to include phenyl and naphthyl and aromatic residues of 5- and 6- membered rings with 1 to 3 heteroatoms or fused 5 or 6 membered bicyclic rings with 1 to 3 heteroatoms of nitrogen, sulfur or oxygen.
  • heterocyclic aromatic rings are pyridine, thiophene, benzothiophene, tetrazole, indole, N-methylindole, dihydroindole, indazole, N-formylindole, benzimidazole, thiazole, furan, pyrimidine, and thiadiazole. Certain of the above defined terms may occur more than once in the above formula and upon such occurrence each term shall be defined independently of the other.
  • the ⁇ v ⁇ 3 antagonist or the growth hormone secretagogue may be administered separately or in conjunction.
  • the administration of one element may be prior to, concurrent to, or subsequent to the administration of the other agent.
  • the elements of the combination of the present invention may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual, or topical (e.g., ocular eyedrop) routes of administration and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.
  • parenteral e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant
  • nasal, vaginal, rectal, sublingual, or topical e.g., ocular eyedrop
  • compositions for the administration of the compounds of this invention may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients.
  • the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • the active object compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases.
  • compositions containing the active ingredient suitable for oral administration may be in the form of discrete units such as hard or soft capsules, tablets, troches or lozenges, each containing a predetermined amount of the active ingredient; in the form of a dispersible powder or granules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid; in the form of syrups or elixirs; or in the form of an oil -in- water emulsion or a water- in-oil emulsion.
  • compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparation.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compounds are admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch.
  • Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate.
  • the dosage forms may also comprise buffering agents.
  • Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients may also be manufactured by known methods.
  • the excipients used may be for example, (1) inert diluents such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintergrating agents such as corn starch, or alginic acid; (3) binding agents such as starch, gelatin or acacia; and (4) lubricating agents such as magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastroinestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl disearate may be employed. They may also be coated by the techniques described in the U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotic therapeutic tablets for controlled release.
  • formulations for oral use may be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
  • Aqueous suspensions normally contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may be
  • suspending agents such as sodium carboxymethyl- cellulose, methylcellulose, hydroxypropylmethyl- cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; (2) dispersing or wetting agents which may be
  • aqueous suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents may be added to provide a palatable oral preparation. These compositions may be prepared by the addition of an antioxidant such as ascorbic acid.
  • Dispersible powders and granules are suitable for the preparation of an aqueous suspension. They provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, those sweetening, flavoring and coloring agents described above may also be present.
  • the pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions.
  • the oily phase may be a vegetable oil such as olive oil or arachis oils, or a mineral oil such as liquid paraffin or a mixture thereof.
  • Suitable emulsifying agents may be (1) naturally-occurring gums such as gum acacia and gum tragacanth, (2) naturally-occurring phosphatides such as soy bean and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, (4) condensation products of said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension or solution.
  • the suspension may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non- toxic paternterally- acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
  • Suitable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspension, or emulsions.
  • non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
  • Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.
  • compositions for nasal or sublingual administration can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • the combination of this invention may also be administered in the form of suppositories for rectal administration.
  • This composition can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials are cocoa butter and polyethylene gylcols.
  • Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
  • the combination of this invention may be formulated in liquid or semi-liquid preparations such as liniments, lotions, applications; oil-in-water or water-in-oil emulsions such as creams, ointments, jellies or pastes, including tooth-pastes; or solutions or suspensions such as drops, and the like.
  • the pharmaceutical composition and method of the present invention may further comprise other therapeutically active compounds usually applied in the treatment of the above mentioned pathological conditions, for instance vitamin D2 and D3 and hydroxylated derivatives, e.g. la-hydroxy- vitamin D3, la-hydroxy- vitamin D2, la-25-dihydroxy- vitamin D3, la-25-dihydroxy-vitamin D2, dehydroepiandrosterone, calcitonin (human, porcine or salmon), mitramycin, sodium fluoride, estrogens, and non-steroid antiinflammatory drugs, such as acetylsalicyclic acid, indomethacin, naprosyn, and timegadine, and bisphosphonates.
  • vitamin D2 and D3 and hydroxylated derivatives e.g. la-hydroxy- vitamin D3, la-hydroxy- vitamin D2, la-25-dihydroxy- vitamin D3, la-25-dihydroxy-vitamin D2, dehydroepiandrosterone, calcitonin (human, porcine or salmon), mitramycin, sodium flu
  • Bisphosphonates useful for treating bone diseases include alendronate, tiludronate, dimethyl-APD, risedronate, etidronate, YM-175, clodronate, pamidronate, and BM-210995, a preferred bisphosphonate being alendronate, and especially alendronate sodium.
  • a preferred bisphosphonate is alendronic acid (alendronate), or a pharmaceutically acceptable salt thereof.
  • An especially preferred bisphosphonate is alendronate sodium, including alendronate sodium trihydrate. Alendronate sodium has received regulatory approval for marketing in the United States under the trademark FOSAMAX®.
  • the dosage of the active ingredients in the compositions of this invention may be varied. However, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained.
  • the selected dosage depends upon the desired therapeutic effect, on the route of administration and on the duration of the treatment.
  • Dosage ranges in the combination for the growth hormone secretogogue and avb3 antagonist are one tenth to one times the clinically effective ranges required to elevate growth hormone and reduce bone resorption respectively when the compounds are used singly.
  • dosage levels of the ⁇ v ⁇ 3 antagonist compound of between about 0.001 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.
  • 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, 250 and 500 milligrams of each of the active ingredients 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 each of the active ingredients, preferably, from about 1 mg to about 100 mg of each of the active ingredients.
  • the most preferred doses will range from about 0.1 to about 10 mg/kg/minute during a constant rate infusion.
  • 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.
  • Dosage levels of the growth hormone secretatogogue of between about 0.001 to 50 mg kg of body weight daily, preferably about 0.005 to about 25 mg/kg per day, and more preferably about 0.01 to about 10 mg/kg per day are administered to a patient to obtain effective treatment or prevention of osteoporosis.
  • the growth hormone secretagoguge is N-[l(R)-[(l,2-dihydro-l- methanesulfonylspiro[3H-indole-3,4'-piperidin]-l'-yl)carbonyl]-2- (phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide, in particular the methanesulfonate salt thereof, and the ⁇ v ⁇ 3 antagonist is 2-Oxo-3(S)- [2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin-l-yl)acetyl- 3(S)-pyridin-3-yl- ⁇ -alanine.
  • the instant combination may also be administered on an intermittent basis.
  • a typical primary oral dose of ⁇ v ⁇ 3 antagonist which lies within the range of from about 0.001 mg to 100 mg per kg body weight and a dose of growth hormone secretatogoue of between 0.001 to 25 mg per kg of body weight may be administered and then, if necessary a sustaining dose of one element or both elements approximately equal to half of the primary dose may be administered at weekly, semiweekly, semimonthly, monthly, bimonthly, quarterly, semiannual, annual or biannual intervals.
  • the avb3 antagonist and the growth hormone secretagogue may be administered in a cyclical manner and it is not necessary that each component be administered concomitantly.
  • the preferred compounds of this combination product are prepared by the references cited above and by the following schemes and examples.
  • R H 2-Oxo-3-(3-oxobutyl)piperidine (1-3)
  • TLC Rf 0.09 (silica, 20% CH3 ⁇ H/EtOAc) 2-Oxo-3-[2-(5,6,7,8-tetrahydro-[l,8]-naphthyridin-2-yl)ethyl]piper- idin-l-yl-acetyl-3(S)-pyridin-3-yl- ⁇ -alanine ethyl ester (1-10)
  • TLC Rf 0.24 (silica, 20% CH3 ⁇ H/EtOAc). (2-Oxo-3-(2-(5.6.7.8-tetrahvdron.81-naphthvridin-2-yl)ethyl)pyrrolidin-l- yl)acetyl-3(S)-ethvnyl- ⁇ -alanine (2-13)
  • TLC RF 0.71 (silica, 70:15:15 CHCl3/EtOAc/CH3 ⁇ H);
  • TLC RF 0.58 (silica, 70:15:15 CHCl3/EtOAc/CH3 ⁇ H);
  • Ethyl 2-oxo-3-r2-naphthyridin-2-yl)ethyllimidazolidin-l-yl-acetate (6-8) A mixture of (1.0 g, 4.1 mmol), 14 (604 mg, 4.9 mmol), L-proline (238 mg), and ethanol (50 mL) was heated at reflux for 20 hr. The cooled reaction mixture was concentrated and the residue purified by flash chromatography (silica, 70:25:5 CHCl3/EtOAc/CH3 ⁇ H) gave 6-8 as a yellow oil.
  • 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 x 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 UN/Vis Diode Array Spectrophotometer. Sample radioactivities were determined in a Packard A5530 gamma counter.
  • osteoclasts 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.
  • 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, II). Bone slices are pooled, placed in a 10% ethanol solution and refrigerated until further use. Prior to experimentation, bovine bone slices are ultrasonicated twice, 20 minutes each in H2O. 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.
  • the bone slices Prior to incubation with osteoclasts, the bone slices are hydrated by the addition of 0.1 ml ⁇ MEM, pH 6.9 containing 5% fetal bovine serum and 1% penicillin/streptomycin. Long bones from 7-14 day old rabbits (New Zealand White
  • Hare are dissected, cleaned of soft tissue and placed in ⁇ 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 rube 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 - 2 x 10 cells/ml).
  • a cell suspension consisting of 5 x 10 /ml in ⁇ MEM containing 5% fetal bovine serum, 10 nM l,25(OH) 2 D3, and pencillin- streptomycin is prepared. 200 ⁇ l aliquots are added to bovine bone slices (200 mm x 6 mm) and incubated for 2 hrs. at 37°C in a humidified 5% CO2 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 ⁇ l chain of type I collagen) by Crosslaps for culture media (Herlev, Denmark).
  • 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 H2O, 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 NH4OH followed by 2 X 15 min ultrasonication in H2O. The bone slices are immediately stained for 6-8 min with filtered 1% toluidine blue and 1% borax.
  • 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 IC50 values are determined for each compound tested.
  • TBS buffer 50 mM Tris-HCl pH 7.2, 150 mM NaCl, 1% BSA, 1 mM CaCl2, 1 mM MgCl2).
  • the reaction mixture was then incubated for 1 h at room temp.
  • the unbound and the bound ⁇ v ⁇ 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, ImM CaC /MgC , pH 7.2).
  • the filter was then counted in a gamma counter.
  • SPA Assay MATERIALS 1. Wheatgerm agglutinin Scintillation Proximity Beads (SPA):
  • n b3 was purified from 293 cells overexpressing a n b3 (Duong et al., J. Bone Min. Res., S:S378, 1993) according to Pytela (Methods in Enzymology, 144:475, 1987) 12.
  • Binding buffer 50 mM HEPES, pH 7.8, 100 mM NaCl, 1 mM Ca 2+ Mg 2 +, 0.5 mM PMSF
  • PROCEDURE 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.
  • each assay tube 2.5 ml (40 mg/ml) of pretreated beads were suspended in 97.5 ml of binding buffer and 20 ml of 50- OG buffer. 5 ml (-30 ng/ml) 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°C. The pellets were then resuspended in 50 ml of binding buffer and 25 ml of 50-OG buffer.
  • Binding buffer ( 125 ml) (v) Each plate was sealed with plate sealer from PACKARD and incubated overnight with rocking at 4°C
  • Osteoblast-like cells (1.8 cells), originally derived from mouse calvaria, were plated in CORNING 24 well tissue culture plates in a 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:
  • 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 x 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 x 10 ⁇ cells/mL.
  • test compounds were added with fresh medium containing D3 to quadruplicate wells. Compounds were added again after 48 h with fresh medium containing D3. 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:
  • 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 were counted in each well.
  • Fluorescent Octform Assay OCTFORM is set up as above. Instead of fixing, staining and counting the multinucleated TRAP stained cells, quantitation is achieved as follows.
  • assay solution 0.5 mL is added to each well and the plate is incubated at 37°C for 1 hr.
  • Assay solution contains 50 mM sodium acetate, 30 mM sodium tartrate, 0.1% Triton X- 100, 5 mM Naphthol AS-BI phosphate, pH 5.0.
  • the frequency of dosing with N-[l(R)-[(l,2-dihydro- l-methane-sulfonylspiro[3H-indole-3,4'-piperidin]-l'-yl)carbonyl]-3- phenylpropyl]-2-amino-2-methyl-propanamide is once daily, seven days a week.
  • the frequency of dosing with 2-Oxo-3(S)-[2-(5,6,7,8- tetrahydro[ l,8]-naphthyridin-2-yl)ethyl]pvrrolidin- l-yl)acetyl-3(S)- pyridin-3-yl- ⁇ -alanine is every other day.
  • N-[l(R)-[(l,2-dihydro-l-methane-sulfonylspiro[3H-indole-3,4'-piperidin]- l'-yl)carbonyl]-3-phenylpropyl]-2-amino-2-methyl-propanamide is orally by gavage and 2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2- yl)ethyl]pyrrolidin-l-yl)acetyl-3(S)-pyridin-3-yl- ⁇ -alanine is by subcutaneous injection (e.g., in carboxy methyl cellulose carrier).
  • the control article is distilled water and the carrier is distilled water.
  • the dosing volume of N-[l(R)-[(l,2-dihydro-l-methane-sulfonylspiro[3H- indole-3,4'-piperidin]-l'-yl)carbonyl]-3-phenylpropyl]-2-amino-2-methyl- propanamide is 5 ml/kg
  • the dosing volume of 2-Oxo-3(S)-[2-(5,6,7,8- tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin-l-yl)acetyl-3(S)- pyridin-3-yl- ⁇ -alanine is 0.5 ml/kg.
  • the test system is the female rat of a strain Sprague-Dawley Crl:CD® (SD) BR, which were of an approximate age at the start of the study of greater than 6 months, and which are of an approximate weight at the start of the study of 250-350 g.
  • SD strain Sprague-Dawley Crl:CD®
  • the rats are housed in individual stainless steel wire cages.
  • Drug Day 1 Blood sampling (approximately 1.5 ml) is from orbital sinus on non-fasted rats, all groups, for measurements of GH; bleeding is done 15 minutes post dosing in control groups and groups receiving N-[l(R)-[(l,2-dihydro-l-methane-sulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-3-phenylpropyl]-2-amino-2-methyl- propanamide alone or in combination.
  • Blood sampling (approximately 1.5 ml) is from orbital sinus on non-fasted rats, all groups except control group 1, for measurement of GH; bleeding is done 15 minutes post dosing in control group 2 and groups receiving N-[l(R)-[(l,2-dihydro-l-methane-sulfonylspiro[3H- indole-3,4'-piperidin]-l'-yl)carbonyl]-3-phenylpropyl]-2-amino-2-methyl- propanamide alone or in combination; Blood sampling (volume: as much as possible) is from the vena cava at necropsy, on non-fasted rats, all groups for measurement of IGF-1.
  • All rats receive bone labelling agents (oxytetracycline and calcein): 9 days (oxytetracycline) and 2 days (calcein), before necropsy.
  • Oxytetracycline is injected subcutaneously at a dose of 25 mg/kg, and calcein is injected subcutaneously at a dose level of 10 mg/kg.
  • Tibiae are processed through increasing concentrations of ethanol followed by methyl methacrylate embedding, using an automated Hypercenter XP tissue processor (Shandon-Lipshaw,
  • MS/BS Mineralizing surface
  • % is calculated as one-half sLS/BS plus dLS/BS, expressed as a percent of bone surface. All measurements are made in the metaphyseal secondary spongiosa 1mm below the epiphyseal growth plate. Length of the tibia is measured and diaphyseal cortical cross sections are cut using a Buehler ISOMET saw 1 - 1.5 cm proximal to the tibiofibular junction. Results are expressed as mean ⁇ SEM.
  • Statistical analysis is performed using the package STATVIEW for Macintosh (Abacus Concepts, Berkeley, CA). Differences between treatment groups are tested by one-way analysis of variance and Fisher PLSD (protected least significant difference). A value of p ⁇ 0.05 is considered significant.
  • the purpose of this study is to evaluate the combined effects of twelve weeks treatment with the growth hormone secretagogue ("GHS"), N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2- methylpropanamide methanesulfonate, and the avb3 antagonist, 2-Oxo- 3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin-l- yl)acetyl-3(S)-pyridin-3-yl- ⁇ -alanine, on bone mineral density and bone formation in dogs.
  • GSS growth hormone secretagogue
  • One year old female (9.1-13 kg) dogs are assigned four per dose group and treated for twelve weeks with either vehicle, or 1.25 mg/kg/day GHS + 1.25 mg/kg/day avb3 antagonist, 2.5 mg/kg/day GHS + 2.5 mg/kg/day ⁇ v ⁇ 3 antagonist, or 5.0 mg/kg/day GHS + 5.0 mg/kg/day avb3 antagonist.
  • To identify formation surfaces dogs receive fluorochrome bone labels prior to necropsy by i.v. administration of oxytetracycline (15 mg/kg i.v.) and calcein (15 mg/kg i.v.) with a 10 day interval between the administration of the two labels.
  • the fourth and fifth lumbar vertebrae and right tibiae are dissected free of muscle and other connective tissue and fixed in cold 70% ethanol (4°C).
  • Spinous and transverse processes of vertebra L5 are removed, the vertebral body is submerged in two inches of water, and bone mineral content is measured using dual energy x-ray absorptiometry (QDR 4500A, HOLOGIC, Waltham Mass.).
  • the L5 vertebral body is then cut in cross-section and a central sagittal piece of the proximal portion is cut using a high speed Dremmel tool.
  • the vertebral cross and sagittal sections are processed and embedded in methylmethacrylate without prior decalcification using a Hypercenter XP tissue processor (Shandon, Pittsburgh, PA). Sagittal and cross-sections of 6-10 mm thickness are cut using a Polycut S microtome (Leica, Deerfield, IL).
  • N, I/mm), trabecular thickness (TbTh., mm), trabecular separation (Tb Sp., mm) are directly measured or calculated from primary measurements of tissue area, trabecular bone area, trabecular bone perimeter and boundary length, using Masson's trichrome stained sections. Osteoid surface (OS/BS, %) or the unmineralized matrix is also measured on Masson's trichrome stained sections in the same area and expressed as a percent of the trabecular bone surface. Osteoid thickness (O.Th, mm) is calculated as the product of osteoid width and the correction factor p/4. Mineralization lag time (Mlt, days) is calculated as O.Th/Aj.AR.
  • Adjusted apposition rate (Aj.AR, mm d) or effective apposition rate is calculated as the product of MAR*(MS/OS).
  • the vertebral anterior (ventral) cortical width is also directly measured from stained mid-vertebral cross-sections.
  • Dynamic labeled parameters are assessed in the same area of 10 mm thick sections viewed under epifluorescence by measuring the length of the oxytetracycline and calcein labels on trabecular bone surface and the interlabel distance.
  • the mineralizing surface (MS/BS, %) is calculated as one-half the length of single labels plus the length of double labels expressed as a percent of bone surface.
  • the mineral appositional rate (MAR, mm day) is calculated as the mean distance between the first and second label at equidistant points divided by the labeling interval (14 days) and then multiplied by the correction factor (p/4) to account for the obliquity of the sectioning plane.
  • Surface based bone formation rate (BFR/BS) is calculated as the product of MS/BS*MAR and expressed per year.

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Abstract

The combination of an αvβ3 antagonist and a growth hormone secretagogue is useful in the treatment or prevention of diseases involving bone resorption, especially osteoporosis.

Description

TITLE OF THE INVENTION
COMBINATION THERAPY FOR THE PREVENTION AND
TREATMENT OF OSTEOPOROSIS
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority of U.S. provisional application Serial No. 60/043,815, filed April 14, 1997, now abandoned. -
FIELD OF THE INVENTION
The present invention provides a combination therapy for the treatment and prevention of osteoporosis. More particularly, the combination of the present invention comprises an αvβ3 antagonist compound and a growth hormone secretogogue.
BACKGROUND OF THE INVENTION
Osteoclasts are multinucleated cells of up to 400 mm in diameter that resorb mineralized tissue in vertebrates. They are actively motile cells that migrate along the surface of bone. They can bind to bone, secrete necessary acid and proteases and thereby cause the actual resorption of mineralized tissue from the bone.
More specifically, osteoclasts are believed to exist in at least two physiological states. In the secretory state, osteoclasts attach to the bone matrix via a tight attachment zone (sealing zone), become highly polarized, form a ruffled border, and secrete lysosomal enzymes and acid to resorb bone. The adhesion of osteoclasts to bone surfaces is an important initial step in bone resorption. In the migratory or motile state, the osteoclasts migrate across bone matrix and do not take part in resorption until they attach again to bone. Integrins are transmembrane, heterodimeric, glycoproteins which interact with extracellular matrix and are involved in osteoclast attachment, activation and migration. The most abundant integrin in osteoclasts (rat, chicken, mouse and human) is the vitronectin receptor, or αvβ3, thought to interact in bone with matrix proteins that contain the RGD sequence. Antibodies to αvβ3 block bone resorption in vitro and m vivo indicating that this integrin plays a key role in the resorptive process. There is increasing evidence to suggest that αvβ3 ligands can be used effectively to inhibit osteoclast mediated bone resoption in vivo in mammals. The current major bone diseases of public concern are osteoporosis, hypercalcemia of malignancy, osteolysis due to bone metastases, periodontal disease, hyperparathyroidism, periarticular erosions in rheumatoid arthritis, Paget's disease, immobilization- induced osteopenia, loosening of bone prostheses and glucocorticoid induced osteoporosis.
All these conditions are characterized by bone loss, resulting from an imbalance between bone resorption (breakdown) and bone formation, which continues throughout life past about age 30 at the rate of about 0.5 - 1% per year on the average. However, the rate of bone turnover differs from site to site, for example, it is higher in the trabecular bone of the vertebrae and the alveolar bone in the jaws than in the cortices of the long bones. The potential for bone loss is directly related to turnover and can amount to over 5% per year in vertebrae immediately following menopause, a condition which leads to increased fracture risk.
There are currently 20 million people with detectable fractures of the vertebrae due to osteoporosis in the United States. In addition, there are 250,000 hip fractures per year attributed to osteoporosis. This clinical situation is associated with a 12% mortality rate within the first two years, while 30% of the patients require nursing home care after the fracture.
Individuals suffering from all the conditions listed above would benefit from treatment with agents which inhibit bone resorption. The literature discloses a variety of αvβ3 antagonist compounds which are useful in the treatment and prevention of diseases involving bone resorption. Representative examples may be found in the following: PCT Patent Pub. Nos. WO 95/32710; WO 96/00574; WO 96/00730; WO 96/26190; WO 96/37492; EPO Patent Publication Nos. EP 0,578,083; EP 0,711,770; EP 0,727,425; EP 0,546,548. The preparation of αvβ3 antagonists is well known in the art. Representative examples may be found in the above mentioned references which disclose the compounds as being useful for the treatment of bone diseases, in particular, as inhibitors of bone resorption. The preparation of additional αvβ3 antagonists which are useful in the combinations and methods of the present invention is described in detail in the schemes and examples which follow.
The treatment of osteoporosis with calcitonin, alone and in combination with human growth hormone ("GH") was examined by Aloia, et al., Metabolism. 34(2) 124-129 (1985). This publication ascribes no benefit in the treatment of osteoporosis from combining calcitonin therapy with the administration of growth hormone and noted that the addition of growth hormone to calcitonin therapy appeared to have a deleterious effect on cortical bone mass. Combined treatment of growth hormone and the bisphosphonate pamidronate has been examined in growth hormone deficient adults (Valk, et al., Clin. Endocrinol.. 43. 317- 324 (Sept. 1995)) and in women with osteoporosis (Erdtsieck, et al., Clin. Endocrinol.. 43, 557-565 (Sept. 1995)). The effects of growth hormone itself in the treatment of osteoporosis was studied by Aloia, et ai., J. Clin. Endocrinol. Metab.. ≤4, 992-999 (1976). This publication noted that under the conditions of the study, growth hormone administration did not result in an increment in skeletal mass. The effects of growth hormone on human bone biology have been reviewed by Inzucchi, et al. J. Clin. Endocrinol. Metab.. 79(3), 691-694 (1994). General reviews of human growth hormone also discuss the role of growth hormone on bone (Strobl, fit al. Pharmacol. Reviews. 46(1), 1-34 (1994); Chipman, <L Pediatric Encocrinol.. 6(3-4), 325-328 (1993)). Bone turnover and bone mineral density in young adult patients with panhypopituitarism following long-term growth hormone therapy was examined by Balducci, et al. Eur. J. Endocrinol.. 132(1), 42-46 (Jan 1995). Also, the effect of growth hormone replacement on bone has been examined in boys with and without classic growth hormone deficiency (Zadik, et al. , J. Pediatrics. 125(2), 189-195 (1994)) and in adults with adult onset growth hormone deficiency (Holmes, et al., Clin. Endocrinol.. 42, 627-633 (1995)). There is a difference in the literature between effects in GH-deficient children, where improvement with GH is seen, and in adults, where most reports show both, increased bone resorption and formation, but no positive balance. Certain non-peptidal growth hormone secretagogues are known to stimulate the pituitary gland to increase its secretion of growth hormone with utility in growth hormone deficient children and adults, in severe burn victims, in the treatment of Turners syndrome, for reversing the adverse effects of glucocorticoid treatment, for treating muscle and exercise tolerance deficiencies in growth hormone deficient adults, and for the treatment of osteoporosis. Certain compounds have been developed which stimulate the release of endogenous growth hormone. Peptides which are known to stimulate the release of endogenous growth hormone include growth hormone releasing hormone, the growth hormone releasing peptides GHRP-6 and GHRP-1 (described in U.S. Patent No. 4,411,890, PCT Patent Pub. No. WO 89/07110, and PCT Patent Pub. No. WO 89/07111) and GHRP-2 (described in PCT Patent Pub. No. WO 93/04081), as well as hexarelin (J. Endocrinol Invest.. 15(Suppl 4), 45 (1992)). Other compounds possessing growth hormone secretagogue activity are disclosed in the following: U.S.
Patent No. 3,239,345; U.S. Patent No. 4,036,979; U.S. Patent No. 4,411,890; U.S. Patent No. 5,206,235; U.S. Patent No. 5,283,241; U.S. Patent No. 5,284,841; U.S. Patent No. 5,310,737; U.S. Patent No. 5,317,017; U.S. Patent No. 5,374,721; U.S. Patent No. 5,430,144; U.S. Patent No. 5,434,261; U.S. Patent No. 5,438,136; U.S. Patent No. 5,494,919; U.S. Patent No. 5,494,920; U.S. Patent No. 5,492,916; U.S. Patent No. 5,536,716; EPO Patent Pub. No. 0,144,230; EPO Patent Pub. No. 0,513,974; PCT Patent Pub. No. WO 94/07486; PCT Patent Pub. No. WO 94/08583; PCT Patent Pub. No. WO 94/11012; PCT Patent Pub. No. WO 94/13696; PCT Patent Pub. No. WO 94/19367; PCT Patent Pub. No. WO 95/03289; PCT Patent Pub. No. WO 95/03290; PCT Patent Pub. No. WO 95/09633; PCT Patent Pub. No. WO 95/11029; PCT Patent Pub. No. WO 95/12598; PCT Patent Pub. No. WO 95/13069; PCT Patent Pub. No. WO 95/14666; PCT Patent Pub. No. WO 95/16675; PCT Patent Pub. No. WO 95/16692; PCT Patent Pub. No. WO 95/17422; PCT Patent Pub. No. WO 95/17423; PCT Patent Pub. No. WO 95/34311; PCT Patent Pub. No. WO 96/02530; PCT Patent Pub. No. WO 96/22997; Science. 260. 1640-1643 (June 11, 1993); Ann. Ren. Med. Chem.. 28, 177-186 (1993); Bioorg. Med. Chem. Ltrs.. 4(22), 2709- 2714 (1994); and Proc. Natl. Acad. Sci. USA <g, 7001-7005 (July 1995). Additional compounds with growth hormone secretagogue activity are described herein.
SUMMARY OF THE INVENTION
The present invention provides a combination which comprises an avb3 antagonist and a growth hormone secretagogue.
In one embodiment of the invention is the combination wherein the growth hormone secretagogue is of the Formula I or II:
Figure imgf000007_0001
Formula Formula II wherein: Ri is selected from the group consisting of: -Ci-Cio alkyl, -aryl, aryl-(Cι-C6 alkyl)-, C3-C7 cycloalkyl-(Ci-C6alkyl)-, -Cl-Cδalkyl-K-Ci-Cδ alkyl, aryl(Co-C5alkyl)-K-(Cι-C5 alkyl)-, and C3-C7 cycloalkyl(Co-C5 alkyl)-K-(Cι-C5 alkyl)-, wherein K is O, S(0)m, N(R2>C(0), C(0)N(R2), OC(O), C(0)0, or - CR2=CR2-, or -CjC-, and wherein the aryl groups are as defined below and the R2 and alkyl groups may be further substituted by 1 to 9 halogen, S(0)mR2a> 1 to 3 OR2a> or C(0)OR2a> and the aryl groups may be further substituted by phenyl, phenoxy, halophenyl, 1-3 C1-C6 alkyl, 1 to 3 halogen, 1 to 2 -OR2, methylenedioxy, -S(0)mR2, 1 to 2 -CF3, -OCF3, nitro, -N(R2)(R2), - N(R2)C(0)R2, -C(0)OR2, -C(0)N(R2)(R2), -Sθ2N(R2)(R2), -N(R2)S(0)2 aryl, and -N(R2)Sθ2R2; R2 is selected from the group consisting of: hydrogen, Cχ-C6 alkyl, C3-C7 cycloalkyl, and where two Cl-Cβ alkyl groups are present on one atom, they may be optionally joined to form a C3-C8 cyclic ring optionally including oxygen, sulfur or NR2a;
R2a is hydrogen, or Cl-Cβ alkyl;
R3a and R3 are independently selected from the group consisting of: hydrogen, halogen, -C1-C6 alkyl, -OR2, cyano, -OCF3, methylenedioxy, nitro, -S(0)mR> -CF3 or -C(0)OR2 and when R3a and R3b are in an ortho arrangement, they may be joined to form a C5 to Cδ aliphatic or aromatic ring optionally including 1 or 2 heteroatoms selected from oxygen, sulfur or nitrogen;
R4 and R5 are independently selected from the group consisting of: hydrogen, -Cχ-C6 alkyl, substituted Cχ-C6 alkyl wherein the substituents are selected from 1 to 5 halo, 1 to 3 hydroxy, 1 to 3
Cl-Cio alkanoyloxy, 1 to 3 Cl-Cβ alkoxy, phenyl, phenoxy, 2-furyl, Ci-Cβ alkoxycarbonyl, -S(0)m(Cl-C6 alkyl); or R4 and R5 can be taken together to form -(CH2)rLa (CH2)s- where La is -C(R2)2-, -0-, -SCOW, or -N(R2)-, where r and s are independently 1 to 3 and R2 is as defined above;
R6 is hydrogen or Ci-Cβ alkyl;
A is: (CH2) -C — (CH2)y-
R7a or
Z-(CH2), •C "(CH2)V
R7a
wherein x and y are independently 0-3; Z is N-R2 or 0; R7 and R7a are independently selected from the group consisting of: hydrogen, -C\-CQ alkyl, -OR2, trifluoromethyl, phenyl, substituted Ci-Cβ alkyl where the substituents are selected from imidazolyl, phenyl, indolyl, p-hydroxyphenyl, -OR2, 1 to 3 fluoro, -S(0)mR2, -C(0)OR2, -C3- C7 cycloalkyl, -N(R2)(R2), -C(0)N(R2)(R2); or R7 and R7a can independently be joined to one or both of R4 and R5 groups to form alkylene bridges between the terminal nitrogen and the alkyl portion of the R7 or R7a groups, wherein the bridge contains 1 to 5 carbons atoms;
B, D, E, and F are independently selected from the group consisting of: -C(R8)(Rlθ)-, -0-, C=0, -S(0)m-, or -NR9., such that one or two of B, D, E, or F may be optionally absent to provide a 5, 6, or 7 membered ring; and provided that B, D, E and F can be -C(Rs)(Rlθ)- or C=0 only when one of the remaining B, D, E and F groups is simultaneously -0-, -S(O)m-, or - NR9-, or B and D, or D and E taken together may be -N=CRιo- or -CRιo=N-, or B and D, or D and E taken together may be
Figure imgf000009_0001
provided one of the other of B and E or F is simultaneously -0-, -S(0)m-, or -NR9-;
R8 and Rχo are independently selected from the group consisting of:
-7- hydrogen, -R2, -OR2, (-CH2)q-aryl, -(CH2)q-C(0)OR2, -(CH2)q- C(0)0(CH2)q-aryl, or -(CH2)q-(lH-tetrazol-5-yl), where the aryl may be optionally substituted by 1 to 3 halo, 1 to 2 Ci-Cs alkyl, 1 to 3 -OR2 or 1 to 2 -C(0)OR2;
R9 is selected from the group consisting of:
-R2, -(CH2)q-aryl, -C(0)R2, -C(0)(CH2)q-aryl, -SO2R2,
-Sθ2(CH2)q-aryl, -C(0)N(R2)(R2)> -C(0)N(R2)(CH2)q-aryl, -C(0)OR2, 1-H- tetrazol-5-yl, -SO3H, -SO2NHCJN, -Sθ2N(R2)aryl, -Sθ2N(R2)(R2), and wherein the (CH2)q may be optionally substituted by 1 to 2 C1-C4 alkyl, and the R2 and aryl may be optionally further substituted by 1 to 3 - OR2a, -0(CH2)q aryl, 1 to 2 -C(0)OR2a, 1 to 2 -C(0)0(CH2)q aryl, 1 to 2 - C(0)N(R2a)(R2a), 1 to 2 -C(O)N(R2a)(CH2)q aryl, 1 to 5 halogen, 1 to 3 Ci- C4 alkyl, 1,2,4-triazolyl, l-H-tetrazol-5-yl, -C(0)NHSθ2R2a, -S(0)mR2a, - C(0)NHS02(CH2)q-aryl, -SO2NHCJN, -Sθ2NHC(0)R2a, - S02NHC(0)(CH2)qaryl, -N(R2)C(0)N(R2a)(R2a), - N(R2a)C(0)N(R2a)(CH2)q-aryl, -N(R2a)(R2a), -N(R2a)C(0)R2a> - N(R2a)C(0)(CH2)q aryl, -OC(0)N(R a)(R2a), -OC(0)N(R2a)(CH2)q aryl, - Sθ2(CH2)qCONH-(CH2)wNHC(0)Rn, wherein w is 2-6 and Rn may be biotin, aryl, or aryl substituted by 1 or 2 OR2, 1-2 halogen, azido or nitro;
m is 0, 1 or 2; n is 1, or 2; q may optionally be 0, 1, 2, 3, or 4; and
G, H, I and J are carbon, nitrogen, sulfur or oxygen atoms, such that at least one is a heteroatom and one of G, H, I or J may be optionally missing to afford a 5 or 6 membered heterocyclic aromatic ring; and pharmaceutically acceptable salts and individual diastereomers thereof.
In a class of the invention is the combination wherein the growth hormone secretagogue is of the Formula V:
Figure imgf000011_0001
wherein:
Rl is selected from the group consisting of:
Figure imgf000011_0002
R3a is H, or fluoro;
D is selected from the group consisting of:
-0-, -S-, -S(OW, N(R2), NS02(R2), NS02(CH2)taryl, NC(0)(R2), NSθ2(CH2)qOH, NS02(CH2)qCOOR2, NS02(CH2)qC(0)-N(R2)(R2), N- Sθ2(CH2)qC(0)-N(R2)(CH2) OH, N-S02(CH2)qC(0)-N(R2)(CH2)w
Figure imgf000012_0001
N-S02(CH2)qC(0)-N(R2)(CH2)w - Nj
Figure imgf000012_0002
N-NH N-S02(CH2)q-^/
N=N '
and the aryl is phenyl or pyridyl and the phenyl may be substituted by 1-2 halogen;
R2 is H, or C1-C4 alkyl; m is 1 or 2; t is 0, 1, or 2; q is 1, 2, or 3; w is 2, 3, 4, 5, or 6; and the pharmaceutically acceptable salts and individual diastereomers thereof.
In a subclass of the invention is the combination wherein the growth hormone secretagogue is selected from the group consisting of:
1) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(lH-indol-3-yl)ethyl]-2-amino-2-methyl- propanamide; 2) N-[l(R)-[(l,2-dihydro-l-methanecarbonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(lH-indol-3-yl)ethyl]-2-amino-2-methyl- propanamide;
3) N-[l(R)-[(l,2-dihydro-l-benzenesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(lH-indol-3-yl)ethyl]-2-amino-2-methyl- propanamide;
4) N-[l(R)-[(3,4-dihydro-spiro[2H-l-benzopyran-2,4'-piperidin]-l'-yl) carbonyl]-2-(lH-indol-3-yl)ethyl]-2-amino-2-methylpropanamide;
5) N-[l(R)-[(2-acetyl-l,2,3,4-tetrahydrospiro[isoquinolin-4,4'-piperidin]- l'-yl)carbonyl]-2-(indol-3-yl)ethyl]-2-amino-2-methyl-propanamide;
6) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2- methylprop anamide ;
7) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2- methylpropanamide methanesulfonate;
8) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(2',6'-difluorophenylmethyloxy)ethyl]-2- amino-2-methylpropanamide;
9) N-[l(R)-[(l,2-dihydro-l-methanesulfonyl-5-fluorospiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2- methylpropanamide;
10) N-[l(S)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(phenylmethylthio)ethyl]-2-amino-2- methylpropanamide; 11) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-3-phenylpropyl]-2-amino-2-methyl- propanamide;
12) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-3-cyclohexylpropyl]-2-amino-2-methyl- propanamide;
13) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]- l'-yl)carbonyl]-4-phenylbutyl]-2-amino-2-methyl- propanamide;
14) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(5-fluoro-lH-indol-3-yl)ethyl]-2-amino-2- methylpropanamide;
15) N-[l(R)-[(l,2-dihydro-l-methanesulfonyl-5-fluorospiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(5-fluoro-lH-indol-3-yl)ethyl]-2-amino-2- methylpropanamide;
16) N-[l(R)-[(l,2-dihydro-l-(2-ethoxycarbonyl)methylsulfonylspiro-[3H- indole-3,4'-piperidin]-l'-yl)carbonyl]-2-(lH-indol-3-yl)ethyl]-2-amino-2- methylpropanamide;
17) N-[l(R)-[(l,2-dihydro-l,l-dioxospiro[3H-benzothiophene-3,4'- piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2- methylpropanamide; and
18) l-[2(R)-(2-Amino-2-methylpropionylamino)-3-(lH-indol-3- yl)propionyl]-3(S)-benzyl-piperidine-3-carboxylic acid ethyl ester;
or a pharmaceutically acceptable salt thereof.
Preferably, the growth hormone secretagogue used in the combination is N-[l(R)-[(l,2-dihydro-l-methanesulfonyl-spiro[3H-indole- 3,4'-piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)-ethyl]-2-amino-2- methylpropanamide, or a pharmaceutically acceptable salt thereof. Most preferably, the growth hormone secretagogue is N-[l(R)-[(l,2-dihydro-l- methanesulfonyl-spiro[3H-indole-3,4'-piperidin]-l'-yl)carbonyl]-2- (phenylmethyloxy)-ethyl]-2-amino-2-methylpropanamide methanesulfonate.
Illustrative of the invention is the combination wherein the αvβ3 antagonist is a compound of the formula
Figure imgf000015_0001
wherein X is a 9- to 10-membered polycyclic ring system, wherein one or more of the rings is aromatic, and wherein the polycyclic ring system contains 0, 1, 2, 3 or 4 heteroatoms selected from N, O or S, and wherein the polycyclic ring system is either unsubstituted or substituted on a carbon atom with Rl and R^;
Y is selected from
—
Figure imgf000015_0002
-N-(CH2)-
R-'
O II
-N-c-(CH2)- -S(0)q-(CH2fc , - 0-(CH2)- or - (CH2)r- I
R3
Z is a 5-11 membered aromatic or nonaromatic mono- or polycyclic ring system containing 0 to 6 double bonds, and containing 0 to 6 heteroatoms chosen from N, O and S, and wherein the ring system is either unsubstituted or substituted on a carbon or nitrogen atom with one or more groups independently selected from R4, R5? R6 and R^; provided that Z is not a 6-membered monocyclic aromatic ring system;
Rl, R2, R4, R5} R13 and Rl4 are each independently selected from hydrogen, halogen, Ci-10 alkyl, C3-8 cycloalkyl, aryl, aryl Ci-8 alkyl, amino, amino Ci-8 alkyl, Cl-3 acylamino, Ci-3 acylamino Ci-8 alkyl, Cl-6 alkylamino, Cl-6 alkylamino- Ci-8 alkyl, Cl-6 dialkylamino, Cl-6 dialkylamino Ci-8 alkyl, Ci-4 alkoxy, Ci-4 alkoxy Cl-6 alkyl, hydroxycarbonyl, hydroxycarbonyl Cl-6 alkyl, Ci-3 alkoxycarbonyl,
Cl-3 alkoxycarbonyl Cl-6 alkyl, hydroxycarbonyl- Ci-6 alkyloxy, hydroxy, hydroxy Cl-6 alkyl, Cl-6 alkyloxy- Ci-6 alkyl, nitro, cyano, trifluoromethyl, trifluoromethoxy, trifluoroethoxy, Cl-8 alkyl-S(0)q, Ci-8 aminocarbonyl, Cl-8 dialkylaminocarbonyl, Cl-8 alkyloxycarbonylamino,
Cl-8 alkylaminocarbonyloxy or Cl-8 alkylsulfonylamino;
R^ is selected from hydrogen, aryl,
-(CH2)p-aryl, hydroxyl,
Ci-5 alkoxycarbonyl, aminocarbonyl, C3-8 cycloalkyl, amino Cl-6 alkyl, arylaminocarbonyl, aryl Ci-5 alkylaminocarbonyl, hydroxycarbonyl Cl-6 alkyl, Cl-8 alkyl, aryl Cl-6 alkyl, Cl-6 alkylamino Cl-6 alkyl, aryl Cl-6 alkylamino Cl-6 alkyl, Cl-6 dialkylamino Cl-6 alkyl, Ci-8 alkylsulfonyl, Ci-8 alkoxycarbonyl, aryloxy carbonyl, aryl Cl-8 alkoxycarbonyl, Ci-8 alkylcarbonyl, arylcarbonyl, aryl Cl-6 alkylcarbonyl,
Ci-8 alkylaminocarbonyl , amino sulfonyl, Ci-8 alkylaminosulfonyl, arylaminosulfonylamino, aryl Cl-8 alkylaminosulfonyl, Cl-6 alkylsulfonyl, aryl sulfonyl, aryl Cl-6 alkylsulfonyl, aryl Cl-6 alkylcarbonyl, Cl-6 alkylthiocarbonyl, arylthiocarbonyl, or aryl Cl-6 alkylthiocarbonyl, wherein any of the alkyl groups may be unsubstituted or substituted with
R13 and R14;
R6, R?, R8, R9, RlO and R11 are each independently selected from hydrogen, aryl,
-(CH2)p-aryl, halogen, hydroxyl,
Ci-8 alkylcarbonylamino, aryl Ci-5 alkoxy,
Ci-5 alkoxycarbonyl, aminocarbonyl,
Ci-8 alkylaminocarbonyl,
Cl-6 alkylcarbonyloxy, C3-8 cycloalkyl, oxo, amino,
Cl-6 alkylamino, amino Cl-6 alkyl, arylaminocarbonyl, aryl C1-5 alkylaminocarbonyl, aminocarbonyl, aminocarbonyl Cl- alkyl, hydroxycarbonyl, hydroxycarbonyl Cl-6 alkyl,
Ci-8 alkyl, either unsubstituted or substituted, with one or more groups selected from: halogen, hydroxyl, Ci-5 alkylcarbonylamino, aryl Ci-5 alkoxy, Ci-5 alkoxycarbonyl, aminocarbonyl, Ci-5 alkylaminocarbonyl, Ci-5 alkylcarbonyloxy, C3-8 cycloalkyl, oxo, amino, Cl-3 alkylamino, amino Ci-3 alkyl, arylaminocarbonyl, aryl Ci-5 alkylaminocarbonyl, aminocarbonyl, aminocarbonyl Ci-4 alkyl, hydroxycarbonyl, or hydroxycarbonyl Ci-5 alkyl,
-(CH2)s C/CH,
-(CH2)s CjC-Ci-6 alkyl,
-(CH2)s CIC-C3.7 cycloalkyl,
-(CH2)s CJC-aryl, -(CH2)s CjC-Ci-6 alkylaryl,
-(CH2)s CH=CH2,
-(CH2)s CH=CH Ci-6 alkyl,
-(CH2)S CH=CH-C3_7 cycloalkyl,
-(CH2)S CH=CH aryl, -(CH2)S CH=CH Cl-6 alkylaryl,
-(CH2)s SO2C1-6 alkyl, or
-(CH2)s SO2C1-6 alkylaryl;
Ci-6 alkoxy, aryl Ci-6 alkoxy, aryl Cι_6 alkyl,
Ci-6 alkylamino Ci-6 alkyl, arylamino, arylamino Cl-6 alkyl, aryl Ci-6 alkylamino, aryl Ci-6 alkylamino Cl-6 alkyl, arylcarbonyloxy, aryl Cl-6 alkylcarbonyloxy,
Ci-6 dialkylamino, Ci-6 dialkylamino Cl-6 alkyl,
Ci-6 alkylaminocarbonyloxy,
Ci-8 alkylsulfonylamino,
Ci-8 alkylsulfonylamino Cl-6 alkyl, arylsulfonylamino Cl-6 alkyl, aryl Ci- alkylsulfonylamino, aryl Ci-6 alkylsulfonylamino Ci-6 alkyl,
Ci-8 alkoxycarbonylamino,
Ci-8 alkoxycarbonylamino Cl-8 alkyl, aryloxycarbonylamino Cl-8 alkyl, aryl Ci-8 alkoxycarbonylamino, aryl Cl-8 alkoxycarbonylamino Cl-8 alkyl,
Ci-8 alkyl carbonylamino,
Ci-8 alkylcarbonylamino Cl-6 alkyl, arylcarbonylamino Cl-6 alkyl, aryl Cl-6 alkylcarbonylamino, aryl Ci-6 alkylcarbonylamino Ci-6 alkyl, aminocarbonylamino Ci-6 alkyl,
Ci-8 alkylamino carbonylamino ,
Ci-8 alkylaminocarbonylamino Ci-6 alkyl, arylaminocarbonylamino Cl-6 alkyl, aryl Ci-8 alkylaminocarbonylamino, aryl Ci-8 alkylaminocarbonylamino Ci-6 alkyl, aminosulfonylamino Ci-6 alkyl,
Ci-8 alkylaminosulfonylamino, Cl-8 alkylaminosulfonylamino Ci-6 alkyl, arylamino sulfonylamino Ci-6 alkyl, aryl Ci-8 alkylaminosulfonylamino, aryl Cl-8 alkylaminosulfonylamino Ci-6 alkyl, Cl-6 alkylsulfonyl,
Cl-6 alkylsulfonyl Cl-6 alkyl, arylsulfonyl Ci-6 alkyl, aryl Cl-6 alkylsulfonyl, aryl Cl-6 alkylsulfonyl Cl-6 alkyl, Ci- alkylcarbonyl,
Cl-6 alkylcarbonyl Cl-6 alkyl, arylcarbonyl Ci-6 alkyl, aryl Cl- alkylcarbonyl, aryl Cl-6 alkylcarbonyl Ci-6 alkyl, Cl-6 alkylthiocarbonylamino,
Cl-6 alkylthiocarbonylamino Ci-6 alkyl, arylthiocarbonylamino Cl-6 alkyl, aryl Cl-6 alkylthiocarbonylamino, aryl Cl-6 alkylthiocarbonylamino Ci-6 alkyl, Cl-8 alkylaminocarbonyl Ci-6 alkyl, arylaminocarbonyl Cl-6 alkyl, aryl Cl-8 alkylaminocarbonyl, or aryl Cl- alkylaminocarbonyl Cι_ alkyl, wherein any of the alkyl groups may be unsubstituted or substituted with Rl3 and Rl4; and provided that the carbon atom to which R and R^ are attached is itself attached to no more than one heteroatom; and provided further that the carbon atom to which RlO and R^ are attached is itself attached to no more than one heteroatom;
l2 is selected from hydrogen, Cl-8 alkyl, aryl, aryl Cl-8 alkyl, hydroxy, Cl-8 alkoxy, aryloxy, aryl Cl-6 alkoxy, Cl-8 alkylcarbonyl oxy Cl-4 alkoxy, aryl Cl-8 alkyl carbonyl oxy Cl-4 alkoxy, Cl-8 alkylaminocarbonylmethyleneoxy, or Cl-8 dialkylaminocarbonylmethyleneoxy;
m is an integer from 0 to 3; n is an integer from 1 to 3; p is an integer from 1 to 4; q is an integer from 0 to 2; r is an integer from 0 to 6; and s is an integer from 0 to 3; and the pharmaceutically acceptable salts thereof.
Preferably, the αvβ3 antagonist is a compound wherein Z is selected from
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000022_0002
and all other variables are as defined above; and the pharmaceutically acceptable salts thereof.
An illustration of the invention is the combination wherein the αvβ3 antagonist is a compound of the formula
Figure imgf000022_0003
wherein X is selected from
Figure imgf000022_0004
Y is selected from -(CH2)r- or -(CH2WNR3-; R3 is selected from hydrogen, -(CH2)p-aryl, Ci-5 alkoxycarbonyl, C3-8 cycloalkyl, arylaminocarbonyl, aryl Ci-5 alkylaminocarbonyl, Cl-8 alkyl, aryl Cl-6 alkyl, Cl-8 alkylsulfonyl, aryl sulfonyl, aryl Cl-6 alkylsulfonyl,
Cl-8 alkoxycarbonyl, aryloxycarbonyl, aryl Cl-8 alkoxycarbonyl,
Ci-8 alkylcarbonyl, arylcarbonyl, aryl Cl-6 alkylcarbonyl, Ci-8 alkylaminocarbonyl, Cl- alkylsulfonyl, or aryl Cl-6 alkylcarbonyl, wherein any of the alkyl groups may be unsubstituted or substituted with R13 and R14; r is an integer from 0 to 3; wherein all other variables are as defined above; and the pharmaceutically acceptable salts thereof. Exemplifying the invention is the combination wherein the αvβ3 antagonist is a compound of the formula
Figure imgf000023_0001
wherein Z is selected from
Figure imgf000024_0001
R^ is selected from hydrogen,
Figure imgf000024_0002
-(CH2)p indolyl,
-(CH2)s CjCH, -(CH2)s C≡C-Ci-6 alkyl, -(CH2)s C≡C-C3-7 cycloalkyl, -(CH2)s C≡C-aryl, -(CH2)s C≡C-Ci-6 alkyl aryl,
-(CH2)s CH=CH2, -(CH2)s CH=CH Cl-6 alkyl, -(CH2)s CH=CH-C3-7 cycloalkyl, -(CH2)s CH=CH aryl, -(CH2)s CH=CH Cl-6 alkyl aryl,
-(CH2)s SO2C1-6 alkyl, or -(CH2)s SO2C1-6 alkylaryl; Rl2 is selected from hydrogen or Cl-8 alkyl; s is an integer from 0 to 3; wherein all other variables are as defined above; and the pharmaceutically acceptable salts thereof.
An example of the invention is the combination wherein the αvβ3 antagonist is selected from 2-Oxo-3-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]piperidin-l-yl- acetyl-3(S)-pyridin-3-yl-β-alanine ethyl ester;
2-Oxo-3-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]piperin- l-yl-acetyl-3(S)-pyridin-3-yl-β-alanine trifluoroacetate;
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-alkynyl-β-alanine ethyl ester;
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-alkynyl-β-alanine;
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]-pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine ethyl ester;
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine;
2-Oxo-3(R)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-alkynyl-β-alanine ethyl ester;
2-Oxo-3(R)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-alkynyl-β-alanine;
2-Oxo-3(R)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]-pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine ethyl ester;
2-Oxo-3(R)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine;
Ethyl 2-oxo-3-[2-(5,6,7,8-tetrahydro[l,8]naphthyridin-2-yl)ethyl]- tetrahydropyrimidin-l-yl-acetyl-3(S)-pyridin-3-yl-β-alanine;
2-Oxo-3-[2-(5,6,7,8-tetrahydro[l,8]naphthyridin-2-yl}ethyl]- tetrahydropyrimidin- 1 -yl-acetyl- 3 ( S)-pyridin- 3 -yl- β-alanine ; Ethyl 2-oxo-3-[2-(5,6,7,8-tetrahydro[l,8]naphthyridin-2- yl)ethyl]imidazolidin-l-yl-acetyl-3(S)-pvridin-3-yl-β-alanine;
2-Oxo-3-[2-(5,6,7,8-tetrahydro[l,8]naphthyridin-2-yl}ethyl]-imidazolidin- l-yl-acetyl-3(S)-pyridin-3-yl-β-alanine;
Ethyl 2-oxo-3(R)-[2-(5,6,7,8-tetrahydro[l,8]naphthyridin-2- yl)ethyl]pyrrolidin-l-yl)acetyl-3(R)-(2-ethylindol-3-yl)-β-alanine;
2-Oxo-3(R)-[2-(5,6,7,8-tetrahydro[l,8]naphthyridin-2-yl)ethyl]pyrrolidin-l- yl)acetyl-3(R)-(2-ethylindol-3-yl)-β-alanine;
Ethyl 3-(S)-(2-{2-oxo-3-[(5,6,7,8-tetrahydro-[l,8]naphthyridin-2-ylmethyl)- amino]-pyrrolidin-l-yl}-acetylamino)-3-(R)-pyridin-3-yl-propionic acid;
3-(S)-(2-{2-Oxo-3-[(5,6,7,8-tetrahydro-[l,8]naphthyridin-2-ylmethyl)- amino]pyrrolidin-l-yl}-acetylamino)-3-(R)-pyridin-3-yl-propionic acid;_
3-{2-[6-Oxo-l-(5,6,7,8-tetrahydro-[l,8]naphthyridin-2-ylmethyl)- hexahydro-(3aS, 6aS)pyrrolo[3,4-b]pyrrol-5-yl]-acetylamino}-3-(S)-pyridin- 3-yl-propionic acid; or
3-{2-[6-Oxo-l-(5,6,7,8-tetrahydro-[l,8]naphthyridin-2-ylmethyl)- hexahydro-(3aR, 6aR)pyrrolo[3,4-b]pyrrol-5-yl]-acetylamino}-3-(S)- pyridin-3-yl-propionic acid; and the pharmaceutically acceptable salts thereof.
Preferably, the αvβ3 antagonist used in the combination is selected from
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]-pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine ethyl ester;
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine; or 4-[2-(l,2,3,4-Tetrahydro-l,8-naphthyridin-7-yl)ethyl]benzoyl- 2(S)-[l(S)10-camphorsulfonylamino] β-alanine; or a pharmaceutically acceptable salt thereof.
More particularly illustrating the invention is the combination wherein the growth hormone secretagogue is N-[1(R)-[(1,2- dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperidin]-l'- yl)carbonyl]-2-(phenylmethyloxy)-ethyl]-2-amino-2-methylpropanamide, and the αvβ3 antagonist is a compound of the formula
Figure imgf000027_0001
wherein X is selected from
rtf R2
Figure imgf000027_0002
Y is selected from -(CH2)r- or -(CH2 WNR3- R3 is selected from hydrogen,
-(CH2)p-aryl,
Cl-5 alkoxycarbonyl,
C3-8 cycloalkyl, arylamino carb onyl , aryl Cl-5 alkylaminocarbonyl,
Cl-8 alkyl, aryl Cl-6 alkyl, Cl-8 alkylsulfonyl, aryl sulfonyl, aryl Cl-6 alkylsulfonyl,
Cl-8 alkoxycarbonyl, aryloxycarbonyl, aryl Cl-8 alkoxycarbonyl, Ci-8 alkylcarbonyl, arylcarbonyl, aryl Cl-6 alkylcarbonyl, Cl-8 alkylaminocarbonyl,
Cl-6 alkylsulfonyl, or aryl Cl-6 alkylcarbonyl, wherein any of the alkyl groups may be unsubstituted or substituted with R13 and R14; and r is an integer from 0 to 3; wherein all other variables are as defined above; or a pharmaceutically acceptable salt thereof. Preferably, the growth hormone secretagogue is N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)- ethyl]-2-amino-2-methylpropanamide methanesulfonate.
Further exemplifying the invention is a pharmaceutical composition which comprises any of the combinations described above and a pharmaceutically acceptable carrier. Another example of the invention is a pharmaceutical composition made by combining an αvβ3 ligand, a growth hormone secretagogue and a pharmaceutically acceptable carrier. Further illustrating the invention is a process for making a pharmaceutical composition comprising combining an vβ3 antagonist, a growth hormone secretagogue and a pharmaceutically acceptable carrier. Additional illustrations of the invention are methods of treating or prevention a disease involving bone resorption which comprises administering to a patient in need of such treatment a therapeutically effective amount of any of the combinations or any of the pharmaceutical compositions described above. Preferably, the disease is osteoporosis.
Another example of the invention is the use of a growth hormone secretagogue and an αvβ3 antagonist for the manufacture of a medicament for the treatment or prevention of a disease involving bone resorption which comprises an effective amount of a growth hormone secretagogue and an effective amount of an αvβ3 antagonist, together or separately.
Another illustration of the invention is a product containing a growth hormone secretagogue medicament and an αvβ3 antagonist medicament as a combined preparation for simultaneous, separate or sequential use in osteoporosis.
DESCRIPTION OF THE INVENTION
The present invention is concerned with the combination of an αvβ3 antagonist compound and a growth hormone secretagogue for the treatment and the prevention of disturbances of calcium and phosphate metabolism, in particular, the treatment and prevention of diseases involving bone resorption, especially, osteoporosis, Paget's disease, malignant hypercalcemia, and metastatic bone disease. This particular combination produces unexpected results in the treatment and the prevention of such clinical disturbances. Thus, it is an object of the instant invention to describe the combination of the two drugs in the treatment and prevention of diseases involving bone resorption, especially, osteoporosis. In addition, it is an object of the instant invention to describe the preferred compounds from each type of compounds which are used in the instant combination. It is a still further object of this invention to describe compositions containing each of the compounds for use in the treatment of osteoporosis. Further objects will become apparent from a reading of the following description. The instant combination for the treatment and prevention of diseases involving bone resorption, especially osteoporosis in elderly patients, contains as a first element a growth hormone secretagogue.
Representative growth hormone secretagoues are disclosed in U.S. Patent No. 5,206,235 is as follows:
Figure imgf000030_0001
(CH2)Q
Figure imgf000030_0002
wherein the various substituents are as defined in U.S. Patent 5,206,235.
Preferred growth hormone secretagogues for use in the present invention identified therein include:
Figure imgf000030_0003
or
Figure imgf000030_0004
Representative growth hormone secretagoues are disclosed in U.S. Patent No. 5,283,241 and PCT Patent Publication No. 94/05634 as benzolactam compounds of the following structural formula:
Figure imgf000031_0001
wherein the various substituents are as defined in U.S. Patent 5,283,241 and PCT Patent Publication No. 94/05634.
Preferred growth hormone secretagogues for use in the present invention identified therein include:
2-amino-2-methyl-N-[2,3,4,5-tetrahydro-l-[[2'- [[[(methylamino)-carbonyl]amino]methyl][l,l'-biphenyl]-4-yl]methyl]-2- oxo-LfiT-benzazepin-3(R)-yl]propanamide and pharmaceutically acceptable salts thereof, in particular, the hydrochloride salt thereof. Additional representative growth hormone secretagoues are disclosed in U.S. Patent No. 5,536,716 as spiro compounds of the following structural Formulas I and II:
Figure imgf000032_0001
Formula Formula II
wherein the various substituents are as defined in U.S. Patent No. 5,536,716.
Preferred growth hormone secretagogues for use in the present invention include:
N-[l(R)-[(l,2-Dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2- methylpropanamide; and
N-[l(R)-[(l,2-Dihydro-l-methanesulfonylspiro[3H-indole-3,4*- piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2- methylpropanamide methanesulfonate.
Especially preferred growth hormone secretagogues for use in the present invention specifically include:
Figure imgf000033_0001
and pharmaceutically acceptable salts thereof; and
Figure imgf000033_0002
Additional representative growth hormone secretagoues are disclosed in U.S. Patent No. 5,492,916 as being compounds of the structural formula:
Figure imgf000033_0003
wherein the various substituents are as defined in U.S. Patent No. 5,492,916. Preferred growth hormone secretagogues for use in the present invention identified therein include l-[2(R)-(2-Amino-2- methylpropionylamino)-3-(lH-indol-3-yl)propionyl]-3(S)-benzyl- piperidine-3-carboxylic acid ethyl ester:
Figure imgf000034_0001
or a pharmaceutically acceptable salt thereof, especially the hydrochloride salt.
The preparation of growth hormone secretagogues is well known in the literature. Full descriptions of the preparation of the growth hormone secretagoues is found in e.g., U.S. Patent No. 3,239,345;
U.S. Patent No. 4,036,979; U.S. Patent No. 4,411,890; U.S. Patent No.
5,206,235; U.S. Patent No. 5,283,241; U.S. Patent No. 5,284,841; U.S.
Patent No. 5,310,737; U.S. Patent No. 5,317,017; U.S. Patent No. 5,374,721;
U.S. Patent No. 5,430,144; U.S. Patent No. 5,434,261; U.S. Patent No. 5,438,136; U.S. Patent No. 5,494,919; U.S. Patent No. 5,494,920; U.S.
Patent No. 5,492,916; U.S. Patent No. 5,536,716; EPO Patent Pub. No.
0,144,230; EPO Patent Pub. No. 0,513,974; PCT Patent Pub. No. WO
94/07486; PCT Patent Pub. No. WO 94/08583; PCT Patent Pub. No. WO
94/11012; PCT Patent Pub. No. WO 94/13696; PCT Patent Pub. No. WO 94/19367; PCT Patent Pub. No. WO 95/03289; PCT Patent Pub. No. WO
95/03290; PCT Patent Pub. No. WO 95/09633; PCT Patent Pub. No. WO
95/11029; PCT Patent Pub. No. WO 95/12598; PCT Patent Pub. No. WO
95/13069; PCT Patent Pub. No. WO 95/14666; PCT Patent Pub. No. WO
95/16675; PCT Patent Pub. No. WO 95/16692; PCT Patent Pub. No. WO 95/17422; PCT Patent Pub. No. WO 95/17423; PCT Patent Pub. No. WO
95/34311; PCT Patent Pub. No. WO 96/02530; Science. 260. 1640-1643 (June 11, 1993); Ann. Rep. Med. Chem.. 28, 177-186 (1993); Bioorg. Med. Chem. Ltrs.. 4(22), 2709-2714 (1994); and Proc. Natl. Acad. Sci. USA <£, 7001-7005 (July 1995).
Methods to obtain the growth hormone releasing peptides GHRP-6 and GHRP-1 are described in U.S. Patent Nos. 4,411,890 and PCT Patent Publications WO 89/07110, WO 89/07111, methods to obtain the growth hormone releasing peptide GHRP-2 are described in PCT Patent Publication WO 93/04081, and methods to obtain hexarelin are described in J. Endocrinol Invest.. 15(Suppl 4), 45 (1992). The identification of a compound as a "growth hormone secretagogue" and thus able to directly or indirectly stimulate or increase the endogenous release of growth hormone in an animal may be readily determined without undue experimentation by methodology well known in the art, such as the assay described by Smith, et al., Science, 260, 1640-1643 (1993) (see text of Figure 2 therein). In a typical experiment pituitary glands are aseptically removed from 150-200 g Wistar male rats and cultures of pituitary cells are prepared according to Cheng et al. Endocrinol, 124, 2791-2798 (1989). The cells are treated with the subject compound and assayed for growth hormone secreting activity and intracellular cAMP levels as described by Chang et al. In particular, the intrinsic growth horomone secretagogue activity of a compounds which may be used in the present invention may be determined by this assay.
In the instant combination for the treatment of osteoporosis, the second element is composed of an αvβ3 antagonist compound or a pharmaceutically acceptable salt thereof.
The production of αvβ33 antagonists is well known in the literature. Representative examples of various αvβ3 antagonist compounds and methods for the preparation may be found in the following: PCT Patent Pub. Nos. WO 95/32710; WO 96/00574; WO 96/00730; WO 96/26190; WO 96/37492; EPO Patent Publication Nos. EP 0,578,083; EP 0,711,770; EP 0,727,425; EP 0,546,548. The utility of a compound as an avb3 antagonist may be demonstrated by the methodology known in the art, such as the assays described in WO 95/32710, published 7 December 1995. Additional αvβ3 antagonist compounds and assays for identifying αvβ3 antagonist compounds are described in detail herein.
The preferred αvβ3 antagonist compounds for use in the combinations and methods of the instant invention are compounds of the formula
Figure imgf000036_0001
wherein X is a 9- to 10-membered polycyclic ring system, wherein one or more of the rings is aromatic, and wherein the polycyclic ring system contains 0, 1, 2, 3 or 4 heteroatoms selected from N, 0 or S, and wherein the polycyclic ring system is either unsubstituted or substituted on a carbon atom with Rl and R^;
Y is selected from
R' O
T' II O
-N- (CH2)— , _(CH2)-N- , -C-(CH2)^ , -C-N-(CH2);
R:
O
-N-C-(CH2)- -S(0)q-(CH2)^ , - 0-(CH2)- or - (CH2)r-
R
Z is a 5-11 membered aromatic or nonaromatic mono- or polycyclic ring system containing 0 to 6 double bonds, and containing 0 to 6 heteroatoms chosen from N, 0 and S, and wherein the ring system is either unsubstituted or substituted on a carbon or nitrogen atom with one or more groups independently selected from R4, R5, R6 and R^; provided that Z is not a 6-membered monocyclic aromatic ring system;
Rl, R2, R4; R5? R13 and R^4 are each independently selected from hydrogen, halogen, Ci-io alkyl, C3-8 cycloalkyl, aryl, aryl Ci-8 alkyl, amino, amino Ci-8 alkyl, Ci-3 acylamino, Ci-3 acylamino Cl-8 alkyl, Cι_6 alkylamino, Cl-6 alkylamino- Ci-8 alkyl, Ci-6 dialkylamino, Ci-6 dialkylamino Ci-8 alkyl, Ci-4 alkoxy, Cl-4 alkoxy Ci-6 alkyl, hydroxycarbonyl, hydroxycarbonyl Ci-6 alkyl, Ci-3 alkoxycarbonyl, Ci-3 alkoxycarbonyl Cl-6 alkyl, hydroxycarbonyl - Ci-6 alkyloxy, hydroxy, hydroxy Ci-6 alkyl, Ci-6 alk loxy- Ci-6 alkyl, nitro, cyano, trifluoromethyl, trifluoromethoxy, trifluoroethoxy, Ci-8 alkyl-S(0)q, Ci-8 aminocarbonyl,
Ci- dialkylaminocarbonyl, Ci-8 alkyl oxycarbonylamino, Cl-8 alkylaminocarbonyloxy or Ci-8 alkylsulfonylamino;
elected from hydrogen, aryl, -(CH2)p-aryl, hydroxyl,
Ci-5 alkoxycarbonyl, aminocarbonyl,
C3-8 cycloalkyl, amino Ci-6 alkyl, arylaminocarbonyl, aryl Cl-5 alkylaminocarbonyl, hydroxycarbonyl Ci-6 alkyl,
Ci-8 alkyl, aryl Ci-6 alkyl,
Cl-6 alkylamino Ci-6 alkyl, aryl Ci-6 alkylamino Ci-6 alkyl, Ci-6 dialkylamino Cl-6 alkyl,
Ci-8 alkylsulfonyl,
Cl-8 alkoxycarbonyl, aryl oxy carbonyl , aryl Ci-8 alkoxycarbonyl, Cl-8 alkylcarbonyl, arylcarbonyl, aryl Cl- alkylcarbonyl,
Cl-8 alkylaminocarbonyl, aminosulfonyl,
Cl-8 alkylaminosulfonyl, arylaminosulfonylamino, aryl Cl-8 alkylaminosulfonyl, Cl-6 alkylsulfonyl, aryl sulfonyl, aryl Cl-6 alkylsulfonyl, aryl Cl-6 alkylcarbonyl, Cl-6 alkylthiocarbonyl, arylthiocarbonyl, or aryl Cl-6 alkylthiocarbonyl, wherein any of the alkyl groups may be unsubstituted or substituted with R13 and R14;
R6> R"7, R8; R9? R10 an(j R11 are each independently selected from hydrogen, aryl, -(CH2)p-aryl, halogen, hydroxyl, Cl-8 alkylcarbonylamino, aryl Cl-5 alkoxy, Cl-5 alkoxycarbonyl, aminocarbonyl, Cl-8 alkylaminocarbonyl , Ci-6 alkylcarbonyloxy,
C3-8 cycloalkyl, oxo, amino, Cl- alkylamino, amino Ci-6 alkyl, arylaminocarb onyl , aryl Ci-5 alkylaminocarbonyl, aminocarbonyl, aminocarbonyl Ci-6 alkyl, hydroxycarbonyl, hydroxycarbonyl Ci-6 alkyl,
Ci-8 alkyl, either unsubstituted or substituted, with one or more groups selected from: halogen, hydroxyl, Cl-5 alkylcarbonylamino, aryl Ci-5 alkoxy,
Ci-5 alkoxycarbonyl, aminocarbonyl, Ci-5 alkylaminocarbonyl, Ci-5 alkylcarbonyloxy, C3-8 cycloalkyl, oxo, amino, Cl-3 alkylamino, amino Ci-3 alkyl, arylamino- carbonyl, aryl Ci-5 alkylaminocarbonyl, aminocarbonyl, aminocarbonyl Ci-4 alkyl, hydroxycarbonyl, or hydroxycarbonyl Cl-5 alkyl, -(CH2)s CiCH, -(CH2)s CjC-Ci-6 alkyl, -(CH2)s CIC-C3-7 cycloalkyl, -(CH2)s ClC-aryl,
-(CH2)s CjC-Ci-6 alkylaryl, -(CH2)s CH=CH2, -(CH2)s CH=CH Ci-6 alkyl, -(CH2)s CH=CH-C3_7 cycloalkyl, -(CH2)S CH=CH aryl,
-(CH2)s CH=CH Cl-6 alkylaryl, -(CH2)s SO2C1-6 alkyl, or -(CH2)s SO2C1-6 alkylaryl; Ci-6 alkoxy, aryl Ci-6 alkoxy, aryl Ci-6 alkyl, Ci-6 alkylamino Ci-6 alkyl, arylamino, arylamino Ci-6 alkyl, aryl Ci-6 alkylamino, aryl Ci- alkylamino Cl-6 alkyl, arylcarbonyloxy, aryl Ci-6 alkylcarbonyl oxy, Ci-6 dialkylamino,
Ci-6 dialkylamino Cl-6 alkyl, Ci-6 alkylaminocarbonyloxy, Ci-8 alkylsulfonylamino, Ci-8 alkylsulfonylamino Ci-6 alkyl, arylsulfonylamino Cι_6 alkyl, aryl Ci-6 alkylsulfonylamino, aryl Ci-6 alkylsulfonylamino Ci-6 alkyl, Ci-8 alkoxycarbonylamino, Ci-8 alkoxycarbonylamino Ci-8 alkyl, aryloxycarbonylamino Ci-8 alkyl, aryl Cι_8 alkoxycarbonylamino, aryl Ci-8 alkoxycarbonylamino Cl-8 alkyl, Ci-8 alkylcarbonylamino, Ci-8 alkylcarbonylamino Ci-6 alkyl, arylcarbonylamino Ci-6 alkyl, aryl Ci-6 alkylcarbonylamino, aryl Ci-6 alkylcarbonylamino Ci-6 alkyl, aminocarbonylamino Cl-6 alkyl, Ci-8 alkylaminocarbonylamino, Ci-8 alkylaminocarbonylamino Cl-6 alkyl, arylaminocarbonylamino Cl-6 alkyl, aryl Cl-8 alkylaminocarbonylamino, aryl Ci-8 alkylaminocarbonylamino Cl-6 alkyl, amino sulfonylamino Cl-6 alkyl, Ci-8 alkylaminosulfonylamino,
Ci-8 alkylaminosulfonylamino Ci-6 alkyl, arylamino sulfonylamino Ci-6 alkyl, aryl Ci-8 alkylaminosulfonylamino, aryl Ci-8 alkylaminosulfonylamino Cl-6 alkyl, Ci-6 alkylsulfonyl,
Ci-6 alkylsulfonyl Ci-6 alkyl, arylsulfonyl Ci-6 alkyl, aryl Ci-6 alkylsulfonyl, aryl Ci-6 alkylsulfonyl Ci-6 alkyl,
Ci-6 alkylcarbonyl,
Ci-6 alkylcarbonyl Ci-6 alkyl, arylcarbonyl Ci-6 alkyl, aryl Cι_6 alkylcarbonyl, aryl Cι_6 alkylcarbonyl Ci-6 alkyl,
Ci-6 alkylthiocarbonylamino,
Ci-6 alkylthiocarbonylamino Ci-6 alkyl, arylthiocarbonylamino Ci-6 alkyl, aryl Ci-6 alkylthiocarbonylamino, aryl Cl-6 alkylthiocarbonylamino Ci-6 alkyl,
Ci-8 alkylaminocarbonyl Ci-6 alkyl, arylaminocarbonyl Ci-6 alkyl, aryl Ci-8 alkylaminocarbonyl, or aryl Ci-8 alkylaminocarbonyl Ci-6 alkyl, wherein any of the alkyl groups may be unsubstituted or substituted with R13 and R14; and provided that the carbon atom to which R^ and R^ are attached is itself attached to no more than one heteroatom; and provided further that the carbon atom to which R^u and R^ are attached is itself attached to no more than one heteroatom;
Rl2 is selected from hydrogen,
Ci-8 alkyl, aryl, aryl Ci-8 alkyl, hydroxy, Ci-8 alkoxy, aryloxy, aryl Cι_6 alkoxy, Cl-8 alkylcarbonyloxy Ci-4 alkoxy, aryl Ci-8 alkylcarbonyloxy Ci-4 alkoxy, Ci-8 alkylaminocarbonylmethyleneoxy, or Ci-8 dialkylaminocarbonylmethyleneoxy ;
m is an integer from 0 to 3; n is an integer from 1 to 3; p is an integer from 1 to 4; q is an integer from 0 to 2; r is an integer from 0 to 6; and s is an integer from 0 to 3; and the pharmaceutically acceptable salts thereof. Preferably, Z is selected from
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000043_0002
and all other variables are as defined above.
More preferred αvβ3 antagonists for use in the present invention are compounds of the formula
Figure imgf000043_0003
wherein X is selected from
Figure imgf000043_0004
Y is selected from -(CH2)r- or -(CH2)m-NR3-; R3 is selected from hydrogen,
-(CH2)p-aryl,
Cl-5 alkoxycarbonyl,
C3-8 cycloalkyl, arylaminocarbonyl, aryl Cl-5 alkylaminocarbonyl,
Cl-8 alkyl, aryl Ci-6 alkyl, Ci-8 alkylsulfonyl, aryl sulfonyl, aryl Ci-6 alkylsulfonyl, Cl-8 alkoxycarbonyl, aryloxycarbonyl , aryl Ci-8 alkoxycarbonyl,
Ci-8 alkylcarbonyl, arylcarbonyl, aryl Ci-6 alkylcarbonyl,
Ci-8 alkylaminocarbonyl, Ci-6 alkylsulfonyl, or aryl Ci-6 alkylcarbonyl, wherein any of the alkyl groups may be unsubstituted or substituted with R13 and R14; and r is an integer from 0 to 3; where Z, R1, R2, R4, R6, R7, R8, R12, R13, R14 and s are as defined above; and the pharmaceutically acceptable salts thereof.
Desirably, the αvβ3 antagonist is a compound of the formula
Figure imgf000044_0001
wherein Z is selected from
Figure imgf000044_0002
R8 is selected from hydrogen,
Figure imgf000045_0001
-(CH2)p indolyl, -(CH2)s C≡CH,
-(CH2)s C≡C-Ci-6 alkyl,
-(CH2)s C≡C-C3-7 cycloalkyl,
-(CH2)s C≡C-aryl,
-(CH2)s C≡C-Ci-6 alkyl aryl, -(CH2)s CH=CH2,
-(CH2)s CH=CH Ci-6 alkyl,
-(CH2)s CH=CH-C3-7 cycloalkyl,
-(CH2)s CH=CH aryl,
-(CH2)s CH=CH Cl-6 alkyl aryl, -(CH2)s S02C 1-6 alkyl, or
-(CH2)s SO2C1-6 alkylaryl; Rl2 is selected from hydrogen or Cl-8 alkyl; s is an integer from 0 to 3; and R4 is as defined above; and the pharmaceutically acceptable salts thereof. Especially preferred αvβ3 antagonist compounds include:
4-[2-(l,2,3,4-Tetrahydro-l,8-naphthyridin-7-yl)ethyl]benzoyl- 2(S)-[l(S)10-camphorsulfonylamino] β-alanine (34-9), the synthesis of which is described in detail in WO 95/32710, published 7 December 1995:
Figure imgf000045_0002
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]-pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine ethyl ester:
Figure imgf000046_0001
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine:
Figure imgf000046_0002
or pharmaceutically acceptable salts thereof.
The instant combination of an αvβ3 antagonist and a growth hormone secretagogue are useful 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), osteogenesis imperfecta, 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.
Combined therapy to inhibit bone resorption, prevent osteoporosis and enhance the healing of bone fractures may be illustrated by the combination of this invention of αvβ3 antagonists and growth hormone secretagogues.
The combination of an αvβ3 antagonist provides an unexpected effect in the treatment and prevention of diseases involving bone resorption when used in combination with a growth hormone secretagogue. While not being bound to any particular theory of operation, that is, an enhanced effect at reducing and reversing the rate of bone loss that occurs during the aging process, the process known as osteoporosis, is observed with the combination of drugs than would be expected from either drug alone. In particular, combination therapy of a growth hormone secretagogue and an αvβ3 antagonist increase bone mass. This increase in bone mass is possibly a result of increased bone turnover or bone formation produced by elevated growth hormone/IGF- 1 levels resulting from the growth hormone secretagogue and decreased bone resorption produced by the αvβ3 antagonist. In mammals, bone formation and bone resorption generally respond to physiological stimuli and therapeutic intervention by changing in the same direction in a relationship referred to as "coupling". Treatment with an αvβ3 antagonist alone is known to decrease bone turnover by decreasing bone resorption with a concomitant ("coupled") reduction in bone formation surface. In accordance with the present invention, it is possible to promote a positive bone balance by uncoupling bone formation and bone resorption by using a combined therapeutic regime. Such uncoupling of bone formation and bone resorption would not have been predicted based on the disclosures in the art. 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 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 include the following:
Acetate, Benzenesulfonate, Benzoate, Bicarbonate, Bisulfate, Bitartrate, Borate, Bromide, Calcium, 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, Methyl sulfate, 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 may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts. The compounds of the present invention, may have chiral centers and occur as racemates, racemic mixtures and as individual diastereomers, or enantiomers with all isomeric forms being included in the present invention. Therefore, where a compound is chiral, the separate enantiomers, substantially free of the other, are included within the scope of the invention; further included are all mixtures of the two enantiomers. Also included within the scope of the invention are polymorphs and hydrates of the compounds of the instant invention.
The present invention includes within its scope prodrugs of the compounds of this invention. In general, such prodrugs will be functional derivatives of the compounds of this invention which are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term "administering" shall encompass the treatment of the various conditions described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs," ed. H. Bundgaard, Elsevier, 1985. Metabolites of these compounds include active species produced upon introduction of compounds of this invention into the biological milieu.
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.
The term "bone resorption," as used herein, refers to the process by which osteoclasts degrade bone.
In the above structural formulas and throughout the instant specification, the following terms have the indicated meanings: The alkyl groups specified above are intended to include those alkyl groups of the designated length in either a straight or branched configuration which may optionally contain double or triple bonds. Exemplary of such alkyl groups are methyl, ethyl, propyl, ethinyl, isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl, isohexyl, allyl, propenyl, butenyl, butadienyl and the like.
The alkoxy groups specified above are intended to include those alkoxy groups of the designated length in either a straight or branched configuration which may optionally contain double or triple bonds. Exemplary of such alkoxy groups are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy allyloxy, propinyloxy, isobutenyloxy, 2-hexenyloxy, and the like.
The term "halogen" is intended to include the halogen atom fluorine, chlorine, bromine and iodine.
The term "aryl" is intended to include phenyl and naphthyl and aromatic residues of 5- and 6- membered rings with 1 to 3 heteroatoms or fused 5 or 6 membered bicyclic rings with 1 to 3 heteroatoms of nitrogen, sulfur or oxygen. Examples of such heterocyclic aromatic rings are pyridine, thiophene, benzothiophene, tetrazole, indole, N-methylindole, dihydroindole, indazole, N-formylindole, benzimidazole, thiazole, furan, pyrimidine, and thiadiazole. Certain of the above defined terms may occur more than once in the above formula and upon such occurrence each term shall be defined independently of the other.
In the combination of the present invention the αvβ3 antagonist or the growth hormone secretagogue may be administered separately or in conjunction. In addition, the administration of one element may be prior to, concurrent to, or subsequent to the administration of the other agent.
The elements of the combination of the present invention may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual, or topical (e.g., ocular eyedrop) routes of administration and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.
The pharmaceutical compositions for the administration of the compounds of this invention may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition the active object compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases.
The pharmaceutical compositions containing the active ingredient suitable for oral administration may be in the form of discrete units such as hard or soft capsules, tablets, troches or lozenges, each containing a predetermined amount of the active ingredient; in the form of a dispersible powder or granules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid; in the form of syrups or elixirs; or in the form of an oil -in- water emulsion or a water- in-oil emulsion. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparation.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compounds are admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients may also be manufactured by known methods. The excipients used may be for example, (1) inert diluents such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintergrating agents such as corn starch, or alginic acid; (3) binding agents such as starch, gelatin or acacia; and (4) lubricating agents such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastroinestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl disearate may be employed. They may also be coated by the techniques described in the U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotic therapeutic tablets for controlled release.
In some cases, formulations for oral use may be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
Aqueous suspensions normally contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may be
1) suspending agents such as sodium carboxymethyl- cellulose, methylcellulose, hydroxypropylmethyl- cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; (2) dispersing or wetting agents which may be
(a) a naturally- occurring phosphatide such as lecithin,
(b) a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene sterate,
(c) a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethyleneoxycetanol,
(d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol such as polyoxyethylene sorbitol monooleate, or
(e) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, for example polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose or saccharin. Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents may be added to provide a palatable oral preparation. These compositions may be prepared by the addition of an antioxidant such as ascorbic acid.
Dispersible powders and granules are suitable for the preparation of an aqueous suspension. They provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, those sweetening, flavoring and coloring agents described above may also be present. The pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions. The oily phase may be a vegetable oil such as olive oil or arachis oils, or a mineral oil such as liquid paraffin or a mixture thereof. Suitable emulsifying agents may be (1) naturally-occurring gums such as gum acacia and gum tragacanth, (2) naturally-occurring phosphatides such as soy bean and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, (4) condensation products of said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension or solution. The suspension may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non- toxic paternterally- acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspension, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. The combination of this invention may also be administered in the form of suppositories for rectal administration. This composition can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene gylcols. Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
For topical administration the combination of this invention may be formulated in liquid or semi-liquid preparations such as liniments, lotions, applications; oil-in-water or water-in-oil emulsions such as creams, ointments, jellies or pastes, including tooth-pastes; or solutions or suspensions such as drops, and the like.
The pharmaceutical composition and method of the present invention may further comprise other therapeutically active compounds usually applied in the treatment of the above mentioned pathological conditions, for instance vitamin D2 and D3 and hydroxylated derivatives, e.g. la-hydroxy- vitamin D3, la-hydroxy- vitamin D2, la-25-dihydroxy- vitamin D3, la-25-dihydroxy-vitamin D2, dehydroepiandrosterone, calcitonin (human, porcine or salmon), mitramycin, sodium fluoride, estrogens, and non-steroid antiinflammatory drugs, such as acetylsalicyclic acid, indomethacin, naprosyn, and timegadine, and bisphosphonates. The use of bisphosphonates for utility in bone diseases has been reviewed, for example, by Hamdy, N.A.T., Role of Bisphosphonates in Metabolic Bone Diseases, Trends in Endocrinol. Metab.. 4, 19-25 (1993). Bisphosphonates useful for treating bone diseases, such as osteoporosis, include alendronate, tiludronate, dimethyl-APD, risedronate, etidronate, YM-175, clodronate, pamidronate, and BM-210995, a preferred bisphosphonate being alendronate, and especially alendronate sodium. A preferred bisphosphonate is alendronic acid (alendronate), or a pharmaceutically acceptable salt thereof. An especially preferred bisphosphonate is alendronate sodium, including alendronate sodium trihydrate. Alendronate sodium has received regulatory approval for marketing in the United States under the trademark FOSAMAX®.
The dosage of the active ingredients in the compositions of this invention may be varied. However, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained. The selected dosage depends upon the desired therapeutic effect, on the route of administration and on the duration of the treatment. Dosage ranges in the combination for the growth hormone secretogogue and avb3 antagonist are one tenth to one times the clinically effective ranges required to elevate growth hormone and reduce bone resorption respectively when the compounds are used singly. Generally, dosage levels of the αvβ3 antagonist compound of between about 0.001 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, 250 and 500 milligrams of each of the active ingredients 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 each of the active ingredients, preferably, from about 1 mg to about 100 mg of each of the active ingredients. 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. Dosage levels of the growth hormone secretatogogue of between about 0.001 to 50 mg kg of body weight daily, preferably about 0.005 to about 25 mg/kg per day, and more preferably about 0.01 to about 10 mg/kg per day are administered to a patient to obtain effective treatment or prevention of osteoporosis.
An especially preferred combination is that wherein the growth hormone secretagoguge is N-[l(R)-[(l,2-dihydro-l- methanesulfonylspiro[3H-indole-3,4'-piperidin]-l'-yl)carbonyl]-2- (phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide, in particular the methanesulfonate salt thereof, and the αvβ3 antagonist is 2-Oxo-3(S)- [2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin-l-yl)acetyl- 3(S)-pyridin-3-yl-β-alanine. In this especially preferred combination dosage levels of each component are as noted above, however, it is even more preferred that 2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2- yl)ethyl]pyrrolidin-l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine be administered at a dosage rate of about 0.01 to about 10 mg/kg/day, especially about 0.05 to about 5.0 mg/kg/day, and more particularly about 0.1 to about 5 mg/kg day, and that N-[l(R)-[(l,2-dihydro-l- methanesulfonylspiro[3H-indole-3,4'-piperidin]-l'-yl)carbonyl]-2- (phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate be administered at a dosage level of about 0.001 to about 20 mg/kg/day, especially about 0.005 to about 10 mg/kg/day, and more particularly about 0.01 to about 5 mg/kg/day.
The instant combination may also be administered on an intermittent basis. For the treatment or prophylaxis of diseases involving bone resorption a typical primary oral dose of αvβ3 antagonist which lies within the range of from about 0.001 mg to 100 mg per kg body weight and a dose of growth hormone secretatogoue of between 0.001 to 25 mg per kg of body weight may be administered and then, if necessary a sustaining dose of one element or both elements approximately equal to half of the primary dose may be administered at weekly, semiweekly, semimonthly, monthly, bimonthly, quarterly, semiannual, annual or biannual intervals. Similarly, the avb3 antagonist and the growth hormone secretagogue may be administered in a cyclical manner and it is not necessary that each component be administered concomitantly. The preferred compounds of this combination product are prepared by the references cited above and by the following schemes and examples.
The following schemes and examples are provided for the purpose of further illustration only and are not intended to be limitations on the spirit or scope of the present invention.
The following Schemes and Examples describe procedures for making representative αvβ3 antagonist compounds useful in the combinations and methods of the present invention. Moreover, by utilizing the procedures described in detail in PCT International Application Publication Nos. WO 95/32710, published 7 December 1995, and WO 95/17397, published 29 June 1995, in conjunction with the disclosure contained herein, one of ordinary skill in the art can readily prepare additional αvβ3 antagonist compounds useful in the combinations and methods of the present invention. More specifically, procedures for preparing the N-terminus of the αvβ3 antagonist compounds of the present invention are described in WO 95/32710. Additionally, for a general review describing the synthesis of β-alanines which can be utilized as the C-terminus of the αvβ3 antagonist compounds of the present invention, see Cole, D.C., Recent Stereoselective Synthetic Approaches to -Amino Acids, Tetrahedron, 1994, 50, 9517-9582; Juaristi, E, et al., Enantioselectiυe Synthesis of -Amino Acids, Aldrichemica Ada, 1994, 27, 3. In particular, synthesis of the 3 -methyl β-alanine is taught in Duggan, M.F. et al., J. Med. Chem., 1995, 38, 3332-3341; the 3-ethynyl β-alanine is taught in Zablocki, J.A., et al., J. Med. Chem., 1995, 38, 2378-2394; the 3- pyrid-3-yl β-alanine is taught in Rico, J.G. et al., J. Org. Chem., 1993, 58, 7948-7951; and the 2-amino and 2-toslylamino β-alanines are taught in Xue, C-B, et al., Biorg. Med. Chem. Letts., 1996, 6, 339-344.
SCHEME 1
EDA, THF, -78°C
Figure imgf000059_0001
1 -3
1 -4 L-proline, ethanol, reflux
Figure imgf000059_0002
Figure imgf000059_0003
NaN(TMS)2, DMF, ethyl bromoacetate
Figure imgf000059_0004
SCHEME 1 (CONT'D)
Figure imgf000060_0001
10% Pd/C, EtOAc, H2
Figure imgf000060_0002
1-10, R = Et
6NHCI
1-11, R = H 2-Oxo-3-(3-oxobutyl)piperidine (1-3)
A stirred solution of TMEDA (3.0 g, 20 mmol), 0.5 M LDA (6 mL, in THF), and THF (10 mL) at -78°C was treated with _\Λ (1.7 g, 10 mmol) (for preparation, see: JOC, 1990, 55, 3682) to effect an orange solution.
After 1 h, the iodide (2.4 g, 10 mmol) (J. Org. Chem., 1983, 48, 5381) was added to the orange solution and the resulting solution stirred for 2 h at -78°C, 3 h at -15°C and then 16 h at ambient temperature. The reaction mixture was concentrated and then treated with IN HCl (30 mL). The mixture was then basified with IN
NaOH/brine followed by extraction with EtOAc (3x). The combined extracts were dried (MgSθ4) and concentrated to give a yellow oil. Flash chromatography (silica, EtOAc 10% CH3θH/EtOAc) gave as a colorless solid. TLC Rf 0.42 (silica, 10% CH3θH/EtOAc)
!H NMR (300 MHz, CDCI3) δ 5.75 (bs, IH), 3.28 (m, 2H), 2.64 (t, 7Hz, 2H), 2.30-1.50 (m, 7H), 2.16 (s, 3H).
2-Oxo-3-r2-(ri.81-naphthyridin-2-yl)ethyllpineridine (l-5) A solution of ___3 (0.25 g, 1.5 mmol), L-proline (85 mg, 0.75 mmol), ______ (0.18 g, 1.5 mmol) (for preparation see: Synth. Commun.
1987, 17, 1695), and ethanol (10 mL) was refluxed for 24 hr. The cooled solution was concentrated and the residue purified by flash chromatography (silica, EtOAc JE 20% CH3θH/EtOAc) to give 1-5 as a solid.
TLC Rf = 0.32 (silica, 20% CH3θH/EtOAc)
!H NMR (300 MHz, CDCI3) δ 9.08 (m, IH), 8.16 (m, IH), 8.10 (d, J=8Hz,
IH), 7.50 (d, J=8Hz, IH), 7.45 (m, IH), 5.64 (bs, IH), 3.31 (m, 2H), 3.18 (m,
2H), 2.50-1.60 (m, 7H).
Ethyl 2-Oxo-3-[2-([l,8]-naphthyridin-2-yl)ethyl]piperidin-l-yl-acetate £ )
A solution of (0.28 g, 1.1 mmol) and DMF (10 mL) at - 15°C was treated with NaN(TMS)2 (1.2 mL, 1.2 mmol, 1M in hexanes) to give a red solution. After 30 min, the red solution was treated with ethyl bromoacetate (128 mL, 1.2 mmol), followed by continued stirring for 1 h. The reaction mixture was then quenched with sat. NH4CI and then extracted with EtOAc (3x). The combined extracts were washed with brine, dried (MgSθ4), and concentrated. Flash chromatography (silica,
10% CH3θH/EtOAc) gave _\__H as a yellow gum.
TLC Rf = 0.50 (silica, 10% CH3θH/EtOAc) iH NMR (300 MHz, CDCI3) δ 9.07 (m, IH), 8.16 (m, IH), 8.10 (d, J=8Hz,
IH), 7.50 (d, J=8Hz, IH), 7.44 (m, IH), 4.30-3.90 (m, 4H), 3.50-3.30 (m, 2H),
3.17 (m, 2H), 2.46 (m, 2H), 2.20-1.70 (m, 5H), 1.28 (t, J=7Hz, 3H).
Ethyl 2-Oxo-3-[2-(5,6,7,8-tetrahydro-[l,8]-naphthyridin-2- yl)ethvnpiperidin-l-yl-acetate (1-7)
A mixture of ±£ (102 mg, 0.3 mmol), 10% Pd/C (50 mg), and
EtOAc (25 mL) was stirred under a hydrogen atmosphere (1 atm) for 24 h. The catalyst was then removed by filtration through celite and the filtrate concentrated. Flash chromatography (silica, 20%
CH3θH/EtOAc) gave 1J_ as a yellow gum.
TLC Rf = 0.45 (silica, 30% CH3θH/EtOAc) iH NMR (300 MHz, CDCI3) δ 7.05 (d, J=6Hz, IH), 6.41 (d, J=6Hz, IH), 4.80 (bs, IH), 4.18 (q, J=7Hz, 2H), 4.08 (m, 2H), 3.37 (m, 4H), 2.80-1.60 (m,
13H), 1.26 (t, 7Hz, 3H).
2-Oxo-3-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]piperidin-l;yL acetic acid (1-8) A solution of (71 mg, 0.21 mmol) and 6N HCl (15 mL) was stirred at 55°C for 2h, followed by concentration to give 1-8 as a pale yellow gum. TLC Rf = 0.09 (silica, 20% CH3θH/EtOAc) 2-Oxo-3-[2-(5,6,7,8-tetrahydro-[l,8]-naphthyridin-2-yl)ethyl]piper- idin-l-yl-acetyl-3(S)-pyridin-3-yl-β-alanine ethyl ester (1-10)
A stirred mixture of 1^ (71 mg, 0.20 mmol), J 9 (59 mg, 0.22 mmol) (Zablocki et al, J. Med. Chem., 1995, 38, 2378), NMM (88 mL, 0.8 mmol), and CH3CN (25 mL) was treated with BOP (97 mg, 0.22 mmol).
After 24h, the reaction mixture was concentrated to dryness, dissolved in EtOAc, and then washed with H2O, dried (MgSθ4), and concentrated. Flash chromatography (silica, 10% (NH3/EtOH EtOAc) gave VΛQ as a colorless gum. TLC Rf = 0.9 (silica, 10% (NH3/EtOH)/EtOAc) iH NMR (300 MHz, CD3OD) δ 8.55 (m, IH), 8.43 (m, IH), 7.83 (m, IH), 7.40 (m, IH), 7.11 (m, IH), 6.37 (m, IH), 5.38 (m, IH), 4.08 (q, J=7Hz, 2H), 4.00 (m, 2H), 3.37 (m, 4H), 2.90 (m, IH), 2.70-1.60 (m, 14H), 1.14 (t, J=7Hz, 3H).
2-Oxo-3-[2-(5,6,7,8-tetrahydro-[l,8]-naphthyridin-2-yl)ethyl]piperin- l-yl-acetyl-3(S)-pyridin-3-yl-β-alanine trifluoro acetate (1-11)
A stirred solution of 1J (52 mg, 0.10 mmol) and 6N HCl (10 mL) was heated at 55°C for 2 h, followed by concentration. Preparative HPLC (VYDAC Ci8 semiprep column, gradient elution: [95:5 (0.1% TFA/H2θ/0.1% TFA/CH3CN) to 50:50 (0.1% TFA H2θ/0.1% TFA/CH3CN) 80 min] gave _\A1 as a colorless solid. !H NMR (300 MHz, CD3OD) δ 8.90 (s, IH), 8.74 (d, J=5Hz, IH), 8.61 (d, J=8Hz, IH), 8.03 (m, IH), 7.56 (d, J=7Hz, IH), 6.59 (d, J=7Hz, IH), 5.43 (m, IH), 4.03 (m, 2H), 3.40 (m, 5H), 3.00 (m, 2H), 2.78 (m, 4H), 2.40-1.60 (m, 12H).
SCHEME 2
Figure imgf000064_0001
2-3
Figure imgf000064_0002
pTSA, acetone, reflux
Figure imgf000064_0003
2-5 SCHEME 2 (CONT'D)
Figure imgf000065_0001
2-5
1-4 proline, ethanol, reflux
Figure imgf000065_0002
Figure imgf000065_0003
10% Pd/C, Ho, ethanol
Figure imgf000065_0004
6N HCl, 50°C
Figure imgf000065_0005
2-8 SCHEME 2 (CONT'D)
Figure imgf000066_0001
2-8
R
,CO Et BOP, NMM,
HOH2N' CH3CN 2-9, R= -C≡CH 2-10, R= 3-pyridyl
Figure imgf000066_0002
2-11, R = -C≡CH 2-12, R= 3-pyridyl
1N NaOH, ethanol
Figure imgf000066_0003
2-13, R = -C≡CH 2-14, R= 3-pyridyl (2-Oxo-3-(3-(ethylendioxy)butyl)pyrrolidin-l-yl)benzyl (2-2)
To a stirred solution of 2Λ (5.3 g, 30 mmol) and THF (100 mL) at -78°C was added LDA (17.5 mL, 35 mmol, 2.0 M in hexanes) dropwise over a 10 minute period. After 30 min, 1-2 (5.0 g, 21 mmol) was added followed by removal of the cooling bath. After 1 h, the reaction was quenched with AcOH (10 mL) and then diluted with EtOAc, washed with sat. NaHC03 and brine, dried (MgSθ4) and concentrated. Flash chromatography (silica, 25% JE 75% EtOAc/hexanes) gave 2__2 as an oil. TLC Rf = 0.38 (silica, EtOAc) !H NMR (300 MHz, CDCI3) δ 7.25 (m, 5H), 4.48 (d, J=15Hz, IH), 4.40 (d, J=15Hz, IH), 3.94 (s, 4H), 3.18 (m, 2H), 2.44 (m, IH), 2.30-1.30 (m, 9H).
2-Oxo-3-(3-(ethylendioxy)butyl)pyrrolidine (2-3)
To a stirred solution of 2 (4.1 g, 14.2 mmol) in THF (100 mL) at -78°C was added a solution of Li 4,4'-di-tert-butylbiphenyl (188 mL, 0.5 M in THF) in 4 portions. After 1 h, the reaction was quenched with AcOH (25 mL). The resulting mixture was diluted with EtOAc and then washed with H2O, sat. NaHCθ3, and brine, dried (MgSθ4) and concentrated. Flash chromatography (silica, EtOAc JE 10% CH3θH/EtOAc) gave as a yellow oil. TLC Rf = 0.1 (silica, EtOAc)
!H NMR (300 MHz, CDCI3) δ 6.23 (bs, IH), 3.94 (s, 4H), 3.30 (m, 2H), 2.70 (m, 2H), 2.10-1.30 (m, 9H).
Ethyl (2-Oxo-3-(3-(ethylendioxy)butyl)pyrrolidin- l-yl)acetate (2-4)
To a rapidly stirred solution of 2^ (0.86 g, 4.3 mmol) and THF (25 mL) at -78°C was added NaN(TMS)2 (5.2 mL, 5.2 mmol,
1.0 M in THF). After 20 min, ethyl bromoacetate (0.58 mL, 5.2 mmol) was added followed by removal of the cooling bath. After 1 h, the reaction mixture was diluted with EtOAc and then washed with H2O, sat. NaHC03 and brine, dried (MgSθ4), and concentrated to give 2Λ as a yellow oil.
TLC Rf = 0.53 (silica, EtOAc) !H NMR (300 MHz, CDCI3) δ 4.18 (q, J=7Hz, 2H), 4.04 (m, 2H), 3.93 (s, 4H), 3.39 (m, 2H), 2.44 (m, IH), 2.23 (m, IH), 2.00-1.30 (m, 9H), 1.25 (t, J=7H, 3H).
Ethyl (2-Oxo-3-(3-oxobutyl)pyrrolidin-l-yl)acetate (2-5)
A solution of ______ (1.1 g, 3.9 mmol), p-TSA (5 mg) and acetone
(50 mL) was heated at reflux for 1 hr. The cooled reaction mixture was diluted with EtOAc and then washed with sat. NaHCθ3 and brine, dried (MgSθ4), and concentration to afford 2r5 as a yellow oil. TLC Rf = 0.48 (silica, EtOAc)
!H NMR (300 MHz, CDCI3) δ 4.18 (q, J=7Hz, 2H), 4.01 (s, 2H), 3.40 (m, 2H), 2.67 (t, J=7Hz, 2H), 2.48 (m, IH), 2.30-1.60 (m, 4H), 2.15 (s, 3H), 1.25 (t, J=7Hz, 3H).
Ethyl (2-Oxo-3-(2-([l,8]naphthyridin-2-yl)ethyl)pyrrolidin-l-yl)- acetate (2-6)
A mixture of (0.77 g, 3.0 mmol), _\Λ (0.55 g, 4.5 mmol, for preparation see Ret, 1993, 36, 2513), L-proline (0.17 g, 1.5 mmol) and ethanol (25 mL) was heated at reflux for 20 hr. The cooled reaction mixture was concentrated and the residue purified by flash chromatography (silica, EtOAc JE 5% CH3θH/EtOAc) to give 2-6 as a yellow oil.
TLC Rf = 0.13 (silica, 10% CH3θH/EtOAc)
!H NMR (300 MHz, CDCI3) δ 9.08 (m, IH), 8.17 (m, IH), 8.12 (d, J=8Hz, IH), 7.49 (d, J=8Hz, IH), 7.46 (m, IH), 4.15 (q, J=7Hz, 2H), 4.04 (m, 2H), 3.42 (m, 2H), 3.21 (t, J=8Hz, 2H), 2.60-1.80 (m, 5H), 1.25 (t, J=7Hz, 3H).
Ethyl (2-Oxo-3-(2-(5,6,7,8-tetrahydro-[l,8]-naphthyridin-2- vPethvPpyrrolidin- l-yl)acetate (2-7) A mixture of 2___\ (0.87 g, 2.6 mmol), 10% Pd/C (0.5 g), and
CH3OH (25 mL) was stirred under a hydrogen atmosphere (1 atm) for 2 hr. The catalyst was then removed by filtration through a celite pad followed by concentration of the filtrate. Flash chromatogrphy (silica, EtOAc JE 5% CH3θH/EtOAc) gave 2J_ as a yellow oil. TLC Rf = 0.18 (silica, 5% CH3θH/EtOAc) iH NMR (300 MHz, CDCI3) δ 7.05 (d, J=7Hz, IH), 6.40 (d, J=7Hz, IH), 4.83 (bs, IH), 4.17 (q, J=7Hz, 2H), 4.03 (m, 2H), 3.40 (m, 4H), 2.80-1.60 (m, 11H), 1.27 (t, J=7Hz, 3H).
(2-Oxo-3-(2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl)pyrro- lidin-l-yl)acetic acid hydrochloride (2-8)
A stirred mixture of (0.45 g, 1.4 mmol) and 6N HCl (10 mL) was heated at 50°C for 1 h, followed by concentration to give _____ as a yellow oil.
!H NMR (300 MHz, CD3OD) δ 7.60 (d, J=7Hz, IH), 6.66 (d, J=7Hz, IH),
4.05 (s, 2H), 3.50 (m, 4H), 2.83 (m, 4H), 2.54 (m, IH), 2.32 (m, IH), 2.10 (m, IH), 2.00-1.75 (m, 4H).
(2-Oxo-3-(2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl)pyrro-lidinzl; yl)acetyl-3(S)-ethynyl-β-alanine ethyl ester (2-11)
To a stirred solution of (50 mg, 0.15 mmol), 2S. (29 mg, 0.17 mmol) (Zablocki et al., J. Med Chem., 1995, 38, 2378), NMM (83 mL, 0.75 mmol), and CH3CN (1 mL) was added BOP (74 mg, 0.17 mmol). After 20 h, the reaction mixture was diluted with EtOAc and then washed with sat. NaHCθ3, H2O and brine, dried (MgSθ4), and concentrated to give 2-11 as a yellow oil. TLC Rf = 0.24 (silica, 10% CH3θH/EtOAc).
(2-Oxo-3-(2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl)pyrro-lidinJ^ yl)acetyl-3(S)-pyridin-3-yl-β-alanine ethyl ester (2-12)
To a stirred solution of 2_S (50 mg, 0.15 mmol), 2JJ3 (44 mg, 0.17 mmol) (Rico et al., J. Org. Chem., 1993, 58, 7948), NMM (83 mL, 0.75 mmol), and CH3CN (1 mL) was added BOP (74 mg, 0.17 mmol). After 20 h, the reaction mixture was diluted with EtOAc and then washed with sat. NaHCθ3, H2O and brine, dried (MgSθ4), and concentrated to give 2r
12 as a brown oil.
TLC Rf = 0.24 (silica, 20% CH3θH/EtOAc). (2-Oxo-3-(2-(5.6.7.8-tetrahvdron.81-naphthvridin-2-yl)ethyl)pyrrolidin-l- yl)acetyl-3(S)-ethvnyl-β-alanine (2-13)
A mixture of 2 (0.1 g, 0.15 mmol), IN NaOH (300 mL, and ethanol (1 mL) was stirred at ambient temperature for 1 hr. Concentration and then flash chromagraphy (silica, 25:10:1:1 JE 15:10:1:1 EtOAc EtOH/NH4θH/H2θ) gave 213 as a white solid. TLC Rf = 0.18 (silica, 10:10:1:1 EtOAc/EtOH/NH4θH/H2θ) iH NMR (300 MHz, CD3OD) δ 7.45 (m, IH), 6.50 (m, IH), 4.53 (m, IH), 3.80-3.30 (m, 5H), 3.05 (m, IH), 2.80-2.15 (m, 9H), 2.00-1.75 (m, 4H).
(2-Oxo-3-(2-(5,6,7,8-tetrahydro[l,8]-naphth ridin-2-yl)ethyl)pyrro-HdinJiI yl)acetyl-3(S)-pyridin-3-yl-β-alanine (2-14)
A mixture of 2 (0.1 g, 0.15 mmol), IN NaOH (300 mL) and ethanol (1 mL) was stirred at ambient temperature for 1 hr. Concentration and the flash chromatography (silica, 25:10:1:1 JE 15:10:1:1 EtOAc/EtOH/NH4θH/EΪ2θ) gave 244 as a white solid. TLC Rf = 0.10 (silica, 10:10:1:1 EtOAc/EtOH/NH4θH/H2θ) !H NMR (300 MHz, CD3OD) δ 8.57 (m, IH), 8.40 (m, IH), 7.86 (m, IH), 7.40 (m, 2H), 6.50 (m, IH), 5.28 (m, IH), 4.65-4.40 (m, IH), 3.90-1.80 (M, 19H).
SCHEME 3
Figure imgf000071_0001
Figure imgf000071_0002
SCHEME 3 (CONT'D)
Figure imgf000072_0001
SCHEME 3 (CONT'D)
Figure imgf000073_0001
EDC, HOBT NMM
Figure imgf000073_0002
4-(Propyl-2-ene)butyric acid (3-2)
To a stirred suspension of methyltriphenylphosphonium bromide (67.7 g, 190 mmol) in 1 L THF at 0°C was added a solution of sodium bis(trimethylsilyl)amide (190 mL, 190 mmol, IM THF). After an additional 30 minutes, 31, ethyl 4-acetylbutyrate (Aldrich Chemical Co.)(25.0 g, 158 mmol) was added, and the mixture stirred for 18 h. The mixture was filtered, and the filtrate concentrated. The residue was triturated with hexanes, and then filtered. Following evaporative removal of the solvent, the residue was chromatographed on silica gel, eluting with 10% ethyl acetate/hexanes to give the olefin as a colorless oil. TLC Rf = 0.52 (10% ethyl acetate/hexanes). !H NMR (300 MHz, CHCI3) δ 4.71 (d, 2H, J=13 Hz), 4.13 (q, 2H, J=7 Hz), 2.29 (t, 2H, J=7 Hz), 2.05 (t, 2H, J= 8 Hz), 1.77 (m, 2H), 1.72 (s, 3H), 1.26 (t, 3H, J=7Hz).
A solution of the above olefin (15.4 g, 98.6 mmol), 1 N NaOH (150 mL), and EtOH (300 mL) was stirred at ambient temperature for 2 h. Following acidification with 1 N HCl, the mixture was extracted with ether. The ether layer was washed with brine, dried over magnesium sulfate, and concentrated to give 3^ as a colorless oil. XH NMR (300 MHz, CHCI3) δ 4.70 (d, 2H, J=13 Hz), 2.27 (t, 2H, J=7 Hz), 2.06 (t, 2H, J= 7
Hz), 1.72 (m, 5H).
(4-(Propyl-2-ene)butanoyl)-4(R)-benzyl-2-oxazolidinone (3-3)
To a solution of (6.0 g , 46.8 mmol) in THF (200 ml) at - 78°C was added triethylamine (7.19 mL, 51.5 mmol) followed by pivaloyl chloride (6.35 mL, 51.5 mmol). The mixture was warmed to 0°C for 1 h, then recooled to -78°C. In a separate flask, of (R)-(+)-4-benzyl-2- oxazolidinone (9.15 g, 51.5 mmol) was dissolved in THF (100 mL), cooled to -78°C, and n-BuLi (32.3 mL, 51.5 mmol; 1.6 M hexanes) was added dropwise. After 10 minutes, the lithium oxazolidinone was added to the pivalic anhydride. After 10 minutes, the mixture was warmed to 0°C for 1.5 h. The mixture was then poured into ethyl acetate, washed with aqueous sodium bicarbonate, and dried over magnesium sulfate. Following evaporative removal of the solvent, the residue was chromatographed (silica gel, dichloromethane) to give ϊ___3 as a slightly yellow oil. TLC Rf = 0.8 (CH2Cl2). iH NMR (300 MHz, CHCI3) δ 7.40-7.18 (m, 5H), 4.80-4.60 (m, 3H), 4.18 (m,
2H), 3.30 (dd, IH, J=3.2, 13.2 Hz), 2.95 (m, 2H), 2.76 (dd, IH, J=9.5, 13.1 Hz), 2.11 (t, 2H, J=7.5 Hz), 1.87 (m, 2H), 1.74 (s, 3H). 2-Chloroethyltriflate (3-4)
To a solution of 1.67 mL (24.8 mmol) of 2-chloroethanol and 3.47 mL (29.8 mmol) of 2,6-lutidine in 20 mL of dichloromethane at 0°C was added 4.59 mL (27.3 mmol) of triflic anhydride. After 1 h, the mixture was diluted with hexanes, washed with ice-cold IN HCl, and dried over sodium sulfate. The solvents were evaporated to give 3-4 as a pink oil. iH NMR (300 MHz, CHCI3) δ 4.69 (t, 2H, J=5.3 Hz), 3.78 (t, 2H, J=5.6 Hz).
2(S)-Chloroethvl-4-(propvl-2-ene)butanovl-(4(R)-benzvl-2-oxazolidinone)
(3-5)
To a solution of (11.0 g, 38.3 mmol) in THF (60 mL) at -78° C was added a solution of sodium bis(trimethylsilyl)amide (42.1 mL, 42.1 mmol; 1M/THF). After 20 min, 34 (16.2 ml, 115 mmol) was added over 5 min, and the resulting mixture stirred for 1.5 h at -78°C, then 2 h at -15°C. The mixture was diluted with hexanes, washed with sat. ammonium chloride, and dried over sodium sulfate. Following evaporative removal of the solvent, the residue was chromatographed (silica gel, 14% ethyl acetate/hexanes) to give 3 5 as a colorless oil. TLC Rf = 0.5 (20% ethyl acetate/hexanes). iH NMR (300 MHz, CHCI3) δ 7.30-7.18 (m, 5H), 4.67 (m, 3H), 4.19 (m, 2H),
3.99 (m, IH), 3.58 (m, 2H), 3.33 (dd, IH, J=3.2, 12.0 Hz), 2.75 (dd, IH, J=9.7, 13.5 Hz), 2.23 (m, IH), 2.18-1.82 (m, 4H), 1.77-1.60 (m, IH), 1.71 (s, 3H).
Ethyl 2-oxo-3(S)-(3-methylenebutyl)pyrrolidine (3-6)
A mixture of (8.15 g, 23.3 mmol) and NaN3 (4.54 g, 69.8 mmol) in DMSO (120 mL) was heated at 75°C for 2 h. After cooling, the mixture was diluted with ether and hexanes, washed with water, and dried over sodium sulfate. Evaporative removal of the solvent gave the azide as a colorless oil. TLC Rf = 0.5 (20% ethyl acetate/hexanes). !H NMR (300 MHz, CHCI3) δ 7.30-7.22 (m, 5H), 4.69 (m, 3H), 4.17 (d, 2H,
J=5.1 Hz), 3.89 (m, IH), 3.38 (m, 3H), 2.74 (m, IH), 2.13-1.63 (m, 6H), 1.71 (s, 3H). To a solution of this azide (8.0 g , 22.4 mmol) in THF (250 mL) and water (40 mL) was added triphenylphosphine (8.24 g, 31.4 mmol) in 4 portions over 5 minutes. This mixture was heated at reflux for 2 h, cooled, and evaporated. The residue was chromatographed (silica gel, 10% chloroform/ethyl acetate) to give 3 . as a colorless oil. TLC Rf = 0.40 (20% chloroform/ethyl acetate). !H NMR (300 MHz, CHCI3) δ 6.47 (br s, IH), 4.73 (m, 2H), 3.31 (m, 2H),
2.33 (m, 2H), 2.08 (m, 3H), 1.81 (m, IH), 1.74 (s, 3H), 1.44 (s, IH).
Ethyl 2-oxo-3(S)-(3-methylenebutyl)pyrrolidin-l-yl)acetate (3-7)
To a solution of (2.50 g, 16.3 mmol) in THF (40 mL) at - 78°C was added sodium bis(trimethylsilyl)amide (17.1 mL, 17.1 mmol; IM THF) dropwise. After an additional 20 min, ethyl bromoacetate (2.17 mL, 19.6 mmol) was added dropwise over 3 min. After an additional 20 min, 20 mL sat. aqueous NH4CI was added, and the cooling bath removed. The layers were separated, the aqueous layer washed with ether, and the combined organic extracts were dried over sodium sulfate. Following evaporative removal of the solvent, the residue was chromatographed (silica gel, 40% ethyl acetate/hexanes) to give 3 7 as a colorless oil.
TLC Rf = 0.85 (50% chloroform/ethyl acetate).
!H NMR (300 MHz, CHCI3) δ 4.73 (m, 2H), 4.18 (q, 2H, J=7.1Hz), 4.06 (dd,
2H, J=17.6, 20.8 Hz), 3.42 (m, 2H), 2.44 (m, IH), 2.27 (m, IH), 2.12 (m, 3H), 1.75 (m, IH), 1.74 (s, 3H), 1.50 (m, IH), 1.28 (t, 3H, J=7.3 Hz).
Ethyl 2-oxo-3(S)-(3-oxo-butyl)pyrrolidin-l-yl)acetate (3-8)
To a solution of 3 7 (3.35 g,14.0 mmol) and N- methylmorpholine-N-oxide (3.27 g, 28.0 mmol) in THF (10 mL) and water (1 mL) was added Osθ4 (5.7 mL, 0.56 mmol; 2.5% t-butanol). After 1 h, NaI04 (5.99 g, 28 mmol) in warm water (30 mL) was added over 2 min, and the resulting mixture stirred for 1 h. Water was then added, and the aqueous layer washed with ether and ethyl acetate, and the combined organic extracts were dried over sodium sulfate. Evaporative removal of the solvent gave ___{_[ as a dark oil containing residual Osθ4. TLC Rf = 0.78 (70:20:10 chloroform ethyl acetate/MeOH). !H NMR (300 MHz, CHCI3) δ 4.19 (m, 2H, J=7.2 Hz), 4.03 (s, 2H), 3.41 (m, 2H), 2.68 (t, 2H, J=9.4 Hz) 2.45 (m, IH), 2.27 (m, IH), 2.17 (s, 3H), 1.97 (m, IH), 1.78 (m, 2H), 1.28 (t, 3H, J=7.2 Hz).
Ethyl 2-oxo-3(S)-[2-([l,8]-naphthyridin-2-yl)ethyl]pyrrolidin-l-yl}acetate
(3-9)
A mixture of (3.25 g, 13.5 mmol), Λ, 2-amino-3- formylpyridine (2.2 g, 18.2 mmol; for preparation see Synth. Commun.
1987, 17, 1695) and proline (0.62 g, 5.39 mmol) in absolute ethanol (45 mL) was heated at reflux for 15 h. Following evaporative removal of the solvent, the residue was chromatographed (silica gel, 70:25:5 chloroform/ethyl acetate/MeOH to give 3^9 as a colorless oil.
TLC Rf = 0.24 (70:25:5 chloroform/ethyl acetate/MeOH).
!H NMR (300 MHz, CHCI3) δ 9.08 (m, IH), 8.16 (m, 2H), 7.47 (m, 2H), 4.17 (m, 4H), 3.42 (m, 2H), 3.21 (t, 2H, J=6.0 Hz), 2.56 (m, IH), 2.39 (m, 2H), 2.08 (m, IH), 1.87 (m, IH), 1.27 (t, 3H, J=7.1 Hz).
Ethyl 2-oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2- vDethyllpyrrolidin-l-vDacetate (3-10) A mixture of 3_ (3.33 g, 10.2 mmol) and 10% Pd/carbon (1.5 g) in EtOH (50 mL) was stirred under a balloon of hydrogen for 13 h.
Following filtration and evaporative removal of the solvent, the residue was chromatographed (silica gel, 70:20:10 chloroform ethyl acetate/MeOH to give 3-10 as a colorless oil. TLC Rf = 0.20 (70:20: 10 chloroform/ethyl acetate/MeOH).
!H NMR (300 MHz, CHCI3) δ 7.05 (d, IH, J=7.3 Hz), 6.38 (d, IH, J=7.3
Hz), 4.88 (br s, IH), 4.17 (dd, 2H, J=7.0, 14.4 Hz), 4.04 (dd, 2H, J=17.6, 27.3 Hz), 3.40 (m, 4H), 2.69 (m, 4H), 2.51 (m, IH), 2.28 (m, 2H), 1.90 (m, 2H), 1.78 (m, 2H), 1.27 (t, 3H, J=6.9 Hz).
2-Oxo-3(S)-r2-(5.6.7.8-tetrahvdrori.81-naphthvridin-2-yl)ethyllpyrrolidin- l-yl)acetic acid (3-11) A mixture of O (0.60 g, 1.81 mmol) and 6N HCl (25 mL) was heated at 60°C for 1 h. Evaporative removal of the solvent gave 3-11 as a yellow oil. !H NMR (300 MHz, DMSO-dβ) δ 8.4 (br s, IH), 7.60 (d, IH, J=7.3 Hz), 6.63 (d, IH, J=7.3 Hz), 3.92 (dd, 2H, J=17.6, 25.9 Hz), 3.43 (m, 2H), 3.35 (m, 2H), 2.74 (m, 4H), 2.28 (m, 2H), 2.03 (m, IH), 1.82 (m, 2H), 1.67 (m, 2H).
2-Oxo-3(S)-r2-(5.6.7.8-tetrahvdrori.81-naphthvridin-2-yl)ethvnpyrrolidin- l-yl)acetyl-3(S)-alkvnyl-β-alanine ethyl ester (3-12) A mixture of 311 (0.20 g, 0.588 mmol), 9 (0.157 g, 0.882 mmol), EDC (0.147 g, 0.765 mmol), HOBT (0.095 g, 0.706 mmol) and NMM (0.453 mL, 4.12 mmol) in CH3CN (3 mL) and DMF (2 mL) was stirred for 20 h. The mixture was diluted with ethyl acetate, washed with water, brine, and dried over sodium sulfate. Following evaporative removal of the solvent, the residue was chromatographed (silica gel, 70:20:10 chloroform/ethyl acetate/MeOH to give 3-12 as a colorless foam. TLC Rf = 0.44 (70:20:10 chloroform/ethyl acetate/MeOH). !H NMR (300 MHz, CHCI3) δ 7.06 (d, IH, J=7.3 Hz), 6.39 (d, IH, J=7.3
Hz), 5.07 (m, IH), 4.94 (br s, IH), 4.18 (q, 2H, J=6.1 Hz), 3.95 (q, 2H, J=16.1 Hz), 3.39 (m, 4H), 2.90 (s, IH), 2.68 (m, 6H), 2.50 (m, IH), 2.27 (m, 3H), 1.82 (m, 4H), 1.27 (t, 3H, J=7.1 Hz).
2-Oxo-3(S)-r2-(5.6.7.8-tetrahvdrori.81-naphthvridin-2-yl)ethvnpyrrolidin- l-yl)acetyl-3(S)-alkvnyl-β-alanine (3-13) To a solution of 2 (0.050 g, 0.117 mmol) in EtOH (1 mL) was added IN NaOH (0.164 ml, 0.164 mmol). After stirring for 2 h, the solvents were evaporated and the residue was chromatographed (silica gel, 25:10:1:1 ethyl acetate/EtOH/water/NH40H to give 3 as a colorless foam. TLC Rf = 0.26 (25:10:1:1 ethyl acetate/EtOH/water/NH40H).
!H NMR (300 MHz, DMSO-de) δ 7.75 (br s, IH), 7.14 (d, IH, J=7.3 Hz), 6.31 (d, IH, J=7.3 Hz), 4.74 (m, IH), 3.90 (d, IH, J=16.6 Hz), 3.67 (d, IH, J=16.6 Hz), 3.23 (m, 4H), 2.57 (m, 7H), 2.30 (m, IH), 2.11 (m, 2H), 1.73 (m, 2H), 1.59 (m, 2H). 2-Oxo-3(S)-r2-(5.6.7.8-tetrahvdrori.81-naphthvridin-2-vl)ethvn-pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine ethyl ester (3-14)
A mixture of 311 (0.30 g, 0.882 mmol), O (0.354 g, 1.32 mmol) , EDC (0.220 g (1.15 mmol), HOBT (0.143 g, 1.05 mmol) and NMM (0.680 mL (6.18 mmol) in CH3CN (5 mL) and DMF (3 mL) at 0°C was stirred for 10 min, then allowed to warm and stir for 20 h. The mixture was diluted with ethyl acetate, washed with water, brine, and dried over sodium sulfate. Following evaporative removal of the solvent, the residue was chromatographed (silica gel, 70:20:10 chloroform/ethyl acetate/MeOH to give 3-14 as a colorless foam.
TLC Rf = 0.31 (70:20:10 chloroform/ethyl acetate/MeOH). iH NMR (300 MHz, CHCI3) δ 8.55 (d, IH, J=2.2 Hz), 8.50 (dd, IH, J= 1.5,
4.6 Hz), 7.64 (m, 2H), 7.23 (m, IH), 7.05 (d, IH, J=7.3 Hz), 6.38 (d, IH, J=7.3 Hz), 5.40 (m, IH), 4.98 (br s, IH), 4.01 (m, 4H), 3.39 (m, 4H), 2.85 (m, 2H), 2.68 (m, 4H), 2.49 (m, IH), 2.25 (m, 2H), 1.83 (m, 4H), 1.16 (t, 3H, J=7.2 Hz).
2-Oxo-3(S)-r2-(5.6.7.8-tetrahvdron.81-naphthvridin-2-yl)ethvnpyrrolidin- l-yl)acetvI-3(S)-pyridin-3-yl-β-alanine (3-15) To a solution of 4 (0.049 g, 0.102 mmol) in THF (1 mL) and water (0.3 mL) at 0°C was added IM LiOH (0.112 ml, 0.112 mmol). After warming to ambient temperature and stirring for 2 h, the solvents were evaporated and the residue was chromatographed (silica gel, 25:10:1:1 ethyl acetate/EtOH/water/NH40H to give 3-15 as a colorless foam. TLC Rf = 0.15 (25:10:1:1 ethyl acetate/EtOH/water/NH40H).
!H NMR (300 MHz, DMSO-de) δ 8.74 (d, IH, J=8.3 Hz), 8.51 (m, IH), 8.42 (m, 2H), 7.70 (d, IH, J=8.1 Hz), 7.33 (m, IH), 7.21 (d, IH, J=7.3 Hz), 6.36 (d, IH, J=7.3 Hz), 5.14 (m, IH), 4.00 (d, IH, J=16.8 Hz), 3.70 (d, IH, J=16.6 Hz), 3.30 (m, 4H), 2.68 (m, 7H), 2.20 (m, 3H), 1.71 (m, 4H). SCHEME 4
Figure imgf000080_0001
EDC, HOBT NMM
Figure imgf000080_0002
NaOH
Figure imgf000080_0003
SCHEME 4 (CONT'D)
Figure imgf000081_0001
2-Oxo-3(R)-r2-(5.6.7.8-tetrahvdrori.81-naphthvridin-2-yl)ethvnpyrrolidin- l-yl)acetyl-3(S)-alkynyl-β-alanine ethyl ester (4-2)
Prepared from 41 (prepared by the method used to prepare 3-11. utilizing (S)-(-)-4-benzyl-2-oxazolidinone) and 2-9. by the method used to prepare 3-12. !H NMR (300 MHz, CHCI3) δ 7.06 (d, IH, J=7 Hz), 6.39 (d, IH), J=7 Hz), 5.06 (m, IH), 4.84 (br s, IH), 4.16 (q, 2H, J=6 Hz), 3.93 (m, 2H), 3.38 (m, 4H), 2.68 (m, 6H), 2.52 (m, IH), 2.25 (m, 2H), 1.90 (m, 2H), 1.78 (m, 2H), 1.26 (t, 3H, J=7Hz).
2-Oxo-3(R)-r2-(5.6.7.8-tetrahvdrori.81-naphthvridin-2-yl)ethyllpyrrolidin- l-yl)acetyl-3(S)-alkvnyl-β-alanine (4-3)
Prepared from 4-2 (0.05 g, 0.11 mmol) by the method used to prepare 3-13. iH NMR (300 MHz, CD3OD, 1 drop IN NaOD) δ 7.11 (d, IH, J=7 Hz), 6.40
(d, IH, J=7 Hz), 4.90 (m, IH), 3.94 (q, 2H, J= 17 Hz), 3.39 (m, 4H), 2.69 (d, 2H, J= 6 Hz), 2.60 (m, 2H), 2.52 (d, J=7 Hz), 2.49 (m, IH), 2.27 (m, IH), 2.13 (m, IH), 1.85 (m, 4H), 1.68 (m, IH).
2-Oxo-3(R)-r2-(5.6.7.8-tetrahvdrori.81-naphthvridin-2-vl)ethvl1-pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine ethyl ester (4-4)
Prepared from 41 (0.35 g, 1.0 mmol) and MQ (0.33 g, 1.2 mmol) by the method used to prepare 3-14. !H NMR (300 MHz, CHCI3) δ 8.55 (d, IH, J= 2 Hz), 8.55 (dd, IH, J= 2, 5 Hz), 7.61 (m, IH), 7.54 (m, IH), 7.06 (d, IH), 6.38 (d, IH, J=7 Hz), 5.40 (m, IH), 4.90 (br s, IH), 4.05 (q, 2H, J=7 Hz), 3.95 (m, 2H), 3.42 (m, 4H), 2.85 (dd, 2H, J=2, 6 Hz), 2.67 (m, 4H), 2.53 (m, IH), 2.27 (m, 2H), 1.90 (m, 2H), 1.78 (m, 2H), 1.16 (m, 3H, J=7 Hz).
2-Oxo-3(R)-r2-(5.6.7.8-tetrahvdro-ri.81-naphthvridin-2-yl)ethyllpyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine (4-5)
Prepared from 44 (0.16 g, 0.33 mmol) by the method used to prepare 3-15. !H NMR (300 MHz, CD3OD) δ 8.57 (s, IH), 8.42 (m, IH), 7.86 (d, IH, J=6 Hz), 7.43 (m, 2H), 6.51 (d, IH, J=7 Hz), 5.28 (m, IH), 4.63 (d, IH, J=17 Hz), 3.60 (m, 2H), 3.47 (d, IH, J= 17 Hz), 3.35 (m, 3H), 3.14 (td, IH, J=5, 13 Hz), 2.75 (m, 5H), 2.42 (m, IH), 2.23 (m, IH), 1.90 (m, 4H). Scheme 5
O o ΛN'H BOC2Q, DMAP, B0C-N^N"B0C
CH3CN, 65°C
5- 1 5-2
Figure imgf000083_0001
5-5 Scheme 5 continued
Figure imgf000084_0001
EDC, HOBT, NMM, DMF, 1-9
l
Figure imgf000084_0002
1.3-Di-tert-buyloxycarbonyl-tetrahvdropyrimidine (5-2)
A heterogeneous mixture of 51 (10.0 g, 100 mmol), BOC2O (48 g, 220 mmol), DMAP (20 mg), and CH3CN (500 mL) was heated for 40 hr at 65°C followed by addition of DMF (100 mL) and then continued heating for 24 hr. The cooled reaction mixture was diluted with EtOAc and then washed with H2O, sat. NaHCθ3, IN HCl, and brine, dried (MgSθ4), and concentrated. The residue was triturated with hexanes to give i___2 as a yellow solid. TLC RF = 0.93 (EtOAc); iHNMR (300 MHz, CDCI3) δ 3.68 (t, J = 7 Hz, 4H), 2.00 (m, 2H), 1.48 (s, 18
H).
Tert-Butyloxycarbonyl-tetrahvdropyrimidine (5-3 )
A solution of J*2 (19.0 g, 63 mmol), Mg(C104)2 (2.8 g, 12.7 mmol), and CH3CN was heated at 50°C for 2 hr. The cooled solution was diluted with CHCI3 and then washed with IN HCl, sat. NaHCθ3, and brine, dried (MgSθ4), and concentrated. Flash chromatography (silica, 75% EtOAc/ hexanes ϊtOAc) gave i 3 as a brown solid. TLC RF = 0.26 (silica, EtOAc); iHNMR (300 MHz, CDCI3) δ 5.50 (bs, IH), 3.70 (m, 2H), 3.29 (m, 2H), 1.97
(m, 2H), 1.48 (s, 9H).
Tert-Butyloxycarbonyl-2-oxo-3-(3-ethylene glycolbutyl)- tetrahvdropyrimidine (5-4) To a stirred solution of (3.2g, 16.1 mmol) and DMF (50 mL) was added LiN(TMS)2 (21 mL, lM/hexanes). After 20 minutes, the iodide \__ (8-6 g, 35.2 mmol) in DMF (10 mL) was added and the reaction mixture heated at 50 °C for 2 hours. The cooled solution was diluted with CHCI3 and then washed with H2O and brine, dried (MgSθ4), and concentrated. Flash chromatography (silica, 60% to 75%
EtOAc/hexanes) gave 54 as an orange oil.
TLC RF = 0.74 (silica, 70:15:15 CHCl3/EtOAc/CH3θH); iH NMR (300 MHz, CDCI3) δ 3.93 (s, 4H), 3.66 (t, J= 6Hz, 2H), 3., 44 (m,
2H), 3.30 (m, 2H), 1.96 (m, 2H), 1.48 (s, 9H), 1.32 (s, 3H). l-Oxo-2-(3-ethylene glvcol-butyl) tetrahvdro-pyrimidine (5-5)
A mixture of f»4 (3.0 g, 9.5 mmol), TFA (1.5 mL, and toluene (30 mL) was stirred at ambient temperature for 20 minutes, concentrated and the residue azeotroped with toluene to remove excess TFA. The residue was then dissolved in toluene (30 mL) and treated with NaHC03 (3g), filtered, and the filtrate concentrated to give a yellow oil. Flash chromatography (silica, 70:15:15 CHCl3/EtOAc/ CH3OH) gave
5-5 as a yellow oil.
TLC RF = 0.63 (silica, 70:15:15 CHCl3/EtOAc/CH3θH); iH NMR (300 MHz, CDCI3) δ 5.16 (bs, IH), 3.94 (s, 4H), 3.40 (m, 2H), 3.24 (m, 4H), 1.90 (m, 2H), 1.34 (s, 3H).
Ethyl 2-oxo-3-[3-ethylene glycol-butyl]tetrahydropyrimidin-l-yl- acetate (5-6) To a stirred solution of j>5 (2.0 g, 9.3 mmol) and DMF (50 mL) was added LiN(TMS)2 (12.1 mL, 1.0 M/THF). After 20 min, ethyl iodoacetate (1.66 mL, 14.0 mmol) was added followed by heating at 60°C for 1 hr. The cooled solution was diluted with EtOAc and then washed with H2O, sat. NaHCθ3, and brine, dried (MgSθ4), and concentrated. Flash chromatography (silica, 50% to 75% EtOAc/hexanes) gave ___ . as a colorless oil.
TLC RF = 0.72 (silica, 70:15:15 CHCl3/EtOAc/CH3θH); iH NMR (300 MHz, CDCI3) δ 4.18 (q, J=7Hz, 2H), 3.93 (s, 4H), 3.42 (m,
2H), 3.34 (m, 4H), 1.98 (m, 2H), 1.92 (m, 2H), 1.34 (s, 3H), 1.25 (t, J=7Hz, 3H).
Ethyl 2-oxo-3-F3-oxo-butvHtetrahvdro-pyrimidin-l-yl-acetate (5-7)
A solution of j 6 (750 mg, 2.5 mmol), p-TSA (10 mg), and acetone (30 mL) was refluxed for 1 hr. The cooled solution was diluted with CHCI3 and then washed with sat. NaHCθ3 and brine, dried
(MgS04), and concentrated to give _ _l_ as a yellow oil.
TLC RF = 0.36 (silica, 10% CH3θH/EtOAc);
!H NMR (300 MHz, CDCI3) δ 4.17 (q, J=7Hz, 2H), 3.56 (m, 2H), 3.34 (m,
4H), 2.76 (t, J=7Hz, 2H), 2.17 (s, 3H), 2.00 (m, 2H), 1.27 (t, J=7Hz, 3H). Ethyl 2-oxo-3-[2-naphthyridin-2-yl)ethyl]-tetrahydropyrimidin-l-yl-acetate
&£}
A mixture of 5 7 (600 mg, 2.3 mmol), 14 (343 mg, 2.8 mmol), L-proline (175 mg), and ethanol (25 mL) was heated at reflux for 18 hr. The cooled reaction mixture was concentrated and the residue purified by flash chromatography (silica, 10% CH3θH/EtOAc) gave 5___[ as a yellow solid.
TLC RF = 0.21 (silica, 10% CH3θH/EtOAc); iH NMR (300 MHz, CDCI3) δ 9.10 (m, IH), 8.19 (m, IH), 8.14 (d, J=8Hz, IH), 7.52 (d, J=8Hz, IH), 7.44 (m, IH), 4.18 (q, J=7Hz, 2H), 3.83 (m, 2H), 3.32 (m, 6H), 1.93 (m, 2H), 1.24 (t, J=7Hz, 3H).
Ethyl 2-oxo-3-[2-(5,6,7,8-tetrahydro-[l,8]-naphthyridine-2-yl)ethyl] tetrahvdropyrimidine-1-yl-acetate (5-9) A mixture of £8 (600 mg, 1.75 mmol), 10% Pd/C (300 mg), and ethanol (10 mL) was stirred at ambient temperature under a hydrogen atmosphere (1 atm) for 20 hr. The catalyst was removed by filtration through a celite pad and the filtrate concentrated to give 5^9 as a yellow oil. iH NMR (300 MHz, CDCI3) δ 7.04 (d, J=8Hz, IH), 6.42 (d, J=8Hz, IH), 4.80
(bs, IH), 4.22-4.03 (m, 4H), 3.60 (m, 2H), 2.78 (m, 2H), 2.66 (m, 2H), 1.96 (m, 4H), 1.24 (t, J=7Hz, 3H).
2-Oxo-3-[2-(5,6,7,8-tetrahydro-[l,8]naphthridin-2-yl)tetrahydro- pyrimidin-1-yl-acetic acid (5-10)
A solution of 5 (600 mg, 1.73 mmol) and 6N HCl (20 mL) was heated at 50°C for 2 hr. The solution was concentrated followed by azeotropic removal of H2O with CH3CN to give 5-10 as a yellow solid. iH NMR (300 MHz, CD3OD) δ 7.58 (d, J=8Hz, IH), 6.63 (d, J=8Hz, IH), 3.98 (s, 2H), 3.62 (t, J=7Hz, 2H), 3.50 (m, 2H), 3.36 (m, 4H),
2.93 (m, 2H), 2.80 (m, 2H), 2.00 (m, 4H).
Ethyl 2-oxo-3-[2-(5,6,7,8-tetrahydro-[l,8]naphthyridin-2-yl)ethyl]- tetrahvdropyrimidin-l-yl-acetyl-3(S)-pyridin-3-yl-β-alanine (5-11) To a stirred solution of 510 (250 mg, 0.70 mmol), (210 mg, 0.77 mmol), EDC (148 mg, 0.77 mmol), HOBT (95 mg, 0.70 mmol), CH3CN (2 mL), and DMF (2 mL) was added NMM (542 μL, 4.9 mmol).
After stirring at ambient temperature for 20 hr, the reaction mixture was diluted with EtOAc and then washed with H2O, sat. NaHC03, brine, dried (MgSθ4), and concentrated. Flash chromatography (silica, 70:15:15 CHCl3/EtOAc CH3θH) gave 541 as a colorless oil. TLC RF = 0.31 (silica, 70:15:15 CHCI3/ EtOAc/CH3θH); iH NMR (300 MHz, CDCI3) δ 8.58 (m, IH), 8.50 (m, IH), 7.94 (m, IH), 7.66 (m, IH), 7.22 (m, IH), 7.05 (d, J=8Hz, IH), 6.40 (d, J=8Hz, IH), 5.43 (m, IH), 4.06 (q, J=7Hz, 2H), 4.02 (m, IH), 3.90 (m, IH), 3.60 (m, 2H), 3.39 (m, 2H), 3.29 (m, 2H), 3.19 (m, 2H), 2.88 (m, 2H), 2.77 (m, 2H), 2.70 (m, 2H), 1.90 (m, 4H), 1.16 (t, J=7Hz, 3H).
2-Oxo-3-[2-(5,6,7,8-tetrahydro-[l,8]naphthvridin-2-yl}ethyl]- tetrahvdropyrimidin-l-yl-acetyl-3-(S)-pyridin-3-yl-β-alanine (5-12)
A mixture of 511 (100 mg, 0.22 mmol), IN NaOH (300 μL), and ethanol (1 mL) was stirred at ambient temperature for 1 hr, followed by concentration. Flash chromatography (silica, 25:10:1:1 to 15:10:1:1 EtOAc/EtOH/NH4θH/H2θ) gave 542 as a white solid.
TLC RF = 0.22 (silica, 10:10:1:1 EtOAc/ethanol NH4θH, H2O); !H NMR (300 MHz, CD3OD) δ 8.66 (m, IH), 8.39 (m, IH), 7.95 (m, IH), 7.53 (d, J=8Hz, IH), 7.40 (m, IH), 6.66 (d, J=8Hz, IH), 5.18 (m, IH), 4.27 (d, J=7Hz, IH), 4.16 (m, IH), 3.64 (d, J=7Hz, IH), 3.50-3.10 (m, 8H), 3.00- 2.65 (m, 6H), 1.95 (m, 4H).
Scheme 6
Figure imgf000089_0001
6-1
6-2
Figure imgf000089_0002
6-6
6-5
Scheme 6 continued
Figure imgf000090_0001
1.3-Di-tert-buyloxycarbonyl-imidazolidin-2-one (6-2)
A heterogeneous mixture of 64 (10.0 g, 116 mmol), BOC2O (56 g, 255 mmol), DMAP (20 mg), and CH3CN (400 mL) was heated for 18 hr at 60°C. The cooled reaction mixture was diluted with EtOAc and then washed with H2O, sat. NaHCθ3, IN HCl, and brine, dried
(MgSθ4), and concentrated. The residue was triturated with hexanes to give 6_2 as a white solid.
TLC RF = 0.91 (EtOAc); iH NMR (300 MHz, CDCI3) δ 3.73 (s, 4H), 1.53 (s, 18 H).
Tert-Butyloxycarbonyl-imidazolidin-2-one (6-3)
A solution of (28.0 g, 98 mmol), Mg(C104)2 (4.3 g, 20 mmol), and CH3CN (400 mL) was heated at 50°C for 3 hr. The cooled solution was diluted with CHCI3 and then washed with IN HCl, sat. naHC03, and brine, dried (Mg SO4), and concentrated. Flash chromatography (silica, 50% EtOAc/ hexanes ^EEtOAc) gave 6-3 as a yellow solid.
TLC RF = 0.31 (silica, EtOAc); iH NMR (300 MHz, CDCI3) δ 6.27 (bs, IH), 3.86 (m, 2H), 3.47 (m, 2H), 1.50 (s, 9H).
l-Tert-Butyloxycarbonyl-3-(3-ethylene glycol-butyl)imidazolidin-2-one (6-
4)
To a stirred solution of &_3 (4.5 g, 24 mmol) and DMF (50 mL) was added LiN(TMS)2 (26.6 mL, IM/hexanes). After 20 minutes, the iodide J 2 (8.6 g, 35.2 mmol) in DMF (10 mL) was added and the reaction mixture heated at 60°C for 4 hours. The cooled solution was diluted with CHCI3 and then washed with H2O and brine, dried (MgSθ4), and concentrated. Flash chromatography (silica, 75% EtOAc/hexanes) gave 64 as an yellow solid.
TLC RF = 0.71 (silica, 70:15:15 CHCl3/EtOAc/CH3θH);
!H NMR (300 MHz, CDCI3) δ 3.93 (s, 4H), 3.75 (m, 2H), 3.36 (m, 4H), 1.90
(m, 2H), 1.53 (s, 9H), 1.34 (s, 3H).
l-(3-Ethylene glvcol-butyl)imidazolidin-2-one (6-5) A mixture of 64 (4.0 g, 13.3 mmol), TFA (3 mL, and toluene (60 mL) was stirred at 50°C for 60 minutes, concentrated and the residue azeotroped with toluene to remove excess TFA. The residue was then dissolved in toluene (30 mL) and treated with NaHCθ3 (3g), filtered, and the filtrate concentrated to give a yellow oil. Flash chromatography
(silica, 70:25:5 CHCl3/EtOAc/CH3θH) gave as a white solid.
TLC RF = 0.58 (silica, 70:15:15 CHCl3/EtOAc/CH3θH);
!H NMR (300 MHz, CDCI3) δ 4.25 (bs, IH), 3.94 (s, 4H), 3.44 (m, 4H), 3.32
(m, 2H), 1.90 (m, 2H), 1.35 (s, 3H).
Ethyl 2-oxo-3-r3-ethylene glvcol-butyllimidazolidin-1-yl-acetate (6-6)
To a stirred solution of 6J5 (2.0 g, 10 mmol) and DMF (50 mL) was added LiN(TMS)2 (11 mL, 1.0 M/THF). After 20 min, ethyl iodoacetate (3.5 mL, 30 mmol) was added at ambient temperature. After 3 hr the solution was diluted with EtOAc and then washed with H2O, sat. NaHCθ3, and brine, dried (MgSθ4), and concentrated. Flash chromatography (silica, 50% to 75% EtOAc/hexanes) gave 6 , as a colorless oil.
TLC RF = 0.71 (silica, 70:15:15 CHCl3/EtOAc/CH3θH); iH NMR (300 MHz, CDCI3) δ 4.18 (q, J=7Hz, 2H), 3.93 (s, 4H), 3.91 (m,
2H), 3.50-3.30 (m, 6H), 1.90 (m, 2H), 1.92 (m, 2H), 1.35 (s, 3H), 1.25 (t,
J=7Hz, 3H).
Ethyl 2-oxo-3-r3-oxo-butyllimidazolidin-l-yl-acetate (6-7) A solution of 6£ (1.4 g, 4.9 mmol), p-TSA (10 mg), and acetone (30 mL) was refluxed for 1 hr. The cooled solution was diluted with CHCI3 and then washed with sat. NaHCθ3 and brine, dried
(MgS04), and concentrated to give 6 7 as a yellow oil. TLC RF = 0.34 (silica, EtOAc); iH NMR (300 MHz, CDCI3) δ 4.17 (q, J=7Hz, 2H), 3.94 (s, 2H),
3.48 (m, 2H), 3.42 (m, 4H), 2.72 (t, J=7Hz, 2H), 2.17 (s, 3H), 1.27 (t, J=7Hz, 3H).
Ethyl 2-oxo-3-r2-naphthyridin-2-yl)ethyllimidazolidin-l-yl-acetate (6-8) A mixture of (1.0 g, 4.1 mmol), 14 (604 mg, 4.9 mmol), L-proline (238 mg), and ethanol (50 mL) was heated at reflux for 20 hr. The cooled reaction mixture was concentrated and the residue purified by flash chromatography (silica, 70:25:5 CHCl3/EtOAc/CH3θH) gave 6-8 as a yellow oil.
TLC RF = 0.42 (silica, 70:15:15 CHCl3/EtOAc/CH3θH); iH NMR (300 MHz, CDCI3) δ 9.10 (m, IH), 8.19 (m, IH), 8.14 (d, J=8Hz, IH), 7.52 (d, J=8Hz, IH), 7.44 (m, IH), 4.17 (q, J=7Hz, 2H)„ 3.81 (m, 2H), 3.42 (m, 4H), 3.32 (m, 4H), 1.24 (t, J=7Hz, 3H).
Ethyl 2-oxo-3-[2-(5,6,7,8-tetrahydro-[l,8]-naphthyridine-2- vDethyllimidazolidin- 1-yl-acetate (6-9)
A mixture of 6£ (1.1 g, 3.35 mmol), 10% Pd/C (500 mg), and ethanol (30 mL) was stirred at ambient temperature under a hydrogen atmosphere (1 atm) for 20 hr. The catalyst was removed by filtration through a celite pad and the filtrate concentrated to give &_9 as a colorless oil.
TLC RF = 0.11 (silica, 70:25:5 CHCl3 EtOAc/CH3θH); iH NMR (300 MHz, CDCI3) δ 7.04 (d, J=8Hz, IH), 6.42 (d, J=8Hz, IH), 4.80 (bs, IH), 4.224.03 (m, 4H), 3.96 (s, 2H), 3.55 (m, 2H), 3.40 (m, 2H), 2.78 (m, 2H), 2.68 (m, 2H), 1.90 (m, 2H), 1.24 (t, J=7Hz, 3H).
2-Oxo-3-[2-(5,6,7,8-tetrahydro-[l,8]naphthridin-2-yl)imidazolidin-l-yl- acetic acid (6-10)
A solution of 6£ (1.0 g, 3.0 mmol) and 6N HCl (40 mL) was heated at 60°C for 1 hr. The solution was concentrated followed by azeotropic removal of H2O with CH3CN to give 6-10 as a yellow solid. !H NMR (300 MHz, CD3OD) δ 7.58 (d, J=8Hz, IH), 6.63 (d, J=8Hz, IH), 3.98 (s, 2H), 3.50 (m, 4H), 3.36 (m, 4H), 2.93 (m, 2H), 2.82 (m, 2H), 1.97 (m, 4H). Ethyl 2-oxo-3-[2-(5,6,7,8-tetrahydro-[l,8]naphthyridin-2- yl)ethvnimidazolidin-l-yl-acetyl-3(S)-pyridin-3-yl-β-alanine (6-11)
To a stirred solution of Q (240 mg, 0.70 mmol), (207 mg, 0.77 mmol), EDC (269 mg, 1.4 mmol), HOBT (95 mg, 0.70 mmol), and CH3CN (3 mL) was added NMM (619 μL, 5.6 mmol). After stirring at ambient temperature for 20 hr, the reaction mixture was diluted with EtOAc and then washed with H2O, sat. NaHCθ3, brine, dried (MgSθ4), and concentrated. Flash chromatography (silica, 70:15:15 CHCl3/EtOAc/CH3θH) gave 641 as a colorless oil. TLC RF = 0.41 (silica, 70:15:15 CHCI3/ EtOAc/CH3θH);
!H NMR (300 MHz, CDCI3) δ 8.58 (m, IH), 8.50 (m, IH), 7.94 (m, IH), 7.66 (m, IH), 7.22 (m, IH), 7.05 (d, J=8Hz, IH), 6.40 (d, J=8Hz, IH), 5.43 (m, IH), 4.06 (q, J=7Hz, 2H), 3.85 (m, IH), 3.55 (m, 2H), 3.40 (m, 2H), 3.33 (m, 4H), 2.90 (m, 2H), 2.77 (m, 2H), 2.70 (m, 2H), 1.90 (m, 2H), 1.77 (m, 2H), 1.18 (t, J=7Hz, 3H).
2-Oxo-3-r2-(5.6.7.8-tetrahvdro-n.81naphthvridin-2-vliethvll-imidazolidin- l-yl-acetyl-3-(S)-pyridin-3-yl-β-alanine (6-12)
A mixture of 611 (160 mg, 0.33 mmol), IN NaOH (500 μL), and ethanol (1 mL) was stirred at ambient temperature for 1 hr, followed by concentration. Flash chromatography (silica, 25:10:1:1 to 15:10:1:1
EtOAc/EtOH/NH4θH/H2θ) gave 612 as a white solid.
TLC RF = 0.21 (silica, 10:10:1:1 EtOAc/ethanol NH4θH, H2O);
!H NMR (300 MHz, CD3OD) δ 8.66 (m, IH), 8.39 (m, IH), 7.95 (m, IH), 7.53 (d, J=8Hz, IH), 7.40 (m, IH), 6.66 (d, J=8Hz, IH), 5.22 (m, IH), 3.93
(d, J=17 Hz, IH), 3.74 (d, J=17Hz, IH), 4.00-3.20 (m, 9H), 3.00-2.65 (m, 6H),
1.89 (m, 4H).
Scheme 7
Figure imgf000095_0001
7-2
Figure imgf000095_0002
7-3 Ethyl 2-oxo-3(R)-[2-(5,6,7,8-tetrahydro-[l,8]naphthyridin-2- yl)ethyllpyrrolidin-l-yl)acetyl-3(R)-(2-ethylindol-3-yl)-β-alanine (7-2)
To a stirred solution of 44 (175 mg, 0.52 mmol), 1_Λ (214 mg, 0.72 mmol; for preparation see US 5,321,034), EDC (197 mg, 1.0 mmol), HOBT (70 mg, 0.52 mmol), and CH3CN (3 mL) was added NMM (498 μL,
4.1 mmol). After stirring at ambient temperature for 20 hr, the reaction mixture was diluted with EtOAc and then washed with H2O, sat. NaHC03, brine, dried (MgSθ4), and concentrated. Flash chromatography (silica, 70:25:5 CHCl3/EtOAc/CH3θH) gave 1__2 as a white solid.
TLC RF = 0.11 (silica, 70:25:5 CHCI3/ EtOAc/CH3θH); !H NMR (300 MHz, CDCI3) δ 8.29 (bs, IH), 7.55 (d, J = 7 Hz, IH), 7.36 (d, J = 7 Hz, IH), 7.20-7.00 (m, 3H), 6.63 (d, J = 7 Hz, IH), 6.39 (d, J = 7 Hz, IH), 4.30 (m, IH), 4.10 (q, J=7Hz, 2H), 3.94 (d, J = 17 Hz, IH), 3.83 (d, J = 17 Hz, IH), 3.36 (m, 4H), 2.80 (m, 2H), 2.69 (m, 3H), 2.53 (d, J= 6 Hz, 2H), 2.50 (m, IH), 2.24 (m, 2H), 1.93 (m, 4H), 1.75 (m, 2H), 1.18 (t, J=7Hz, 3H).
2-Oxo-3(R)-r2-(5.6.7.8-tetrahvdro-ri.81naphthvridin-2-yl)ethyllpyrrolidin- l-yl)acetyl-3(R)-(2-ethylindol-3-yl)-β-alanine (7-3) A mixture of (60 mg, 0.11 mmol), IN NaOH (132 μL), and ethanol (1 mL) was stirred at ambient temperature for 1 hr, followed by concentration. Flash chromatography (silica, 25:10:1:1 to 15:10:1:1
EtOAc/EtOH/NH4θH/H2θ) gave W as a white solid.
TLC RF = 0.12 (silica, 10:10:1:1 EtOAc/ethanol/NH4θH/H2θ); iH NMR (300 MHz, CD3OD) δ 7.52 (d, J = 7 Hz, IH), 7.43 (d, J = 7 Hz, IH),
7.30 (d, J = 8 Hz, IH), 7.05 (m, 2H), 6.92 (m, IH), 6.48 (d, J = 7 Hz, IH),
4.54 (d, J = 17 Hz, IH), 4.27 (m, IH), 3.50-1.70 (m, 22H).
SCHEME 8
Figure imgf000097_0001
8-1
H, dioxane
Figure imgf000097_0002
Figure imgf000097_0003
SCHEME 8 (Cont'd)
Figure imgf000098_0001
8-5
Figure imgf000098_0002
8-7
Figure imgf000099_0001
N-(4-Iodo-phenylsulfonylamino)-L-asparagine (8-2)
To a stirred solution of acid 84 (4.39 g, 33.2 mmol), NaOH (1.49 g, 37.2 mmol), dioxane (30 ml) and H2O (30 ml) at 0°C was added pipsyl chloride (10.34 g, 34.2 mmol). After ~5 minutes, NaOH (1.49, 37.2 mmol) dissolved in 15 ml H2O, was added followed by the removal of the cooling bath. After 2.0 h, the reaction mixture was concentrated. The residue was dissolved in H2O (300 ml) and then washed with EtOAc.
The aqueous portion was cooled to 0°C and then acidified with concentrated HCl. The solid was collected and then washed with Et2θ to provide acid &2 as a white solid. iH NMR (300 MHz, D2O) δ 7.86 (d, 2H, J=8HZ), 7.48 (d, 2H, J=8Hz) 3.70
(m, IH), 2.39 (m, 2H).
2(S)-(4-Iodo-phenylsuIfonylamino)-β-alanine (8-3) To a stirred solution of NaOH (7.14 g, 181.8 mmol) and H2O
(40 ml) at 0°C was added Br2 (1.30 ml, 24.9 mmol) dropwise over a ten minute period. After ~5 minutes, acid &2 (9.9 g, 24.9 mmol), NaOH (2.00 g, 49.8 mmol) and H2O (35 ml) were combined, cooled to 0°C and then added in a single portion to the reaction. After stirring for 20 minutes at 0°C, the reaction was heated to 90°C for 30 minutes and then recooled to 0°C. The pH was adjusted to ~7 by dropwise addition of concentrated HCl. The solid was collected, washed with EtOAc, and then dried in vacuo to provide acid 8___\ as a white solid. !H NMR (300 MHz, D2O) δ 8.02 (d, 2H, J=8Hz), 7.63 (d, 2H, J=8Hz), 4.36 (m, IH), 3.51 (dd, IH, J=5Hz, 13Hz) 3.21 (m, IH).
Ethyl 2(S)-(4-iodo-phenylsulfonylamino)-β-alanine-hvdrochloride (8-4)
HCl gas was rapidly bubbled through a suspension of acid &; 3 (4.0 g, 10.81 mmol) in EtOH (50 ml) at 0°C for 10 minutes. The cooling bath was removed and the reaction was heated to 60°C. After 18 h, the reaction was concentrated to provide ester 84 as a white solid. iH NMR (300 MHz, CD3OD) δ 7.98 (d, 2H, J=8Hz), 7.63 (d, 2H, J=8Hz),
4.25 (q, IH, J=5Hz), 3.92 (m, 2H), 3.33 (m, IH), 3.06 (m, IH), 1.01 (t, 3H, J=7Hz). Ethyl 4-r2-(2-Aminopyridin-6-yl)ethyllbenzoate (8-5) A mixture of ester 8-5a (700 mg, 2.63 mmol), (for preparation, see: Scheme 29 of PCT International Application Publication No. WO 95/32710, published December 7, 1995) 10% Pd/C (350 mg) and EtOH were stirred under 1 atm H2- After 20 h, the reaction was filtered through a celite pad and then concentrated to provide ester 8 5 as a brown oil.
TLC Rf = 0.23 (silica, 40% EtOAc/hexanes) iH NMR (300 MHz, CDCI3) δ 7.95 (d, 2H, J=8Hz), 7.26 (m, 3H), 6.43 (d, IH, J=7Hz), 6.35 (d, IH, J=8Hz), 4.37 (m, 4H), 3.05 (m, 2H), 2.91 (m, 2H), 1.39 (t, 3H, J=7Hz).
4-r2-(2-Aminopyridin-6-yl)ethyl"lbenzoic acid hydrochloride (8-6)
A suspension of ester 8 5 (625 mg, 2.31 mmol) in 6N HCl (12 ml) was heated to 60°C. After -20 h, the reaction was concentrated to give acid 8 as a tan solid. iH NMR (300 MHz, CD3OD) δ 7.96 (d, 2H, J=8Hz), 7.80 (m, IH), 7.33 (d,
2H, J=8Hz), 6.84 (d, IH, J=9Hz), 6.69 (d, IH, J=7Hz), 3.09 (m, 4H).
Ethyl 4-[2-(2-Aminopyridin-6-yl)ethyl]benzoyl-2(S)-(4-iodo- phenylsulfonylamino)-β-alanine (8-7)
A solution of acid 8 (400 mg, 1.43 mmol), amine 84
(686 mg, 1.57 mmol), EDC (358 mg, 1.86 mmol), HOBT (252 mg, 1.86 mmol), NMM (632 μl, 5.72 mmol) and DMF (10 ml) was stirred for -20 h.
The reaction was diluted with EtOAc and then washed with sat NaHC03, brine, dried (MgSθ4) and concentrated. Flash chromatography (silica, EtOAC JE 5% isopropanol EtOAc) provided amide 8 7 as a white solid. TLC Rf = 0.4 (silica, 10% isopropanol/EtOAc)
!H NMR (300 MHz, CD3OD) δ 7.79 (d, 2H, J=9Hz) 7.61 (d, 2H, J=8Hz), 7.52 (d, 2H, J=9Hz), 7.29 (m, IH), 7.27 (d, 2H, J=8Hz), 4.20 (m, IH), 3.95 (q, 2H, J=7Hz), 3.66 (dd, IH, J=6Hz, 14Hz), 3.49 (dd, IH, J=8Hz, 13Hz), 3.01 (m, 2H), 2.86 (m, 2H), 1.08 (t, 3H, J=7Hz).
4-[2-(2-Aminopyridin-6-yl)ethyl]benzoyl-2(S)-(4-iodophenyl- sulfonylamino)-β-alanine (8-8) A solution of ester 8 7 (200 mg, 0.3213 mmol) and 6N HCl (30 ml) was heated to 60 °C. After -20 h, the reaction mixture was concentrated. Flash chromatography (silica, 20:20:1:1 EtOAc/EtOH/ NH4OH/H2O) provided acid as a white solid. TLC Rf = 0.45 (silica, 20:20:1:1 EtOAc/EtOH/NH4θH/H2θ) iH NMR (400 MHz, DMSO) δ 8.40 (m, IH), 8.14 (Bs, IH), 7.81 (d, 2H, J=8Hz), 7.62 (d, 2H, J=8Hz), 7.48 (d, 2H, J=8Hz), 7.27 (m, 3H), 6.34 (d, IH, J=7Hz), 6.25 (d, IH, J=8Hz), 5.85 (bs, 2H), 3.89 (bs, IH), 3.35 (m, 2H), 2.97 (m, 2H), 2.79 (m, 2H).
4-[2-(2-Aminopyridin-6-yl)ethyl)benzoyl-2(S)-(4-trimethylstannyl- phenylsulfonylamino-β-alanine (8-9)
A solution of iodide &8 (70 mg, 0.1178 mmol), (CH3Sn)2 (49 μl, 0.2356 mmol), Pd(PPh3)4 (5 mg) and dioxane (7 ml) was heated to 90°C. After 2 h, the reaction was concentrated and then purified by prep HPLC (Delta-Pak Cl8 15 μM 100A°> 40 x 100 mm; 95:5 JE 5:95 H2O/CH3CN) provided the trifluoroacetate salt. The salt was suspended in H2O (10 ml), treated with NH4OH (5 drops) and then lyophilized to provide amide &_9 as a white solid. iH NMR (400 MHz, DMSO) δ 8.40 (m, IH), 8.18 (d, IH, J=8Hz), 7.67 (m, 5H), 7.56 (d, 2H, J=8Hz), 7.29 (d, 2H, J=8Hz), 6.95-7.52 (m, 2H), 6.45 (bs, 2H), 4.00 (m, IH), 3.50 (m, IH), 3.33 (m, IH), 2.97 (m, 2H), 2.86 (m, 2H).
4-[2-(2-Aminopyridin-6-yl)ethyl]benzoyl-2(S)4-125iodo- phenylsulfonylamino-β-alanine (8-10)
An iodobead (Pierce) was added to a shipping vial of 5 mCi of Nal25χ (Amersham, IMS30) and stirred for five minutes at room temperature. A solution of 0.1 mg of &_9 in 0.05 mL of 10% H2Sθ4/MeOH was made and immediately added to the Nal25ι/i0(j0bea(i γ aι After stirring for three minutes at room temperature, approximately 0.04-0.05 mL of NH4OH 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 x 250 mm, linear gradient of 10% acetonitrile (0.1% (TFA):H2θ (0.1% TFA) to 90% acetonitrile (0.1% TFA):H2θ (0.1% TFA) over 30 minutes, 1 mL/min]. The retention time
400- of 8-10 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 8-10. which coeluted on HPLC analysis with an authentic sample of 8-8. 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 x 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 x 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 UN/Vis Diode Array Spectrophotometer. Sample radioactivities were determined in a Packard A5530 gamma counter.
SCHEME 9
Figure imgf000104_0001
Boc20, THF
Figure imgf000104_0002
402- SCHEME 9 (Cont'd)
Figure imgf000105_0001
9-9 (S )-(3-amino-2-oxo-pyrrolidin-l-yl)-acetic acid (9-2)
A solution of 21 (0.50 g, 1.84 mmol) (prepared as described by Freidinger, R. M.; Perlow, D. S.; Veber, D. F.; J. Org. Chem. , 1982, 26, 104) in anhydrous ethyl acetate (50 mL) was cooled to 0°C and saturated with HCl gas, then stirred at 0°C for 2 h. The resulting colorless solution was concentrated at reduced pressure and the residue triturated with anhydrous diethyl ether giving 9_2 as a hygroscopic white solid.
!H NMR (300 MHz, CD3OD) δ 4.16 (d, 2H); 4.2 (m, IH); 3.68 (s, 3H); 3.53 (m, 2H); 2.58 (m, IH); 2.09 (m, IH).
2-oxo-3-(S)-[l,8]naphthyridin-2-ylmethyl)-amino]-pyrrolidin-l-yl]- acetic acid (9-4)
A solution of (232 mg, 1.11 mmol) and 9£ (176 mg, 1.11 mmol) (prepared as reported by Weissenfels, M.; Ulrici, B.; Z. Chem. 1978, 18, 20.) in anhydrous methanol (10 mL) was treated with NaOAc (91 mg, 1.11 mmol) , NaBH3CN (70 mg, 1.11 mmol) and powdered 4 A molecular sieves (450 mg). The resulting mixture was stirred at 0° for 3.5 h, then concentrated and the residue subjected to flash chromatography on silica gel (95:4.5:0.5 CH2Cl2/MeOH/NH4θH) to afford 9^4 as a colorless glass.
FAB MS (315, M+l); iH NMR (300 MHz, CD3OD) δ 9.04 (d, IH); 8.41 (dd, IH); 8.38(d, IH); 7.72
(d, IH); 7.62 (dd, IH); 4.31 (d, 2H); 4.21 (m, 2H); 3.68 (s, 3H);3.63 (m, IH); 3.53 (m, 2H); 2.52 (m, IH); 1.95 (m, IH).
Methyl-[3-(S)-[te^butoxycarbonyl-[l,8]naphthvri aminol-2-oxo-pyrrolidin-l-yll-acetic acid (9-5)
A solution of amine 94 (69 mg, 0.22 mmol) in THF (5 mL) was treated with Boc2θ (83 mg, 0.24 mmol) and stirred at room temperature for 18 h. The solvent was removed in vacuo and the resulting residue isolated by chromatography on silica gel (5% MeOH/CH2Cl2) to afford as a yellow glass.
FAB MS (415, M+l); iH NMR (300 MHz, CD3OD) δ 9.04 (d, IH); 8.20 (m, 2H); 7.88 (d, 0.5H (rotamer a)); 7.82 (d, 0.5H (rotamer b)); 7.46(m, IH); 5.1-4.3 (m, 5H); 3.81 (m, 2H); 3.72 (s, 3H); 3.41 (m, 2H); 2.36 (m, 2H); 1.47 (s, 4.5 H (rotamer a)); 1.30 (s, 4.5 H , (rotamer b)).
Methyl-3-(S)-[έe^-butoxycarbonyl-(5,6,7,8-tetrahydro-[l,8]naphthyridin-2- ylmethyl)-amino]-2-oxo-pyrrolidin-l-yl]- acetic acid (9-6)
A solution of (40 mg, 0.097mmol) in EtOH (5 mL) was treated with 10% Pd on C (8 mg) and then stirred under a H2 filled balloon for 16 h. The catalyst was removed by filtration through celite and the filtrate concentrated to afford Q as a colorless glass. iH NMR (300 MHz, CD3OD) δ 7.10 (d, IH) 6.78 (d, 0.5H (rotamer a)); 6.62
(d, 0.5H (rotamer b)); 4.8-3.9 (m, 5H); 3.81 (m, 2H); 3.72 (s, 3H); 3.38 (m, 2H); 2.36 (m, 2H); 1.21(s, 4.5 H (rotamer a)); 1.15 (s, 4.5 H , (rotamer b)).
3-(S)-[ er£-butoxycarbonyl-(5,6,7,8-tetrahydro-[l,8]naphthyridin-2- ylmethyl)-aminol-2-oxo-pyrrolidin-l-yll-acetic acid (9-7)
A solution of 9__o (38 mg, 0.091 mmol) in 50% aqueous THF (2 mL) was treated with 1.0 N NaOH (95 mL, 0.095 mmol) and stirred at room temperature for 2 h. The reaction was neutralized with IN HCl, evaporated, and the residue dissolved in MeOH (2.5 mL), filtered and evaporated to afford 9^7 as a colorless glass. iH NMR (300 MHz, CD3OD) δ 7.31 (d, IH) 6.78 (br, d, IH); 4.8-3.9 (m, 5H); 3.81 (m, 2H); 3.38 (m, 2H); 2.36 (m, 2H); 1.21(s, 4.5 H (rotamer a)); 1.15 (s, 4.5 H, (rotamer b)).
Ethyl -3-(S)-(2-{2-oxo-3-[(5,6,7,8-tetrahydro-[l,8]naphthyridin-2-ylmethyl)- amino1-pvrrolidin-l-vll-acetvlamino)-3-(R)-pvridin-3-vl-propionic acid (9- 8}
9J_ (43 mg, 0.093 mmol), V_9 (25 mg, 0093 mmol), EDC (18 mg, 0.093 mmol), HOBT (13 mg, 0.093 mmol), and N-methyl morpholine (31 mL, 0.28 mmol) in anhydrous DMF (5 mL) was stirred at room temperature for 18 h, then concentrated in vacuo and the residue chromatographed on silica gel using 5% MeOH/CH2Cl2 as eluent affording 9J3 as a colorless glass. iH NMR (300 MHz, CDCI3) δ 8.61 (s, IH); 8.45 (d, IH); 8.00 (m, IH); 7.68,
(d, IH); 7.21 (m, IH); 7.17 (d, IH); 5.56 (m, IH); 4.75 (s, 2H); 4.45 (m, 2H); 4.05 (q, 2H); 3.95 (m, IH); 3.5-3.3 (m, 4H); 2.92 (m, IH); 2.87 (m, IH); 2.74 (m, 2H); 2.35 (m, 2H); 1.92 (m, 2H); 1.36 (s, 9H); 1.21 (t, 3H).
3-(S)-(2-{2-oxo-3-[(5,6,7,8-tetrahydro-[l,8]naphthyridin-2-ylmethyl)- amino]-pyrrolidin-l-yl}-acetylamino)-3-(R)-pyridin-3-yl-propionic acid (9-9)
9-8 (25 mg, 0.043 mmol) was dissolved in 6 N HCl (2 mL) and stirred at room temperature for 16 h, then evaporated to afford 9^) as a pale yellow solid.
FAB MS (453, M+l); iH NMR (300 MHz, CD3OD) δ 9.00 (s, IH); 8.81 (d, IH); 8.79(m, IH); 8.10
(m, IH); 7.71 (d, IH); 7.01 (m, IH); 5.56 (m, IH); 4.75 (s, 2H); 4.61 (m, IH); 4.50 (m, IH); 4.35 (m, IH); 4.10 (s, 2H); 3.62 (m, 4H); 3.4 -3.0 (m, 2H); 2.8 (m, 2H); 2.70 (m, IH); 2.45 (m IH); 1.98 (m, 2H).
Following the procedure described in Scheme 10, bicyclic compounds such as 10-6 are readily prepared by one of ordinary skill in the art.
SCHEME 10
Figure imgf000109_0001
10-2 10-1 (Fluka)
Figure imgf000109_0002
10-6 The test procedures employed to measure αvβ3 binding and the bone resorption inhibiting activity of the compounds of the present invention are described below.
EXAMPLE 1
Bone Resorption-Pit Assay
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.
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, II). Bone slices are pooled, placed in a 10% ethanol solution and refrigerated until further use. Prior to experimentation, bovine bone slices are ultrasonicated twice, 20 minutes each in H2O. 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 αMEM, pH 6.9 containing 5% fetal bovine serum and 1% penicillin/streptomycin. Long bones from 7-14 day old rabbits (New Zealand White
Hare) are dissected, cleaned of soft tissue and placed in αMEM 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 rube 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 - 2 x 10 cells/ml). A cell suspension consisting of 5 x 10 /ml in αMEM containing 5% fetal bovine serum, 10 nM l,25(OH)2D3, and pencillin- streptomycin is prepared. 200 μl aliquots are added to bovine bone slices (200 mm x 6 mm) and incubated for 2 hrs. at 37°C in a humidified 5% CO2 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 αl chain of type I collagen) by Crosslaps for culture media (Herlev, Denmark).
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 H2O, 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 NH4OH followed by 2 X 15 min ultrasonication in H2O. The bone slices are immediately stained for 6-8 min with filtered 1% toluidine blue and 1% borax.
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 IC50 values are determined for each compound tested.
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, 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.
EXAMPLE 2
409- EIB Assay
Duong et al., J. Bone Miner. Res.. 8:S 378, 1993, describe a system for expressing the human integrin αvβ3. 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.
Reaction Mixture:
1. 175 ml TBS buffer (50 mM Tris-HCl pH 7.2, 150 mM NaCl, 1% BSA, 1 mM CaCl2, 1 mM MgCl2).
2. 25 ml cell extract (dilute with 100 mM octylglucoside buffer to give 2000 cpm/25 ml). 3. 125i-echistatin (25 ml/50,000 cpm) (see EP 382 451).
4. 25 ml buffer (total binding) or unlabeled echistatin (nonspecific binding).
The reaction mixture was then incubated for 1 h at room temp. The unbound and the bound αvβ3 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, ImM CaC /MgC , pH 7.2). The filter was then counted in a gamma counter.
EXAMPLE 3
SPA Assay MATERIALS: 1. Wheatgerm agglutinin Scintillation Proximity Beads (SPA):
Amersham
2. Octylglucopyranoside: Calbiochem
3. HEPES: Calbiochem
4. NaCl: Fisher 5. CaCl2: Fisher 6. MgCl2: SIGMA
7. Phenylmethylsulfonylfluoride (PMSF): SIGMA
8. Optiplate: PACKARD
9. 84Q (specific activity 500-1000 Ci/mmole) 10. test compound
11. Purified integrin receptor: anb3 was purified from 293 cells overexpressing anb3 (Duong et al., J. Bone Min. Res., S:S378, 1993) according to Pytela (Methods in Enzymology, 144:475, 1987) 12. Binding buffer: 50 mM HEPES, pH 7.8, 100 mM NaCl, 1 mM Ca2+ Mg2+, 0.5 mM PMSF
13. 50 mM octylglucoside in binding buffer: 50-OG buffer
PROCEDURE: 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.
2. Preparation of SPA beads and receptor mixture
In each assay tube, 2.5 ml (40 mg/ml) of pretreated beads were suspended in 97.5 ml of binding buffer and 20 ml of 50- OG buffer. 5 ml (-30 ng/ml) 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°C. The pellets were then resuspended in 50 ml of binding buffer and 25 ml of 50-OG buffer.
3. Reaction
The following were sequentially added into Optiplate in corresponding wells:
411- (i) Receptor/beads mixture (75 ml)
(ii) 25 ml of each of the following: compound to be tested, binding buffer for total binding or 8-8 for non-specific binding (final concentration 1 mM) (iii) 8-10 in binding buffer (25 ml, final concentration 40 pM)
(iv) Binding buffer ( 125 ml) (v) Each plate was sealed with plate sealer from PACKARD and incubated overnight with rocking at 4°C
4. Plates were counted using PACKARD TOPCOUNT
5. % inhibition was calculated as follows: A = total counts B = nonspecific counts C = sample counts
% inhibition = [{(A-B)-(C-B)}/(A-B)]/(A-B) x 100
EXAMPLE 4
Ocform Assay
Osteoblast-like cells (1.8 cells), originally derived from mouse calvaria, were plated in CORNING 24 well tissue culture plates in a 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:
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 x 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 x 10^ cells/mL. 50 mL was added to each well of 1.8 cells to yield 50,000 cells/well and 1,25-dihydroxy-vitamin D3(D3) was added to each well to a final concentration of 10 nM. The cultures were incubated at 37°C in a humidified, 5% Cθ2 atmosphere.
After 48 h, the medium was changed. 72 h after the addition of bone marrow, test compounds were added with fresh medium containing D3 to quadruplicate wells. Compounds were added again after 48 h with fresh medium containing D3. 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:
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 were counted in each well.
Fluorescent Octform Assay OCTFORM is set up as above. Instead of fixing, staining and counting the multinucleated TRAP stained cells, quantitation is achieved as follows.
1. Plates are washed once with 150 mM NaCL, 10 mM HEPES pH 7.1 (HBS).
2. 0.25 mL of 0.05% trypsin, 0.53 mM EDTA is added to each well and the plate is incubated at 37°C for 5 minutes.
3. Plates are washed twice with HBS.
4. 0.5 mL of assay solution is added to each well and the plate is incubated at 37°C for 1 hr. Assay solution contains 50 mM sodium acetate, 30 mM sodium tartrate, 0.1% Triton X- 100, 5 mM Naphthol AS-BI phosphate, pH 5.0. 50 μL of IM NaOH is added to each well and the fluorescence is measured at excitation = 360 nm, emission = 530 nm.
EXAMPLE 5
Combined Therapy with N-[l(R)-[(l,2-Dihydro-l-methane- sulfonylspiro[3H-indole-3,4'-piperidin]-l'-yl)carbonyl]-3-phenylpropyl]-2- amino-2-methyl-propanamide and 2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]- naphthyridin-2-yl)ethyl]pyrrolidin-l-yl)acetyl-3(S)-pyridin-3-yl-β- alanine: Exploratory Nine(9)-Week Bone Study in Old Female Rats The purpose of this study is to evaluate the effect of N-[1(R)-
[(l,2-dihydro-l-methane-sulfonylspiro[3H-indole-3,4'-piperidin]-l?- yl)carbonyl]-3-phenylpropyl]-2-amino-2-methyl-propanamide, alone and in combination with the αvβ3 antagonist 2-Oxo-3(S)-[2-(5,6,7,8- tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin-l-yl)acetyl-3(S)- pyridin-3-yl-β-alanine, on the bone in old female rats. The duration of the study is 9 weeks. The frequency of dosing with N-[l(R)-[(l,2-dihydro- l-methane-sulfonylspiro[3H-indole-3,4'-piperidin]-l'-yl)carbonyl]-3- phenylpropyl]-2-amino-2-methyl-propanamide is once daily, seven days a week. The frequency of dosing with 2-Oxo-3(S)-[2-(5,6,7,8- tetrahydro[ l,8]-naphthyridin-2-yl)ethyl]pvrrolidin- l-yl)acetyl-3(S)- pyridin-3-yl-β-alanine is every other day. The route of administration of N-[l(R)-[(l,2-dihydro-l-methane-sulfonylspiro[3H-indole-3,4'-piperidin]- l'-yl)carbonyl]-3-phenylpropyl]-2-amino-2-methyl-propanamide is orally by gavage and 2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2- yl)ethyl]pyrrolidin-l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine is by subcutaneous injection (e.g., in carboxy methyl cellulose carrier). The control article is distilled water and the carrier is distilled water. The dosing volume of N-[l(R)-[(l,2-dihydro-l-methane-sulfonylspiro[3H- indole-3,4'-piperidin]-l'-yl)carbonyl]-3-phenylpropyl]-2-amino-2-methyl- propanamide is 5 ml/kg, and the dosing volume of 2-Oxo-3(S)-[2-(5,6,7,8- tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin-l-yl)acetyl-3(S)- pyridin-3-yl-β-alanine is 0.5 ml/kg. The test system is the female rat of a strain Sprague-Dawley Crl:CD® (SD) BR, which were of an approximate age at the start of the study of greater than 6 months, and which are of an approximate weight at the start of the study of 250-350 g. There are no contaminants in the feed and water that are known to interfere with the purpose and conduct of this study. There are no contaminants in the bedding that are known to interfere with the purpose and conduct of this study. The rats are housed in individual stainless steel wire cages.
Number of animals
Dosage levels Males Females
Control 1 0 7
Control 2 (Distilled water) 0 8
N-[l(R)-[(l,2-Dihydro-l-methane-sulfonyl- 0 11 spiro[3H-indole-3,4'-piperidin]-l'-yl)-carbonyl]- 3-phenylpropyl]-2-amino-2-methyl- propanamide [50 mg/kg/day]
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]- 0 10 naphthyridin-2-yl)ethyl]pyrrolidin-l-yl)acetyl-
3(S)-pyridin-3-yl-β-alanine
[10 mg/kg/every other day]
N-[l(R)-[(l,2-Dihydro-l-methane-sulfonyl- spiro[3H-indole-3,4'-piperidin]-l'-yl)-carbonyl]- 12
3-phenylpropyl]-2-amino-2-methyl- propanamide + 2-Oxo-3(S)-[2-(5,6,7,8- tetrahydro[l,8]-naphthyridin-2- yl)ethyl]pyrrolidin-l-yl)acetyl-3(S)-pyridin-3-yl- β-alanine
[50 mg/kg/day + 10 mg/kg/every other day] HORMONE ANALYSIS
Drug Day 1: Blood sampling (approximately 1.5 ml) is from orbital sinus on non-fasted rats, all groups, for measurements of GH; bleeding is done 15 minutes post dosing in control groups and groups receiving N-[l(R)-[(l,2-dihydro-l-methane-sulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-3-phenylpropyl]-2-amino-2-methyl- propanamide alone or in combination.
Drug Weeks 2*. 9:
Blood sampling (approximately 1.5 ml) is from orbital sinus on non-fasted rats, all groups except control group 1, for measurement of GH; bleeding is done 15 minutes post dosing in control group 2 and groups receiving N-[l(R)-[(l,2-dihydro-l-methane-sulfonylspiro[3H- indole-3,4'-piperidin]-l'-yl)carbonyl]-3-phenylpropyl]-2-amino-2-methyl- propanamide alone or in combination; Blood sampling (volume: as much as possible) is from the vena cava at necropsy, on non-fasted rats, all groups for measurement of IGF-1.
(* for control group 1, only)
Growth Hormone Levels (Day 1) are measured for each treatment group.
BONE ANALYSIS
All rats receive bone labelling agents (oxytetracycline and calcein): 9 days (oxytetracycline) and 2 days (calcein), before necropsy.
Oxytetracycline is injected subcutaneously at a dose of 25 mg/kg, and calcein is injected subcutaneously at a dose level of 10 mg/kg.
Tibiae are processed through increasing concentrations of ethanol followed by methyl methacrylate embedding, using an automated Hypercenter XP tissue processor (Shandon-Lipshaw,
Pittsburgh, PA). Five micron thick sections are cut using a Reichert- Jung Polycut-S microtome and stained and analyzed with Bioquant
Image Analysis System (R&M Biometrics, Nashville, TN). Calculated endpoints included: cancellous bone volume expressed as a percent of tissue volume (BV/TV, %), osteoblast surface expressed as a percent of trabecular bone surface (ObS/BS, %), osteoclastic surface expressed as a percent of trabecular bone surface (Oc.S/BS), the number of osteoclasts per unit bone surface (NOc/BS, #mm), trabecular thickness (TbTh, μm), trabecular separation (TbSp, μm), and trabecular number (TbN, #/mm); doubly labeled surfaces, the extent of surface bearing both bone labels as a percent of trabecular surface (dLS/BS); singly labeled surface, the extent of surface bearing one bone label only as a percent of trabecular surface (sLS/BS); and mineral apposition rate (MAR), the mean distance between the two bone labels (at many sites), divided by seven, the time between the two bone labels. Mineralizing surface (MS/BS, %) is calculated as one-half sLS/BS plus dLS/BS, expressed as a percent of bone surface. All measurements are made in the metaphyseal secondary spongiosa 1mm below the epiphyseal growth plate. Length of the tibia is measured and diaphyseal cortical cross sections are cut using a Buehler ISOMET saw 1 - 1.5 cm proximal to the tibiofibular junction. Results are expressed as mean ± SEM. Statistical analysis is performed using the package STATVIEW for Macintosh (Abacus Concepts, Berkeley, CA). Differences between treatment groups are tested by one-way analysis of variance and Fisher PLSD (protected least significant difference). A value of p < 0.05 is considered significant.
EXAMPLE 6
Combined Therapy with N-[l(R)-[(l,2-Dihydro-l-methane- sulfonylspiro[3H-indole-3,4'-piperidin]-l'-yl)carbonyl]-2- (phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide and 2-Oxo-3(S)- [2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin-l-yl)acetyl- 3(S)-pyridin-3-yl-β-alanine:
Twelve (12) Week Bone Study in Female Dogs
The purpose of this study is to evaluate the combined effects of twelve weeks treatment with the growth hormone secretagogue ("GHS"), N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2- methylpropanamide methanesulfonate, and the avb3 antagonist, 2-Oxo- 3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin-l- yl)acetyl-3(S)-pyridin-3-yl-β-alanine, on bone mineral density and bone formation in dogs. One year old female (9.1-13 kg) dogs are assigned four per dose group and treated for twelve weeks with either vehicle, or 1.25 mg/kg/day GHS + 1.25 mg/kg/day avb3 antagonist, 2.5 mg/kg/day GHS + 2.5 mg/kg/day αvβ3 antagonist, or 5.0 mg/kg/day GHS + 5.0 mg/kg/day avb3 antagonist. To identify formation surfaces dogs receive fluorochrome bone labels prior to necropsy by i.v. administration of oxytetracycline (15 mg/kg i.v.) and calcein (15 mg/kg i.v.) with a 10 day interval between the administration of the two labels. At necropsy, the fourth and fifth lumbar vertebrae and right tibiae are dissected free of muscle and other connective tissue and fixed in cold 70% ethanol (4°C). Spinous and transverse processes of vertebra L5 are removed, the vertebral body is submerged in two inches of water, and bone mineral content is measured using dual energy x-ray absorptiometry (QDR 4500A, HOLOGIC, Waltham Mass.). The L5 vertebral body is then cut in cross-section and a central sagittal piece of the proximal portion is cut using a high speed Dremmel tool. The vertebral cross and sagittal sections are processed and embedded in methylmethacrylate without prior decalcification using a Hypercenter XP tissue processor (Shandon, Pittsburgh, PA). Sagittal and cross-sections of 6-10 mm thickness are cut using a Polycut S microtome (Leica, Deerfield, IL).
All morphometric measurements are performed using the Bone Morphometry software (Bioquant System IV, Nashville, TN), a computer with a digitizing board and a microscope equipped with visible and UV light sources. For each specimen, the following cancellous bone parameters are measured in a mean tissue area of 4.6 mm2 located in the metaphyseal region of the vertebra beginning 2 mm inferior to the growth plate. Using bright field illumination, the following variables of cancellous bone structure: bone volume/tissue volume (BV/TV, %), trabecular number (Tb. N, I/mm), trabecular thickness (TbTh., mm), trabecular separation (Tb Sp., mm) are directly measured or calculated from primary measurements of tissue area, trabecular bone area, trabecular bone perimeter and boundary length, using Masson's trichrome stained sections. Osteoid surface (OS/BS, %) or the unmineralized matrix is also measured on Masson's trichrome stained sections in the same area and expressed as a percent of the trabecular bone surface. Osteoid thickness (O.Th, mm) is calculated as the product of osteoid width and the correction factor p/4. Mineralization lag time (Mlt, days) is calculated as O.Th/Aj.AR. Adjusted apposition rate (Aj.AR, mm d) or effective apposition rate is calculated as the product of MAR*(MS/OS). The vertebral anterior (ventral) cortical width is also directly measured from stained mid-vertebral cross-sections.
Dynamic labeled parameters are assessed in the same area of 10 mm thick sections viewed under epifluorescence by measuring the length of the oxytetracycline and calcein labels on trabecular bone surface and the interlabel distance. The mineralizing surface (MS/BS, %) is calculated as one-half the length of single labels plus the length of double labels expressed as a percent of bone surface. The mineral appositional rate (MAR, mm day) is calculated as the mean distance between the first and second label at equidistant points divided by the labeling interval (14 days) and then multiplied by the correction factor (p/4) to account for the obliquity of the sectioning plane. Surface based bone formation rate (BFR/BS) is calculated as the product of MS/BS*MAR and expressed per year.
Statistical analysis of histomorphometric data is done using the statistical package Statview™ (Macintosh). Differences between treatment group are tested by one-way analysis of variance. If significant differences are indicated by ANOVA, comparison between groups means are tested by the Fisher Protected Least Significant Difference test (PLSD, p < 0.05). Bone densitometry data are analyzed using a Statistical Trend Analysis (NOSTASOT) test to assess the effect of increasing drug doses.
While the invention has been described and illustrated with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various adaptations, changes, modifications, substitutions, deletions, or additions of procedures and protocols may be made without departing from the spirit and scope of the invention. For example, effective dosages other than the particular dosages as set forth herein above may be applicable as a consequence of variations in the responsiveness of the mammal being treated for any of the indications with the compounds of the invention indicated above. Likewise, the specific pharmacological responses observed may vary according to and depending upon the particular active compounds 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 defined by the scope of the claims which follow and that such claims be interpreted as broadly as is reasonable.

Claims

WHAT IS CLAIMED IS:
1. A combination which comprises an αvβ3 antagonist and a growth hormone secretagogue.
2. The combination of Claim 1, wherein the growth hormone secretagogue is of the Formula I or II:
Figure imgf000123_0001
Formula Formula II
wherein: Ri is selected from the group consisting of: -Ci-Cio alkyl, -aryl, aryl-(Cι-C6 alkyl)-, C3-C7 cycloalkyl-(Cι-C6alkyl)-, -Ci-Cδalkyl-K-Ci-Cδ alkyl, aryl(Co-C5alkyl)-K-(Cι-C5 alkyl)-, and C3-C7 cycloalkyl(Co-C5 alkyl)-K-(Ci-C5 alkyl)-, wherein K is O, S(0)m, NOtøtaCO), C(0)N(R2), OC(O), C(0)0, or - CR2=CR2-, or -C≡C-, and wherein the aryl groups are as defined below and the R2 and alkyl groups may be further substituted by 1 to 9 halogen, S(0)mR2a> 1 to 3 OR2a> or C(0)OR2a> and the aryl groups may be further substituted by phenyl, phenoxy, halophenyl, 1-3 Cχ-C6 alkyl, 1 to 3 halogen, 1 to 2 -OR2,
421- methylenedioxy, -S(0)mR2, 1 to 2 -CF3, -OCF3, nitro, -N(R2)(R2), -
N(R2)C(0)R2, -C(0)OR2, -C(0)N(R2)(R2), -S0 N(R2)(R2), -N(R2)S(0)2 aryl, and -N(R2)Sθ2R25
R2 is selected from the group consisting of: hydrogen, Ci-Cβ alkyl, C3-C7 cycloalkyl, and where two Cχ-C6 alkyl groups are present on one atom, they may be optionally joined to form a C3-C8 cyclic ring optionally including oxygen, sulfur or NR2a>
R2a is hydrogen, or Cχ-C6 alkyl;
R3a and R3 are independently selected from the group consisting of: hydrogen, halogen, -Cχ-C6 alkyl, -OR2, cyano, -OCF3, methylenedioxy, nitro, -S(0)mR, -CF3 or -C(0)OR2 and when R3a and R3 are in an ortho arrangement, they may be joined to form a C5 to Cg aliphatic or aromatic ring optionally including 1 or 2 heteroatoms selected from oxygen, sulfur or nitrogen;
R4 and R5 are independently selected from the group consisting of: hydrogen, -Ci-Cβ alkyl, substituted Cχ-C6 alkyl wherein the substituents are selected from 1 to 5 halo, 1 to 3 hydroxy, 1 to 3
C1-C10 alkanoyloxy, 1 to 3 Ci-Cρ alkoxy, phenyl, phenoxy, 2-furyl, Ci-Cβ alkoxycarbonyl, -S(0) (Cι-C6 alkyl); or R4 and R5 can be taken together to form -(CH2)rLa (CH2)S- where La is -C(R2)2-, -0-, -S(0)m-, or -N(R2)-, where r and s are independently 1 to 3 and R2 is as defined above;
R6 is hydrogen or Cχ-C6 alkyl;
A is: ?7
— (CH2) -C — (CH2)V-
R 7a or
- Z-(CH2)X-C — (CH2)V
R 7a
wherein x and y are independently 0-3; Z is N-R2 or 0; R7 and R7a are independently selected from the group consisting of: hydrogen, -Cχ-C6 alkyl, -OR2, trifluoromethyl, phenyl, substituted Cχ-C6 alkyl where the substituents are selected from imidazolyl, phenyl, indolyl, p-hydroxyphenyl, -OR2, 1 to 3 fluoro, -S(0)mR2, -C(0)OR2, -C3- C7 cycloalkyl, -N(R2)(R2), -C(0)N(R2)(R2); or R7 and R7a can independently be joined to one or both of R4 and R5 groups to form alkylene bridges between the terminal nitrogen and the alkyl portion of the R7 or R7a groups, wherein the bridge contains 1 to 5 carbons atoms;
B, D, E, and F are independently selected from the group consisting of: -C(R8)(RlθK -0-, C=0, -S(0)m-, or -NR9-, such that one or two of B, D, E, or F may be optionally absent to provide a 5, 6, or 7 membered ring; and provided that B, D, E and F can be -C(Rs)(Rlθ)- or C=0 only when one of the remaining B, D, E and F groups is simultaneously -0-, -S(0)m-, or - NR9-, or B and D, or D and E taken together may be -N=CRχo- or -CRio=N-, or B and D, or D and E taken together may be -CR8=CRιo-, provided one of the other of B and E or F is simultaneously -0-, -S(0)m-, or -NR9-;
R8 and Rχo are independently selected from the group consisting of: hydrogen, -R2, -OR2, (-CH2)q-aryl, -(CH2)q-C(0)OR2, -(CH2)q- C(0)0(CH2)q-aryl, or -(CH2)q-(lH-tetrazol-5-yl), where the aryl may be optionally substituted by 1 to 3 halo, 1 to 2 Ci-Cδ alkyl, 1 to 3 -OR2 or 1 to 2 -C(0)OR2;
R9 is selected from the group consisting of:
-R2, -(CH2)q-aryl, -C(0)R2, -C(0)(CH2)q-aryl, -S02R2,
-Sθ2(CH2)q-aryl, -C(0)N(R2)(R2), -C(0)N(R2)(CH2)q-aryl, -C(0)OR2, 1-H- tetrazol-5-yl, -SO3H, -Sθ2NHC≡N, -Sθ2N(R2)aryl, -Sθ2N(R2)(R2X and wherein the (CH2)q may be optionally substituted by 1 to 2 C1-C4 alkyl, and the R2 and aryl may be optionally further substituted by 1 to 3 - OR2a, -0(CH2)q aryl, 1 to 2 -C(0)OR2a, 1 to 2 -C(0)0(CH2)q aryl, 1 to 2 - C(0)N(R2aXR2a), 1 o 2 -C(0)N(R2a)(CH2)q aryl, 1 to 5 halogen, 1 to 3 Oχ- C4 alkyl, 1,2,4-triazolyl, l-H-tetrazol-5-yl, -C(0)NHS02R2a, -S(0)mR2a> - C(0)NHS02(CH2)q-aryl, -Sθ2NHC≡N, -Sθ2NHC(0)R2a, - S02NHC(0)(CH2)qaryl, -N(R2)C(0)N(R2a)(R2a), - N(R2a)C(0)N(R2a)(CH2)q-aryl, -N(R2a)(R2a), -N(R2a)C(0)R2a, - N(R2a)C(0)(CH2)q aryl, -OC(0)N(R2a)(R2a), -OC(0)N(R2a)(CH2)q aryl, - Sθ2(CH2)qCONH-(CH2)wNHC(0)Rn, wherein w is 2-6 and Rχι may be biotin, aryl, or aryl substituted by 1 or 2 OR2, 1-2 halogen, azido or nitro;
m is 0, 1 or 2; n is 1, or 2; q may optionally be 0, 1, 2, 3, or 4; and
G, H, I and J are carbon, nitrogen, sulfur or oxygen atoms, such that at least one is a heteroatom and one of G, H, I or J may be optionally missing to afford a 5 or 6 membered heterocyclic aromatic ring; and pharmaceutically acceptable salts and individual diastereomers thereof.
3. The combination of Claim 1, wherein the growth hormone secretagogue is of the Formula V:
Figure imgf000127_0001
V wherein:
Rl is selected from the group consisting of:
Figure imgf000127_0002
R3a is H, or fluoro;
D is selected from the group consisting of: -0-, -S-, -S(0)m-, N(R2), NS02(R2), NSθ2(CH2)taryl, NC(0)(R2), NS02(CH2)qOH, NS02(CH2)qCOOR2, NS02(CH2)qC(0)-N(R2)(R2), N- Sθ2(CH2)qC(0)-N(R2)(CH2)wOH,
Figure imgf000128_0001
N-S02(CH2)qC(0)-N(R2)(CH2)w.
Figure imgf000128_0002
N-NH N-S02(CH2)q-π/ I
N=N '
and the aryl is phenyl or pyridyl and the phenyl may be substituted by 1-2 halogen;
R2 is H, or C1-C4 alkyl; m is 1 or 2; t is 0, 1, or 2; q is 1, 2, or 3; w is 2, 3, 4, 5, or 6; and the pharmaceutically acceptable salts and individual diastereomers thereof.
4. The combination of Claim 1, wherein the growth hormone secretagogue is selected from the group consisting of:
1) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(lH-indol-3-yl)ethyl]-2-amino-2-methyl- propanamide; 2) N-[l(R)-[(l,2-dihydro-l-methanecarbonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(lH-indol-3-yl)ethyl]-2-amino-2-methyl- propanamide;
3) N-[l(R)-[(l,2-dihydro-l-benzenesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(lH-indol-3-yl)ethyl]-2-amino-2-methyl- propanamide;
4) N-[l(R)-[(3,4-dihydro-spiro[2H-l-benzopyran-2,4'-piperidin]-l'-yl) carbonyl]-2-(lH-indol-3-yl)ethyl]-2-amino-2-methylpropanamide;
5) N-[l(R)-[(2-acetyl-l,2,3,4-tetrahydrospiro[isoquinolin-4,4'-piperidin]- l'-yl)carbonyl]-2-(indol-3-yl)ethyl]-2-amino-2-methyl-propanamide;
6) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2- methylprop anamide ;
7) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2- methylpropanamide methanesulfonate;
8) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(2',6'-difluorophenylmethyloxy)ethyl]-2- amino-2-methylpropanamide;
9) N-[l(R)-[(l,2-dihydro-l-methanesulfonyl-5-fluorosρiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2- methylpropanamide;
10) N-[l(S)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(phenylmethylthio)ethyl]-2-amino-2- methylpropanamide; 11) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-3-phenylpropyl]-2-amino-2-methyl- propanamide;
12) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-3-cyclohexylpropyl]-2-amino-2-methyl- propanamide;
13) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]- l'-yl)carbonyl]-4-phenylbutyl]-2-amino-2-methyl- propanamide;
14) N-[l(R)-[(l,2-dihydro-l-methanesulfonylspiro[3H-indole-3,4'- piperidin]-l'-yl)carbonyl]-2-(5-fluoro-lH-indol-3-yl)ethyl]-2-amino-2- methylpropanamide;
15) N-[l(R)-[(l,2-dihydro-l-methanesulfonyl-5-fluorospiro[3H-indole-3,4' piperidin]-l'-yl)carbonyl]-2-(5-fluoro-lH-indol-3-yl)ethyl]-2-amino-2- methylpr op anamide ;
16) N-[l(R)-[(l,2-dihydro-l-(2-ethoxycarbonyl)methylsulfonylspiro-[3H- indole-3,4'-piperidin]-l'-yl)carbonyl]-2-(lH-indol-3-yl)ethyl]-2-amino-2- methylpropanamide;
17) N-[l(R)-[(l,2-dihydro-l,l-dioxospiro[3H-benzothiophene-3,4'- piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2- methylpropanamide; and
18) l-[2(R)-(2-Amino-2-methylpropionylamino)-3-(lH-indol-3- yl)propionyl]-3(S)-benzyl-piperidine-3-carboxylic acid ethyl ester;
or a pharmaceutically acceptable salt thereof.
5. The combination of Claim 4, wherein the growth hormone secretagogue is selected from: N-[l(R)-[(l,2-dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperidin]- l'-yl)carbonyl]-2-(phenylmethyloxy)-ethyl]-2-amino-2- methylpropanamide; or
l-[2(R)-(2-Amino-2-methylpropionylamino)-3-(lH-indol-3-yl)propionyl]- 3(S)-benzyl-piperidine-3-carboxylic acid ethyl ester;
or a pharmaceutically acceptable salt thereof.
6. The combination of Claim 5, wherein the growth hormone secretagogue is N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)- ethyl]-2-amino-2-methylpropanamide methanesulfonate.
7. The combination of Claim 1, wherein the αvβ3 antagonist is a compound of the formula
Figure imgf000131_0001
wherein X is a 9- to 10-membered polycyclic ring system, wherein one or more of the rings is aromatic, and wherein the polycyclic ring system contains 0, 1, 2, 3 or 4 heteroatoms selected from N, O or S, and wherein the polycyclic ring system is either unsubstituted or substituted on a carbon atom with Rl and R^;
Y is selected from
429- R^ O
I R°
■N-(CH2): m -(CH2)-N- , -C-(CH2fe- ,
Figure imgf000132_0001
or - (CH2)r- ;
Figure imgf000132_0002
Z is a 5-11 membered aromatic or nonaromatic mono- or polycyclic ring system containing 0 to 6 double bonds, and containing 0 to 6 heteroatoms chosen from N, O and S, and wherein the ring system is either unsubstituted or substituted on a carbon or nitrogen atom with one or more groups independently selected from R4, R5, R6 and R7; provided that Z is not a 6-membered monocyclic aromatic ring system;
Rl, R2, R4; R5^ R13 ancι R14 are each independently selected from hydrogen, halogen, Cl-lθ alkyl, C3-8 cycloalkyl, aryl, aryl Cl-8 alkyl, amino, amino Cl-8 alkyl, Cl-3 acylamino, Cl-3 acylamino Ci-8 alkyl, Cl-6 alkylamino, Cl-6 alkylamino- Ci-8 alkyl, Ci-6 dialkylamino, Cl-6 dialkylamino Cl-8 alkyl,
Ci-4 alkoxy, Cl-4 alkoxy Cl-6 alkyl, hydroxycarbonyl, hydroxycarbonyl Cl-6 alkyl, Cl-3 alkoxycarbonyl, Cl-3 alkoxycarbonyl Cl-6 alkyl, hydroxycarbonyl- Ci-6 alkyloxy, hydroxy, hydroxy Cl-6 alkyl, Cl-6 alkyloxy- Cl-6 alkyl, nitro, cyano, trifluoromethyl, trifluoromethoxy, trifluoroethoxy, Cl-8 alkyl-S(0)q, Cl-8 aminocarbonyl, Cl-8 dialkylaminocarbonyl, Cl-8 alkyloxycarbonylamino, Cl-8 alkylaminocarbonyloxy or Cl-8 alkylsulfonylamino;
R3 is selected from hydrogen, aryl, -(CH2)p-aryl, hydroxyl, Ci-5 alkoxycarbonyl, aminocarbonyl, C3-8 cycloalkyl, amino Ci-6 alkyl, arylaminocarbonyl, aryl Cl-5 alkylaminocarbonyl, hydroxycarbonyl Ci-6 alkyl, Cl-8 alkyl, aryl Cl-6 alkyl, Cl-6 alkylamino Cl-6 alkyl, aryl Cl-6 alkylamino Cl-6 alkyl, Ci-6 dialkylamino Ci-6 alkyl, Ci-8 alkylsulfonyl, Ci-8 alkoxycarbonyl, aryloxycarbonyl, aryl Cl-8 alkoxycarbonyl, Cl-8 alkylcarbonyl, arylcarbonyl, aryl Cl-6 alkylcarbonyl, Ci-8 alkylaminocarbonyl, amino sulfonyl,
Cl-8 alkylaminosulfonyl, arylaminosulfonylamino, aryl Cl-8 alkylaminosulfonyl, Cl-6 alkylsulfonyl, aryl sulfonyl, aryl Cl-6 alkylsulfonyl, aryl Cl-6 alkylcarbonyl, Cl-6 alkylthiocarbonyl, arylthiocarbonyl, or aryl Cl-6 alkylthiocarbonyl, wherein any of the alkyl groups may be unsubstituted or substituted with Rl3 and Rl ; R6> R?, R8, R9, RlO and RH are each independently selected from hydrogen, aryl, -(CH2)p-aryl, halogen, hydroxyl,
Cl-8 alkylcarbonylamino, aryl Cl-5 alkoxy, Ci-5 alkoxycarbonyl, aminocarbonyl,
Cl-8 alkylaminocarbonyl,
Cl-6 alkylcarbonyloxy,
C3-8 cycloalkyl, oxo, amino ,
Cl-6 alkylamino, amino Cl-6 alkyl, arylaminocarbonyl, aryl Ci-5 alkylaminocarbonyl, aminocarbonyl, aminocarbonyl Cl-6 alkyl, hy dr oxy carb onyl , hydroxycarbonyl Cl-6 alkyl,
Cl-8 alkyl, either unsubstituted or substituted, with one or more groups selected from: halogen, hydroxyl,
Cl-5 alkylcarbonylamino, aryl Cl-5 alkoxy, Cl-5 alkoxycarbonyl, aminocarbonyl, Cl-5 alkylaminocarbonyl, Cl-5 alkylcarbonyloxy, C3-8 cycloalkyl, oxo, amino, Cl-3 alkylamino, amino Cl-3 alkyl, arylamino- carbonyl, aryl Ci-5 alkylaminocarbonyl, aminocarbonyl, aminocarbonyl Cl-4 alkyl, hydroxycarbonyl, or hydroxycarbonyl Cl-5 alkyl, -(CH2)s C≡CH, -(CH2)s C≡C-C 1-6 alkyl, -(CH2)s C≡C-C3-7 cycloalkyl, -(CH2)s C≡C-aryl, -(CH2)s C≡C-Cl-6 alkylaryl, -(CH2)S CH=CH2, -(CH2)s CH=CH Cl-6 alkyl,
-(CH2)S CH=CH-C3-7 cycloalkyl, -(CH2)s CH=CH aryl, -(CH2)s CH=CH Cl-6 alkylaryl, -(CH2)s SO2C1-6 alkyl, or -(CH2)s SO2C1-6 alkylaryl;
Cl-6 alkoxy, aryl Cl-6 alkoxy, aryl Ci-6 alkyl, Cl-6 alkylamino Cl-6 alkyl, arylamino, arylamino Cl-6 alkyl, aryl Ci-6 alkylamino, aryl Cl-6 alkylamino Cl-6 alkyl, arylcarbonyloxy, aryl Cl-6 alkylcarbonyloxy,
Cl-6 dialkylamino, Cl-6 dialkylamino Cl-6 alkyl, Cl-6 alkylaminocarbonyloxy, Cl-8 alkylsulfonylamino, Cl-8 alkylsulfonylamino Cl-6 alkyl, arylsulfonylamino Cl-6 alkyl, aryl Cl-6 alkylsulfonylamino, aryl Cl-6 alkylsulfonylamino Cl-6 alkyl, Cl-8 alkoxycarbonylamino, Cl-8 alkoxycarbonylamino Cl-8 alkyl, aryloxycarbonylamino Cl-8 alkyl, aryl Cl-8 alkoxycarbonylamino, aryl Cl-8 alkoxycarbonylamino Cl-8 alkyl, Cl-8 alkylcarbonylamino, Ci-8 alkylcarbonylamino Ci-6 alkyl, arylcarbonylamino Cl-6 alkyl, aryl Ci-6 alkylcarbonylamino, aryl Ci-6 alkylcarbonylamino Cl-6 alkyl, aminocarbonylamino Ci-6 alkyl,
Ci-8 alkylaminocarbonylamino, Ci-8 alkylaminocarbonylamino Ci-6 alkyl, arylaminocarbonylamino Ci-6 alkyl, aryl Ci-8 alkylaminocarbonylamino, aryl Ci-8 alkylaminocarbonylamino Ci-6 alkyl, amino sulfonyl amino Ci- alkyl, Ci-8 alkylaminosulfonylamino, Ci-8 alkylaminosulfonylamino Cι_6 alkyl, arylamino sulfonylamino Ci-6 alkyl, aryl Ci-8 alkylaminosulfonylamino, aryl Ci-8 alkylaminosulfonylamino Ci-6 alkyl,
Ci-6 alkylsulfonyl,
Ci-6 alkylsulfonyl Cl-6 alkyl, arylsulfonyl Ci-6 alkyl, aryl Ci-6 alkylsulfonyl, aryl Cl-6 alkylsulfonyl Ci-6 alkyl, Ci-6 alkylcarbonyl, Ci-6 alkylcarbonyl Cl-6 alkyl, arylcarbonyl Ci-6 alkyl, aryl Ci-6 alkylcarbonyl, aryl Ci-6 alkylcarbonyl Ci-6 alkyl, Ci-6 alkylthiocarbonylamino, Ci-6 alkylthiocarbonylamino Cl-6 alkyl, arylthiocarbonylamino Ci-6 alkyl, aryl Ci-6 alkylthiocarbonylamino, aryl Ci-6 alkylthiocarbonylamino Ci-6 alkyl, Ci-8 alkylaminocarbonyl Ci-6 alkyl, arylaminocarbonyl Ci-6 alkyl, aryl Ci-8 alkylaminocarbonyl, or aryl Ci-8 alkylaminocarbonyl Ci-6 alkyl, wherein any of the alkyl groups may be unsubstituted or substituted with Rl3 and Rl4; and provided that the carbon atom to which R^ and R^ are attached is itself attached to no more than one heteroatom; and provided further that the carbon atom to which Rlυ and RU are attached is itself attached to no more than one heteroatom;
Rl2 is selected from hydrogen, Ci-8 alkyl, aryl, aryl Ci-8 alkyl, hydroxy, Ci-8 alkoxy, aryloxy, aryl Ci-6 alkoxy,
Ci-8 alkylcarbonyloxy Ci-4 alkoxy, aryl Cι_8 alkylcarbonyloxy Cl-4 alkoxy, Ci-8 alkylaminocarbonylmethyleneoxy, or Ci-8 dialkylaminocarbonylmethyleneoxy; m is an integer from 0 to 3; n is an integer from 1 to 3; p is an integer from 1 to 4; q is an integer from 0 to 2; r is an integer from 0 to 6; and s is an integer from 0 to 3; and the pharmaceutically acceptable salts thereof.
8. The combination of Claim 7, wherein the αvβ3 antagonist is the compound where Z is selected from
Figure imgf000138_0001
Figure imgf000138_0002
and the pharmaceutically acceptable salts thereof.
9. The combination of Claim 8, wherein the αvβ3 antagonist is a compound of the formula
Figure imgf000139_0001
wherein X is selected from
Figure imgf000139_0002
Y is selected from -(CH2)r- or -(CH2)m-NR3-; R3 is selected from hydrogen, -(CH2)p-aryl, Cl-5 alkoxycarbonyl, C3-8 cycloalkyl, arylaminocarbonyl, aryl Ci-5 alkylaminocarbonyl,
Ci-8 alkyl, aryl Ci-6 alkyl, Cl-8 alkylsulfonyl, arylsulfonyl, aryl Ci-6 alkylsulfonyl, Cl-8 alkoxycarbonyl, aryloxy carb onyl , aryl Cl-8 alkoxycarbonyl, Cl-8 alkylcarbonyl, arylcarbonyl, aryl Ci-6 alkylcarbonyl,
Cl-8 alkylaminocarbonyl ,
Cl-6 alkylsulfonyl, or aryl Ci-6 alkylcarbonyl, wherein any of the alkyl groups may be unsubstituted or substituted with Rl3 and R14; and r is an integer from 0 to 3; and the pharmaceutically acceptable salts thereof.
10. The combination of Claim 9, wherein the αvβ3 antagonist is a compound of the formula
Figure imgf000140_0001
wherein Z is selected from
Figure imgf000140_0002
R is selected from hydrogen,
Figure imgf000140_0003
-(CH2)p indolyl, -(CH2)S C≡CH, -(CH2)S C≡C-C 1-6 alkyl, -(CH2)s C≡C-C3-7 cycloalkyl, -(CH2)s C≡C-aryl, -(CH2)s C≡C-Cl-6 alkyl aryl, -(CH2)s CH=CH2, -(CH2)s CH=CH Cl-6 alkyl, -(CH2)s CH=CH-C3-7 cycloalkyl,
-(CH2)s CH=CH aryl,
-(CH2)s CH=CH Cl-6 alkyl aryl,
-(CH2)s S02Ci-6 alkyl, or -(CH2)s SO2C1.6 alkylaryl; l2 is selected from hydrogen or Ci-8 alkyl; and s is an integer from 0 to 3; and the pharmaceutically acceptable salts thereof.
11. The combination of Claim 10, wherein the αvβ3 antagonist is selected from
2-Oxo-3-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]piperidin-l-yl- acetyl-3(S)-pyridin-3-yl-β-alanine ethyl ester;
2-Oxo-3-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]piperin- l-yl-acetyl-3(S)-pyridin-3-yl-β-alanine trifluoroacetate;
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-alkynyl-β-alanine ethyl ester;
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-alkynyl-β-alanine;
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]-pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl- β-alanine ethyl ester;
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-pvridin-3-yl-β-alanine;
2-Oxo-3(R)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-alkynyl-β-alanine ethyl ester; 2-Oxo-3(R)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-alkynyl-β-alanine;
2-Oxo-3(R)-[2-(5,6,7,8-tetrahydro[l,8]-naphthvridin-2-yl)ethyl]-pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine ethyl ester;
2-Oxo-3(R)-[2-(5,6,7,8-tetrahydro[l,8]-naphthvridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine;
Ethyl 2-oxo-3-[2-(5,6,7,8-tetrahydro[l,8]naphthyridin-2-yl)ethyl]- tetrahydropyrimidin-l-yl-acetyl-3(S)-pyridin-3-yl-β-alanine;
2-Oxo-3-[2-(5,6,7,8-tetrahydro[l,8]naphthyridin-2-yl}ethyl]- tetrahydropyrimidin-l-yl-acetyl-3(S)-pyridin-3-yl-β-alanine;
Ethyl 2-oxo-3-[2-(5,6,7,8-tetrahydro[l,8]naphthyridin-2- yl)ethyl]imidazolidin-l-yl-acetyl-3(S)-pyridin-3-yl-β-alanine;
2-Oxo-3-[2-(5,6,7,8-tetrahydro[l,8]naphthyridin-2-yl}ethyl]-imidazolidin- l-yl-acetyl-3(S)-pyridin-3-yl-β-alanine;
Ethyl 2-oxo-3(R)-[2-(5,6,7,8-tetrahydro[l,8]naphthyridin-2- yl)ethyl]pyrrolidin-l-yl)acetyl-3(R)-(2-ethylindol-3-yl)-β-alanine;
2-Oxo-3(R)-[2-(5,6,7,8-tetrahydro[l,8]naphthyridin-2-yl)ethyl]pyrrolidin-l- yl)acetyl-3(R)-(2-ethylindol-3-yl)-β-alanine;
Ethyl 3-(S)-(2-{2-oxo-3-[(5,6,7,8-tetrahydro-[l,8]naphthyridin-2-ylmethyD- amino]-pyrrolidin-l-yl}-acetylamino)-3-(R)-pyridin-3-yl-propionic acid;
3-(S)-(2-{2-Oxo-3-[(5,6,7,8-tetrahydro-[l,8]naphthyridin-2-ylmethyD- amino]pyrrolidin-l-yl}-acetylamino)-3-(R)-pyridin-3-yl-propionic acid;. 3-{2-[6-Oxo-l-(5,6,7,8-tetrahydro-[l,8]naphthyridin-2-ylmethyl)- hexahydro-(3aS, 6aS)pyrrolo[3,4-b]pyrrol-5-yl]-acetylamino}-3-(S)-pyridin- 3-yl-propionic acid; or
3-{2-[6-Oxo-l-(5,6,7,8-tetrahydro-[l,8]naphthyridin-2-ylmethyl)- hexahydro-(3aR, 6aR)pyrrolo[3,4-b]pyrrol-5-yl]-acetylamino}-3-(S)- pyridin-3-yl-propionic acid;
and the pharmaceutically acceptable salts thereof.
12. The combination of Claim 1, wherein the αϊDβ3 antagonist is selected from
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]-pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine ethyl ester;
2-Oxo-3(S)-[2-(5,6,7,8-tetrahydro[l,8]-naphthyridin-2-yl)ethyl]pyrrolidin- l-yl)acetyl-3(S)-pyridin-3-yl-β-alanine; or
4-[2-(l,2,3,4-Tetrahydro-l,8-naphthyridin-7-yl)ethyl]benzoyl- 2(S)-[l(S)10-camphorsulfonylamino] β-alanine; or a pharmaceutically acceptable salt thereof.
13. The combination of Claim 9, wherein the growth hormone secretagogue is selected from:
N-[l(R)-[(l,2-dihydro-l-methanesulfonyl-spiro[3H-indole-3,4'-piperidin]- l'-yl)carbonyl]-2-(phenylmethyloxy)-ethyl]-2-amino-2- methylpropanamide; or
l-[2(R)-(2-Amino-2-methylpropionylamino)-3-(lH-indol-3-yl)propionyl]- 3(S)-benzyl-piperidine-3-carboxylic acid ethyl ester;
or a pharmaceutically acceptable salt thereof.
14. The combination of Claim 13, wherein the growth hormone secretagogue is N-[l(R)-[(l,2-dihydro-l-methanesulfonyl- spiro[3H-indole-3,4'-piperidin]-l'-yl)carbonyl]-2-(phenylmethyloxy)- ethyl]-2-amino-2-methylpropanamide methanesulfonate.
15. A pharmaceutical composition which comprises the combination of Claim 1 and a pharmaceutically acceptable carrier.
16. A pharmaceutical composition made by combining the combination of Claim 1 and a pharmaceutically acceptable carrier.
17. A process for making a pharmaceutical composition comprising combining an αvβ3 antagonist, a growth hormone secretagogue and a pharmaceutically acceptable carrier.
18. A method of treating a disease involving bone resorption which comprises administering to a patient in need of such treatment a therapeutically effective amount of a growth hormone secretagogue and an αvβ3 antagonist.
19. The method of Claim 18 wherein the disease is osteoporosis.
20. A method of treating a disease involving bone resorption which comprises administering to a patient in need of such treatment a therapeutically effective amount of the pharmaceutical composition of Claim 15.
21. The method of Claim 20 wherein the disease is osteoporosis.
PCT/US1998/007065 1997-04-14 1998-04-09 Combination therapy for the prevention and treatment of osteoporosis WO1998046220A1 (en)

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