GB2327609A - A method for eliciting an avß5 or dual avß3/avß5 antagonizing effect - Google Patents

A method for eliciting an avß5 or dual avß3/avß5 antagonizing effect Download PDF

Info

Publication number
GB2327609A
GB2327609A GB9815165A GB9815165A GB2327609A GB 2327609 A GB2327609 A GB 2327609A GB 9815165 A GB9815165 A GB 9815165A GB 9815165 A GB9815165 A GB 9815165A GB 2327609 A GB2327609 A GB 2327609A
Authority
GB
United Kingdom
Prior art keywords
alkyl
aryl
alanine
amino
mmol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
GB9815165A
Other versions
GB9815165D0 (en
Inventor
Mark E Duggan
George D Hartman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck and Co Inc
Original Assignee
Merck and Co Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9800961.6A external-priority patent/GB9800961D0/en
Priority claimed from GBGB9808017.9A external-priority patent/GB9808017D0/en
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of GB9815165D0 publication Critical patent/GB9815165D0/en
Publication of GB2327609A publication Critical patent/GB2327609A/en
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4402—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 2, e.g. pheniramine, bisacodyl
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

A method for eliciting an α v # 5 or dual α v # 3 /α v # 5 antagonizing effect in a mammal which comprises administering to the mammal a therapeutically effective amount of a compound of the formula for example which are useful for inhibiting restenosis, angiogenesis, atherosclerosis, diabetic retinopathy, macular degeneration, inflammation or tumor growth.

Description

TITLE OF THE INVENTION A METHOD FOR ELICITING AN avsss OR DUAL av(33/otvPS ANTAGONIZING EFFECT BACKGROUND OF THE INVENTION The invention relates generally to avssS antagonists useful for inhibiting vascular restenosis, diabetic retinopathy, macular degeneration, angiogenesis, atherosclerosis, inflammation and tumor growth.
The method of the invention can inhibit neovascularization by acting as antagonists of the integrin receptor avssS. A monoclonal antibody for αvss5 has been shown to inhibit VEGF-induced angiogenesis in rabbit cornea and the chick chorioallantoic membrane model; M.C. Friedlander, et. al., Science 270, 1500-1502, 1995. Thus, compounds that antagonize avss5 are useful for treating and preventing macular degeneration, diabetic retinopathy, tumor growth and for inhibiting vascular restenosis, angiogenesis, and inflammation.
SUMMARY OF THE INVENTION The invention is a method for eliciting an avssS or dual αvss3/αvss5 antagonizing effect in a mammal which comprises administering to the mammal a therapeutically effective amount of a compound of the formula:
and pharmaceutically acceptable salts thereof, wherein Xis a 5- or 6-membered monocyclic partially or fully saturated ring system containing 0, 1, 2, 3 or 4 heteroatoms selected from N, 0 and S and either unsubstituted or substituted with R1 or R2, or a 9- to 10-membered polycyclic ring system, wherein one or more of the rings is partially or fully saturated, containing 0, 1, 2, 3 or 4 heteroatoms selected from N, 0 and S and either unsubstituted or substituted with R1 or R2, wherein R1 and R2 are independently selected from the group consisting of hydrogen, F, Cl, Br, I, C1-10 alkyl, C3-8 cycloalkyl, aryl, aryl C1-8 alkyl, amino, amino C1-8 alkyl, Cl -3 acylamino, C1-3 acylamino C1-8 alkyl, C1-6 alkylamino, Cl-6 alkylamino C1-8 alkyl, Cl-6 dialkylamino, C1-6 dialkylamino C1-8 alkyl, C14 alkoxy, C14 alkoxy C1-6 alkyl, carboxy, carboxy C1-6 alkyl, C1-3 alkoxycarbonyl, C1-3 alkoxycarbonyl C1-6 alkyl, carboxy C1-6 alkyloxy, hydroxy, and hydroxy C1 6 alkyl; Yis
where Z is O, NR8, or S; and R8 is defined as R1 above; R3 and R4 are independently hydrogen, a five or six membered mono or nine or ten membered polycyclic partially or fully saturated ring system containing 0, 1, 2, 3, or 4 heteroatoms selected from nitrogen, oxygen and sulfur, either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, Cl-3 alkoxy, C1-5 alkylcarbonyloxy, CI.5 alkoxycarbonyl, C1-5 alkyl, aminoCi-5 alkyl, hydroxycarbonyl, hydroxycarbonylC1-5 alkyl, or hydroxycarbonylCi 5 alkoxy, -(CH2)n-aryl, wherein n=14 and aryl is defined as a five or six membered unsaturated or partially saturated monocyclic ring system, or nine or ten membered polycyclic ring system wherein at least one of the rings is unsaturated or partially saturated and each of the other rings may be unsaturated, partially saturated or fully saturated, said aryl group containing 0, 1, 2, 3, or 4 heteroatoms selected from nitrogen, oxygen and sulfur, either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, C1-3 alkoxy, C1-5 alkylcarbonyloxy, C1-5 alkoxycarbonyl, C1 5 alkyl, aminoC 1-5 alkyl, hydroxycarbonyl, hydroxycarbonylC 1-5 alkyl, or hydroxycarbonylCl S alkoxy, halogen, hydroxyl, C1-5alkylcarbonylamino, arylC1-5 alkoxy, C1-5 alkoxycarbonyl, aminocarbonyl, C1-5 alkylaminocarbonyl, C1-5 alkylcarbonyloxy, C3-8 cycloalkyl, oxo, amino, Cl -3 alkylamino, aminoC 1-3 alkyl, arylaminocarbonyl, arylC1-5alkylaminocarbonyl, aminocarbonyl, aminocarbonyl-C I -4 alkyl, hydroxycarbonyl, hydroxycarbonyl C1-5 alkyl, C1-6alkyl, either unsubstituted or substituted, with one or more groups selected from halogen, hydroxyl, C1-5alkylcarbonylamino, arylC1-5 alkoxy, C1-5 alkoxycarbonyl, aminocarbonyl, C1-5 alkylaminocarbonyl, C1-5 alkylcarbonyloxy, C3-8 cycloalkyl, oxo, amino, C 1-3 alkylamino, aminoC 1-3 alkyl, arylaminocarbonyl, arylC 1-5 alkylaminocarbonyl, aminocarbonyl, aminocarbonyl-Cl 4 alkyl, hydroxycarbonyl, or hydroxycarbonyl C1 5 alkyl, provided that the carbon atom to which R3 and R4 are attached bears only one heteroatom, -(CH2)m C#CH, -(CH2)m C#C-C1-6 alkyl, -(CH2)m C#C-C3-7cycloalkyl, -(CH2)m C#C- aryl, -(CH2)m C#C-C1-6 alkyl aryl, -(CH2)m CH=CH2, -(CH2)m CH=CH C1-6 alkyl, -(CH2)m CH=CH-C3 jcycloalkyl, -(CH2)m CH=CH aryl, -(CH2)m CH=C-H C1-6 alkyl aryl, -(CH2)m SO2C1-6 alkyl, or -(CH2)m SO2C1-6 alkylaryl; R5 is hydrogen, fluorine, C1-8 alkyl, hydroxyl, hydroxy C1-6 alkyl, carboxy, carboxy C1-6 alkyl, C14 alkyloxy.
C3-8 cycloalkyl, aryl Cl -6 alkyloxy, aryl, aryl C1-6 alkyl, Cl -6 alkylcarbonyloxy, amino, C1-6 alkylamino, amino C1-6 alkyl, C1-6 alkylamino C1-6 alkyl, aryl amino, aryl amino C1-6 alkyl, aryl C1-6 alkylamino, aryl C1-6 alkylamino C1-6 alkyl, aryl carbonyloxy, aryl C1-6 alkylcarbonyloxy, C1-6 dialkylamino, C1-6 dialkylamino C1-6 alkyl, C 1-6 alkylaminocarbonyloxy, C1-8 alkylsulfonylamino, C1-8 alkylsulfonylamino C1 6 alkyl, aryl sulfonylamino C 1-6 alkyl, aryl sulfonylamino, aryl C1-6 alkylsulfonylamino, aryl C1-6 alkylsulfonylamino C1-6 alkyl, C 1-8 alkyloxycarbonylamino, C1-8 alkyloxycarbonylamino C1-8 alkyl, aryl C1-8 alkyloxycarbonylamino, aryl oxycarbonylamino, aryl oxycarbonylamino C1-8 alkyl, aryl C1-8 alkyloxycarbonylamino C1-8 alkyl, C1-8 alkylcarbonylamino, C1-8 alkylcarbonylamino C1-6 alkyl, aryl carbonylamino C1-6 alkyl, aryl carbonylamino, aryl C1-6 alkylcarbonylamino, aryl C1-6 alkylcarbonylamino C1-6 alkyl, C1-8 alkylaminocarbonylamino, aminocarbonylamino, aminocarbonylamino C1-6 alkyl, C1-8 alkylaminocarbonylamino C1-6 alkyl, aryl aminocarbonylamino C1-6 alkyl, aryl aminocarbonylamino, aryl C1-8 alkylaminocarbonylamino, aryl C1-8 alkylaminocarbonylamino Cl -6 alkyl, aminosulfonylamino Cl -6 alkyl, aminosulfonylamino, C1-8 alkylaminosulfonylamino, C1-8 alkylaminosulfonylamino C1-6 alkyl, aryl aminosulfonylamino C1-6 alkyl, aryl aminosulfonylamino, aryl C1-8 alkylaminosulfonylamino, aryl C1-8 alkylaminosulfonylamino C1 6 alkyl, C1-6 alkylsulfonyl, C1-6 alkylsulfonyl C1-6alkyl, aryl sulfonyl, aryl sulfonyl C1-6alkyl, aryl alkylsulfonyl, aryl Cl -6 alkylsulfonyl, aryl C 1-6 alkylsulfonyl Cl -6alkyl, C1-6 alkylcarbonyl, C1-6 alkylcarbonyl C1-6 alkyl, aryl carbonyl C1-6alkyl, aryl carbonyl, aryl C1-6 alkylcarbonyl, aryl C1-6 alkylcarbonyl Cl-6alkyl, C 1-6 alkylthiocarbonylamino, C1-6 alkylthiocarbonylamino C 1-6 alkyl, aryl thiocarbonylamino C1-6 alkyl, aryl thiocarbonylamino, aryl C1-6 alkylthiocarbonylamino, aryl C1-6 alkylthiocarbonylamino C1-6 alkyl, aminocarbonyl C1-6 alkyl, aminocarbonyl, C 1-8 alkylaminocarbonyl, C1-8 alkylaminocarbonyl C1-6 alkyl, aryl aminocarbonyl C1-6 alkyl, aryl aminocarbonyl, aryl C1-8 alkylaminocarbonyl, aryl C1-8 alkylaminocarbonyl C1-6 alkyl, wherein alkyl groups and aryl groups may be unsubstituted or substituted with one or more substituents selected from R1 and R2; and R6, R7, and R9 are independently hydrogen, C1-8 alkyl, aryl, aryl C1-8 alkyl, hydroxy, C1-8 alkyloxy, aryloxy, aryl C1-6 alkyloxy, C1-8 alkylcarbonyloxy C1-4 alkyloxy, aryl C1-8 alkylcarbonyloxy C1-4 alkyloxy, Cl.8 alkylaminocarbonylmethyleneoxy, or C1-8 dialkylaminocarbonylmethyleneoxy, and wherein m and n are integers 0-6.
The invention is also a method for inhibiting a condition selected from the group conistsing of restenosis, angiogenesis, diabetic retinopathy, macular degeneration, inflammation, and tumor growth in a mammal which comprises administering to the mammal a therapeutically effective amount of a compound defined above.
In a class of methods of the invention, the compounds of the formula are
and pharmaceutically acceptable salts thereof, wherein Xis
wherein n is 2-4, and n' is 2 or 3, and wherein R1 and R2 are independently selected from the group consisting of hydrogen, F, Cl, Br, I, C1-10 alkyl, C3-8 cycloalkyl, aryl, aryl C1-8 alkyl, amino, amino C1-8 alkyl, Cl -3 acylamino, C1-3 acylamino C1-8 alkyl, C1-6 alkylamino, C1-6 alkylamino C1-8 alkyl, C1-6 dialkylamino, C1-6 dialkylamino C1-8 alkyl, C1-4 alkoxy, C1-4 alkoxy C1-6 alkyl, carboxy, carboxy C 1-6 alkyl, C 1-3 alkoxycarbonyl, C 1-3 alkoxycarbonyl C 1-6 alkyl, carboxy C1-6 alkyloxy, hydroxy, and hydroxy C1-6 alkyl.
The invention is also a method for inhibiting a condition selected from the group conistsing of restenosis, angiogenesis, diabetic retinopathy, macular degeneration, inflammation, and tumor growth in a mammal which comprises administering to the mammal a therapeutically effective amount of a compound defined above.
In a subclass of the class of methods described above, the compounds of the formula
and pharmaceutically acceptable salts thereof, wherein X is
wherein n' is 2 or 3, and Yis
where Z is 0, NR8, or S; and R8 is defined as R1.
The invention is also a method for inhibiting a condition selected from the group conistsing of restenosis, angiogenesis, diabetic retinopathy, macular degeneration, inflammation, and tumor growth in a mammal which comprises administering to the mammal a therapeutically effective amount of a compound defined above.
In a group of the subclass are methods wherein the compounds have the formula
and pharmaceutically acceptable salts thereof, wherein Xis
wherein R1 and R2 are independently selected from the group consisting of hydrogen or amino, amino C1-8 alkyl; Yis
R8 is hydrogen or aryl CO-8 alkyl; R3 is hydrogen, a six membered monocyclic partially or fully saturated ring system, either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, C1-3 alkoxy, C1-5 alkylcarbonyloxy, C1-5 alkoxycarbonyl, C1-5 alkyl, aminoC1-5 alkyl, hydroxycarbonyl, hydroxycarbonyiC 1-S alkyl, or hydroxycarbonylC1-5 alkoxy, -(CH2)n-aryl, wherein n=1-4 and aryl is defined as a six membered monocyclic unsaturated or partially saturated ring system, either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, C1-3 alkoxy, C1-5 alkylcarbonyloxy, C1-5 alkoxycarbonyl, C1-5 alkyl, aminoCl-S alkyl, hydroxycarbonyl, hydroxycarbonylC 1-S alkyl, or hydroxycarbonylC1-5 alkoxy, C3-8 cycloalkyl, or Cl 6alkyl, either unsubstituted or substituted, with C3-8 cycloalkyl; R4 is hydrogen, -(CH2)n-aryl, wherein n=0-4 and aryl is defined as a six membered monocyclic unsaturated or partially saturated ring or a nine or ten membered polycyclic ring system wherein at least one of the rings is unsaturated or partially saturated and each of the other rings may be unsaturated, partially saturated or fully saturated, said aryl group either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, C1-3 alkoxy, C1-5 alkylcarbonyloxy, C1-5 alkoxycarbonyl, C1-5 alkyl, aminoC1-5 alkyl, hydroxycarbonylC0-5 alkyl, or hydroxycarbonylCl.S alkoxy, C1-6alkyl, or -(CH2)o-4 CZECH; R5is hydrogen, aryl sulfonylamino C1-6 alkyl, aryl sulfonylamino, aryl C1-6 alkylsulfonylamino, aryl C1-6 alkylsulfonylamino C1-6 alkyl, C 1-8 alkylsulfonylamino, C1-8 alkylsulfonylamino C1-6 alkyl, aryl sulfonylamino C1-6 alkyl, aryl sulfonylamino, aryl C1-6 alkylsulfonylamino, aryl C1-6 alkylsulfonylamino C1-6 alkyl, aminosulfonylamino C1-6 alkyl, aminosulfonylamino, C1 8 alkylaminosulfonylamino, C1-8 alkylaminosulfonylamino C1-6 alkyl, aryl aminosulfonylamino C1 6 alkyl, aryl aminosulfonylamino, aryl C1-8 alkylaminosulfonylamino, aryl C1-8 alkylaminosulfonylamino C1-6 alkyl, C1-6 alkylsulfonyl, C1-6 alkylsulfonyl C1-6alkyl, aryl sulfonyl C1-6alkyl, aryl sulfonyl, aryl C1-6 alkylsulfonyl, aryl C1-6 alkylsulfonyl C1-6alkyl, wherein alkyl groups and aryl groups may be unsubstituted or substituted with one or more substituents selected from R1 and R2; and R6 is hydrogen, C1-8 alkyl, or aryl, aryl C1-8 alkyl.
The invention is also a method for inhibiting a condition selected from the group conistsing of restenosis, angiogenesis, diabetic retinopathy, macular degeneration, inflammation, and tumor growth in a mammal which comprises administering to the mammal a therapeutically effective amount of a compound defined above.
In a subgroup of the group are methods wherein the compounds have the formula
and pharmaceutically acceptable salts thereof, wherein Xis
Yis
R3 is hydrogen, methyl,
R4is hydrogen, methyl,
R5 is hydrogen, or
R6 is hydrogen, methyl, ethyl, or t-butyl.
The invention is also a method for inhibiting a condition selected from the group conistsing of restenosis, angiogenesis, diabetic retinopathy, macular degeneration, inflammation, and tumor growth in a mammal which comprises administering to the mammal a therapeutically effective amount of a compound defined above.
Specific examples of this subgroup include methods wherein the compounds are 4-(2-Aminothiazol-4-yl)butanoyl-glycyl-2(S)-phenylsulfonamido-j3- alanine t-butyl ester, 4-(2-Aminothiazol-4-yl)butanoyl-glycyl-2(S)-phenylsulfonamido- alanine, 4-(2-Aminothiazol-4-yl)butanoyl-glycyl-3(R)-(2-phenethy1)--alanine methyl ester, 4-(2-Aminothiazol-4-yl)butanoyl-glycyl-3(R)-(2-phenethyl)- -alanine, 5-(2-Pyridylamino)pentanoylglycyl-2(S)-phenylsulfonamido- -alanine ethyl ester, 5-(2-Pyridylamino)pentanoylglycyl-2(S)-phenylsulfonamido- -alanine, 4-(2-Amino-pyridin-6-yl)butanoyl-sarcosine-3(R)-[(2-indol-3-yl)ethyl] p-alanine ethyl ester, 4-(2-Aminopyridin-6-yl)butanoyl-sarcosine-3(R)-[(2-indol-3-yl)ethyl] -alanine, 4-(2-Aminopyridin-6-yl)butanoyl-glycyl-2(S )-phenylsulfonamido-- alanine t-butyl ester, 4-(2-Aminopyridin-6-yl)butanoyl-glycyl-2(S)-phenylsulfonamido-I3- alanine, 4-(Pyridin-4-yl)butanoyl-sarcosine-3(R)-[2-(indol-3-yl)ethyl]- -alanine ethyl ester, 4-(Pyridin-4-yl)butanoyl-sarcosine-3(R)-[2-(indol-3-yl)ethyl]- -alanine, 4 -(2-Bocamino-pyridin-6-yl)butanoyl -N-cyclopropylglycyl-3 (R)-(2phenethyl)- -alanine ethyl ester, 4-(2-Amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-(2- phenethyl)- -alanine ethyl ester, 4-(2-Amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-(2phenethyl)- -alanine, 4-(Boc-amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-[(2indol-3-yl)ethyl]- -alanine, 4-(2-Amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-[(2-indol3-yl)ethyl]- -alanine, 4-(Bocamino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)methyl- -alanine ethyl ester, 4-(2-Amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-methyl- alanine ethyl ester, 4-(2-Amino-pyridin-6-yl)butanoyl -N-cyclopropylglycyl -3(R)-methyl - - alanine, 4-(Pyridin-4-yl)butanoyl-N-(2-phenylethyl)glycyl-3(R)-(2-phenethyl) (3-alanine ethyl ester, 4-(Pyridin-4-yl)butanoyl-N-(2-phenyl)glycyl-3(R)-(2-phenethyl)- alanine, 4-(2-BOC-Aminopyridin-4-yl)butanoyl-N-(2-phenethyl)glycyl-3(R)methyl- -alanine, 4-(2-Aminopyridin-4-yl)butanoyl-N-(2-phenethyl)glycyl-3(R)-methyl (3-alanine, 4-(Pyridyloxy)butyrate-N-(2-phenethyl)glycyl-3(R)-2-phenethyl- alanine ethyl ester, 4-(Pyridyloxy)butyrate-N-(2-phenethyl)glycyl-3(R)-2-phenethyl- alanine, 3-[(N-Methyl)-N-(4-pyridyl)]aminopropionyl-sarcosine-3(R)-(2 phenethyl)- -alanine ethyl ester, 3-[(N-Methyl)-N-(4-pyridyl)]aminopropionyl-sarcosine-3(R)-(2 phenethyl)-(3-alanine, N- (N'-3-(4-t-Butoxycarbonyl- 1 -piperizinyl)benzoyl)glycyl ) -3(R)- methyl- -alanine benzyl ester, N- [N'-[3-( 1 -Piperazinyl)benzoyl] glycyl] -3 (R)-methyl- -alanine, N-[N'-[3-(4-t-Butoxycarbonyl-1-piperazinyl)benzoyl]glycyl]-3(R)-(2 phenethyl)- -alanine methyl ester, N-[N'-[3-( 1 -Piperazinyl)benzoyl]glycyl]-3(R)-(2-phenethyl)- -alanine, N-[N'-[3 -(4-t-Butoxycarbonyl- 1 -piperazinyl)benzoyl] -N'- (2- phenethyl)glycyl]-3(R)-(2-phenethyl)- -alanine methyl ester, N-[N'-[3-(1-Piperazinyl)benzoyl]-N'-(2-phenethyl)glycyl]-3(R)-(2phenethyl)- -alanine, 4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-yl)butanol-glycyl- -alanine t-butyl ester, 4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-yl)butanoyl-glycyl- alanine, 4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-yl)butanoyl-glycyl-3(S) pyridin-3-yl- -alanine ethyl ester, 4-(1,2,3 ,4-Tetrahydro- 1 ,8-naphthyridin-7-yl)butanoyl-glycyl 3(S)pyridin-3-yl-P-alanine, Ethyl N-pyridin-4-ylisonipectoyl-N-cyclopropylglycine-3(S)-ethynyl (3-alanine, N-Pyridin-ylisonipecotyl -N-cyclopropylglycine-3 (S)-ethynyl- - alanine, Ethyl N-pyridin-4-ylnipecotyl -N-cyclopropylglycine-3(S)-ethynyl-P- alanine, N-Pyridin-4-ylnipecotyl-N-cyclopropylglycine-3(S)-ethynyl- - alanine, 4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-5-yl)butanoyl-N-(cyclo propyl)gly-3 (S)ethynyl-B-alanine ethyl ester, 4-(1 ,2,3,4-Tetrahydro-l ,8-naphthyridin-5-yl)butanoyl-N-(cyclo- propyl)glycyl-3(S)-ethynyl-B-alanine, or 3-{2-[5-(1H-Benzoimidazol-2-yl-amino)-pentanoylamino]-acetylamino}-3(S)-pyridin3-yl- propionic acid, or a pharmaceutically acceptable salt, such as a trifluoroacetate salt (e.g. 4-(2- Aminothiazol-4-yl)butanoyl-glycyl-3(R)-(2-phenethyl)- -alanine trifluoroacetate salt, 5 (2-Pyridylamino)pentaoylglycyl-2(S)-phenylsulfonamido- -alanine trifluoroacetate salt' N-tN'[3-(1-Piperazinyl)benzoylOglycyl]-3(R)-methyl- alanine trifluoroacetic acid salt, N-[N'-t3-(1-Piperazinyl)benzoyl]glycyl]-3(R)-(2-phenethyl)- alanine trifluoroacetic acid salt, N-(N1-f3-(1 -Piperazinyl)benzoylj-N -(2-phenethyl)glycylj- 3(RY(2-phenethyl)-fralanine trifluoroacetic acid salt) or a hydrochloric acid salt (e.g. 4-(2-Amino-pyridin-6-yl)butanoyl-N- cyclopropylglycyl-3(R)-(2-phenethyl)- -alanine ethyl ester hydrochloride).
The invention is also a method for inhibiting a condition selected from the group conistsing of restenosis, angiogenesis, diabetic retinopathy, macular degeneration, inflammation, and tumor growth in a mammal which comprises administering to the mammal a therapeutically effective amount of a compound defined above.
An example of the invention is the method wherein the antagonizing effect is an αv 5 antagonizing effect illustrated by the effects of inhibiting restenosis, atherosclerosis, angiogenesis, diabetic retinopathy, macular degeneration, inflammation or tumor growth.
The methods of the invention are particularly useful for inhibiting tumor growth in mammals. Pharmacologically effective amounts of the compounds, including pharmaceutically acceptable salts thereof, are administered to the mammal, to inhibit tumor growth. The growth of tumors depends on an adequate blood supply, which in turn depends on growth of new vessels into the tumor. New vessels are stimulated by factors secreted by the tumor. Inhibition of angiogenesis can cause tumor regression in animals.
The methods of the invention are also particularly useful for treating and preventing diabetic retinopathy in mammals.
Pharmacologically effective amounts of the compounds, including pharamaceutically acceptable salts thereof, are administered to the mammal, to inhibit diabetic retinopathy.
More particularly illustrating the invention is a method for eliciting an αv 5 or dual αv 3/αv 5 antagonizing effect in a mammal in need thereof, comprising administering to the mammal a pharmaceutical composition comprising any of the compounds described above and a pharmaceutically acceptable carrier.
Further illustrating the invention is a method of treating and/or preventing a condition mediated by antagonism of a vitronectin receptor in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of any of the compounds described above. Preferably, the condition is selected from restenosis, atherosclerosis, diabetic retinopathy, macular degeneration, angiogenesis, inflammation, and tumor growth.
More specifically exemplifying the invention is a method of eliciting an antagonizing effect in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of any of the compounds or any of the pharmaceutical compositions described above, wherein the antagonizing effect is an av S antagonizing effect (e.g. inhibition of restenosis, atherosclerosis, aniogenesis, diabetic retinopathy, macular degeneration, inflammation or tumor growth).
More particularly illustrating the invention is any of the methods of treating and/or preventing osteoporosis and/or of inhibiting bone resorption described above, wherein the compound is administered in combination with a second bone resorption inhibitor; preferably, the second bone resorption inhibitor is alendronate.
More particularly exemplifying the invention is the use of any of the compounds described above in the preparation of a medicament for the treatment and/or prevention of tumor growth, restenosis, atherosclerosis, diabetic retinopathy, macular degeneration, inflammation and/or angiogenesis.
DETAILED DESCRIPTLON OF THE INVENTION The term "pharmaceutically acceptable salts" shall mean 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 salts: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote, palmitate, panthothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, trifluoroacetate, and valerate.
Compounds of the present invention are chiral; included within the scope of the present invention are racemic mixtures and separated enantiomers of the general formula. Furthermore, all diastereomers, including E, Z isomers, of the general formula are included in the present scope. Furthermore, hydrates as well as anhydrous compositions and polymorphs of the general formula are within the present invention.
Prodrugs, such as ester derivatives of described compounds, are compound derivatives which, when absorbed into the bloodstream of a warm-blooded animal, cleave in such a manner as to release the drug form and permit the drug to afford improved therapeutic efficacy. 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 "pharmaceutically effective amount" shall mean that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system or animal that is being sought by a researcher or clinician.
The term "anti-coagulant" shall include heparin, and warfarin.
The term "thrombolytic agent" shall include agents such as streptokinase and tissue plasminogen activator.
The term "platelet anti-aggregation agent" shall include agents such as aspirin and dipyridamole.
The term "antagonist," as used herein, refers to a compound which binds to and antagonizes either the avp3 receptor or the av 5 receptor, or a compound which binds to and antagonizes both the avp3 and av S receptors (i.e., a dual av 3/avp5 receptor antagonist).
The term "bone resorption" means the process by which osteoclasts solubilize bone minerals and increase the activity of enzymes that degrade bone matrix.
The term "alkyl" means straight or branched alkane containing 1 to about 10 carbon atoms, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexy, octyl radicals and the like.
The term "alkenyl" shall mean straight or branched chain alkenes of two to ten total carbon atoms, e.g., propylenyl, buten-l-yl, isobutenyl, pentenylen-l-yl, 2,2-methylbuten- 1 -yl, 3-methylbuten- 1 -yl, hexen-l-yl, hepten-l-yl, and octen-l-yl radicals and the like.
The term "alkynyl" shall mean straight or branched chain alkynes of two to ten total carbon atoms containing 2 to about 10 carbon atoms, e.g., ethynyl, propynyl, butyn-l-yl, butyn-2-yl, pentyn-l-yl, pentyn-2-yl, 3-methylbutyn-1-yl, hexyn-l-yl, hexyn-2-yl, hexyn-3-yl, 3,3-dimethylbutyn-l-yl radicals and the like.
The term "cycloalkyl" shall mean cyclic rings of alkanes of three to eight total carbon atoms, or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl).
The term "cycloheteroalkyl," as used herein, shall mean a 3- to 8-membered fully saturated heterocyclic ring containing one or two heteroatoms chosen from N, O or S. Examples of cycloheteroalkyl groups include, but are not limited to piperidinyl, pyrrolidinyl, azetidinyl, morpholinyl, piperazinyl.
Unless otherwise specifically defined, the term "aryl" means a 5- or 6-membered unsa component of the arylalkyl or alkylaryl unit. Examples of arylalkyl include benzyl, fluorobenzyl, chlorobenzyl, phenylethyl, phenylpropyl, fluorophenylethyl, chlorophenylethyl, thienylmethyl, thienylethyl, and thienylpropyl. Examples of alkylaryl include toluene, ethylbenzene, propylbenzene, methylpyridine, ethylpyridine, propylpyridine, butylpyridine, butenylpyridine, and pentenylpyridine.
The term "halogen" includes fluorine, chlorine, iodine and bromine.
The term "oxy" means an oxygen (0) atom. The term "thio" means a sulfur (S) atom. The term "oxo" means =0.
The term "substituted" shall be deemed to include multiple degrees of substitution by a named substituent. Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independeritly substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally. By independently substituted, it is meant that the (two or more) substituents can be the same or different.
Under standard nomenclature used throughout this disclosure, the terminal portion of the designated side chain is described first followed by the adjacent functionality toward the point of attachment. For example, a Ci -6 alkyl substituted with C1 5 alkylcarbonylamino is equivalent to
HO I II C1-6-alkyl-N-C-C 1-5-alkyl When a substituent includes the definition CO (e.g., aryl CO- 8 alkyl), the group modified by CO is not present in the substituent.
Similarly, when any of the variables such as m or n is zero, the group modified by the variable is not present.
Compounds of the invention where X is a 5-membered monocyclic partially or fully saturated ring system, e.g., a thiazole system, can be prepared by fonning an alkyl ester substituted derivative of the ring, e.g., methyl 4-(2-aminothiazol-4-yl)butanoate, forming the corresponding acid with HCI, and reacting with an amine to form the final product.
Compounds of the invention where X is a 6-membered monocyclic partially or fully saturated ring system, e.g., a pyridine system, can be prepared using 2-aminopyridine, 2-aminopicoline, 4vinyl pyridine, etc., as described in Schemes 3, 4, and 10.
Compounds of the invention where X is a 9-membered polycyclic partially or fully saturated fused ring system can be prepared by reacting a substituted 5-membered ring starting material such as 2 amino -3 -bromo thiophene, 2-nitro-3-bromo thiophene, 2-amino-3bromo pyrrole, and 2-amino-3-bromo furan, with an appropriate compound under suitable ring closure conditions to effect formation of the 9-membered fused ring system.
Compounds of the invention where X is a 10-membered polycyclic partially or fully saturated ring system can be prepared using a starting material such as naphthyridin (Hamada, Y. et al., Chem.
Pharm. Bull. Soc., 1971, 19(9), 1857-1862), or by reacting an aminoaldehyde pyridine with a suitable ketone under suitable ring closure conditions to effect formation of the 10-membered fused ring system.
The examples illustrate procedures for preparing compounds of the invention where Y is -(CH2)04, -0-, and -N(R8)-.
To make compounds where Y is -N(R8)C(O)-, an acid such as compound 1-4 can be subjected to a Curtius reaction to form the amine, and subsequent condensation to give the final product.
In the schemes and examples below, various reagent symbols have the following meanings: BOC or Boc: t-butyloxycarbonyl Pd-C: Palladium on activated carbon catalyst DMF: Dimethylformamide DMSO: Dimethylsulfoxide CH2Cl2: Methylene chloride EtOH: ethanol MeOH: methanol EtOAc: ethyl acetate HOAc: acetic acid BOP: Benzotriazol-lyloxytris(dimethylamino)phosphonium, hexafluorophosphate EDC: 1 -(3 -Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride LDA: Lithium diisopropylamide PYCLU: Chloro-N,N,N',N' bis(pentamethylene)formamidinium hexafluorophosphate NMM: N-methylmorpholine HOBT: 1 -hydroxybenzotriazole hydrate TFA: trifluoroacetic acid NaOH: sodium hydroxide SOC12: thionyl chloride H2SO4: sulfuric acid NaH: sodium hydride LiOH: lithium hydroxide THF: tetrahydrofuran CH3CN: acetonitrile KHSO4: potassium hydrogen sulfate MgSO4: magnesium sulfate Boc2O: di-t-butyl dicarbonate CICH2CH2CI: dichloroethane 9-BBN: (9-borabicyclo[3 .3.1 ]nonane) TEA or NEt3: triethylamine Cs2CO3: cesium carbonate CH3NH2: methylamine NMP: N-methylpyrrolidinone BrCH2CO2tBu: tertbutylbromoacetate iPr2NEt: diisopropylethylamine NaN(TMS)2: sodium hexamethyldisilazide B ocNHCH2COOH:N-tert-butyloxycarbonylglycine In the methods of the present invention, the compounds described above can be administered in such oral forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. Likewise, they may be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, ocular or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed as an avS antagonist.
The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.
Oral dosages of the present invention, when used for the indicated effects, will range between about 0.01 mg per kg of body weight per day (mgRg/day) to about 100 mg/kg/day and preferably 0.01-50 mg/kg/day and more preferably 0.01-20 mg/kg/day, e.g. 0.1 mg/kg/day, 1.0 mgjkg/day, 5.0 mg/kg/day, or 10 mg/kg/day. A once-aday oral dosage is, for example, 10 mg, 100 mg or 500 mg.
Intravenously, the most preferred doses will range from about 1 to about 10 mg/kg/minute during a constant rate infusion.
Advantageously, compounds of the present invention may be administered in divided doses of two, three, or four times daily.
Furthermore, preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather that intermittent throughout the dosage regime.
In ocular formulations such as eyedrops (e.g. aqueous solutions), from about 0.01-5.0% (w/v) of active ingredient can be employed, e.g., from about 0.01-2.0% (w/v) of active ingredient.
Suitable eyedrop volume is, for example, 20, 30, 35, 50 or 100 ,ul. The objective is to administer a dose of between about 0.005-0.5 mg/kg per day to each eye, for a total dosage of between about 0.01-1.0 mg/kg/day, e.g. a dose of about 0.05 mg/kg per day to each eye, for a total dosage of about 0.1 mgAg/day. For example, the eyedrops can be used to provide doses of 1 mg, 10 mg, or 50 mg. These dosage values are based on known and presently understood pharmacology of compounds of the invention. Dosage requirements are variable and must be individualized on the basis of the disease and the response of the patient.
Suitable eyedrop formulations are those which are isotonic and maintain sufficient contact with the eye surface to systemically deliver the active agent to the patient. Such formulations advantageously have a pH approximating neutrality and are nonirritating to the eye, e.g. they do not induce tearing and consequential flow of active agent out of the eye. Pharmaceutically acceptable carriers are, for example, water, mixtures of water and water-miscible solvents such as lower alkanols or arylalkanols, vegetable oils, polyalkylene glycols, petroleum based jelly, ethyl cellulose, hydroxy ethyl cellulose, ethyl oleate, carboxymethylcellulose, polyvinylpyrrolidone, isopropyl myristate and other conventionallyemployed non-toxic, pharmaceutically acceptable organic and inorganic carriers. The pharmaceutical preparation may also contain non-toxic auxiliary substances such as emulsifying, preserving, wetting agents, bodying agents and the like, as for example, polyethylene glycols 200, 300, 400 and 600, carbowaxes 1000, 1500, 4000, 6000 and 10000, antibacterial compounds, phenylmercuric salts known to have cold sterilizing properties and which are non-injurious in use, thimerosal, methyl and propyl paraben, benzyl alcohol, phenyl ethanol, buffering ingredients such as sodium chloride, sodium borate, sodium acetates, gluconate buffers, and other conventional ingredients such as sorbitan monolaurate, triethanolamine, oleate, polyoxyethylene sorbitan monopalmitylate, dioctyl sodium sulfosuccinate, monothioglycerol, thiosorbitol, ethylenediamine tetraacetic acid, and the like.
Additionally, suitable ophthalmic vehicles can be used as carrier media for the present purpose including conventional phosphate buffer vehicle systems, isotonic boric acid vehicles, isotonic sodium chloride vehicles, isotonic sodium borate vehicles and the like.
In the procedure for making eyedrops, formulations are rendered sterile by appropriate means, such as starting the preparation procedure with sterile components and proceeding under sterile conditions, irradiating or autoclaving the finished formulation, and the like. Suitable anti microbial agents are also useful for maintaining sterility of the eyedrop.
The ocular preparation may also be a solid insert such as one which, after dispensing the compound, remains essentially intact, or a bioerodible insert that is soluble in lachrimal fluids, or otherwise disintegrates. For example, one may use a solid water soluble polymer as the carrier for the compound. The polymer used to form the insert may be any water soluble non-toxic polymer, for example, cellulose derivatives such as methylcellulose, sodium carboxymethyl cellulose, hydroxyethylcel lulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose, acrylates such as polyacrylic acid salts, ethylacrylates, polyacrylamides, natural products such as gelatin, alginates, pectins, tragacanth, karaya, chondrus, agar, acacia, starch derivatives such as starch acetate, hydroxyethyl starch ethers, hydroxypropyl starch, as well as other synthetic derivatives such as polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl methyl ether, polyethylene oxide, neutralized carbopol, gellan gum and xanthan gum, and mixtures of said polymers.
The ocular preparation may also be an ointment which is compounded, for example, by mixing finely milled powdered ingredients with a small amount of white petrolatum and levigating or otherwise mixing until a uniform distribution is achieved. The balance of white petrolatum is added by geometric addition until the desired dosage form is made.
In the methods of the present invention, the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with convention pharmaceutical practices.
For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, calcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or betalactose, corn-sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch methyl cellulose, agar, bentonite, xanthan gum and the like.
In the methods of the present invention, the compounds can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
In the methods of the present invention, the compounds may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds may also be coupled with soluble polymers as targetable drug carriers.
Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxy-propyl-methacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels.
The present invention is also directed to the use of the compounds defined above with one or more agents useful in the prevention or treatment of osteoporosis. For example, the compounds of the instant invention may be effectively administered in combination with effective amounts of other agents used in the treatment of osteoporosis such as bisphosphonate bone resorption inhibitors; preferably, the bone resorption inhibitor is the bisphosphonate alendronate, now sold as FOSAMAX. Preferred combinations are simultaneous or alternating treatments of an avp3 receptor antagonist of the present invention and FOSAMAXt).
In addition, the compounds may be effectively administered in combination with a growth hormone secretagogue in the therapeutic or prophylactic treatment of disorders in calcium or phosphate metabolism and associated diseases. These diseases include conditions which can benefit from a reduction in bone resorption. A reduction in bone resorption should improve the balance between resorption and formation, reduce bone loss or result in bone augmentation. A reduction in bone resorption can alleviate the pain associated with osteolytic lesions and reduce the incidence and/or growth of those lesions. These diseases include: osteoporosis (including estrogen deficiency, immobilization, glucocorticoid induced and senile), osteodystrophy, Paget's disease, myositis ossificans, Bechterew's disease, malignant hypercalcemia, metastatic bone disease, periodontal disease, cholelithiasis, nephrolithiasis, urolithiasis, urinary calculus, hardening of the arteries (sclerosis), arthritis, bursitis, neuritis and tetany.
Increased bone resorption can be accompanied by pathologically high calcium and phosphate concentrations in the plasma, which would be alleviated by this treatment. Similarly, the present invention would be useful in increasing bone mass in patients with growth hormone deficiency. Thus, preferred combinations are simultaneous or alternating treatments of the compounds with a growth hormone secretagogue, optionally including a third component comprising FOSAMAX.
In accordance with the method of the present invention, the individual components of the combination can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The instant invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly.
The compounds described above were prepared according to the procedure of the following examples. The most preferred compounds of the invention are any or all of those specifically set forth in these examples. These compounds are not, however, to be construed as forming the only genus that is considered as the invention, and any combination of the compounds or their moieties may itself form a genus. The following examples further illustrate details for the preparation of the compounds of the present invention. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. All temperatures are degrees Celsius unless otherwise noted.
SCHEME 1
HCH2Nm < CO2+BU 2 o H SO2 1 -5 Br2, MeOH O"C to RT Boc-Gly-OH NMM, isobutyl chloroformate, Br)MCO2Me O , EtOAc, -15"C to RT BocHN N > CO2-t-Bu BocHN H H SO, Q EtOH, A H 1=4i H2N,C02MeCH2CI, TF TFA, CH2CI2 i=a -150C 6N HCI TFAH2N,C02-t-Bu H H' 3- S02 H HC H2N CO2H 1-7 gl/ EDC, HOBT, NMM EDC, HOST, NMM DMF, -15 C to RT SCHEME 1 CONTINUED
Methyl 6-bromo-5-oxohexanoate (1-24 5-Oxohexanoic acid (1-1, 5 mL, 42 mmol) was dissolved in 84 mL MeOH and cooled to 00C. Br2 (2.2 mL, 43 mmol) was added dropwise, and the reaction was stirred at RT overnight. After removing the MeOH by rotary evaporation, the residue was dissolved in ether, washed with water, sat. NaHCO3 and brine, dried (Na2S04), filtered and concentrated. Flash chromatography (silica, 10% EtOAc/hexane) provided the bromide-ester 1-2 as a yellow oil.
TLC Rf 0.09 (silica, 15% EtOAc/hexane) 1H-NMR (300 MHz, CDC13): 6 3.88 (s, 2H), 3.67 (s, 3H), 2.75 (t, J = 7Hz, 2H), 2.37 (t, J = 7 Hz, 2H), 1.94 (qn, J = 7 Hz, 2H).
Methyl 4-(2-aminothiazol4-vl)butanoate (1-3) Bromide 1-2 (3.45 g, 15.5 mmol) and thiourea (1.4 g, 18 mmol) were combined in 77 mL EtOH and heated to reflux. After disappearance of 1-2, the EtOH was removed by rotary evaporation and the residue was diluted with EtOAc, washed with water and brine, then dried (MgSO4), filtered and concentrated. The pH of the aqueous phase was adjusted to 7, and the solution was re-extracted with EtOAc (2x).
These organic extracts were washed with brine, dried (MgSO4), filtered and concentrated, combined with the first organic residues, then purified by flash chromatography (silica, EtOAc) providing aminothiazole 1-3 as a white solid.
TLC Rf 0.5 (silica, EtOAc) 1H-NMR (400 MHz, CDC13): s 6.09 (s, 1H), 5.19 (br s, 2H), 3.66 (s, 3H), 2.55 (t, J = 7 Hz, 2H), 2.34 (t, J =7 Hz, 2H), 1.96 (qn, J = 7 Hz, 2H).
4-(2-Aminothiazol4-yl)butanoic acid hydrochloride (1-4) Ester 1 3 (1.3 g, 6.5 mmol) was dissolved in 32 mL 6 N HCl. After stirring overnight, the resulting suspension was concentrated, providing acid 1-4 as a white solid.
1H-NMR (400 MHz, d6-DMSO): 6 9.12 (br s, 1H), 6.51 (s, 1H), 3.50 (br s), 2.51 (t, J = 7 Hz, 2H), 2.24 (t, J = 7 Hz, 2H), 1.77 (qn, J = 7 Hz, 2H).
N-Boc-glycyl-2(S)-phenylsulfonamido- -alanine t-butyl ester (1-6) N-Boc-glycine (255 mg, 1.5 mmol) was dissolved in 7.4 mL EtOAc, cooled to -15 C, then NMM (179 pL, 1.6 mmol) and isobutyl chloroformate (211 pL, 1.6 mmol) were added. After 20 min, amine 1-5 (500 mg, 1.5 mmol) and additional NMM (422 jiL, 3.2 mmol) were added and the reaction was warmed to RT overnight.
Following dilution with EtOAc, the mixture was washed with water, sat.
NaHCO3, 10% KHSO4, and brine, dried (MgSO4), filtered and concentrated, providing amide 1-6 as a white solid.
TLC Rf 0.73 (silica, EtOAc) 1H-NMR (300 MHz, CDC13): 8 7.84 (d, J = 7 Hz, 2H), 7.59 (ABX t, J = 7 Hz, 1H), 7.51 (ABX t, J = 7 Hz, 2H), 6.58 (br m, 1H), 5.58 (d, J = 8 Hz, 1H), 5.11 (br s, 1H), 3.90-3.78 (m, 3H), 3.72 (m, 1H), 3.40 (m, 1H), 1.48 (s, 9H), 1.28 (s, 9H).
Glycyl-2(S)-phenylsulfonamido- -alanine t-butyl ester trifluoroacetate salt (1-7) Protected amide 1-6 (576 mg, 1.26 mmol) was dissolved in 6.3 mL CH2C12, cooled to -150C, and TFA (6.3 mL) was added. After 25 min the reaction was concentrated, providing amine 1-7.
TLC Rf 0.36 (silica, 9:1:1 CH2Cl2/MeOH/HOAc).
4-(2-Aminothiazol-4-yl)butanoyl-glycyl-2(S)-phenylsulfonamido- alanine t-butyl ester (1-8) Acid 1-4 (300 mg, 1.35 mmol), amine 1^7 (600 mg, 1.37 mmol), HOBT (219 mg, 1.14 mmol) and NMM (445 pL, 4.04 mmol) were combined in 13 mL DMF, cooled to -150C, and EDC (310 mg, 1.61 mmol) was added. The reaction was warmed to RT, stirred ovemight, then diluted with EtOAc, washed with water, sat. NaHCO3, and brine, dried (MgSO4), filtered and concentrated. Flash chromatography (silica, 20% MeOH/EtOAc) provided 1-8 as yellow solid.
TLC Rf 0.55 (silica, 20% MeOFVEtOAc) 1H-NMR (400 MHz, CDCl3): 57.80 (d; J = 7 Hz, 2H), 7.55 (ABX t, J = 7 Hz, 1H), 7.47 (ABX t, J = 8 Hz, 2H), 7.35 (br s, 1H), 7.04 (br m, 1H), 6.12 (s, 1H), 5.41 (br s, 2H), 4.05-3.95 (m, 3H), 3.69 (m, 1H), 3.39 (ddd, 1H), 2.70-2.55 (m, 2H), 2.33 (m, 2H), 2.01 (qn, J = 7 Hz, 2H), 1.27 (s, 9H).
4-(2-Aminothiazol-4-yl)butanoyl-glycyl-2(S)-phenylsulfonamido- alanine (1-9) Ester 1-8 (365 mg, 0.69 mmol) was dissolved in CH2C12 (3.5 mL), then TFA (3.5 mL) was added. After 5 h the reaction mixture was concentrated, azeotroped with toluene, then purified by sequential flash chromatography (silica, 22:20:1:1 EtOAc/EtOH/H2O/NH4OH, then silica, 4:1:1 CH2Cl2/MeOH/HOAc, then 7:1:1 CH2Cl2/MeOH/HOAc), providing 1-9 as a white solid.
TLC Rf 0.33 (silica, 7:1:1 CH2Cl2/MeOHJHOAc) H-NMR (400 MHz, CD30D): 6 7.86 (d, J = 7 Hz, 2H), 7.58 (ABX t, J = 7 Hz, 1H), 7.52 (ABX t, J = 8 Hz, 2H), 6.27 (s, lH), 3.89 (AB d, J = 17 Hz, 1H), 3.77 (AB d, J = 17 Hz, 1H), 3.64 (t, J = 6 Hz, 1H), 3.53 (AB dd, 1H), 3.41 (AB dd, 1H), 2.57 (t, J = 7 Hz, 2H), 2.35-2.25 (m, 2H), 1.95 (m, 2H).
L-asparagine NaOH, H20, PhSO2CI 0 H2NC"0N0H)o2Ph H 1-la NaOH, dioxane, Br2 OOC to 900C
CO2H H2N ,4C > 02H H N HSO2Ph 1-2a isobutylene, H2S04, dioxane SOCIO 12 b. 1 N HCllether ethanol COVET HOl. CO2tBu 2 ~ & 2 > > 11 NHSO2Ph H NHSO2Ph 3-4 1-5 N-Phenylsulfonyl-L-asparagine (1-1a) To a stirred solution of L-asparagine (Aldrich) (10 g, 76 mmol), NaOH (3.4 g, 85 mmol), H20 (50 mL), and dioxane (50 mL) at 0 C was added PhS02Cl (10.6 mL, 84 mmol). After 1 min, NaOH (3.4 g) in H20 (50 mL) was added and the reaction mixture stirred for 30 min. The reaction mixture was then concentrated to remove the dioxane then washed with EtOAc. The aqueous phase was then cooled to 0 C and acidified to pH 5.0 with conc. HCl to effect product precipitation. The resulting solid was collected by filtration, washed with H2O (20 mL) and dried at 50 C under vacuum to give N phenylsulfonyl-L-asparagine (1-la) as a white solid.
Rf 0.40 (silica, 10:1:1 ethanol/H2O/NH4OH). 1H NMR (300 MHz, D2O) 6 7.59 (m, 2H), 7.26 (m, 3H), 3.92 (m, 1H), 3.02 (m, 1H), 2.35 (m, 1H).
3-Amino-2(S)-phenylsulfonylaminopropionic acid (1-2b) To stirred solution of NaOH (15.6 g, 0.4 mol) in H20 (70 mL), cooled with an icebath, was added bromine (3.6 mL, 0.07 mol) dropwise. After S min, a cold solution of N-phenylsulfonyl-Lasparagine, 1-la (14.6 g, 54 mmol) and NaOH (4.3 g, 0.1 mol) in H20 (50 mL) was added in one portion. The solution was stirred for 20 min at 0 C then 30 min at 900C. The reaction mixture was recooled to OOC, and the pH adjusted to 7 through dropwise addition of conc. HCI. The white precipitate formed was collected by filtration and then dried to give (1-2b) as a white solid. 1H NMR (300 MHz, D20) 6 8.00, 7.50 (m, SH), 3.88 (m, 1H), 3.37 (m, 1H), 3.12 (m, 1H).
Ethyl 3-Amino-2(S)-phenylsulfonylaminopropionate hydrochloride (34) Amino acid 1-2a (1.0 g, 4.1 mmol) was suspended in 20 mL EtOH, cooled to OOC, and SOC12 (1.5 mL, 21 mmol) was added dropwise. After stirring at RT overnight the mixture was concentrated, triturated with Et20 (2x), and dried, providing 9 (1.26 g) as a hygroscopic yellow solid.
H-NMR (300 MHz, d6-DMSO): 8 8.30 (br s), 7.79 (d, J = 8 Hz, 2H), 7.70-7.60 (m, 3H), 4.21 (t, J Hz, 1H), 3.90-3.80 (m, 2H), 3.09 (ABX dd,J=13,6Hz, 1H),2.90(ABXdd,J= 13,8Hz,2H),0.97(t,J=7 Hz, 3H).
tert-Butyl 3-Amino-2(S)-phenylsulfonylaminopropionate hydro-chloride (1 -S) In a Fischer-Porter tube, a mixture of 1-2a (10.2 g, 42 mmol) and DME (150 mL) was sequentially treated with H2S04 (6A mL, 0.12 mol), cooled to -780C, and then condensed isobutylene (75 mL). The cooling bath was removed. After 24 h, ice/water (250 mL) was added followed by washing with ether (2x). The aqueous phase was basified with aq 6N NaOH, then saturated with NaCl, followed by extraction with EtOAc (3x). The combined extracts were washed with brine, dried (MgSO4), and concentrated to give a white solid. This was dissolved in CH2C12 and treated with 1N HCI/ether (22 mL), and then concentrated to give 1-5 as a glassy yell
SCHEME 2
4-(2-Aminothiazol -4-yl)butanoyl-glycyl-3 (R)-(2-phenethyl)-13-alanine methyl ester (2-2) Acid 14 (300 mg, 1.35 mmol), amine 2-1 (405 mg, 1.35 mmol) (prepared as described in Duggan et al., U.S. Patent 5,264,420) HOBT (219 mg, 1.62 mmol) and NMM (445 pL, 4.04 mmol) were combined in 7 mL DMF, cooled to -15 C, and EDC (310 mg, 1.61 mmol) was added. The reaction was warmed to RT, stirred overnight, then diluted with EtOAc, washed with water, sat. NaHCO3, and brine, dried (MgS04), filtered and concentrated. Flash chromatography (silica, 10% MeOH/EtOAc) provided 2-2 as a yellow oil.
TLC Rf 0.32 (silica, 10 % MeOH/EtOAc) 1H-NMR (400 MHz, CDCl3): 6 7.82 (d, J = 7 Hz, 1H), 7.65 (d, J = 8 Hz, 1H), 7.40-7.10 (m, SH), 6.93 (d, J = 8 Hz, 1H), 6.10 (s, 1H), 4.31 (m, 1H), 3.96 (ABX dd, J = 17, 6 Hz, 1H), 3.89 (ABX dd, J = 17, 5 Hz, 1H), 3.64 (s, 3H), 2.68-2.54 (m), 2.32-2.17 (m, 2H), 2.25-1.80 (m).
4-(2-Aminothiazol-4-yl)butanoyl-glycyl-3(R)-(2-phenethyl)-ss-alanine trifluoroacetate salt (2-34 Ester 2-2 (220 mg, 0.51 mmol) and 1 N NaOH (1.3 mL, 1.3 mmol) were combined in 5 mL MeOH. After 3 d the reaction mixture was concentrated, purified by flash chromatography (silica, 9:1:1 CH2C12/MeOH/HOAc), then preparative HPLC (C1g, 0.1 % TFA in CH3CN/H20), providing, after lyophilization, acid 2-3 as a white solid.
TLC Rf 0.54 (silica, 4:1:1 CH2Cl2/MeOH/HOAc) 1H-NMR (400 MHz, CD30D): # 7.26-7.10 (m, 5H), 6.52 (s, 1H), 4.23 (m, 1H), 3.88 (AB d, J = 17 Hz, 1H), 3.79 (AB d, J = 17 Hz, 1H), 2.722.55 (m, 4H), 2.51 (d, J = 7 Hz, 2H), 2.34 (t, J = 7 Hz, 2H), 1.99-1.75 (m, 4H).
SCHEME 3
,NH2 C02Et 3-1 Cbz-Gly-OH, NaH, DMF, 0 C to RT; isobutyl chloroformate, Ethyl 5-bromopentanoate NMM RT to 750C EtOAc, -1 5 C to RT H 0 )N \CO2Et CbzNH/tN > CO2Et 3-2 H H HN- SO24 1 N NaOH, EtOH H,, H % NCO2H 0 H2N) & CO2Et 11- HN H N- SO2 H BOP, NMM DMF SCHEME 3 CONTINUED
Ethvl 5-(2-pyridylamino)pentanoate (3-2) 2-Aminopyridine (3-1 1.97 g, 20.9 mmol) in 10 mL DMF was added to a suspension of NaH (60 % in oil, 1.00 g, 25 mmol) in 80 mL DMF cooled to 0 C. After warming to RT for 45 min, ethyl 5bromopentanoate (4.2 mL, 25 mmol) was added dropwise. This mixture was heated at 750C overnight, then cooled to RT, diluted with EtOAc, washed with water (2x), sat. NaHCO3, and brine, dried (MgSO4), filtered and concentrated. Flash chromatography (silica, 50% then 70 % EtOAc/hexane) provided 3-2 as a yellow oil.
TLC Rf 0.55 (silica, 70 % EtOAc/hexane) 1H-NMR (400 MHz, CDC13): 6 8.07 (dd, J = 5, 1 Hz, 1H), 7.40 (m, 1H), 6.55 (m, 1H), 6.37 (d, J = 8 Hz, 1H), 4.48 (br s, 1H), 4.13 (q, J = 7 Hz, 2H), 3.29 (q, J =7 Hz, 2H), 2.35 (t, J = 7 Hz, 2H), 1.80-1.55 (m, 4H), 1.25 (t, J = 7 Hz, 3H).
S-(2-Pyridvlamino)pentanoic acid (3-3) Ester 3-2 (0.41 g, 1.84 mmol) was dissolved in 18 mL EtOH, 1 N NaOH (4.6 mL, 4.6 mmol) was added, and the reaction was stirred overnight. The pH of the solution was adjusted to 7 with 1 N HCl, and concentration provided a white solid containing acid 3-3 and NaCl.
TLC Rf 0.06 (silica, 19:1:1 CH2C12/MeOH/HOAc) 1H-NMR (400 MHz, D20): 6 7.81 (m, 1H), 7.77 (d, J = 6 Hz, 1H), 6.96 (d,J=9Hz, 1H),6.82 (t,J=7Hz, iH),3.36 (t,J=7Hz,2H),2.24 (m, 2H), 1.72-1.50(m, 4H).
N-Cbz-glycyl-2(S)-phenylsulfonamido-ss-alanine ethvl ester (3-5) N-Cbz-glycine (339 mg, 1.62 mmol) was dissolved in 8 mL EtOAc, cooled to -15 C, then NMM (196 pL, 1.8 mmol) and isobutyl chloroformate (230 uL, 1.8 mmol) were added. After 20 min, the mixed anhydride solution was added to amine 3-4 (0.50 mg, 1.6 mmol) suspended in S mL EtOAc and the reaction was warmed to RT for 90 min. Following dilution with EtOAc, the mixture was washed with water, sat. NaHCO3, 5% KHSO4, and brine, dried (MgSO4), filtered and concentrated. Flash chromatography (silica, 75 % EtOAc/hexane) provided amide 3-5 as a colorless oil.
TLC Rf 0.29 (silica, 75 % EtOAc/hexane) 1H-NMR (300 MHz, CDCl3): # 7.65-7.45 (m, 3H), 7.40-7.25 (m, 5H), 6.68 (t, J = 6 Hz, 1H), 5.83 (d, J = 8 Hz, 1H), 5.49 (t, J = 6 Hz, 1H), 5.15 (s, 2H), 4.04-3.95 (m, 3H), 3.89-3.85 (m, 2H), 3.73 (m, 1H), 3.46 (m, 1H), 1.11 (t, J = 7 Hz, 3H).
Glycyl-2(S)-phenylsulfonamido-ss-alanine ethvl ester (3-6) Protected amine 3-5 (0.47 g, 1.01 mmol) was dissolved in 10 mL EtOH, 10 % Pd/C (94 mg) was added, and the reaction was stirred under an H2 balloon. After 4 h, additional 10 % Pd/C was added (94 mg), and the reaction was continued for 3 d. The mixture was filtered through Celite, concentrated, and azeotroped with CHCl3, providing amine 3-6 as a gum.
1H-NMR (300 MHz, CDCl3): 6 7.95 (m), 7.86 (d, J = 7 Hz, 2H), 7.607.45 (m, 3H), 4.05 (dd, J = 5, 6 Hz 1H), 3.96 (q, J = 7 Hz, 2H), 3.803.55 (m), 1.07 (t, J = 7 Hz, 3H).
5-(2-Pyridylamino)pentanoylglycyl-2(S)-phenylsulfonamido-ss-alanine ethyl ester (3-7) Acid X (186 mg, 0.55 mmol), amine 3-6 (150 mg, 0.46 mmol), NMM (0.20 mL, 1.8 mmol) and BOP (302 mg, 0.68 mmol) were combined in 3 mL DMF. After 5 d the DMF was removed on a rotary evaporator, the residue was diluted with EtOAc, then washed with water, sat. NaHCO3, and brine, dried (MgSO4), filtered and concentrated. Flash chromatography (silica, 25 % i-PrOH/EtOAc) provided 3-7 as a colorless oil.
TLC Rf 0.30 (silica, 25 % i-PrOH/EtOAc) 1H-NMR (400 MHz, CDC13): 6 8.05 (d, J = 4 Hz, 1H), 7.85 (d, J = 7 Hz, 2H), 7.57 (t, J = 7 Hz, 1H), 7.55-7.45 (m, 2H), 7.42 (m, 1H), 6.80 (br t, 1H), 6.54 (dd, J = 6, 4 Hz, lH), 6.45 (m, lH), 6.39 (d, J = 8 Hz, 1H), 5.19 (m, 1H), 4.16 (ABX dd, J = 17, 7 Hz, 1H), 4.08-3.95 (m), 3.85-3.75 (m, 2H), 3.29 (q, J = 6 Hz, 2H), 2.40-2.32 (m, 2H), 1.85 (m, J = 7 Hz, 2H), 1.75 (m, 2H), 1.10 (t, J = 7 Hz, 3H).
5-(2-Pyridylamino)pentanoylglycyl-2(S)-phenylsulfonamido-ss-alanine trifluoroacetate salt (3-8) Ester 3-7 (59 mg, 0.12 mmol) was dissolved in 1 mL THF, then 1 N LiOH (0.29 mL, 0.29 mmol) was added. After stirring overnight the reaction was concentrated, the mixture was concentrated.
Flash chromatography (silica, 22:20:1:1 EtOAc/EtOH/H20/NH40H), followed by prep. HPLC (C-18, 0.1 % TFA/CH3CN/H20) and lyophilization provided 3-8 as a white solid.
TLC Rf 0.26 (silica, 22:20:1:1 EtOAc/EtOH/H2O/NH4OH) 1H-NMR (400 MHz, D20): 8 7.83-7.75 (m, 3H), 7.70 (d, J = 6 Hz, 1H), 7.67 (d, J = 7 Hz, 1H), 7.58 (t, J = 7 Hz, 2H), 6.96 (d, J = 9 Hz, 1H), 6.80 (t, J = 7 Hz, 1H), 3.86-3.80 (m, 3H), 3.55 (dd, J = 14,4 Hz, 1H), 3.36 (m, 2H), 3.29 (dd, J = 14, 8 Hz, 1H), 2.39 (m, 2H), 1.72 (m, 4H).
SCHEME 4
SCHEME 4 CONTINUED
4-(2-N-Boc-aminopyridin-4.ylThutanoic acid (4-1) The protected picoline (90 g, 0.43 mol) was dissolved in 3 L THF under N2, cooled to -780C, and n-BuLi (1.6 M, 675 mL, 1.08 mol) was added during 30 min. The mixture was allowed to warm to RT for 1 h, then the resulting orange suspension was cooled to -780C.
Methyl 3-bromopropionate (79 g, 0.47 mol) was added during 2 min.
After 15 min the cooling bath was removed and the mixture was allowed to warm to -20 C at which point it was quenched with 60 mL HOAc in 250 mL THF. The solution was diluted with 2L EtOAc, washed with water, sat. NaHCO3, and brine, dried (MgS04). The aqueous layers were re-extracted with EtOAc (2x), and these organic layers were filtered, concentrated, and dissolved in 1.5 L EtOH and 1.5 L 1 N NaOH (1.5 mol). After 1 h the reaction was concentrated by 1/3, diluted with 4 L EtOAc, the aqueous layer was removed. The pH of the aqueous layer was adjusted to 4-5 with 10% KHSO4, then extracted with EtOAc (2 x 3L). The EtOAc layers were washed with brine, dried (MgSO4), filtered and concentrated, providing 4-1 as a yellow oil.
4-(2-Boc-amino-pvridin-6-ylAbutanovl-sarcosme ethyl ester (4-2) Acid 4-1(200 mg, 0.71 mmol), H-Sar-OEtoHCl (130 mg, 0.84 mmol), NMM (314 pL, 2.9 mmol) and BOP (378 mg, 0.86 mmol) were combined in S mL DMF. After stirring overnight the reaction mixture was diluted with EtOAc, washed with water (5x), sat. NaHCO3, and brine, dried (MgSO4), filtered and concentrated. Flash chromatography (silica, 50-70 % EtOAc/hexane) provided 4-2 as a colorless oil.
TLC Rf 0.54 (silica, 80% EtOAc/hexane) 1H-NMR (400 MHz, CDCl3): 4:1 mixture of amide rotomers, major rotomer 6 8.12 (d, J = S Hz, 1H), 7.79 (s, 1H), 7.48 (br s, 1H), 6.83 (d, J = 6 Hz, 1H), 4.19 (q, J = 7 Hz, 2H), 4.11 (s, 2H), 3.03 (s, 3H), 2.68 (t, J = 7 Hz, 2H), 2.39 (t, J = 7 Hz, 2H), 2.02 (qn, J = 7 Hz, 2H), 1.53 (s, 9H) 1.26 (t, J = 7 Hz, 3H).
4-(2-Boc-amino-pyridin-4-yl)butanoyl-sarcosine (4-3) Ester 4-2 (91 mg, 0.24 mmol) was dissolved in 2.4 mL EtOH, and 1 N NaOH (0.60 mL, 0.60 mmol) was added. After 45 min the mixture was concentrated, then diluted with EtOAc, washes with 10 % KHSO4 and brine, dried (MgSO4) filtered and reconcentrated, providing acid 4-3 as a glass.
TLC Rf0.18 (silica, 18:1:1, CH2C12/MeOH/HOAc) 1H-NMR (400 MHz, CDC13): 1:1 mixture of amide rotomers # 8.037.82 (m, 3H), 6.86 (br s, 1H), 4.15/3.96 (s, 2H), 3.06/3.02 (s, 3H), 2.752.65 (m, 2H), 2.40 (m, 2H), 2.22-2.00 (m, 2H), 1.53 (s, 9H).
4-(2-Boc-amino-pyridin-4-yl)butanoyl-sarcosine-3(R)-[2-(indol-3 yl)ethvl1--alanine ethyl ester (4-5) Acid 4-3 (84 mg, 0.24 mmol), amine 4-4 (see Duggan et al., U.S. Patent 5,264,420) (68 mg, 0.26 mmol), NMM (104 11L, 0.95 mmol) and BOP (127 mg, 0.29 mmol) were combined in 2.4 mL CH3CN. After stirring overnight the mixture was diluted with EtOAc, washed with water (4x), sat. NaHCO3, and brine, dried (MgSO4) and concentrated. Flash chromatography (silica, EtOAc) provided 4-5 as a colorless oil TLC Rf 0.66 (silica, 20 % MeOH/EtOAc) 1H-NMR (400 MHz, CDC13): 4:1 mixture of amide rotomers, major rotomer 8 8.12 (s, 1H), 8.09 (d, J = 5 Hz, 1H), 7.78 (s, 1H), 7.55 (d, J = 8 Hz, 1H), 7.39 (s, 1H), 7.34 (d, J = 8 Hz, 1H), 7.17 (t, J = 8 Hz, 1H), 7.08 (t, J = 7 Hz, 1H), 7.03 (d, J = 2 Hz, 1H), 6.79 (dd, J = 5, 1 Hz, 1H), 6.71 (d, J = 9 Hz, 1H), 4.32 (m, 1H), 4.16-4.05 (m, 3H), 4.00 (AB d, J = 15 Hz, 1H), 3.94 (AB d, J = 15 Hz, 1H), 3.04 (s, 3H), 2.77 (m, 2H), 2.63 (t, J = 8 Hz, 2H), 2.53 (m, 2H), 2.36 (m, 2H), 2.02-1.90 (m, 4H), 1.53 (s, 9H), 1.22 (t, J = 7 Hz, 3H).
4-(2-Aminopyridin-4-yl)butanoyl-sarcosine-3(R)-[2-(indol-3-yl)ethyl] ss-alanine (4-6) Ester 4-5 (20 mg, 34 ymol) was dissolved in 350 pL EtOH, then 1 N NaOH (85 pL, 85 rnol) was added. After 2 h the reaction was diluted with EtOAc, washed with 10 % KHSO4 and brine, dried (MgSO4), filtered and concentrated. The residue was dissolved in 1 mL CH2C12, treated with 1 mL TFA for 1 h, then concentrated and azeotroped with toluene. Flash chromatography (silica, 50:1:1, EtOH/H2O/NH4OH) provided 4-6 as an off-white solid.
TLC Rf 0.55 (silica, 20:1:1 EtOH/H20/NH40H) H-NMr (400 MHz, CD30D): 2:1 mixture of amide rotomers # 7.72/7.67 (d, J = 6 Hz, 1H), 7.52 (t, J = 8 Hz, 1H), 7.30 (t, J = 8 Hz, 1H), 7.07-6.90 (m, 4H), 6.55/6.54 (s, 1H), 6.49 (s, 1H), 4.36-4.25 (m, 1H), 4.14-3.93 (m, 2H), 3.06/2.93 (s, 3H), 2.60 (t, J = 8 Hz, 2H), 2.552.45 (m, 4H), 2.34 (t, J =7 Hz, 1H), 2.05-1.84 (m).
SCHEME 5
H2N rCH3 5-1 Boc20 I ClCH2CH2CI, BocN HUNCH3 ,C02-t-Bu S 24 i) HCl'H2N H N-SO2 H 1) LDA, THF, -230C 2) allyl bromide, -78 C Cbz-Gly-OH BOP, NMM BocNH N N > < / > DMF I 0' 2) H202, NaOH H H N- 502 ' 2) H202, NaOH H H H-SO2t BocNH .OH 5-6 v 10% Pd/C 5-4 EtOAc Jones Reagent acetone 0 BocNH N CO2H H2N)k N CO2-t-Bu I H H' NHSO2\/ DMF < , DMF SCHEME S CONTINUED
2-(B oc-amino)-6-methvlpvridine (5-2) 2-Amino-6-picoline (5.0 g, 46.2 mmol) and Boc2O (11.1 g, 50.8 mmol) were combined in 150 mL dichloroethane. After heating at reflux for 6 h, additional Boc20 (2.0 g, 9.2 mmol) was added, and the reaction was heated ovemight. After concentration, the reaction mixture was flash filtered (silica, CH2C12), providing 5-2 as a waxy solid.
TLC Rf 0.21 (silica, CH2C12) 1H-NMR (400 MHz, CDC13): 6 7.70 (d, J = 8 Hz, 1H), 7.54 (t, J = 8 Hz, 1H), 7.19 (br s, 1H), 6.80 (d, J = 7 Hz, 1H), 2.42 (s, 3H), 1.51 (s, 9H).
2-Boc-amino-6-(4-butenyl)pyridine (5-3) Methylpyridine 5-2 (4.0 g, 19.2 mmol) was dissolved in 40 mL THF, cooled to -230C, and LDA (2 M, 24 mL, 48 mmol) was added dropwise. After 30 min the mixture was cooled to -78 C and allyl bromide (2.49 mL, 2.88 mmol) was added dropwise. After 15 min more, the reaction was quenched with sat. NH4Cl, warmed to RT, diluted with EtOAc, and the organic layer was washed with brine.
After drying (MgSO4), filtration and concentration, flash chromatography provided 5-3 as a yellow oil.
TLC Rf 0.40 (silica, 75 % CH2C12/hexane) 1H-NMR (300 MHz, CDCl3): 6 7.72 (d, J = 8 Hz, 1H), 7.56 (t, J = 8 Hz, 1H), 7.16 (br s, 1H), 6.80 (d, J = 7 Hz, 1H), 5.85 (m, 1H), 5.03 (dm, J = 17 Hz, 1H), 4.97 (dm, J = 10 Hz, 1H), 2.74 (t, J = 7 Hz, 2H), 2.42 (qm, J = 7 Hz, 2H), 1.52 (s, 9H).
2-(Boc-amino)-6-(4-hydroxybutyl)pyridine (5-4) A solution of alkene 5-3 (558 mg, 2.25 mmol) in 2 mL ThF was added dropwise to a solution of 9-BBN (0.5 M in THF, 4.95 mL, 2.48 mmol). After stirring overnight, and additional portion of 9 BBN (0.5 M, 1.1 mL, 0.55 mmol) was added and the reaction was continued 1 h more. The reaction was quenched by the successive addition of EtOH (1.5 mL), 6 N NaOH (0.5 mL), and 30 % H2O2 (1.0 mL, exothermic), and heating to 500C for 1h. The cooled mixture was saturated with K2C03, then partitioned between EtOAc and water. The aqueous phase was reextracted with EtOAc, the combined organic phases were washed with brine, dried (MgSO4), filtered and concentrated. Flash chromatography (silica, 40 % EtOAc/hexane) provided alcohol 5-4 as a colorless oil.
TLC Rf 0.26 (silica, 40 % EtOAc/hexane) 1H-NMR (400 MHz, CDC13): 6 7.73 (d, J = 8 Hz, 1H), 7.56 (t, J = 8 Hz, 1H), 7.20 (br s, 1H), 6.80 (d, J = 7 Hz, 1H), 3.67 (t, J = 7 Hz, 2H), 2.70 (t, J = 7 Hz, 2H), 1.77 (qn, J = 7 Hz, 2H), 1.61 (m, 2H), 1.51 (s, 9H).
4-(2-Boc-aminopyridin-6-yl)butanoic acid (5-5) A solution of alcohol 5-4 (247 mg, 0.93 mmol) in 5 mL acetone was cooled to 0 C and a solution of Jones Reagent was added dropwise. As the color of the reaction changed from brown to green, additional Jones Reagent was added, until the alcohol was no longer detected by TLC (3.5 h). After quenching with i-PrOH the mixture was diluted with EtOAc, washed with S % KHSO4 and brine, dried (MgSO4), filtered and concentrated, providing 5-5 as an off-white waxy solid.
1H-NMR (400 MHz, CDC13): 6 9.13 (br s, 1H), 7.90 (d, J = 8 Hz, 1H), 7.64 (t, J = 8 Hz, 1H), 6.85 (d, J = 8 Hz, 1H), 2.80 (t, J = 8 Hz, 2H), 2.46 (t, J = 7 Hz, 2H), 2.01 (qn, J = 7 Hz, 2H), 1.54 (s, 9H).
N-Cbz-glycyl-2(S)-phenylsulfonamido-ss-alanine t-butvl ester ($-6) Amine 1-5 (0.42 g, 1.25 mmol), Cbz-Gly-OH (288 mg, 1.38 mmol), NMM (0.53 mL, 5.0 mmol) and BOP (829 mg, 1.88 mmol) were combined in 6 mL DMF. After stirring overnight the solvent was evaporated, the residue was taken up in EtOAc, the organic solution was washed with water (2x), 5 % KHSO4, sat. NaHCO3 and brine, dried (MgSO4), filtered and concentrated. Flash chromatography (silica, 60 % EtOAc/hexane) provided 5-6 as a white glass.
TLC Rf 0.27 (silica, 60 % EtOAc/hexane) 1H-NMR (400 MHz, CDC13): 6 7.83 (d, J = 7 Hz, 2H), 7.58 (t, J = 7 Hz, 1H), 7.50 (t, J = 8 Hz, 2H), 7.42-7.30 (m, SH), 6.55 (br s, 1H), 5.59 (d, J = 7 Hz, 1H), 5.40 (br s, 1H), 5.16 (s, 2H), 3.95-3.70 (m, 4H), 3.34 (m, 1H), 1.27 (s, 9H).
Glycyl-2(S)-phenylsulfonamido-ss-alanine t-butvl ester (5-7) A solution of 5-6 (0.54 g, 1.10 mmol) in 11 mL EtOAc was treated with 10 % Pd/C (108 mg) and stirred under a H2 balloon overnight. After addition of more 10% Pd/C (100 mg) and hydrogenation for S d the mixture was filtered through Celite and concentrated, providing 5-6 as a white glass.
1H-NMR (400 MHz, CD30D): 6 7.84 (dm, J = 8 Hz, 2H), 7.61 (tm, J = 8 Hz, lH), 7.54 (tm, J = 8 Hz, 2H), 4.00 (dd, J = 8, 5 Hz, lH), 3.59 (dd, J = 14, 5 Hz, 1H), 3.37 (s, 2H), 1.25 (s, 9H).
4-(2-Boc-aminopyridin-6-yl)butanoyl-glycyl-2(S)-phenylsulfonamido-ssalanine t-butyl ester (5-8) Acid 5-5 (144 mg, 0.51 mmol), amine 5-7 (202 mg, 0.56 mmol), NMM (226 11L, 2.1 mmol) and BOP (241 mg, 0.77 mmol) were combined in 2.6 mL DMF. After stirring overnight the solvent was evaporated, the residue was dissolved in EtOAc, washed with water, sat.
NaHCO3, 5 % KHSO4, and brine, dried (MgSO4), filtered and concentrated. Flash chromatography (silica, 10 % CHC13/EtOAc) provided 5-8 as an off-white glass.
TLC Rf 0.22 (silica, 10 % CHC13/EtOAc) 1H-NMR (400.MHz, d6-DMSO): 89.53 (s, 1H), 8.23 (br d, J = 7 Hz, 1H), 7.98 (t, J = 6 Hz, 1H), 7.91 (t, J = 6 Hz, 1H), 7.76 (d, J = 7 Hz, 2H), 7.65-7.53 (m, 5H), 6.87 (d, J = 7 Hz, 1H), 3.85 (br s, 1H), 3.60 (t, J = 5 Hz, 2H), 3.20-3.10 (m, 2H), 2.60 (t, J = 7 Hz, 2H), 2.15 (t, J = 7 Hz, 2H), 1.85 (qn, J = 7 Hz, 2H), 1.46 (s, 9H), 1.18 (s, 9H).
4-(2-Aminopyridin-6-yl)butanoyl -glycyl -2(S)-phenylsulfonamido-ss- alanine (5-9) A solution of 5-8 (138 mg, 0.22 mmol) in 1 mL CH2C12 was cooled to 0 C, treated with 1 mL TFA, and warmed to RT for 5 h.
After concentration and azeotroping with toluene the residue was purified by flash chromatography (silica, 12:20:1:1, EtOAc/EtOH/H20/NH40H), providing 5-9 as a colorless glass.
TLC Rf 0.34 (silica, 12:20:1:1, EtOAc/EtOH/H20/NH40H) 1H-NMR (400 MHz, D20): # 7.76 (dm, J = 7 Hz, 2H), 7.55-7.48 (m, 4H), 6.69 (d, J = 7 Hz, 1H), 6.57 (d, J = 8 Hz, 1H), 3.72-3.62 (m, 2H), 3.55 (dd, J = 8,5 Hz, 1H), 3.37 (dd, J = 13, 8 Hz, 1H); 3.13 (dd, J = 13, 8 Hz, 1H), 2.63 (t, J = 7 Hz, 2H), 2.34 (t, J = 7 Hz, 2H), 1.96 (qn, J = 7 Hz, 2H). SCHEME 6
SCHEME 6 CONTINUED
4-(Pyridin-4-yl)butanoyl-sarcosine ethyl ester (6-1) 4-(4-Pyridyl)butanoic acid 10-5 (100 mg, 1.8 mmol), H Sar-OEt-HCI (300 mg, 2.0 mmol), BOP (965 mg, 2.2 mmol) and NMM (700 pL, 6.4 mmol) were combined in 9 mL DMF. After stirring overnight the mixture was diluted with EtOAc, washed with water (4x), sat. NaHCO3, and brine, dried (MgSO4), filtered and concentrated.
Flash chromatography (silica, 80% to 100 % EtOAc/hexane) provided 6rl as a colorless oil.
TLC Rf 0.44 (silica, 20 % MeOHlEtOAc) 1H-NMR (400 MHz, CDCl3): 4:1 mixture of amide rotomers, major rotomer 8 8.50 (d, J = 5 Hz, 2H), 7.14 (d, J = 5 Hz, 2H), 4.20 (q, J = 7 Hz, 2H), 4.12 (s, 2H), 3.03 (s, 3H), 2.70 (t, J = 8 Hz, 2H), 2.39 (t, J = 7 Hz, 2H), 2.02 (qn, J = 7 Hz, 2H), 1.23 (t, J = 7 Hz, 3H).
4-TPvridin-4-Yllbutanovl-sarcosine (6-2) Ester 6-1 (324 mg, 1.22 mmol) was dissolved in 6 mL EtOH, then 1 N NaOH (2.4 mL, 2.4 mmol) was added. After stirring overnight the mixture was concentrated, rediluted with EtOAc, extracted into 10 % KHSO4, then concentrated, providing acid X, along with inorganic salts.
TLC Rf 0.16 (silica, 4:1:1 CH2C12/MeOH/HOAc) 1H-NMR (400 MHz, CD30D): 1:1 mixture of amide rotomers, 6 8.41 (br s, 2H), 7.33 (m, 2H), 4.00/3.90 (s, 2H), 3.05/2.95 (s, 3H), 2.77-2.67 (m, 2H), 2.48/2.37 (t, J = 7 Hz, 2H), 2.00-1.90 (m, 2H).
4-(Pyridin-4-yl)butanoyl-sarcosine-3(R)-[2-(indol-3-yl)ethyl]-ss-alanine ethyl ester (6-3) Acid 6-2 (288 mg, 1.22 mmol), amine 4-4 (318 mg, 1.22 mmol), BOP (647 mg, 1.5 mmol), and NMM (462 pL, 4.2 mmol) were combined in 6 mL DMF. After stirring overnight the mixture was diluted with EtOAc, washed with water (4x), sat. NaHCO3, and brine, dried (MgSO4), filtered and concentrated. Flash chromatography (silica, EtOAc then 5 % MeOH/EtOAc) provided 6-3 as an orange oil.
TLC Rf 0.4 (20 % MeOH/EtOAc) 1H-NMR (400 MHz, CDC13): 4:1 mixture of amide rotomers, major rotomer 6 8.47 (br d, J = 5 Hz, 2H), 8.02 (br d, J = 6 Hz, 2H), 7.58 (dd, J = 16, 8 Hz, 1H), 7.34 (dd, J = 8,4 Hz, 1H), 7.20-7.03 (m, 3H), 7.01 (s, 1H), 6.72 (d, J = 9 Hz, 1H), 4.33 (m, 1H), 4.1 (t, J = 7 Hz, 3H), 3.98 (s, 2H), 3.05 (s, 3H), 2.90-2.45 (m), 2.39 (t, J = 7 Hz, 2H), 2.02-1.70 (m), 1.23 (t, J = 7 Hz, 3H).
4-(Pyridin-4-yl)butanoyl-sarcosine-3(R)-[2-(indol-3 -yl)ethyl] -ssalanine (6-4) Ester 6-3 (400 mg, 0.84 mmol) was dissolved in 4 mL EtOH, then 1 N NaOH (1.7 mL, 1.7 mmol) was added. After 90 min the reaction was neutralized with 1 N HC1 (1.7 mL, 1.7 mmol) and concentrated to an oil. Flash chromatography (silica, 50:1:1 EtOH/H20/NH4OH, then again with 12:10:1:1 EtOAc/EtOH/H2O/NH4OH) provided 6-4 TLC Rf 0.17 (silica, 12:10:1:1 EtOAc/EtOEVH20/NH40H) 1H-NMR (400 MHz, CD30D): 2:1 mixture of amide rotomers, 6 8.388.28 (m, 2H), 7.54-7.48 (m, 1H), 7.30-7.25 (m, 2H), 7.21-7.19 (m, 1H), 7.07-6.92 (m, 3H), 4.364.27 (m, 1H), 4.03-3.98 (m, 2H), 3.06/2.93 (s, 3H), 2.86-2.60 (m, 4H), 2.52-2.32 (m), 2.05-1.85 (m).
SCHEME 7
7 NH2 Ethyl bromoacetate, TEA CH2C12, O"C to RT BocNH Y CO2H 7 C02H BOP NMM DMF 57 BocNH ,N, Ns,CO2Et 0 SCHEME 7 (CONTD)
SCHEME 7 (CONTD)
N-Cvclopropylglycine ethvl ester (7-2) Cyclopropylamine (12.1 mL, 175 mmol) and TEA (42 mL, 385 mmol) were combined at 0 C in 350 mL CH2C12, then ethyl bromoacetate (19A mL, 175 mmol) was added dropwise. The reaction was warmed to RT for 3 h, then diluted with additional CH2C12, washed with water, sat. NaHCO3, and brine, dried (Na2S04), filtered and concentrated. Flash filtration (silica, 30 % EtOAc/hexane) provided 7-2 as a light yellow oil.
TLC Rf 0.70 (silica, EtOAc) 1H-NMR (400 MHz, CDCl3): 6 4.20 (q, J = 7 Hz, 2H), 3.45 (s, 2H), 2.23 (tt, J = 6, 3 Hz, 1H), 1.29 (t, J = 7 Hz, 3H), 0.43 (m, 2H), 0.36 (m, 2H).
4-(2-Boc-amino-pyridin-4-yl)butanoyl-N-cyclopropylglycine ethyl ester (7-3) Acid 4-1 (0.86 g, 3.1 mmol), amine 7-2 (0.48 g, 3.4 mmol), NMM (1.35 mL, 12.3 mmol) and BOP (2.04 g, 4.61 mmol) were combined in 15 mL DMF. After stirring ovemight the mixture was concentrated, redissolved in EtOAc, washed with water, 5 % KHSO4, sat. NaHCO3 and brine, dried over MgSO4, filtered and concentrated. Flash chromatography (silica, 50 % EtOAc/hexane) provided 7-3 as a colorless oil.
TLC Rf 0.29 (silica, 50 Go EtOAc/hexane) 1H-NMR (400 MHz, CDCl3: 8 8.14 (d, J =5 Hz, 1H), 7.81 (s, 1H), 7.77 (br s, 1H), 6.84 (dd, J = 5, 1 Hz, 1H), 4.18 (q, J = 7 Hz, 2H), 4.08 (s, 2H), 2.80 (tt, J = 7, 4 Hz, 1H), 2.69 (t, J = 7 Hz, 2H), 2.60 (t, J=7 Hz, 2H), 2.02 (qn, J = 7 Hz, 2H), 1.53 (s, 9H), 1.27 (t, J = 7 Hz, 3H), 0.83 (m, 2H), 0.72 (m, 2H).
4-(2-Boc-amino-pyridin-4-yl)butanoyl-N-cyclopropylglycine (7-4) Ester 7-3 (1.07 g, 2.64 mmol) was dissolved in 26 mL MeOH, then treated with 1 N NaOH (6.6 mL, 6.6 mmol). After stirring overnight the reaction was concetrated, redissolved in water, the pH was adjusted to 1 with 10 % KHSO4, then extracted with EtOAc (5x).
The aqueous phase was adjusted to pH 3 with aq. NaOH, then reextracted with EtOAc (2x). The combined organic phases were washed with brine, dried (MgS04), filtered and concentrated, providing 1=4 as a white foam.
TLC Rf 0.24 (silica, 19:1:1, CH2Cl2/MeOH/HOAc) 1H-NMR (300 MHz, CDCl3): 8 9.15 (br s, 1H), 7.97 (d, J = 5 Hz, 1H), 7.94 (s, 1H), 6.89 (dd, J = 5, 1 Hz, 1H), 4.14 (s, 2H), 2.81 (tt, J = 7, 3 Hz, 1H), 2.73 (t, J = 7 Hz, 2H), 2.61 (t, J = 7 Hz, 2H), 2.04 (qn, J = 7) Hz, 2H), 1.51 (s, 9H), 0.85 (m, 2H), 0.76 (m, 2H).
4-(2-Boc-amino-pyridin-4-yl)butanoyl-N-cyclopropylglycyl-3(R)-(2- phenethvl)-ss-alanine ethyl ester (7-6) Acid 7-4 (415 mg, 1.1 mmol), amine hydrochloride 7-5 (see procedure in EP 478 362 utilizing Boc-Gly(OEt) as starting material) (284 mg, 1.1 mmol), NMM (0.48 mL, 4.4 mmol) and BOP (729 mg, 1.65 mmol) were combined in 5 mL DMF. After stirring overnight the reaction was concentrated, redissolved in EtOAc, washed with water, 5 % KHSO4, sat. NaHCO3, and brine, dried (MgSO4), filtered and concentrated. Flash chromatography (silica, EtOAc) provided 7-6 as a colorless waxy solid.
TLC Rf 0.39 (silica, EtOAc) 1H-NMR (400 MHz, d6-DMSO): 6 9.66 (s, 1H), 8.11 (d, J = S Hz, 1H), 7.76 (d, J = 9 Hz, 1H), 7.68 (s, 1H), 7.25 (t, J = 7 Hz, 2H), 7.20-7.12</RT
4-(2-Amino-pyridin-4-yl)butanoyl-N-cyclopropylglycyl-3(R)-(2- phenethvl)-(3-alanine ethvl ester hvdrochloride (7-7) A solution of 7-6 (530 mg, 0.91 mmol) in 4.6 mL CH2Cl2 was cooled to OOC, 4.6 mL TFA was added, and after 1 h the reaction was warmed to RT for 90 min. After concentration and azeotroping with toluene the residue was purified by flash chromatography (silica, 10:1, EtOAc:NH3-saturated EtOH). The residue was dissolved in EtOAc, treated with 1 N HCl in ether, concentrated, then lyophilized from aq. acetonitrile, providing 7-7 as a glassy solid.
TLC Rf 0.25 (10:1, EtOAc:NH3-saturated EtOH) 1H-NMR (400 MHz, d6-DMSO): # 7.94 (br s, 1H), 7.86-7.82 (m, 2H), 7.25 (t, J = 7 Hz, 2H), 7.20-7.13 (m, 3H), 6.80-6.75 (m, 2H), 4.05 (m), 4.02 (q, J = 7 Hz, 2H), 3.93 (AB d, J = 16 Hz, iH),3.85(ABd,J= 16 Hz, 1H), 2.78 (qn, 1H), 2.65 (t, J = 7 Hz, 2H), 2.59 (t, J = 7 Hz, 2H), 2.55-2.40 (m), 1.82 (qn, J = 7 Hz, 2H), 1.80-1.70 (m, 2H), 1.15 (t, J = 7 Hz, 3H), 0.80-0.70 (m, 4H).
4-(2-Amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-(2 phenethvl)-ss-alanine (7-8) Ester 7-7 (100 mg, 0.18 mmol) was dissolved in 4 mL THF, then treated with 1 N LiOH (0.9 mL, 0.9 mmol). After stining overnight the mixture was concentrated and purified by flash chromatography (silica, 15:20:1:1 EtOAc/EtOH/H2O/NH4OH) to provide 7-8 as a white solid.
TLC Rf 0.36 (silica, 15:20:1:1 EtOAc/EtOH/H20/NH40H) 1H-NMR (400 MHz, d6-DMSO): 6 7.81 (d, J = 9 Hz, 1H), 7.77 (d, J = S Hz, 1H), 7.27 (t, J = 7 Hz, 2H), 7.20-7.12 (m, 3H), 6.34 (dd, J = 5, 1 Hz, 1H), 6.28 (s, 1H), 5.76 (br s, 2H), 4.03 (m, 1H), 3.91 (AB d, J = 16 Hz, 1H), 3.85 (AB d, J = 16 Hz, 1H), 2.76 (m, 1H), 2.65-2.50 (m), 2.45 (t, J = 7 Hz, 2H), 2.37 (d, J = 7 Hz, 2H), 1.82-1.60 (m), 0.77-0.68 (m, 4H).
SCHEME 8
ed BocNH ,NJt Ntco2H 8-2 TFA Anisole CH2CI2 V Y1 H2N > C No)t N 4 co2H H2N 8-3
4-(2-Boc-amino-pyridin-4-yl)butanoyl-N-cyclopropylglycyl-3 (R)-[2 (indol-3-vlethvll-8-alanine ethyl ester (8-1) Acid 7-4 (180 mg, 0.48 mmol), amine 4-4 (130 mg, 0.50 mmol), NMM (183 pL, 1.7 mmol) and BOP (253 mg, 0.57 mmol) were combined in S mL DMF. After stirring overnight the reaction was concentrated, redissolved in EtOAc, washed with water (3x), 10 % KHSO4, sat. NaHCO3, and brine, dried (MgSO4), filtered and concentrated. Flash chromatography (silica, 80 % EtOAc/hexane) provided 8-1 as a glassy solid.
TLC Rf 0.34 (silica, EtOAc) 1H-NMR (400 MHz, CDC13): 6 8.10-8.00 (m, 2H), 7.79 (s, 1H), 7.56 (d, J = 8 Hz, 1H), 7.35-7.30 (m, 2H), 7.16 (t, J = 8 Hz, 1H), 7.08 (t, J = 8 Hz, 1H), 7.04 (s, 1H), 6.80 (d, J = 5 Hz, 1H), 6.71 (d, J = 9 Hz, 1H), 4.29 (m, 1H), 4.09 (q, J = 7 Hz, 2H), 3.99 (s, 2H), 2.85-2.70 (m, 4H), 2.66 (t, J = 7 Hz, 2H), 2.61 (t, J = 7 Hz, 2H), 2.51 (m), 2.05-1.87 (m, 4H), 1.53 (s, 9H), 1.21 (t, J = 7 Hz, 3H), 0.90-0.75 (m, 4H).
4-(2-Boc-amino-pyridin-4-yl)butanoyl-N-cyclopropylglycyl-3(R)-[2 (indol-3-yl)ethyll - -alanine (8-2) Ester 8-1 (223 mg, 0.36 mmol) was dissolved in 4 mL EtOH, then 1 N NaOH (0.90 mL, 0.90 mmol) was added. After a few hours the reaction was diluted with EtOAc, extracted with water and the pH of the aq. phase was adjusted to 1 with 10 % KHSO4. The aqueous layer was extracted with EtOAc (2x), the combined organic layers were washed with brine, dried (MgSO4), filtered and concentrated, providing 8-2 as an oil.
TLC Rf 0.64 (silica, 9:1:1 CH2Cl2/MeOH/HOAc)
4-(2-Amino-pyridin-4-yl)butanoyl-N-cyclopropylglycyl-3 (R)- [2-(indol3-vl)ethvll -alanine (8-3) Acid 8-2 (144 mg, 0.24 mmol) was dissolved in 3 mL CH2C12, then anisole (120 pL, 0.96 mmol) and TFA (3 mL) were added. After ca 1 h the reaction was concentrated. Flash chromatography (silica, 18:10:1:1 EtOAc/EtOH/H2O/NH4OH, twice) provided 8-3 as a white solid.
TLC RfO.29 (silica, 18:10:1:1 EtOAc/EtOH/H20/NH40H) 1H-NMR (400 MHz, D20): 6 7.88 (m, 1H) 7.70 (m, 1H), 7.53 (m, 1H), 7.30-7.10 (m, 3H), 6.69 (m, 1H), 6.58 (m, 1H), 4.23 (m, 1H), 3.99 (m, 2H), 2.84 (m, 3H), 2.70 (m, 2H), 2.62 (m, 2H), 2.44 (m), 2.10-1.82 (m), 0.88-0.72 (m, 4H).
SCHEME 9
4-(2-Boc-amino-pyridin-4-yl)butanoyl-N-cyclopropylglycyl-3 (R)methyl-ss-alanine ethvl ester (9-2) Acid 7-4 (100 mg, 0.26 mmol), amine hydrochloride EL (see United States Patent 5,281,585) (49 mg, 0.29 mmol), NMM (117 pL, 1.1 mmol) and BOP (176 mg, 0.40 mmol) were combined in 1.3 mL DMF. After 3 d the mixture was concentrated, redissolved in EtOAc, washed with water (2x), 5 % KHSO4, sat. NaHCO3, and brine, dried (Mg SO4), filtered and concentrated. Flash chromatography (silica, EtOAc) provided 2=2 as a colorless oil.
TLC Rf 0.27 (silica, EtOAc) 1H-NMR (400 MHz, CDC13): # 8.10 (d, J = 5 Hz, 1H), 8.00 (br S, 1H), 7.85 (s, 1H), 6.90 (dd, J = 5, 1 Hz, 1H), 6.63 (d, J = 8 Hz, 1H), 4.30 (m, 1H), 4.12 (q, J = 7 Hz, 2H), 4.02 (AB d, J = 15 Hz, 1H), 3.93 (AB d, J = 15 Hz, 1H), 2.77 (m, 1H), 2.73 (t, J = 7 Hz, 2H), 2.62 (t, J = 7 Hz, 2H), 2.47 (m, 2H), 2.04 (m, 2H), 1.53 (s, 9H), 1.25 (t, J=7 Hz, 3H), 1.20 (d, J = 7 Hz, 3H), 0.88-0.78 (m, 4H).
4-(2-Amino-pyridin-4-yl)butanoyl-N-cyclopropylglycyl-3(R)-methyl-ss alanine ethvl ester (9-3) A solution of 2A (84 mg, 0.17 mmol) in 1 mL CH2C12 at 0 C was treated with 1 mL TFA. After 3 h the mixture was warmed to RT for 1 h, then concentrated and azeotroped with toluene. Flash chromatography (silica, 15 % NH3 satd. i-PrOH/EtOAc) and lyophilization from aq. acetonitrile provided 9-3 as a semi-solid.
TLC Rf 0.19 (silica, 15 % NH3 satd. i-PrOH/EtOAc) 1H-NMR (400 MHz, d6-DMSO): 87.78 (d, J = 5 Hz, 1H), 7.76 (d, J = 8 Hz, 1H), 6.37 (dd, J = 5, 1 Hz, 1H), 6.30 (s, 1H), 5.90 (br s, 1H), 4.09 (m,J=7Hz, lH),4.04(q,J=7Hz,2H),3.86(ABd,J= 16Hz, 1H), 3.79 (AB d, J = 16 Hz, 1H), 2.73 (m, 1H), 2.45 (t, J = 7 Hz, 2H), 2.35 (ABXdd,J= 15,7Hz, 1H), 1.77(qn,J=7Hz,2H), 1.17(t,J=7Hz, 3H), 1.07 (d, J = 7 Hz, 3H), 0.76-0.67 (m, 4H).
4-(2-Amino-pyridin-4-yl)butanoyl-N-cyclopropylglycyl-3(R)-methyl-ssalanine (944 Ester 9-3 (44 mg, 0.11 mmol) was dissolved in 1.1 mL THF, then 1 N LiOH (0.28 mL, 0.28 mmol) was added. After stirring ovemight the reaction mixture was loaded directly onto a flash chromatography column (silica, eluting with 7:20:1:1 EtOAc/EtOH/H20/NH40H) providing 9-4 as a white solid.
TLC Rf 0.62 (silica, 7:20:1:1 EtOAc/EtOH/H2O/NH4OH) 1H-NMR (300 MHz, D20): 6 7.72 (d, J = 7 Hz, 1H), 6.90-6.80 (m, 2H), 4.16 (hex, J = 7 Hz, 1H), 4.04 (s, 2H), 2.86 (tt, J = 7, 4 Hz, 1H), 2.802.65 (m, 4H), 2.41 (ABX dd, J = 14, 6 Hz, 1H), 2.31 (ABX dd, J = 14, 7 Hz, 1H), 1.98 (qn, J = 7 Hz, 2H), 1.16 (d, J = 7 Hz, 3H), 0.89 (m, 2H), 0.80 (m, 2H).
SCHEME 10
SCHEME 10 CONTINUED
Methyl 2-(methoxycarbonyl)-4-(pyridin-4-yl)butyrate (10-3) To a stirred solution of elemental sodium (20 g, 840 mmol) and CH30H (600 ml) was added dimethyl malonate 10-1 (135 ml, 1120 mmol). After S minutes, 4-vinyl pyridine 10-2 (15.3 ml, 140 mmoles) was added and the solution was heated to 50 C for 18 h. The reaction was diluted with EtOAc and then washed with sat NaHCO3, brine, dried (MgSO4) and concentrated. Flash chromatoraphy (silica, 60% EtOAc/hexanes) furnished the diether 10-3 (19.1 g) as a yellow oil TLC Rf = 0.43 (silica, EtOAc) 1H NMR (400 MHz, CDC13)6 852 (d,J = 6 Hz, 2H), 7.12 (d,J = 6 Hz, 2H), 3.75 (s, 6H), 3.38 (t, J = 8 Hz, 1H), 2.64 (t, J = 8 Hz, 2H), 2.24 (m, 2H).
Methyl 4-(Pyridin-4-vl)butyrate (10-4) A solution of diester 10-3 (19.0 g, 80.1 mmol), H20 (1.45 ml, 80.1 mmol), NaCI (10.5 g, 160.2 mmol) and DMF was heated to 1700C for 18 h. The reaction was diluted with EtOAc and then washed with sat. NaHCO3, brine, dried (MgSO4) and concentrated. Flash chromatography (silica, 60% EtOAc/hexanes) afforded ester 10-4 as a brown oil.
TLC Rf 0.32 (silica, EtOAc) 1H NMR (400 MHz, CD30D) 6 8.40 (d, J = 6 Hz, 2H), 7.28 (d, J = 6 Hz, 2H), 3.64 (s, 3H), 2.67 (t, J = 8 Hz, 2H), 2.36 (t, J = 8 Hz, 2H), 1.94 (m, 2H).
4-(Pyridin-4-yl )butanoic acid (10-5) A solution of ester 10-4(10.0 g, 56 mmol), 1N NaOH (84 ml, 84 mmole) and CH30H (200 ml) was stirred at ambient temperature for 1.0 h. Concentrated HCl (7.0 ml, 84 mmol) was added followed by concentration. The residue was dissolved in CHC13, dried (MgSO4) and concentrated to give acid 10-S as a yellow solid.
TLC Rf 0.41 (silica 10:1:1 CH2C12/MeOH/AcOH) 1H NMR (400 MHz, CD30D) # 8.40 (d, J = 6 Hz, 2H), 7.30 (d, J = 6 Hz, 2H), 2.71 (t, J = 8 Hz, 2H), 2.32 (t, J = 7 Hz, 2H), 1.93 (m, 2H).
N-(2-Phenethvl)glvcine methvl ester (10-8) A solution of amine methyl ester 10-6 (1.0 g, 7.96 mmol), bromide 10-7 (1.09 ml, 7.96 mmole), NEt3 (3.33 ml, 23.9 mmol) and DMSO (25 ml) was heated to 600C for 16 h. The reaction mixture was diluted with EtOAc and then washed with sat. NaHCO3, brine, dried (MgSO4) and concentrated. Flash chromatography (silica, 80% EtOAc/hexanes) furnished ester 10-8 as a yellow oil.
TLC Rf 0.29 (silica, EtOAc) 1H NMR (400 MHz, CDCl3) 6 7.29 (m, 2H), 7.22 (m, 3H), 3.71 (s, 3H), 3.43 (s, 2H), 2.89 (m, 2H), 2.82 (m, 2H).
4-(PYridin-4-yllbutanovl-N-(2-phenethYl)glvcine methvl ester (10-9) To a stirred solution of acid 10-5 (342 mg, 2.07 mmol), NMM (910 pl, 8.28 mmol) and CH3CN (15 ml) was added BOP reagent (1.01 g, 2.28 mmol). After 30 minutes, amine 10-S (400 mg, 2.07 mmol) was added and stirring continued for an additional 18 h. The reaction was diluted with EtOAc and then washed with sat. NaHCO3, brine, dried (MgSO4) and concentrated. Flash chromatography (silica, EtOAc) furnished ester 10-9 as a yellow oil.
TLC Rf =0.23 (silica, EtOAc) 1H NMR (CD3OD) 8.39 (d, J = 8 Hz, 2H), 7.14-7.29 (m, 7H), 4.84 (s, 2H), 3.70 (s, 3H), 3.58 (t, J = 7 Hz, 2H), 2.82 (t, J = 7 Hz, 2H), 2.66 (t, J = 8 Hz, 0.56 H), 2.55 (t, J = 8 Hz, 1.44 H), 2.28 (t, J = 7 Hz, 0.56 H), 2.10 (t, J = 7Hz, 1.44 H).
4-(Pyridin-4-yl)butanoyl-N-(2-phenethyl)glycyl (10-10) A solution of ester 10-9 (500 mg, 1.47 mmole), 1N NaOH (2 ml, 2 mmol) and CH30H (5 ml) was stirred at ambient temperature for 2.0 h. Concentrated HCI (167 l, 2.0 mmol) was added followed by concentration. The residue was dissolved in CHC13, dried (MgSO4) and concentrated to give acid 10-10 as a white solid.
*1H NMR (400 MHz, CD30D) & 8.47 (d, J =5 Hz, 2H), 7.44 (m, 2H), 7.25 (m, SH), 4.02 (s, 1.44 H), 3.96 (s, 0.56 H), 3.58 (m, 2H), 2.84 (m, 2H), 2.74 (t, J = 8Hz, 0.56 H), 2.63 (t, J = 8Hz, 1.44 H), 2.33 (t, J = 7Hz, 0.56 H), 2.14 (t, J = 7 Hz, 1.44 H), 1.94 (m, 0.56 H), 1.79 (m, 1.44 H).
4-(Pyridin-4-yl)butanoyl-N-(2-phenylethyl)glycycl-3(R)-(2-phenethyl) ss-alanine ethyl ester (10-11) A solution of acid 10-10 (160 mg, 0.4903 mmol) amine :Z (164 mg, 0.49 mmol), NMM (216 pl, 1.96 mmol), BOP reagent (239 mg, 0.539 mmol) and CH3CN (5 ml) was stirred at ambient temperature for 18 h. The reaction was diluted with EtOAc and then washed with sat. NaHCO3, brine, dried (MgSO4) and concentrated. Flash chromatography (silica, EtOAc) furnished ester 10-11 (220 mg) as a colorless oil.
TLC Rf = 0.21 (silica, EtOAc) 1H NMR (400 MHz, CD30D) # 8.37 (m, 2H), 7.24 (m, 12H), 4.23 (m, 1H), 4.06 (m, 2H), 3.95 (m, 2H), 2.84 (m, 2H), 2.56 (m, 6H), 2.34 (t, J = 7 Hz, 0.56H), 2.16 (t, J = 7 Hz, 1.44 H), 1.83 (m, 0.56 H), 1.81 (m, 1.44 H), 1.91 (m, 3H).
4-(Pyridin-4-yl)butanoyl-N-(2-phenyl)glycyl-3(R)-(2-phenethyl)-ssalanine (10-12) A solution of ester 10-11 (200 mg, 0.3778 mmole), 1N NaOH (0.5 ml, 0.5 mmol) and CH30H was stirred at ambient temperature for 1.5 h followed by concentration. The crude acid was dissolved in H20, acidified with conc. HCl, concentrated and then azeotroped with toluene. Flash chromatography (silica, 20:20:1:1 EtOAc/EtOH/NH4OH/H2O) furnished acid 10-12 (100 mg) as a white solid.
TLC Rf 0.18 (20:20:1:1 EtOAc/EtOH/NH40H/H20) 1H NMR (400 MHz, D20)6 8.47 (d, J = 6 Hz, 1.36 H), 8.43 (d, J = 6 Hz, 0.64 H), 7.71 (d, J = 6 Hz, 0.64 H), 7.66 (d, J = 6 Hz, 1.36 H), 7.20 (m, 10 H), 4.07 (m, 1H), 3.81 (s, 1.36 H), 3.73 (d, J = 6 Hz, 0.64 H), 3.51 (bt, 1.36 H), 3.43 (m, 0.64 H), 2.73 (m, 3H), 2.60 (t, J = 7 Hz, 1.36 H), 2.53 (m, 3.64 H), 2.18 (t, J = 7 Hz, 0.64 H), 1.78 (m, 5.36 H).
SCHEME 11
SCHEME 11 CONTINUED
N-(2-Phenethyl)glycine effivl ester (11-3) A solution of amine 11-1(20.0 g, 165 mmol), NEt3 (47 ml, 330 mmol) in CH2Cl2 at 0 C was treated with bromide 11-2 (22.4 ml, 182 mmol) followed by the removal of the cooling bath. After 1.0 h, the solution was washed with sat. NaHCO3, brine, dried (MgSO4) and concentrated. Flash chromatography (silica, 50% EtOAc/hexanes) afforded ester 11-3 as a yellow oil.
TLC Rf 0.25 (silica, 50% EtOAchexanes) 1H NMR (400 MHz, CD30D) 7.25 (m, 5H), 4.15 (q, J = 7 Hz, 2H), 3.37 (s, 2H), 2.81 (m, 4H), 1.23 (t, J = 7 Hz, 3H).
[4-(2-Boc-aminopyridin-4-yl)butanoyl]-N-(2-phenethyl)glycine ethyl ester (11-4) A solution of acid 4-1 (1.5 g, 5.35 mmol), amine 11-3 (1.66 g, 8.03 mmol), BOP reagent (2.61 g, 5.89 mmol), NMM (3.0 ml, 21.4 mmol) and CH3CN (30 ml) was stirred at ambient temperature for 18 h. The solution was diluted with EtOAc and then washed with H20, sat. NaHCO3, 10% KHSO4, brine, dried (MgSO4) and concentrated.
Flash chromatography (silica, 50% EtOAc/hexanes 80% EtOAc/hexanes) fumished ester 11-4 as a yellow solid.
TLC Rf 0.35 (silica, 50% EtOAc/hexanes) 1H NMR (400 MHz, CDC13) s 8.12 (d, J = 5 Hz, 1H), 7.77 (m, 2H), 7.21 (m, 4H), 7.10 (d, J = 7 Hz, 1H), 6.79 (m, 1H), 4.18 (q, J = 7 Hz, 2H), 4.02 (s, 2H), 3.58 (m, 2H), 2.82 (m, 2H), 2.62 (t, J = 7 Hz, 0.64 H), 2.57 (t, J = 7 Hz, 1.36 H), 2.15 (m, 2H), 1.91 (m, 2H), 1.52 (s, 9H), 1.27 (m, 3H).
[4-(2-BOC-Aminopyridin-4-yl)butanoyl]-N-(2-phenethyl)glycine (11-5) A solution of ester 11-4 (1.8 g, 3.84 mmol), 1N NaOH (6 ml, 6 mmol) and EtOH (10 ml) was stirred at ambient temperature for 30 minutes. The solution was acidified with 10% KHSO4 and then extracted with EtOAc. The EtOAc phase was washed with brine, dried (MgSO4) and concentrated to furnish acid 11-5 as a yellow solid.
TLC Rf 0.80 (silica, 20:1:1 CH2C12/MeOH/AcOH) 1H NMR (400 MHz, CD30D) 6 8.12 (m, 1H), 7.17-7.29 (m, 7H), 4.06 (m, 2H), 3.61 (t, J = 7 Hz, 2H), 2.85 (t, J = 7 Hz, 2H), 2.81 (m, 0.64 H), 2.63 (t, J = 8 Hz, 1.36 H), 2.35 (t, J = 7 Hz, 0.64 H), 2.14 (t, J = 7 Hz, 1.36 H), 1.79 (m, 2H), 1.57 (s, 9H).
4-(2-BOC-Aminopyridin-4-yl)butanoyl-N-(2-phenethyl)glycyl-3 (R) methvl-p-alanine benzvl ester (11-7) A solution of acid 11-5 (400 mg, 0.91 mmol), amine 11-6 (available from Celgene) (285 mg, 1.09 mmol), BOP reagent (440 mg, 0.997 mmol), NMM (502 pl, 3.63 mmol) and CH3CN (20 ml) was stirred at ambient temperature for 18 h. The solution was diluted with EtOAc and then washed with H20, sat. NaHCO3, 10% KHSO4, brine, dried (MgSO4) and then concentrated. Flash chromatography (silica, 80% EtOAc/hexanes) furnished benzyl ester 11-7 as a yellow oil.
TLC Rf 0.49 (silica, EtOAc) 1H NMR (400 MHz, CD30D) 6 8.06 (d, J = 5 Hz, 1H), 7.7 (s, 0.32 H), 7.68 (s, 0.68 H), 7.09-7.36 (m, 10 H), 6.83 (m, 1H), 5.16 (s, 1.36 H), 5.08 (s, 0.64 H), 4.29 (m, 1H), 3.93 (m, 2H), 3.51 (t, J = 7 Hz, 2H), 2.79 (q, J = 7 Hz, 2H), 2.50-2.61 (m, 4H), 2.25 (t, J = 8 Hz, 0.64 H), 2.09 (t, J = 7 Hz, 1.36 H), 1.73-1.84 (m, 4H), 1.51 (s, 9H), 1.25 (d, J = 7 Hz, 3H).
4-(2-BOC-Aminopyridin-4-yl)butanoyl-N-(2-phenethyl)glycyl-3 (R)methyl-ss-alanine (11-8) A solution of benzyl ester 11-7 (380 mg, 0.597 mmol), 1N NaOH (1 ml, 1.0 mmol) and EtOH (5 ml) was stirred at ambient temperature for 1.0 h. The solution was acidified with 10% KHSO4 and then extracted with EtOAc. The EtOAc phase was washed with brine, dried (MgSO4) and concentrated to furnish acid 11-8 as a yellow oil.
1H NMR (400 MHz, CD30D) s 8.13 (m, lH), 7.15-7.35 (m, 7H), 4.24 (m, 1H), 3.91 (m, 2H), 3.58 (m, 2H), 2.81 (m, 2.64 H), 2.62 (t, J = 8 Hz, 1.36 H), 2.36 (t, J = 7 Hz, 0.64 H), 2.14 (t, J = 7 Hz, 1.36 H), 1.79 (m, 2H), 1.57 (s, 9H), 1.19 (m, 3H).
4-(2-Aminopyridin-4-yl)butanoyl-N-(2-pheneffiyl)glycyl-3 (R)-methyl ss-alanine (11-9) A solution of acid 11-8 (320 mg, 0.59 mmol) in CH2C12 (5 ml) was treated with TFA (5 ml). After 1.0 h, the solution was concentrated and then azeotroped with toluene. Flash chromatography (silica, 10:10:1:1 EtOAc/EtOH/NH4OH/H2O) furnished amine 11-10 (210 mg) as a white solid.
TLC Rf = 0.28 (silica, 5:5:.5:.5 EtOAc/EtOH/NH4OH/H2O) 1H NMR (400 MHz, CD30DD) 6 7.71 (d, J = 6 Hz, 1H), 7.15-7.32 (m, SH), 6.62-6.69 (m, 2H), 4.25 (m, 1H), 3.99 (m, 2H), 3.58 (t, J = 7 Hz, 2H), 2.81 (m, 2H), 2.61 (t, J = 7 Hz, 0.64 H), 2.31-2.51 (m, 3.36 H), 2.12 (td, J = 3 Hz, 7 Hz, 1.36 H), 1.89 (t, J = 8 Hz, 0.64 H), 1.79 (m, 2H), 1.19 (m, 3H).
SCHEME 12
SCHEME 12 CONTINUED
Ethvl-4-(4-Pvlidvloxv)butyrate (12-2) A mixture of 4-hydroxypyridine (10 g, 105 mmol), ethyl 4-bromobutyrate 12-1 (15.0 ml, 105 mmol) and Cs2C03 (34.2 g, 105 mmol) in DMF (100 ml) was stirred at room temperature for 24 h. The reaction was filtered and the filtrate diluted with ethyl acetate (300 ml) and washed with water (4 x 100 ml) and brine (100 ml) then dried (Na2 SO4), filtered, and evaporated. The resulting oil was purified by chromatography on silica gel (3% CH30H/CH2C12) to give 12-2 as a colorless glass.
TLC Rf 0.45 (silica, 5% CH3OH/CH2Cl2) 1H NMR (300 MHz, CDC13) # 8.41 (d, J=6.8Hz, 2H), 6.83 (d, J=6Hz, 2H), 4.16 (q, J=7Hz, 2H), 4.07 (t, J=7Hz, 2H), 2.52 (t, J=7Hz, 2H), 2.81 (t, J=7Hz, 2H), 1.23 (t, J=7.0Hz, 3H).
Potassium 4-(4-pvridvloxv)butyrate (12-3) The ester 12-1 (2.5 g, 12.0 mmol) was dissolved in 10 ml ThfF and treated with 0.5 N KOH (24 ml, 12.0 mmol) and H20 (10 ml).
The resulting solution was stirred at room temperature for 78 h then evaporated at reduced pressure to give 12-2 as a white solid.
1H NMR (300 MHz, D20)8 8.19 (d, J = 6.8 Hz, 2H), 6.83 (d, J = 6.8 Hz, 2H), 6.83 (d, J = 6.8 Hz, 2H), 3.96 (t, J = 7.1 Hz, 2H), 2.18 (t, J = 7.1 Hz, 2H), 1.93 (m, 2H).
4-(4-Pyridyloxy)butyrate-N-(2-phenethyl)glycine ethyl ester (12-4) The alkoxy pyridine 12-3 (298 mg, 1.36 mmol) and amine 11-3 (450 mg, 1.36 mmol) were combined with EDC (260 mg, 136 mmol), HOBT (208 mg, 136 mmol), in DMF (30 ml) and stirred at room temperature for 16 h. The solution was then diluted with ethyl acetate (200 ml) and washed with sat. NaHCO3 (2 x 100 ml) and brine (100 ml). The organic layer was dried (Na2S04), filtered, and evaporated and the residue chromatographed on silica gel (3% CH3OH/CH2C12) to give 12-4 as a colorless glass.
1H NMR (300 MHz, CDC13) 6 8.41 (d, J = 6.5 Hz, 2H), 7.25 (m, 5H), 6.78 (d, J = 6.5 Hz, 2H), 61.23 (m, 2H), 4.02 (s, 2H), 4.00 (m, 2H), 3.63 (m, 2H), 3.41 (m, 2H), 2.15 (m, 2H), 1.31 (m, 3H).
4-(4-Pyridyloxy)butyrate-N-(2-phenethyl)glycine potassium salt (12-$) Ester 12-4 (360 mg, 0.97 mmol) was hydrolyzed in 0.5 N KOH (1.94 ml, 0.97 mmol) to give the potassium salt 12-S as a white solid.
1H NMR (300 MHz, DMSO-d6) s 8.38 (d, J = 6.5 Hz, 2H), 7.25 (m, SH), 6.93 (d, J = 6.5 Hz, 2H), 4.016 m, 2H), 3.45 (m, 2H), 3.25 (s, 2H), 2.68 (m, 2H), 2.21 (m, 2H), 1.86 (m, 2H).
4-(Pyridyloxy)butyrate-N-(2-phenethyl)glycyl-3 (R)-2-phenethyl-P- alanine ethyl ester (12-64 Acid salt 12-5 (352 mg, 0.93 mmol) and amino ester 7-5 (240 mg, 193 mmol), HOBT (142 mg, 0.93 mmol), EDC (198 mg, 0.93 mmol), and triethylamine (130 pl, 0.93 mmol) was dissolved in DMF (15 ml) and stirred at room temperature for 18 h. The solution was diluted with ethyl acetate (200 ml) washed with sat. NaHCO3, water and brine (100 ml each), dried (Na2S04) and concentrated to give a colorless oil. Chromatography on silica gel afforded 12-6 as a colorless glass.
TLC Rf 0.50 (silica, 3% CH30H/CH2C12) 1H NMR (300 MHz, CDC13) 6 8.40 (d, J = 6.6 Hz, 2H), 7.25 (m, 10H), 6.85 (t, J = 7.4 Hz, lH), 7.25 (d, J = 6.6 Hz, 2H), 4.25 (m, 1H), 4.18 (m, 2H), 4.00 (m, 2H), 3.60 (m, 2H), 2.95 (m, 2H), 2.63 (m, 2H), 2.58 (m, 2H), 2.40 (m, 2H), 2.08 (m, 2H), 1.85 (m, 2H), 1.16 (m, 3H).
4-(4-Pyridyloxy)butyrate-N-(2-phenethyl)glycyl-3(R)-2-phenethyl-ssalanine (12-7) Ester 12-6 (123 mg, 0.23 mmol) was hydrolyzed with 0.5 N KOH and the acid was isolated as its TFA salt following preparative reverse phase chromatography.
1H NMR (300 MHz, CD30D) 8 8.63 (d, J = 6.5 Hz, 2H), 7.52 (d, J = 6.5 Hz, 2H), 7.20 (m, 10H), 4.41 (m, 1H), 4.32 (m, 2H), 4.01 (m, 2H), 3.81 (m, 2H), 2.85 (m, 2H), 2.63 (m, 2H), 2.30 (m, 2H), 2.41 (m, 2H), 2.20 (m, 2H), 1.85 (m, 2H).
SCHEME 13
SCHEME 13 CONTINUED
tert-Butyl 3-N-methylaminopropionate (13-1) Tert-butyl acrylate (15 g, 117 mmol) was added to a solution or methanol saturated with CH3NH2 (300 ml) and stirred at room temperature for 16 h. The solution was evaporated to afford 13-1 as a colorless liquid.
1H NMR (300 MHz, CDCI3) 6 2.81 (t, J =7.2 Hz, 2H), 2.43 (t, J = 7.2 Hz, 2H), 1.45 (s, 9H).
tert-Butyl 3-[(N-methyl-N-(4-pyridyl)]aminopropionate (13-2) A mixture of 4-chloropyridine hydrochloride (10 g, 75 mmol), 13=1 (12 g, 75 mmol) and N-methylmorpholine (9.1 nil, 82.5 mmol) in N-methyl pyrrolidinone (100 ml) was heated at 1200C forl6 h. The solvent was removed at reduced pressure and the residue partitioned between EtOAc (100 ml) and water (50 ml). The organic layer was washed with water and brine (50 ml each) then dried (Na2SO4), filtered and evaporated. The ester 13-2 was isolated as a colorless glass following flash chromatography on silica gel (5% CH30H/CH2C12).
1H NMR (300 MHz, CDC13) 6 8.30 (d, J = 6.8 Hz, 2H), 6.91 (d, J = 6.8 Hz, 2H), 3.81 (t, J = 7.1 Hz, 3H), 3.22 (s, 3H), 2.65 (t, J = 7.1 Hz, 2H), 1.41 (s, 9H).
3-[(N-Methyl-N'-(4-pyridyl)]aminopropionate hydrochloride (13-3) A solution of 13-2 (2.2 g, 9.3 mmol) in 75 ml anhydrous EtOAc was cooled to 0 and treated with HC1 gas for 10 min. The solution was warmed to room temperature and stirred. For 16 h the resulting solid was filtered to give 13-3 as a hygroscopic yellow solid. 1H NMR (300 mHz, DMSO-d6) 8.26 (d, J = 6.8 Hz, 2H), 7.0 (br d, 2H), 3.82 (t, J = 7.1 Hz, 2H), 3.21 (s, 3H), 2.60 (t, J = 7.1 Hz, 2H).
3-F (N-methyl-N-(4-pvridyl)l aminopropionyl-sarcosine ethvl ester (13-44 Acid 13-3 (383 mg, 1.5 mmol) was coupled with sarcosine ethyl ester hydrochloride (253 mg, 1.65 mmol) following the EDC/HOBT procedure previously described to give 13-4 as a colorless glass.
TLC Rf 0.45 (silica, 3% CH30H/CH2C12) 1H NMR (300 MHz, CDCl3) 6 8.21 (d, J = 6.8 Hz, 2H), 6.51 (d, J = 6.8 Hz, 2H), 4.20 (q, J = 7.0 Hz, 2H), 4.18 (s, 2H), 3.75 (t, J = 7.0 Hz, 2H), 3.09 (s, 3H), 3.04 (s, 3H), 2.65 (t, J = 7.0 Hz, 2H), 1.31 (t, J = 7.0 Hz, 3H).
3-[(N-Methyl)-N'-(4-pyridyl)laminopropionyl-sarcosine potassium salt !13-5) A solution of 13-4 (353 mg, 1.26 mmol) in THF (5 ml) was treated with 0.5 N KOH (2.52 ml, 1.21 mmol) and H20 (5 ml) and stirred at room temperature for 18 h. The solvent was removed in vacuo to afford the potassium salt 13-S as a white solid.
1H NMR (300 MHz, DMSO-d6) & 8.08 (d, J = 6.7 Hz, 2H), 6.57 (d, J = 6.7 Hz, 2H), 3.61 (t, J = 7 Hz, 2H), 3.51 (s, 2H), 2.86 (s, 3H), 2.75 (s, 3H), 2.42 (t, J = 7.0 Hz, 2H).
3-[(N-Methyl)-N'-(4-pyridyl)]aIIiinopropionyl-sarcosine-3(R)-(2- phenethyl)-ss-alanine ethvl ester (13-6) The acid 13-5 was coupled with 7-5 (229 mg, 0.88 mmol) under the EDC/HOBT procedure to afford 13-6 following chromatography (CH2Cl2/CH3OH/NH2/OH, 90:8:2) 1H NMR (300 MHz, CDCl3) # 8.21 (d, J = 6.8 Hz, 2H) 7.25 (m, 2H), 6.73 (d, J = 7.0 Hz, 1H), 6.51 (d, J = 6.8 Hz, 2H), 4.36 (m, 1H), 61.18 (m, 2H), 3.89 (m, 2H), 3.81 (t, J = 7.0 Hz, 2H), 3.06 (s, 3H), 2.98 (s, 3H), 2.85 (m, 2H), 2.65 (m, 2H), 2.51 (m, 2H), 1.83 (m, 2H), 1.20 (m, 3H).
3-[(N-Methyl)-y-(4-pyridyl)]aminopropionyl-sarcosine-3(R)-(2- phenethyl)-ss-alanine (13-7) A solution of the ester 13-6 (75 mg, 0.16 mmol) in THF ( ml) was treated with 0.5N KOH (320 ml, 0.16 mmol) and H20 (5 ml).
The resulting solution was stirred at room temperature for 7.5 h then evaporated at reduced pressure. The resulting residue was purified by reverse phase a white powder.
1H NMR (300 MHz, DMSO-d6) 6 8.61 (d, J = 6.8 Hz, 2H), 7.25 (m, 5H), 7.18 (d, J = 7.0 Hz, 1H), 6.83 (d, J = 6.8 Hz, 2H), 4.35 (m, 1H), 3.83 (m, 2H), 3.81 (t, 2H), 3.13 (s, 2H), 2.95 (s, 3H), 2.85 (m, 2H), 2.65 (m, 2H), 2.56 (m, 2H), 1.86 (m, 2H).
SCHEME 14
N-(t-Butoxycarbonyl)-N-(2-phenylethyl)glycine ethvl ester (14-1) The amine 11-3 (1.11 g, 5.36 mmol) and (BOC)2O (1.28 g, 5.9 mmol) in 10 ml THF were stirred for 48 h under argon. Removal of the solvent in vacuo gave a yellow oil which was purified by chromatography (silica, hexane/EtOAc 9:1) to afford 14-1 as a colorless oil.
Rf (silica, hexane/EtOAc 9:1) 0.41.
N-(t-Butoxycarbonyl)-N-(2-phenylethyl)glycine (14-2) A solution of the ester 14-1 (1.7 g, 5.5 mmol), 11.1 mL 1N LiOH and 11 mL MeOH was stirred at room temperature for 16 h. The mixture was poured into water/EtOAc and acidified with 1N HCI to pH =3. After extraction with EtOAc (2x), the organic layers were washed with brine, dried (MgSO4) and evaporated to give 14-2 as a foam which was used as such in the next step.
N-[N-(t-Butoxycarbonyl)-N'-(2-phenyleffiyl)glycyl -3 (R)-(2- phenylethyl)-ss-alanine methvl ester (14-5) The acid 14-5 (502 mg, 1.8 mmol), 3(R)-(2-phenylethyl) ss-alanine methyl ester hydrochloride 144 (see U.S. Patent 5,281,585) (482 mg, 2.0 mmol), HOBT (267 mg, 2.0 mmol), EDC hydrochloride (515 mg, 2.7 mmol) and N-methylmorpholine (0.22 ml, 2.0 mmol) were stirred in 10 ml DMF for 16 h under argon. After pouring the solution into EtOAc110% citric acid (aqueous solution) the mixture was extracted twice with EtOAc, washed with water then brine, dried (MgSO4) and the solvent removed in vacuo. The residual yellow oil was subjected to column chromatography (silica, hexane/EtOAc 1:1) to give 14-5 as a colorless oil.
Rf (silica, hexane/EtOAc 1:1) 0.44.
N-[N'-(2-phenethyl)glycyl]-3(R)-(2-phenethyl)-ss-alanine methyl ester hydrochloride (14-7) A solution of 14-5 (719 mg, 1.5 mmol) in 40 mL of EtOAc was treated with HCI (g) until saturated. After 30 min the solvent was removed in vacuo and the residue was triturated with ether from which 14-10 crystallized as a white solid.
1H NMR (CD30D) 6 1.87 (2H, m), 2.5-2.8 (4H, m), 3.05 (2H, m), 3.28 (2H, m), 3.62 (3H, s), 3,78 (1H, d), 3.84 (1H, d), 4.26 (1H, m), 7.1-7.4 (10H, m).
N-EN'-(t-Butoxycarbonyl)glycyl]-3(R)-(2-phenethyl)-ss-alanine methyl ester (14-6) N-(t-butoxycarbonyl)glycine 14-3 (Aldrich) was coupled with 14-4 according to the procedure described for the preparation of 14-5. The title compound 14-6 was purified by column chromatography (silica, hexane/EtOAc 1:1).
Rf (silica, hexane/EtOAc 1:1) 0.22.
N-Glycyl-3(R)-(2-phenethyl)-ss-alanine methyl ester hydrochloride (14-8) Following the procedure described for the preparation of 14-7 compound 14-6 was converted into 14-8. 1H NMR (CD30D) # 1.88 (2H, m), 2.5-2.8 (4H, m), 3.64 (5H, s), 4.25 (1H, m), 7.1-7.3 (SH, m).
SCHEME 15
N-[N'-(t-Butoxycarbonyl)glycyl] 3(R)-methyl -p-alanine benzyl ester (15-14 N-(t-Butoxycarbonyl)glycine 14-3 (Aldrich) was coupled with 3(R)-methyl-ss-alanine benzyl ester 05 H2SO4 11-7 (Celgene) according to the procedure described for the preparation of 14-5. The title product 15-1 was then obtained by chromatography (silica, hexane/EtOAc 2:3).
Rf (silica, hexane/EtOAc 1:1) 03.
N-(Glycyl-3(R)-methyl-ss-alanine benzyl ester hydrochloride (15-2) Following the procedure for the preparation of 14-7, compound 15 - 1 was converted into 15-2 which was isolated as a white solid.
1H NMR (CD30D) # 1.22 (3H, d), 2.58 (2H, m), 3.53 (1H, d), 3.63 (1H, d), 6.3 (2H, s), 7.35 (5H, m).
SCHEME 16
H2N CO2Et 1 HN 4 c32 HCI(1 n-butanol, (Aldrich) 16-1 2) (BOC)20, EtN, CH2C12 3) LiOH/EtOH/H2O
SCHEME 16 CONTINUED
3-(4-t-Butoxycarbonyl-1-piperizinyl)benzoic acid (16-3) Ethyl 3-aminobenzoate 16-1 (Aldrich, 24.3 g, 0.147 mol) and bis (2-chloroethyl)amine hydrochloride 16-2 (Aldrich, 26.3 g, 0.147 mol) were heated at reflux in 500 mL n-butanol for 24 h. The solution was concentrated in vacuo, the residue was taken up in EtOAc and washed successively with saturated aqueous NaHCO3 then brine.
After drying (MgS04), the solvent was removed and the resulting black oil chromatographed (silica, EtOAc then EtOAc/MeOH 1:1 then MeOH) to give the corresponding piperizine derivative as a mixture of ethyl and butyl esters.
This piperazine (17.8 g, 76 mmol) was dissolved in 500 mL dry CH2Cl2 and Et3N (13.3 ml, 95.6 mmol) was then added. To this cooled -50C solution was added (BOC)20 (17.4 g, 79.9 mmol) in 45 ml dry CH2Cl2 and stirring was continued until the reaction was complete (as monitored by TLC). The solution was poured into 10% citric acid solution then the organic layer was washed with water, saturated aqueous NaHC03 and brine. After drying over MgSO4, the solvent was removed in vacuo to give a brown oil. Silica gel chromatography (hexane/EtOAc 1:1) gave the BOC-protected piperazine as a mixture of ethyl and butyl esters.
The BOC-protected piperazine (22.1 g) was dissolved in 150 ml 1N LiOH and 600 ml absolute ethanol and this solution was heated at reflux for 16 h. After removal of the ethanol, EtOAc andiO% citric acid solution were added. The organic layer was washed with 1N NaOH, the aqueous layer was then re-acidified with 1N HC1 and extracted with EtOAc. This EtOAc extract was washed with brine, dried (MgSO4) and concentrated to give 16-3 as a white solid.
Rf (silica, hexane/EtOAc 1:1) 0.22.
1H NMR (CDC13) 6 1A9 (9H, s), 3.21 (4H, br t), 3.61 (4H, br t), 7.16 (1H, dd), 7.36 (1H, t), 7.64 (2H, m).
N- { N'-3-(4-t-Butoxycarbonyl- 1 -piperizinyl)benzoyl)glycyl } -3(R) methyl-j3-alaiine benzvl ester (16-4) The acid 16-3 was coupled with 15-2 according to the procedure described for the preparation of 14-5 to yield 16-4.
Rf (silica, EtOAc) 0.45.
N-[N'-[3-(1-Piperazinyl)benzoyl]glycyl]-3(R)-methyl-ss-alanine trifluoroacetic acid salt (16-6) The ester 16-4 (452 mg, 0.84 mmol) was dissolved in 4 ml MeOH, treated with 1N LiOH (2.5 ml, 2.5 mmol) and stirred for 48 h.
The solvent was removed under reduced pressure and to the residue was added 10 ml 1N HCl. After 10 min, the solution was concentrated and the residue purified by preparative HPLC (H20/CH3CN with 0.1% TFA, gradient) to give 16-6.
FAB mass spectrum m/z = 349 (m + I) 1H NMR (CD30D) # 1.22 (3H, d), 2.43 (1H, dd), 2.57 (1H, dd), 3.38 (4H, m), 3.46 (4H, m), 3.96 (1H, d), 4.04 (1H, d), 4.30 (1H, sextet), 7.25 (1H, m), 7.4 (2H, m), 7.5 (1H, m).
N-[N'-[3-(4-t-Butoxycarbonyl- 1 -piperazinyl)benzoyl]glycyl] -3(R)-(2phenethyl)-ss-alanine methyl ester (16-5) The acid 16 3 was coupled with 14-8 according to procedure described for the preparation 14-5 to yield 16-5.
1H NMR (CDCl3) # 1.49 (9H, s), 1.90 (2H, m), 2.58 (2H, d), 2.63 (2H, m), 3.15 (6H, m), 3.55 (4H, m), 3.62 (3H, s), 4.10 (2H, d), 4.32 (1H, m), 7.0-7.5 (9H, m).
N-[N'-[3-(1-Piperazinyl)benzoyl]glycyl]-3(R)-(2-phenethyl)-ss-alanine trifluoroacetic acid salt (16-7) Following the procedure described for the preparation of 16-6, 16-5 was converted into 16-7.
FAB mass spectrum m/z = 439 (m+1) Anal. calcd. for C24 H30 N4 O4 1.35 TFA 1.0 H20 C, 52.53; H, 5.51; N, 9.18 found: C, 52.57; H, 5.44; N, 9.26
N-[N'-[3 -(4-t-Butoxycarbonyl- 1 -piperazinyl)benzoyl] -N'-(2- phenethyl)glycyl]-3(R)-(2-phenethyl)-ss-alanine methvl ester (16-8) The acid 16-3 was coupled with 14-7 according to the procedure described for the preparation of 14-5 to yield 16-8.
Rf (silcia, EtOAc/hexane 2:1) 0.37.
N-[N'-[3-(1-Piperazinyl)benzoyl]-N'-(2-phenethyl)glycyl]-3(R)-(2phenethyl)-ss-alanine trifluoroacetic acid salt (16-9) Following the procedure described for the preparation of 16-6, 16-8 was converted into 16-9.
FAB mass spectrum m/z = 543 (m + 1) Anal. calcd. for C32 H38 N4 O4 @ @ 1.8 TFA .0.8 H20 C, 56.09; H, 5.47; N, 7.35 found: C, 56.09; H, 5.41; N, 7.74 SCHEME 17
Fm Fm 0 BOCN N 002K BOON N 002H ,"" NHCO?H NH 16-3 17-I 1 [-I' 1. WCIH,CO,Et 17-2 2. deprotection 111 HN N NHCQH NH NH0 17-4 IDJN 1. HCI*H2N ~co2t 2. deprotection 0 HNFmNONH &num;OQH \uQNH 0 17-5
3-(4-t-Butoxycarbonyl-1-piperazinyl)benzoyl glycine (17-1) The acid 16-3 was coupled with glycine ethyl ester followed by hydrolysis of the resulting ester using previously described chemistry to yield 17-1.
1H NMR (300 MHz, CD30D) # 1.48 (9H, s), 3.22 (4H, m), 3.59 (4H, m), 4.08 (2H, s), 7.22 (1H, m), 7.40 (2H, m), 7.55 (1H, s).
N-[N'-[3-(1-Piperazinyl)benzoyl]glycyl]-3(S)-ethynyl-ss-alanine trifluoroacetic acid salt (17-4) The acid 17-1 was coupled with 3(S)-ethynyl-ss-alanine ethyl ester hydrochloride (Zablocki et al., J. Med. Chem., 1995, 38, 2378-2394) using standard peptide coupling conditions. The product was then fully deprotected using previously described methodology to give, after reverse phase chromotography, 17-4 as the trifluoroacetate salt.
FAB mass spectrum m/z = 359 (M+1) Anal. calculated for C18H22N4O4.1.10 TFA.O.30 H20 C, 49.59; H, 4.88; N, 11.45 Found: C, 49.58; H, 4.80; N, 11.57
N- ( N'-[3 -(1 -Piperazinyl)benzoyl] glycyl) -3 (S)-(3 -pyridyl)-ss-alanine trifluoroacetic acid salt (17-5) The acid 17-1 was coupled with 3(S)-(3-pyridyl)-ss-alanine ethyl ester hydrochloride (Rico et al., J. Org Chem., 1993, vol. 58, p.
7948) using standard peptide coupling conditions. The product was then fully deprotected using previously described methodology to give, after reverse phase chromatography, 17-5 as the trifluoroacetate salt.
FAB mass spectrum m/z = 412 (M+1) Anal. calculated for C21H25N504-2.55 TFAs0.75 H20 C, 43.80; H, 4.09; N, 9.79 Found: C, 43.76; H, 3.98; N, 10.15 SCHEME 18
SCHEME 18 (CONT'D)
18-4 II C02Et 1. HCIH2N 2. deprotection NH OO2H HN N)$ N NH NH F ' 1. H2N CO2Et 1. H2N2 2. deprotection C /m 0 002H HN NHC02H 0NH0 18-6
5-Amino-2-fluorobenzoic acid 18-2 2-Fluoro-5-nitrobenzoic acid 18-1 (Aldrich) was reduced using 10% Pd-C catalyst in MeOH under an atmosphere of H2 to give, after filtration of the catalyst and removal of the solvent, 18-2 as a solid.
Rf = 0.54 (silica, 10-1-1 EtOH*NH4OHsH20)
N-(5-Amino-2-Fluorobenzovl )glvcine methvl ester 18-3 The acid 18-2 was coupled with glycine methyl ester using standard peptide coupling conditions to give 18-3.
Rf = 0.65 (silica; EtOAc/MeOH 9:1)
5-(4-t-Butoxycarbonyl-1-piperizinyl)-2-fluoro benzoyl glycine 184 Following the procedure described for the preparation of 16-3, the aniline 18-3 was converted into the piperazine-acid 18-4 1H NMR (300 MHz, CD30D) 6 1 A6 (9H, s), 3.11 (4H, m), 3.58 (4H, m), 4.12 (2H, s), 7.05-7.21 (2H, m), 7.40 (1H, m).
N-{N'-[2-Fluoro-5-(1-piperazinyl)benzoyl]glycyl}-3(S)-ethynyl-ssalanine trifluoro acetic acid salt 18-5 Following the procedure described for the preparation of 17-4, compound 18-4 was converted into 18-5.
FAB mass spectrum m/z = 377 (M+1) Anal. calculated for C18H21N4O4F.1.30TFA.0.50 H20 C, 46.37; H, 4.40; N, 10.50 Found: C, 46.34; H, 4.37; N, 10.58
N-{N'-[2-fluoro-5-(1-piperazinyl)benzoyl]glycyl}-3(S)-(3-pyridyl)-ssalanine trifluoroacetic acid salt 18-6 Following the procedure described for the preparation of 17-5, compound 18-4 was converted into 15-6.
FAB mass spectrum m/z = 430 (M+1) Analysis calculated for C21H24N5O4F.2.65 TFA.0.90H2O C, 42.24; H, 3.83; N, 9.37 Found: C, 42.25; H, 3.81; N, 9.71 SCHEME 19
Nsg NH2 +0C02Et HCIH2N'CO,t-Bu CH3 OHO OH3 19-i Etch, EtOH, Cbz-Glycine tnCO2Et BDOMPF' NMM 19-3
SCHEME 19 CONT'D
Ethyl 4-(1.8-naphthyridin-2-yl)butanoate (19-3) Aminoaldehyde 19-1 (2.02 g, 16.6 mmol, prepared according to Het. 1993, 36, 2513), ketone 19-2 (5.3 mL, 33.1 mmol) and L-proline (0.48 g, 4.17 mmol) were combined in 75 mL EtOH.
After heating at reflux overnight the reaction was concentrated. Flash chromatography (silica, EtOAc) provided 19-3 as an off-white crystalline solid.
TLC Rf 0.23 (silica, EtOAc) 1H NMR (300 MHz, CDC13): 9.09 (dd, J=4, 2Hz, 1H), 8.17 (dd, J=8, 2Hz, 1H), 8.12 (d, J=8Hz, 1H), 7.46 (dd, J=8, 4Hz, 1H), 7.42 (d, J=8Hz, 1H), 4.12 (q, J=7Hz, 2H), 3.11 (t, J=8Hz, 2H), 2.44 (t, J=7Hz, 1H), 2.26 (qn, J=8Hz, 2H), 1.25 (t, J=7Hz, 3H).
Ethyl 4-(1,2,3,4-tetrahydro-1,8-naphthyridin-7-yl)butanoate (19-4) A solution of 19-3 (2.3 g, 9.4 mmol) in 50 mL EtOAc was treated with 10% Pd/C (230 mg) and a hydrogen balloon. After 4 d the reaction filtered through celite, concentrated, and purified by flash chromatography (silica, 70% EtOAc/hexane), providing 194 as a yellow oil.
TLC Rf 0.40 (silica, EtOAc) 1H NMR (300 MHz, CDC13): 6 7.05 (d, J=7Hz, 1H), 6.35 (d, J=7Hz, 1H), 4.73 (br s, 1H), 4.12 (q, J=7Hz, 2H), 2.69 (t, J=6Hz, 2H), 2.57 (t, J=8Hz, 2H), 2.33 (t, J=7Hz, 2H), 1.98 (m, 2H), 1.90 (m, 2H), 1.25 (t, J=7Hz, 3H).
4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-yl)butanoic acid hydrochloride (19-5) Ester 19-4 (1.8 g, 7.25 mmol) in 36 mL 6 N HCl was heated at 500C for 4 h, then concentrated, providing 19-5 as a yellow solid.
1H NMR (300 MHz, CD30D): 6 7.59 (d, J=7Hz, 1H), 6.63 (d, J=7Hz, 1H), 3.50 (t, J=5Hz, 2H), 2.82 (t, J=6Hz, 2H), 2.74 (t, J=8Hz, 2H), 2.38 (t, J=7Hz, 2H), 2.02-1.90 (m, 4H).
N-Cbz-Glvcvl-D-alanine t-butyl ester (19-7) N-CBz-Glycine (1.0 g, 4.78 mmol), amine 19-6 (0.91 g, 5.02 mmol), NMM (2.1 mL, 19.1 mmol) and BOP (3.17 g, 7.17 mmol) were combined in 15 mL DMF. After stirring overnight the mixture was concentrated, diluted with EtOAc, washed with water, sat.
NaHCO3, water, 5% KHSO4 and brine, dried (MgSO4), filtered and concentrated. Flash chromatography (silica, 60% EtOAc/hexane) provided 19-7 as a colorless oil.
TLC Rf 0.24 (silica, 60% EtOAc/hexane) 1H NMR (400 MHz, d6-DMSO): 6 7.89 (br t, J=SHz, 1H), 7.44 (br t, J=6Hz, 1H), 7.40-7.30 (m, 5H), 5.02 (s, 2H), 3.56 (d, J=6Hz, 2H), 3.25 (q, J=6Hz, 2H), 2.35 (t, J=7Hz, 2H), 1.40 (s, 9H).
Glvcvl-D-alanine t-butyl ester hvdrochloride (19-8) A solution of 19-7 (1.51 g, 4.49 mmol) in 40 mL EtOAc was treated with 10% Pd/C (0.30 g), and a H2 balloon. After stirring overnight under a hydrogen atmosphere, an additional 200 mg of 10% Pd/C was added and hydrogenation was continued for 4 h before filtering through Celite and concentrating, providing the free amine as a colorless oil. The amine was dissolved in Et20 and an excess of 1 M HCI in Et2O was added. Concentration provided 19-8 as a waxy solid.
1H NMR (free amine, 400 MHz, d6-DMSO): 6 8.31 (br s, 1H), 5.30 (br s, 2H), 3.29 (q, J=6Hz, 2H), 3.25 (s, 2H), 2.38 (t, J=7Hz, 2H), 1.41 (s, 9H).
4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-yl)butanoyl-glycyl-ss-alanine t-butyl ester (19-9) A mixture of 19-5 (62 mg, 0.24 mmol), 19-8 (69 mg, 0.29 mmol), NMM (130 RL, 1.2 mmol) and BOP (160 mg, 0.36 mmol) in 2 mL CH3CN was stirred overnight. After diluting with EtOAc the mixture was washed with sat. NaHCO3, water (5x) and brine, dried (MgSO4), filtered and concentrated, providing 19-9.
TLC Rf 0.79 (silica, 25% NH3-sat. EtOH/EtOAc) 1H NMR (300 MHz, CDCl3): 8 8.50 (br t, 1H), 7.08 (d, J=7Hz, 1H), 6.64 (br t, 1H), 6.33 (d, J=7Hz, 1H), 5.69 (br s, 1H), 3.99 (d, J=7Hz, 2H), 3.53 (q, J=6Hz, 2H), 3.43 (m, 2H), 2.69 (t, J=6Hz, 2H), 2.60 (t, J=7Hz, 2H), 2.46 (t, J=6Hz, 2H), 2.25 (t, J=7Hz, 2H), 2.05-1.90 (m, 4H), 1.45 (s, 9H).
4-(1,2,3 ,4-Tetrahydro-l ,8-naphthyridin-7-yl)butanoyl-glycyl-P- alanine (19-10) Ester 19-9 (69 mg, 0.17 mmol) was dissolved in 1 mL CH2C12 at OOC, 1 mL TFA was added, and the reaction was wanned to ambient temperature for 6 hr. After concentrating and azeotroping with toluene, flash chromatography (silica, 7:20:1:1 EtOAc/EtOH/H20/NH40H) provided 19-10 as a white solid.
TLC Rf 0.38 (silica, 7:20:1:1 EtOAc/EtOH/H20/NH40H) 1H NMR (400 MHz, D20): 6 7.53 (d, J=7Hz, 1H), 6.59 (d, J=7Hz, 1H) 3.85 (s, 2H), 3.46 (t, J=6Hz, 2H), 3.42 (t, J=7Hz, 2H), 2.78 (t, J=6Hz, 2H), 2.72 (t, J=8Hz, 2H), 2.40 (apparent q, J=7Hz, 4H), 2.00 (qn, J=6Hz, 2H), 1.92 (qn, J=6Hz, 2H).
SCHEME 20
4-(1.23.4-Tetrahydro-1.8-naphtheridin-7-vl)butanovl glycine (20-la) A mixture of 19-5 (1.02 g, 4.0 mmol), glycine benzyl ester (0.8 g, 4.0 mmol), NMM (1.76 ml, 16 mmol) and BOP (2.03 g, 4.6 mmol) in CH3CN (100 ml) was stirred overnight. The reaction was concentrated and the residue was partitioned between EtOAc and H2O.
The organic layer was washed with sat. NaHCO3 solution, brine, dried (MgSO4), filtered and concentrated to a yellow gum which was purified by flash chromatography (silica, 1:1, acetone/CH2C12) to provide the ester as a colorless gum.
A solution of the ester (1.3 g, 3.5 mmol) in EtOH (100 ml) was hydrogenated at 1 atm for 18 hr. The reaction was diluted with EtOAc (200 ml) to dissolve the product, filtered and concentrated to a solid which was sonicated with ether (100 ml) to provide 20-la as a colorless solid.
TLC Rf 0.35 (silica, EtOH/NH3) 1H NMR (300 MHz, CD30D): # 7.50 (d, J=7Hz, 2H), 6.59 (d, J=7Hz, 2H), 3.80 (s, 2H), 3.47 (t, J=6Hz, 2H), 2.79 (t, J=6Hz, 2H), 2.72 (t, J=7Hz, 2H), 2.26 (t, J=7Hz, 2H), 2.02 (m, 2H), 1.94 (m, 2H).
4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-yl)butanoyl-glycyl-3(S)pyridin-3-yl-ss-alanine ethyl ester (20-2) The CH3CN solution (300 mL) of 20-la (164 mg, 0.59 mmol), 20-1 (Rico et al., J. Org. Chem., 1993, 58, 7948) (158 mg, 0.58 mmol), NMM (260 l, 2.36 mmol) and BOP (300 mg, 0.68 mmol) was stirred under ambient conditions for 48 h. The reaction was concentrated to a yellow gum which was purified by flash chromatography (silica, 9:1 CH2Cl2/EtOH.NH3) to provide 20-2 as a colorless gum.
Rf 0.21 (silica, 9:1 CH2Cl2/EtOH.NH3) 1H NMR (300 MHz, CD30D): # 8.53 (bs, 1H), 8.42 (d, J=SHz, 1H), 7.82 (d, J=8Hz, 1H), 7.39 (dd, J=8Hz, 5Hz, 1H), 7.10 (d, J=8Hz, 1H), 6.36 (d, J=7Hz, 1H), 5.40 (t, J=7Hz, 1H), 4.07 (q, d=7Hz, 2H), 3.85 (s, 2H), 3.36 (t, J=6Hz, 2H), 2.91 (m, 2H), 2.68 (t, J=6Hz, 2H), 2.51 (t, J=7Hz, 2H), 2.23 (t, J=7Hz, 2H), 1.89 (m, 4H), 1.16 (t, J=7Hz, 3H).
4-(1,2,3,4-Tetrahydro- 1,8 -naphthyridin-7-yl)butanoyl-glycyl-3 (S) pyridin-3 -vl--alanine (20-3) A methanol solution (10 mL) of 20-2 (190 mg, 0.42 mmol) and 1N NaOH (2.1 mL, 2.1 mmol) was stirred under ambient conditions for 18 h. The reaction was concentrated to dryness and the residue neutralized with 1N HCI and the resultant solution concentrated to a gum which was chromatographed (silica, 38/1/1 EtOH/NH4OH/H2O) to provide a solid which was purified by HPLC using a VyOAC C18 semi prep column with gradient elution [95:5(99.9:0.1 H2O/TFA)/(99.9:0.1 CH3CN/TFA # 50:50 (99.9:0.1 H20/TFA)/(99.9:0.1 CH3CN/TFA)80 min] to provide 20-3 as a hygroscopic solid ditrifluoroacetate salt.
Rf 0.36 (silica 38:1:1 EtOH/NH4OH/H2O) 1H NMR (300 MHz, CD30D): 6 8.79 (bs, 1H), 8.65 (d, J=SHz, 1H), 8.7 (d, J=8Hz, 1H), 7.84 (m, 1H), 7.57 (d, J=7Hz, 1H), 6.61 (d, J=7Hz, 1H), 5.44 (t, J=7Hz, 4H), 3.88 (m, 2H), 3.48 (t, J=SHz, 2H), 2.98 (d, J=7Hz, 2H), 2.81 (t, J=6Hz, 2H), 2.70 (m, 2H), 2.31 (m, 2H), 1.96 (m, 4H). LiOH, THF/H2O HN CO2tBu SCHEME 21 (CONT'D) HCl, E N CO2tBu N CO2H H CO2Et HCl.
H2N 2 PYCLU, iPr2NEt, DMF Ethyl isonipecotate (6.0 g, 38.66 mmol), 4-chloropyridine hydrochloride (5.9 g, 38.66 mmol) and N-methylmorpholine (9.3 mL, 85.0 mmol) were dissolved in N-methylpyrrolidinone (50 mL) and the resulting solution heated at 1000 for 48 h. The solution was concentrated in vacuo and the residue dissolved in ethyl acetate (200 mL), washed with water and brine (2 x 100 mL), then dried (Na2S04) and evaporated. The resulting residue was purified by flash chromatography (5%MeOH/CH2C12) to afford 21-1 as a crystalline solid.
1H NMR (300 MHz, CDCl3) # 8.21 (d, J=6.8Hz, 2H), 6.78 (d, J=6.8Hz, 2H), 4.18 (q, J=7.0Hz, 2H), 3.85 (m, 2H), 3.10 (m, 2H), 2.61 (m, 1H) 2.05 (m, 2H), 1.85 (m, 2H), 1.23 (t, J=7.0Hz, 3H).
N N CO2H 21=2 N-Pyridin-4-ylisonipecotic acid (21-2) A solution of ester 21-1 (10 g, 42.7 mmol) in THF (50 mL) was treated with 1N LiOH (47 mL, 47.0 mmol) and H20 (50 mL). The resulting solution was concentrated and the aqueous residue cooled to 0 C, then adjusted to pH 6 with 1N HCl and the resulting solid 21-2, collected by filtration.
1H NMR (300 MHz, D20) # 7.95 (d, 6.8Hz, 2H), 6.73 (d, 6.8Hz, 2H), 3.76 (d, J=12.8Hz, 2H), 2.81 (m, 2H), 2.20 (m, 1H), 1.85 (d, J=12.8Hz, tert-Butvl-N-cvclopropylglycine (21-3) A mixture of cyclopropylamine (10.0 g, 175.1 mmol) and triethylamine (4.9 ml, 35.5 mmol) in 100 ml CH2C12 was cooled to 0 and treated with tert-butyl bromoacetate (5.25 ml, 35.0 mmol). The resulting mixture was stirred at 0 C for 2 h, refluxed for 1.5 h, then cooled and washed with sat. NaHCO3, and brine (50 ml each) then dried (Na2S04) and evaporated to afford 21-3 a colorless oil.
1H NMR (300 MHz, CDCl3) # 3.35 (s, 2H), 2.19 (m, 1H), 2.08 (br s, 1H), 1.48 (s, 9H), 0.47 (m, 2H), 0.38 (m, 2H).
# # # N N N CO2tBu O 21-4 tert-Butyl N-pyridin-4-ylisonipecotyl-N-cyclopropyl glvcine (21-4) A solution of acid 21-2 (500 mg, 2.36 mmol), ester 21-4 (404 mg, 2.36 mmol), chloro-N,N,N',N'-bis(pentamethylene)- formamidinium hexafluorophosphate (PYCLU) (851 mg, 2.36 mmol), and diisopropylethyl amine (305 mg, 2.36 mmol) in anhydrous DMF (50 mL) was stirred at room temperature for 18 h then concentrated in vacuo to afford a yellow residue. Chromatography on silica gel (1:1 MeOH/EtOAc) afforded 21-4 as a crystalline solid.
1H NMR (300 MHz, CD30D) 88.12 (d, J=6.8 Hz, 2H), 6.75 (d, J=6.8Hz, 2H), 3.94 (d, J=12.8Hz, 2H), 3.85 (s, 2H), 2.81 (m, 2H), 1.95 (m, 2H), 1.85 (m, 2H), 1.55 (m, 2H), 1.42 (s, 9H), 0.47 (m, 2H), 0.38 (m, 2H).
Ester 21-5 (250 mg, 0.70 mmol) was suspended in EtOAc (25 mL), cooled to 0 and treated with HCI gas for 15 min. The resulting solution was stirred at 0 for 3.5 h then evaporated to give 21-S as a yellow glass.
1H NMR (300 MHz, CD30D) 6 8.18 (d, J=6.8Hz, 2H), 7.18 (d, J=6.8Hz, 2H), 4.24 (d, J=12.8Hz, 2H), 3.95 (s, 2H), 3.21 (m, 2H), 1.95 (m, 2H), 1.85 (m, 2H), 1.62 (m, 2H), 0.87 (m, 2H), 0.75 (m, 2H).
H N CO2Et N N N O 21-7 Ethyl N-pyridin-4-ylisonipecotyl-N-cyclopropylglycyl-3(S)-ethynyl ss-alanine (21-7) A solution of acid 21-5 (232 mg, 0.68 mmol), ester 21-6 (121 mg, 0.68 mmol) (21-6 prepared as described in U.S. patent 5,272,162), PYCLU (245 mg, 0.68 mmol), and diisopropylethyl amine (176 mg, 0.68 mmol) in anhydrous DMF (50 ml) was stirred at room temperature for 18 h then concentrated in vacuo to afford a yellow residue. Preparative reverse phase chromatographic purification afforded ester 21-7 as its TFA salt.
1H NMR (300 MHz, CD30D) # 8.48 (d, J=6.8Hz, 1H), 8.08 (d, J=6.8Hz, 2H), 7.18 (d, J=6.8Hz, 2H), 5.01 (m, H), 4.24 (d, J=12.8Hz, 2H), 4.12 (q, J=7Hz, 2H), 3.99 (s, 2H), 3.72 (m, 1H), 3.31 (m, 2H), 2.95 (m, 1H), 2.73 (m, 2H), 1.95 (m, 2H), 1.85 (m, 2H), 1.21 (t, J=7.0Hz, 2H), 0.87 (m, 2H), 0.75 (m, 2H).
H N CO2H N N N H 21-8 N-Pyridin-4-ylisonipecotyl-N-cyclopropylglycyl -3(S)-ethynyl-ssalanine (21-8) A solution of ester 21-7 (180 mg, 0.422 mmol), in ThIF (10 mL) was treated with 1N LiOH (0.84 mL, 0.84 mmol) and stirred at room temperature for 16 h. The mixture was concentrated and the residue purified by preparative reverse phase chromatography to afford 19-8 as its TFA salt.
1H NMR (300 MHz, CD30D) 6 8.40 (d, J=6.8Hz, 1H), 8.21 (d, J=6.8Hz, 2H), 7.21 (d, J=6.8Hz, 2H), 4.81 (m, 1H), 4.22 (d, J=12.8Hz, 2H), 3.99 (m, 2H), 3.72 (m, 1H), 3.31 (m, 2H), 2.95 (m, 1H), 2.76 (m, lH), 2.71 (m, 2H), 1.95 (m, 2H), 1.85 (m, 2H), 0.87 (m, 2H), 0.75 (m, PYCLU, iPr2NEt, DMF SCHEME 22 (CONT'D) Ethyl N-pyridin-4-ylnipecotate (22-1) Ethyl (#) nipecotate (7.0 g, 44.53 mmol) was reacted with 4-chlorpyridine hydrochloride (6.67 g, 44.53 mmol) as described for 21-1 to give the title compound as a yellow solid.
1H NMR (300 MHz, CDCl3) # 8.22 (d, J=6.8Hz, 2H), 6.68 (d, J=6.8Hz, 2H), 4.18 (q, J=7.0Hz, 2H), 3.85 (m, 1H), 3.72 (m, 1H), 3.21 (m, 1H), 3.10 (m, 1H), 2.60 (m, 1H), 2.08 (m, 1H), 1.81 (m, 2H), 1.60 (m, 1H), 1.13 (t, J=7.0Hz, 3H).
CO2H N N 22-2 N-Pyridin-4-ylnipecotic acid (22-2) Prepared from 22-1 (764 mg, 3.25 mmol) in a manner similar to that described for 21-2.
1H NMR (300 MHz, DMSO-d6) # 8.13 (d, J=6.8Hz, 2H), 6.74 (d, J=6.8Hz, 2H), 4.08 (d, 1H), 3.78 (m, 1H), 2.92 (m, 2H), 2.10 (m, 1H), 1.95 (m, 1H), 1.71 (m, 1H), 1.42 (m, 2H).
Prepared from 22-2 (320 mg, 1.51 mmol) and 21-3 (258 mg, 1.51 mmol) in a manner similar to that described for 214.
1H NMR (300 MHz, CDCl3) # 8.12 (d, 6.8Hz, 2H), 6.62 (d, J=6.8Hz, 2H), 3.94 (s, 2H), 3.85 (m, 1H), 3.12 (m, 1H), 3.08 (m, 1H), 2.51 (m, 2H), 1.95 (m, 1H), 1.85 (m, 2H), 1.58 (m, 2H), 1.42 (s, 9H), 0.47 (m, 2H), 0.38 (m, 2H).
### N N N CO2H O N-Pyridin-4-ylnipecotyl-N-cyclopropylglycine hydrochloride (22-4) Ester 22-3 (250 mg, 0.70 mmol) was suspended in EtOAc (25 mL), cooled to 0 and treated with HCl gas for 15 min. The resulting solution was stirred for 3.5 h then evaporated to give 22-4 as a white solid.
1H NMR (300 MHz, DMSO-d6) 6 8.18 (d, J=6.8Hz, 2H), 7.18 (d, J=6.8Hz, 2H), 4.24 (d, J=12.8Hz, 2H), 3.95 (m, 2H), 3.21 (m, 1H), 1.94 (m, 1H), 1.85 (m, 1H), 1.72 (m, 1H), 1.53 (m, 1H), 0.87 (m, 2H), 0.75 Ethyl N-pyridin4-ylnipecotyl-N-cyclopropylglycyl-3 (S)-ethyny1-- alanine (22-5) Prepared from 224 (195 mg, 0.60 mmol) in a manner similar to that described for 21-7.
1H NMR (300 MHz, CD30D) # 8.49 (d, J=6.8Hz, 1H), 8.17 (d, J=6.8Hz, 2H), 7.21 (d, J=6.8Hz, 2H), 5.15 (m, 1H), 4.26 (m, 1H), 4.21 (d, 1H), 4.08 (q, 2H), 3.82 (m, 1H), 3.5-3.3 (m, 3H), 2.95 (m, 1H), 2.76 (m, 1H), 2.71 (m, 2H), 2.15 (m, 1H), 1.95 (m, 1H), 1.81 (m, 1H), 1.72 (m, 1H), 1.21 (t, 3H), 0.87 (m, 2H).
O N N H N CO2H H 22-6 N-Pyridin-4-ylnipecotyl-N-cyclopropylglycyl-3(S)-ethynyl-ssalanine (22-6) Prepared from 224 (20 mg, 0.04 mmol) in a manner similar to that described for 21-8.
FAB mass spectrum m/z = 399 (M + 1).
1H NMR (300 MHz, CD30D) # 8.16 (d, J=6Hz, 2H), 6.91 (d, J=6.8Hz, 2H), 5.05 (m, 1H), 4.26 (d, Hz, 1H), 4.21 (d, 1H), 3.82 (m, lh), 3.5-3.3 (m, 3H), 2.95 (m, 1H), 2.76 (m, 1H), 2.71 (m, 2H), 2.15 (m, 1H), 1.95 (m, 1H), 1.81 (m, 1H), 1.72 (m, 1H), 1.21 (t, 1H), 0.87 (m, 2H).
NaN(TMS)2 THF, -78 X N then N CO2CH3 Br 23-2 # 21-3 N HCl-HN CO2Et N BOP, NMM, DMF O 10% Pd/C N OEt EtOH H2 O SCHEME 23 (CONT'D) 1N NaOH O N EtOH OEt 21-6 H CO2Et HCl H2N O BOP, NMM N OH 1. 1N NaOH N CO2Et EtOH 2. Prep HPLC Methyl 4-(1,8-naphthyridin-4-yl)butyrate (23-2) To a stirred solution of naphthyridine 23-1 (Hamada, Y. et al., Chem. Pharm. Bull. Soc., 1971, 19(9), 1857-1862), (2.2 g, 15.2 mmol) and THF (200 ml) at -780C was added NaN(TMS)2 (1M/rHF, 18 ml, 18 mmol) dropwise over a 20 min period. After 30 minutes at 78 C, methyl 3-bromopropionate was added in a stream. After 30 min, the reaction was quenched with 50 ml 10% KHSO4. The mixture was extracted with Et20. The remaining aqueous portion was basified with sat. NaHCO3 and then extracted with EtOAc. The EtOAc portion was washed with brine, dried (MgSO4) and concentrated. Flash chromotography (silica, 2% EtOH/EtOAc) gave the ester 23-2 (1.61 g) as a yellow oil.
TLC Rf = 0.27 (silica, 2% EtOH/EtOAc) 1H NMR (400 MHz, CDC13) # 9.14 (m, lH), 9.35 (d, J=4Hz, 1H), 8.50 (d, J=7Hz, 1H), 7.52 (q, J=4Hz, 1H), 7.33 (d, J=4Hz, 1H), 3.71 (s, 3H), 3.14 (t, J=8Hz, 2H), 2.46 (t, J=7Hz, 2H), 2.09 (m, 2H).
N CO2H 23-3 4-(1,8-Naphthyridin-4-yl)butanoic acid (1-3) A solution of ester 23-2 (1.60 g, 6.9 mmol), 1N NaOH (7 ml, 7 mmol) and EtOH (20 ml) was stirred at ambient temperature for 1.0 h. The solution was extracted with Et20. The aqueous portion was neutralized with concentrated HCl (583 l, 7.0 mmol). The precipitate was collected, washed with Et20, and dried in vacuo to furnish carboxylic acid 23-3 as a tan solid.
TLC Rf = 0.59 (silica, 20:1:1 CH2C12/MeOH/AcOH) 1H NMR (400 MHz, CD30D) # 9.05 (q, J=2H, 1H), 8.95 (d, J=4H, 1H), 8.77 (dd, J=2Hz, 8Hz, 1H), 7.67 (q, J=4H, 1H), 7.53 (d, J=4Hz, 1H), 3.22 (t, J=8Hz, 2H), 2.46 (t, J=7Hz, 2H), 2.03 (m, 2H).
N' 0 N N OEt O 23-4 4-(1,8-Naphthyridin-4-yl)butanoyl-N-(cyclopropyl)glycine ethyl ester (23-4) A solution of acid 23-3 (400 mg, 1.84 mmol), amine 21-3 (331 mg, 1.84 mmol), BOP reagent (979 mg, 2.21 mmol), NMM (1.03 ml, 7.36 mmol) and DMF (20 ml) was stirred at ambient temperature for 20 h. The solution was diluted with ethyl acetate and then washed with sat. NaHCO3, brine, dried (MgSO4) and concentrated. Flash chromatography (silica, 10:1 EtOAc/sat. NH3-EtOH) furnished ester 234 (600 mg) as an orange solid.
TLC Rf = 0.15 (silica, 10:1 EtOAc/sat. NH3-EtOH) 1H NMR (300 MHz, CDCl3) # 9.12 (m, lH), 9.03 (d, J=4Hz, 1H), 8.62 (dd, J=2Hz, 8Hz, 1H), 7.53 (q, J=4Hz, 1H), 7.38 (d, J=4Hz, 1H), 4.20 (q, J=7Hz, 2H), 4.13 (s, 2H), 3.19 (t, J=8Hz, 2H), 2.79 (m, 1H), 2.70 (m, 2H), 2.13 (m, 2H), 1.29 (t, J=8Hz, 3H), 0.85 (m, 2H), 0.74 (m, 2H).
4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-5-yl)butanoyl-N-(cyclopropyl)glycine ethyl ester (23-5) A mixture of ester 23-4 (600 mg, 1.75 mmol), 10% Pd/C (300 mg) and EtOH (30 ml) was stirred under hydrogen atmosphere (1 atm) at ambient temperature for 20 h. The catalyst was removed by filtration through celite and then the filtrate was concentrated. Flash chromatography (silica, 50%/EtOAc/sat. NH3-EtOH) gave ester 23-5 as a colorless oil.
TLC Rf = 0.25 (silica, 50:1 EtOAc/sat. NH3-EtOH) 1H NMR (400 MHz, CD30D) # 7.58 (d, J=6Hz, 1H), 6.48 (d, J=6Hz, 1H), 4.15 (q, J=7Hz, 2H), 4.08 (s, 2H), 3.36 (t, J=SHz, 2H), 2.86 (m, 1H), 2.75 (t, J=6Hz, 2H), 2.68 (t, J=7Hz, 2H), 2.60 (t, J=8Hz, 2H), 1.90 (m, 4H), 1.25 (t, J=7Hz, 3H), 0.87 (m, 2H), 0.78 (m, 2H).
HN # O N N OH O 23-6 4-(1,2,3,4-Tetrahydro-1,8-napthyridin-5-yl)butanoyl-N-(cyclopropyl)glycine (23-6) A solution of ester 23-S (200 mg, 0.5774 mmole), 1N NaOH (600 l, 0.600 mmole) and CH30H was stirred at ambient temperature for 1.5 h. The solution was concentrated. The residue was dissolved in 1N HC1 (600 l) and then the solution was concentrated.
The residue was dissolved in CHC13, filtered and concentrated to give the carboxylic acid 23-6 (110 mg) as a white solid.
TLC Rf = 0.14 (silica, 10:1:1 CH2C12/MeOH/AcOH) 1H NMR (300 MHz, CD30D) # 7.56 (d, J=6Hz, 1H), 6.64 (d, J=6Hz, 1H), 3.98 (s, 2H), 3.41 (t, J=6Hz, 2H), 2.89 (m, 1H), 2.81 (t, J=6Hz, 2H), 2.71 (m, 4H), 1.88 (m, 4H), 0.82 (m, 4H).
HN N CO2Et N NH O 23-7 4-(1,2,3,4-Tetrahydro-1,8-napthyridin-5-yl)butanoyl-N-(cyclopropyl)glycyl-3(S)-ethynyl-ss-alanine ethyl ester (23-7) To a stirred solution of acid 23-6 (40 mg, 0.1256 mmol), amine 21-6 (33 mg, 0.1884 mmol), NMM (70 l, 0.5024 mmol) and CH3CN (1 ml) was added BOP reagent (61 mg, 0.1382 mmol). After 20 h at ambient temperature, the solution was diluted with ethyl acetate and then washed with sat. NaHCO3, brine, dried (MgSO4) and concentrated. Flash chromatography (silica, 40:1:1 CH2Cl2/MeOH/AcOH) gave the ester 23-7 as a colorless oil.
TLC Rf = 0.23 (silica, 40:1:1 CH2Cl2/MeOH/AcOH) 1H NMR (300 MHz, CD30D) # 7.58 (d, J=6Hz, 1H), 6.66 (d, J=6Hz, 1H), 5.01 (m, 1H), 4.13 (q, J=7Hz, 2H), 4.02 (s, 2H), 3.42 (t, J=6Hz, 2H), 2.72 (m, 10H), 1.95 (m, 4H), 1.24 (t, J=7Hz, 3H), 0.85 (m, 2H), 4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-5-yl)butanoyl-N-(cyclopropyl)glycyl-3(S)-ethynyl-ss-alanine (23-8) A solution of ester 23-7 (32 mg, 0.0725 mmol), 1N NaOH (100 1) and CH30H (500 ml) was stirred at ambient temperature for 1.0 h. The solution was concentrated. The residue was dissolved in 1N HCl (100 l) and then concentrated. Preparative HPLC purification (C18, H2O/CH3CN/TFA) provided acid 23-8 as a TFA salt.
TLC Rf = 0.50 (silica, 10:1:1 EtOH/NH4OH/H2O) 1H NMR (300 MHz, CD30D) & 8.43 (d, J=9Hz, 1H), 7.58 (d, J=7Hz, 1H), 6.76 (d, J=7Hz, 1H), 4.99 (m, 1H), 4.03 (d, J=3Hz, 2H), 3.46 (t, J=SHz, 2H), 2.72 (m, 10H), 1.95 (m, 4H), 0.87 (m, 2H), 0.79 (m, 2H).
NMM, CH3CN # HOBT, EDC BOC O H OC2H5 N N HCl/dioxane N HCl/dioxane H O O SCHEME 24 (CONT'D)
SCHEME 24 (CONT'D)
Preparation of 3-{2-[5-(1H-Benzoimidazol-2-yl-amino)-pentanoylamino]-acetylamino}-3(S)-pyridin-3-yl- propionic acid (24-9) 3-t-Butoxycarbonylaminoacetylamino-3(S)-pyridin-3-yl-propionic acid ethyl ester bis hydrochloride (24-la) A stirred solution of BOC-Gly (645 mg, 3.7 mmol), NMM (452 uL, 4.0 mmol), and EtOAc (35 mL) at 0 C was treated with isobutyl chloroformate (534 uL, 4.0 mmol). After 20 min 20-1(1.0 g, 3.7 mmol) and NMM (1.2 mL, 11 mmol) were added followed by removal of the cooling bath. After 20 hr, the reaction mixture was washed with H20, sat. NaHCO3, and brine, dried (MgSO4), and concentrated. Flash chromatography (silica, EtOAc to 5% MeOH/EtOAc) gave 24-1 as a colorless oil.
TLC: Rf = 0.31 (20% MeOH/EtOAc), 1H NMR (300MHz, CDCL3) 6 8. 58 (bs, 1H), 8.51(m, 1H), 7.62 (m, 1H), 7.49 (m, 1H), 5.48 (m, 1H), 4.13 (m, 1H), 4.08 (q, J=7Hz, 2H), 3.83 (m, 2H), 2.90 (m, 2H), 1.43 (s, 9H), 1.13 (t, J=7Hz, 3H).
3-Aminoacetylamino-3 (S)-pyridin-3-yl-propionic acid ethyl ester bishvdrochloride (24-2) HCl gas was passed through a solution of 24-la (0.84 g, 2.4 mmol) in EtOAc (24 mL) at 0 C for 15 min and the reaction mixture stirred for an additional 15 min. The reaction mixture was concentrated and the residue triturated with ether to give 24-2 as awhite solid.
TLC: Rf = 0.29 (10:1:1 ethanoVH20/NH40H).
3-[2-(5-t-Butoxycarbonylarminopentanoylamino)acetylamino]-3(S)- pyridin-3.yl-propionic acid ethvl ester (24-3) A CH3CN solution (20 mL) of 24-I (71.7 mg, 0.33mmol), 24-2 (97 mg, 0.30 mmol), HOBT (50.5 mg, 0.33mmol), EDC (63.3 mg, 0.33 mmol) and NMM (132 ml, 1.2 mmol) was stirred under ambient conditions for 18 hr. The reaction solution was concentrated to a yellow gum which was partitioned between EtOAc and sat. NaCO3 solution. The EtOAc layer was washed with H20, brine, dried(MgSO4) and concentrated to provide 24-3 as a colorless gum.
TLC: Rf = 0.41 (50% CH2Cl2/acetone), 1H NMR (300MHz, CDCL3) # 8.56(bs, 1H), 8.51(m, 1H), 7.62(m, 1H), 7.49(m, 1H), 5.43(m, 1H), 4.08(q, J=7Hz, 2H), 3.94(m, 2H), 3.12(m, 2H), 2.90(m, 2H), 2.28(m, 2H), 1.64(m, 4H), 1.43(s, 9H), 1.13(t, J=7Hz, 3H).
3-[2-(5-Aminopentanoylamino)-acetylamino]-3(S)-pyridin-3-ylpropionic acid ethyl ester dihydrochloride (24-4) A 4M HCl/dioxane solution(I0 mL) of 24-3 (101 mg, 0.24 mmol) was stirred under ambient conditions for 18 hr. The solution was concentrated to provide 24-4 as a pale yellow gum which was used in the next step without further purification.
1H NMR (300 MHz, CD30D) 6 8.93(bs, 1H), 8.79(m, 1H), 8.69(m, 1H), 8.10(m, 1H), 5.48(m, 1H), 4.14(q, J=7Hz, 2H), 3.88(m, 2H), 3.07(m, 2H), 2.89(m, 2H), 2.33(m, 2H), 1.68(m, 4H), 1.23(t, J=7Hz, 3H).
3-(2-{5-[3-(2-Nitrophenyl)-thioureido]-pentanoylamino}acetylamino)-3(S)-pyridin-3-yl-propionic acid ethvl ester (24-6) An ethanol solution(20 mL) of 24-5 (40 mg, 0.224 mmol) and 24-4 (95 mg, 0.224 mmol) was refluxed for 2 hr and concentrated to a yellow gum which was purified by flash chromatography (80% EtOAc/EtOH-NH3) to provide 24-6 as a yellow gum.
TLC: Rf = 0.41 (80% EtOAc/EtOH-NH3), 1H NMR (300 MHz, CD30D) # 8.54(m, 1H), 8.42(m, 1H), 8.03(m, 2H), 7.83(m, 1H), 7.63(m, 1H), 7.41(m, 1H), 7.32(m, 1H), 5.39(m, 1H), 4.09(q, J=7 Hz, 2H), 3.86(s, 2H), 3.61(m, 2H), 2.91(m, 2H), 2.33(m, 2H), 1.69(m, 4H), 1.16(t, J=7Hz, 3H).
3-(2- ( 5-[3-(2-Aminophenyl)-thioureido] -pentanoylamino 1 acetylamino)-3(S)-pyridin-3-yl-propionic acid methyl ester (24-7) 10% Pd/C (50 mg) and 24-6 (103 mg, 0.194 mmol) were added to methanol saturated with ammonia and the mixture hydrogenated at 1 atm. for 18 hr. The reaction was filtered and concentrated to provide 24-7 as a pale yellow gum which was used in the next step without further purification.
1H NMR (300MHz, CD30D) # 8.53(m, 1H), 8.41(m, 1H), 7.84(m, 1H), 7.40(m, 1H), 7.07(m, 1H), 6.97(m, 1H), 6.82(m, 1H), 6.67(m, 1H), 5.38(m, 1H), 3.85(m, 2H), 3.62(s, 3H). 3.54(m, 2H), 2.93(m, 2H), 2.30(m, 2H), 1.60(m, 4H).
3- ( 2-[5-(1 H-Benzoimidazol-2-yl-amino)-pentanoylamino]-acetyl amino }-3(S)-pyridin-3-yl-propionic acid methvl ester (24-8) An ethanol mixture(20 ml) of 24-7 (89 mg, 0.18 mmol), mercuric oxide (78.8 mg, 0.36 mmol) and sulfur (1.8 mg, 0.056 mmol) was refluxed for 2 hr. After cooling, the mixture was filtered and the filtrate concentrated to a semi-solid which was purified by flash chromatography (20 % MeOH/CH2C12) to provide 24-8 as a solid.
TLC: Rf = 0.13 (20% MeOH/CH2C12), 1H NMR (300 MHz, CD30D) # 8.52(m, 1H), 8.41(m, 1H), 7.81(m, 1H), 7.38(M, 1H), 7.23(m, 2H), 7.06(m, 2H), 5.38(m, 1H), 3.85(s, 2H), 3.62(s, 3H), 3.37(m, 2H), 2.95(m, 2H), 2.33(m, 2H), 1.71(m, 4H).
3-{2-[5-(1H-Benzoimidazol-2-yl-amino)-pentanoylamino]-acetyl amino }-3(S)-pyridin-3-yl-propionic acid (24-9) A 6N HCI solution (5 ml) of 24-8 (33 mg, 0.073 mmol) was stirred under ambient conditions for 18 hr. The reaction was concentrated to give a viscous gum which was purified by prep HPLC (Delta-Pak C18, gradient elution over 40 min., 5-50% CH3CN/H200.1% TFA) to give 24-9.
1H NMR (300MHz, CD30D) # 8.78(m, 1H), 8.65(m, 1H), 8.40(m, 1H), 7.86(m, 1H), 7.34(m, 2H), 7.27(m, 2H), 5.40(m, 1H), 3.87(m, 2H), 3.42(m, 2H), 2.98(m, 2H), 2.34(M, 2H), 1.74(m, 4H).
SCHEME 25
?N 002H t\H4Ac H2 25-2 CHO Ac 25-1 | 1. Ph > COCI 2. Penicilin acylace resolution 3. 3. 6N HCI, 4. EtOH/HCI q Y > N 2 HOle H2| > CO2Et 25-3 BocNHCH2002 H EDC, EDC, HOBT, NMM, DMF t SCHEME 25 (Cont'd)
SCHEME 25 (Cont'd)
SCHEME 25 (Cont'd)
3(S)-Ouinolin-3-vl-ss-alanine ethyl ester hvdrochlonde (25-3) A solution containing 25-1 3-carboxaldehyde ( 5 g, 31.8 mmol), malonic acid (3.6 g, 35.0 mmol), and ammonium acetate (5.0 g, 63.6 mmol) in anhydrous ethanol (125 mL) was heated at reflux for 12 h. After cooling to room temperature, the resulting white solid was collected by filtration and washed with cold ethanol (50 mL) and then dried under vacuum to provide 25-2 as a white solid.
H NMR (300 MHz, D20): d 8.91 (d, J = 2 Hz 1H), 8.21 (d, J = 2Hz, 1H), 8.12 (d, J = 8Hz, 1H), 7.84 (d, J = 7 Hz, 1H), 7.72 (t, J= 7Hz, 1H), 7.54 (t, J = 7 Hz, 1,H), 4.72 (M, 1H), 2.73 (M, 2H). The (S)enantiomer of 25-2 was prepared using the enzymatic resolution described by Soloshonok et. al. (Tetrahedron: Asymmetry, 6, 1601, 1995). The resolved material was converted to 25-3 by refluxing in ethanolic HCl.
1H NMR (300 MHz, CD30D): d 9.25 (d, J = 2Hz 1H), 8.31 (d, J = 2Hz, lH), 8.15 (d, J = 8Hz, IH), 7.84 (d, J =7 Hz, 1H), 7.72 (t, J = 7Hz, 1H), 7.54 (t, J = 7 Hz, 1,H), 4.72 (M, 1H), 4.15 (q, J = 6 Hz, 2H), 2.73 (M, 2H) 1.18(t,J=6Hz,3H).
N-Boc-Glycyl-2(S)-quinolin-3-yl-ss-alanine ethvl ester (254) N-Boc-Glycine (60.7 mg, 0.35 mmol), amine 25-3 (0.10 mg, 3.15 mmol), NMM (0.13 mL, 1.26 mmol) and EDC (78.5 mg, 0.41 mmol) were combined in 3 mL DMF. After stirring overnight the mixture was concentrated, diluted with EtOAc, washed with water, sat.
NaHCO3, water, and brine, dried (MgS04), filtered and concentrated.
Flash chromatography (silica, EtOAc) provided 25-4 as a colorless oil.
TLC Rf 0.45 (silica, EtOAc) 1H NMR (300 MHz, CDC13): d 8.88 (d, J = 2Hz 1H), 8.09 (d, J = 2Hz, 1H), 8.05 (d, J = 8Hz, 1H), 7.79 (d, J =6 Hz, 1H), 7.72 (t, J = 6Hz, 1H), 7.68 (br d, 1H), 7.54 (t, J = 7 Hz, 1,H), 5.65 (M, 1H), 5.18 (br, t, 1H), 4.15 (q, J = 6Hz, 2H), 3.00 (M, 2H), 1.21 (s, 9H), 1.08 (t, J= 7Hz, 3H).
Glycyl-3(S)-quinolin-3-yl-ss-alanine-dihadrochloride (25-5) The ester 25-4 (92 mg, 0.23 mmol)was dissolved in HC1 saturated ethanol and stirred at room temperature for 3.5 h then concentrated at reduced pressure to afford 25-5 as a white solid.
1H NMR (300 MHz, CD30D): d 9.38 (s, 1H), 9.15 (s, 1H), 8.45 (d, J = 6Hz, 1H), 8.22 (d, J = 6 Hz, 1H), 8.17 (t, J = 6Hz, 1H), 8.00 (t, J = 7 Hz, 1,H), 5.65 (M, 1H), 5.18 (br, t, 1H), 4.15 (q, J = 6 Hz, 2H), 3.00 (M, 2H), 1.21 (s, 9H), 1.08 (t, J= 7 Hz, 3H).
4-(1,2,3,4-Tetrahydro-1,8-naphtyridin-7-yl)butanoyl-glycyl-3(S) quinolin-3-vl-f3-alanine ethyl ester (25-6).
A mixture of 19-5 (57 mg, 0.22 mmol), 25-5 (84 mg, 0.22 mmol), NMM (123 mL, 1.12 mmol) and, HOBT (39 mg, 0.29 mmol) and EDC (55 mg, 0.29 mmol) in 2 mL DMF was stirred overnight.
After diluting with EtOAc the mixture was washed with sat. NaHCO3, water, and brine, dried (MgSO4), filtered and concentrated,and chromatographed on silica (30% MeOH/EtOAc) providing 25-6.
1H NMR (300 MHz, CDCl3): d 9.91(s, 1H), 8.56 (br t, 1H), 8.18 (br d, 1H), 8.16 (s, 1HI, 8.05 (d, J = 6 Hz, 1H), 7.82 (d, J= 6 Hz, 1H), 7.73 (t, J = 6 Hz, 1H), 7.54 (t, J = 6 Hz, 1H), 7.08 (d, J = 7Hz, 1H), 6.33 (d, J = 7Hz, 1H), 5.71 (m, 1H), 5.69 (br s, 1H), 4.15 (d, J = 7Hz, 2HI, 4.05, (t, J = 7 Hz, 2H), 3.53 (q, J=6Hz, 2H), 3.43 (m, 2H), 3.00 (m, 2H), 2,69 (t, J = 6Hz, 2H), 2.60 (t, J = 7Hz, 2H), 2.46 (t, J = 6Hz, 2H), 2.25 (t, J = 2H), 2.05-1.90 (m, 4H), 1.08 (t, J = 7 Hz, 3H).
4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-yl)butanoyl-glycyl-3(S) quinolin-3-yl--alanine. (25-7).
Ester 25-6 was dissolved in 5 mL 6N HCl and warmed to 50 C for 1.5 h then concentrated and the residue chromatographed on silica (75% EtOHNH3/EtOAc) to afford 25-7.
1H NMR (300 MHz, D20): d 8.71(s, 1H), 8.16 (s, 1H), 7.71 (d, J = 6 Hz, 1H), 7.64 (d, J = 6 Hz, 1H), 7.54 (t, J = 6 Hz, 1H), 7.54 (t, J = 6 Hz, 1H), 7.08 (d, J = 7Hz, 1H), 6.06 (d, J = 7Hz, 1H), 5.35 (m, 1H), 4.81 (s, 2H), 3.53 (q, J= 6Hz, 2H), 3.43 (m, 2H), 3.00 (m, 2H), 2.69 (t, J = 6Hz, 2H), 2.60 (t, J = 7Hz, 2H), 2.46 (t, J = 6Hz, 2H), 2.25 (t, J = 7Hz, 2H), 2.05-1.90 (m, 4H).
EXAMPLE 26 Tablet Preparation Tablets containing 25.0, 50.0, and 100.0 mg., respectively, of the following active compounds are prepared as illustrated below: 4-(2-Amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3 (R)-(2 phenethyl)-ss-alanine; 4-(2-Bocamino-pyridin-6-yl)butanoyl-N-cyclopropylgycyl-3(R)-[(2indol-3-yl)ethyl]-ss-alanine; and 4-(2-Amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-[[2-indol3-yl)ethyl]-ss-alanine.
TABLE FOR DOSES CONTAINING FROM 25-100MG OF THE ACTIVE COMPOUND Amount-mg Active Compound 25.0 50.0 100.0 Microcrystalline cellulose 37.25 100.0 200.0 Modified food corn starch 37.25 4.25 8.5 Magnesium stearate 0.50 0.75 1.5 All of the active compound, cellulose, and a portion of the corn starch are mixed and granulated to 10% corn starch paste. The resulting granulation is sieved, dried and blended with the remainder of the corn starch and the magnesium stearate. The resulting granulation is then compressed into tablets containing 25.0, 50.0, and 100.0 mg, respectively, of active ingredient per tablet.
EXAMPLE 27 Intravenous formulations An intravenous dosage form of the above-indicated active compound is prepared as follows: Active Compound 0,5-10.0mg Sodium Citrate 5-50mg Citric Acid l-15mg Sodium Chloride 1-8mg Water for Injection (USP) q.s. to 1 L Utilizing the above quantities, the active compound is dissolved at room temperature in a previously prepared solution of sodium chloride, citric acid, and sodium citrate in Water for Injection (USP, see page 1636 of United States Pharmacopoeia/National Formulary for 1995, published by United States Pharmacopoeial Convention, Inc., Rockville, Maryland, copyright 1994.
EXAMPLE 28 Intravenous formulation A pharmaceutical composition was prepared at room temperature using 4-(2-Aminothiazol-4-yl)butanoyl-glycyl -2(S)- phenylsulfonamido-P-alanine t-butyl ester, a citrate buffer, and sodium chloride, to obtain a concentration of 4-(2-Aminothiazol4-yl)butanoyl- glycyl-2(S)-phenylsulfonamido-ss-alanine t-butyl ester of 0.25 mg/ml.
800 grams of water was introduced into a standard pharmaceutical mixing vessel. 0.25 grarns of the ester was dissolved in the water. 2.7 grams sodium citrate and 0.16 grams citric acid were added to obtain a finished citrate concentration of 10 mM. 8 grams of sodium chloride was added. 200 grams of water was then added to achieve the desired final concentrations of ingredients. The resulting aqueous formulation had the following concentrations: Ingredient Amount 4-(2-Aminothiazol4-yl)butanoyl-glycyl-2(S)- phenylsulfonamido-ss-alanine t-butyl ester 0.25 mg/ml citrate buffer 10 mM sodium chloride 8 mg/ml The finished concentrated formulation is stored in a standard USP Type I borosilicate glass container at 3040 degrees C.
Prior to compound administration, the concentrated formulation is diluted in a 4:1 ratio resulting in a finished concentration of 0.05 mg/ml and transfered to an infusion bag.
Therapeutic Treatment Oral dosages of the compounds used to elicit a vitronectin receptor antaginizing effect will range between about 0.01 mg per kg of body weight per day (mg/kg/day) to about 100 mg/kg/day and preferably 0.01-50 mg/kg/day and more preferably 0.01-20 mg/kg/day, e.g. 0.1 mg/kg/day, 1.0 mg/kg/day, 5.0 mg/kg/day, or 10 mg/kg/day.
Advantageously, compounds of the present invention may be administered in divided doses of two, three, or four times daily.
Intravenously, the most preferred doses will range from about 1 to about 10 mg/kg/minute during a constant rate infusion. Furthermore, preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather that intermittent throughout the dosage regime.
EIB ASSAY Duong et al., J. Bone Miner. Res., 8:S 378, describe a system for expressing the human integrin avss3. It has been suggested that the integrin is involved in the 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 yl TBS buffer (50 mM Tris.HCl pH 7.2, 150 mM NaCl, 1% BSA, 1 mM CaC12, 1 mM MgC12).
2. 25 Ill cell extract (dilute with 100 mM octylglucoside buffer to give 2000 cpm/25 cm1).
3. 125I-echistatin (25 Sly50,000 cpm) (see EP 382 451).
4. 25 1 buffer (total binding) or unlabeled echistatin (non specific binding).
The reaction mixture was then incubated for 1 h at room temp. The unbound and the bound avss3 were separated by filtration using a Skatron Cell Harvester. The filters (prewet in 1.5% polyethyleneimine for 10 mins) were then washed with the wash buffer (50 mM Tris HCl, lmM CaC12/MgC12, pH 7.2). The filter was then counted in a gamma counter.
SPA ASSAY Materials: 1. Wheatgerm agglutinin Scintillation Proximity Beads (SPA): Amersham 2. Octylglucopyranoside: Calbiochem 3. HEPES: Calbiochem 4. NaCl: Fisher 5. CaC12: Fisher 6. MgC12: SIGMA 7. Phenylmethylsulfonylfluoride (PMSF): SIGMA 8. Optiplate: PACKARD 9. 29-10 (see preparation below)(specific activity 500-1000 Ci/mmole) 10. test compound 11. Purified integrin receptor: &alpha;#ss3 was purified from 293 cells overexpressing avp3 (Duong et al., J. Bone Min. Res., 8: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 l (40 mg/ml) of pretreated beads were suspended in 97.5 Ill of binding buffer and 20 l of 50-OG buffer. S p (-30 ng/pl) 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 1ll of binding buffer and 25 ,ul of 50-OG buffer.
3. Reaction The following were sequentially added into Optiplate in corresponding wells: (i) Receptor/beads mixture (75 ,ul) (ii) 25 1ll of each of the following: compound to be tested, binding buffer for total binding or 29-8 (see preparation below) for nonspecific binding (final concentration 1 ,uM) (iii) 29-10 in binding buffer (25 l, final concentration 40 pM) (iv) Binding buffer (125 Rl) (v) Each plate was sealed with plate sealer from PACKARD and incubated ovemight with rocking at 40C 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 SCHEME 29
H2N C02H O H NH2 29-1 oSO2CI NaOH, dioxane H20 H2N < co2H O H Nv SO 29-2 n I 1. Sr25NaOH, H20 2. HCI H2Nts çCO2H H Nv H 29-3 1 EtOH SCHEME 29 (Cont'd)
SCHEME 29 (Cont'd)
SCHEME 29 (Cont'd)
H2N JiX QS11 21 HNH N H2C H3 o 29-7 6N HCI 6000 025, H2 N "'" N CO2H 29-8 (CH3Sn)25 Pd(PPh3)4, H2N/ Sn(CH3)3 H2N NN H H 025, NH N CO2H H2N i) Oiyw$Ii25l 29-9 N O CO2H 0 29-10 N-(4-Iodo-phenylsulfonylamino)-L-asparagine (29-2) To a stirred solution of acid 29-1 (4.39 g, 33.2 mmol), NaOH (1.49 g, 37.2 mmol), dioxane (30 ml) and H20 (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 Et20 to provide acid 29-2 as a white solid.
1H NMR (300 MHz, D20) 6 7.86 (d, 2H, J=8Hz), 7.48 (d, 2H, J=8Hz) 3.70 (m, 1H), 2.39 (m, 2H).
2(S)-(4-Ioso-phenylsufonylamino)-ssalanine (29-3) 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 29-2 (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 29-3 as a white solid.
1H NMR (300 MHz, D20) 8 8.02 (d, 2H, J=8Hz), 7.63 (d, 2H, J=8Hz), 4.36 (m, 1H), 3.51 (dd, 1H, J=5Hz, 13Hz) 3.21 (m, 1H).
Ethvl 2(S)-*-iodo-phenvlsulfonylamino)-ss-alanine-hydrochlonde (29 44 HCl gas was rapidly bubbled through a suspension of acid 29-3 (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 29-4 as a white solid.
1H NMR (300 MHz, CD30D) # 7.98 (d, 2H, J=8Hz), 7.63 (d, 2H, J=8Hz), 4.25 (q, 1H, J=SHz), 3.92 (m, 2H), 3.33 (m, 1H), 3.06 (m, 1H), 1.01 (t, 3H, J=7Hz).
Ethyl 4-[2-(2-Aminopyridin-6-yl)ethyl]benzoate (29-5) A mixture of ester 29-5a (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 29-5 as a brown oil.
TLC Rf = 0.23 (silica, 40% EtOAc/hexanes) 1H NMR (300 MHz, CDC13) # 7.95 (d, 2H, J=8Hz), 7.26 (m, 3H), 6.43 (d, 1H, J=7Hz), 6.35 (d, 1H, J=8Hz), 4.37 (m, 4H), 3.05 (m, 2H), 2.91 (m, 2H), 1.39 (t, 3H, J=7Hz).
4-[2-(2-Aminopyridin-6-yl)ethy]benzoic acid hydrochloride (29-6) A suspension of ester 29-5 (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 29-6 as a tarn solid.
1H NMR (300 MHz, CD30D) # 7.96 (d, 2H, J=8Hz), 7.80 (m, 1H), 7.33 (d, 2H, J=8Hz), 6.84 (d, 1H, J=9Hz), 6.69 (d, 1H, J=7Hz), 3.09 (m, 4H).
Ethyl 4-[2-(2-Aminopyridin-6-yl)ethyl]benzoyl-2(S)-(4-iodo phenylsuffonv1amino)--alanine (29-7) A solution of acid 29-6 (400 mg, 1.43 mmol), amine 29-4 (686 mg, 1.57 mmol), EDC (358 mg, 1.86 mmol), HOBT (252 mg, 1.86 mmol), NMM (632 p1, 5.72 mmol) and DMF (10 ml) was stirred for -20 h. The reaction was diluted with EtOAc and then washed with sat NaHCO3, brine, dried (MgSO4) and concentrated. Flash chromatography (silica, EtOAC # 5% isopropanoVEtOAc) provided amide 29-7 as a white solid.
TLC Rf = 0.4 (silica, 10% isopropanoVEtOAc) 1H NMR (300 MHz, CD30D) # 7.79 (d, 2H, J=9Hz) 7.61 (d, 2H, J=8Hz), 7.52 (d, 2H, J=9Hz), 7.29 (m, 1H), 7.27 (d, 2H, J=8Hz), 4.20 (m, 1H), 3.95 (q, 2H, J=7Hz), 3.66 (dd, 1H, J=6Hz, 14Hz), 3.49 (dd, 1H, 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)-ss-alanine (29-8) A solution of ester 29-7 (200 mg, 0.3213 mmol) and 6N HCl (30 ml) was heated to 600 C. After -20 h, the reaction mixture was concentrated. Flash chromatography (silica, 20:20:1:1 EtOAc/EtOH/ NH4OH/H2O) provided acid 29-8 as a white solid.
TLC Rf =0.45 (silica, 20:20:1:1 EtOAc/EtOH/NH4OH/H2O) 1H NMR (400 MHz, DMSO) # 8.40 (m, 1H), 8.14 (Bs, 1H), 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, 1H, J=7Hz), 6.25 (d, 1H, J=8Hz), 5.85 (bs, 2H), 3.89 (bs, 1H), 3.35 (m, 2H), 2.97 (m, 2H), 2.79 (m, 2H).
4-[2-(2-Aminopyridin-6-yl)ethyl)benzoyl-2(S)-(4-trimethylstannyl- phenylsulfonylamino-ss-alanine (29-9) A solution of iodide 29-8 (70 mg, 0.1178 mmol), ((CH3)3Sn)2 (49 p1, 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 C18 15 M 100A , 40 x 100 mm; 95:5 # 5:95 H20/CH3CN) provided the trifluoroacetate salt. The salt was suspended in H20 (10 ml), treated with NH40H (5 drops) and then lyophilized to provide amide 29-9 as a white solid.
1H NMR (400 MHz, DMSO) # 8.40 (m, 1H), 8.18 (d, 1H, 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, 1H), 3.50 (m, 1H), 3.33 (m, 1H), 2.97 (m, 2H), 2.86 (m, 2H).
4-[2-(2-Aminopyridin-6-yl)ethyl]benzoyl-2(S)-4- 125iodo- phenrlsulfonylamino-ss-alanine (29-10) An iodobead (Pierce) was added to a shipping vial of 5 mCi of Nal25I (Amersham, IMS30) and stirred for five minutes at room temperature. A solution of 0.1 mg of 29-9 in 0.05 mL of 10% H2S04/MeOH was made and immediately added to the Na125yiodobead vial. After stirring for three minutes at room temperature, approximately 0.04-0.05 mL of NH40H 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 (O.lto (TFA):H20 (0.1% TFA) to 90% acetonitrile (0.1% TFA):H20 (0.1% TFA) over 30 minutes, I mUmin]. The retention time of 29-10 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 29-10, which coeluted on HPLC analysis with an authentic sample of 29-8.
* 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 Git son 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 UV/Vis Diode Array Spectrophotometer.
Sample radioactivities were determined in a Packard A5530 gamma counter.
&alpha;vss5 ATTACHMENT ASSAY Duong et al., J. Bone Miner. Res., 11:S 290, describe a system for expressing the human &alpha;vss5.
Materials: 1. Media and solutions used in this assay are purchased from BRL/Gibco, except BSA and the chemicals are from Sigma.
2. Attachment medium: HBSS with 1 mg/ml heat-inactivated fatty acid free BSA and 2 mM CaCl2.
3. Glucosaminidase substrate solution: 3.75 mM p-nitrophenyl-N acetyl-beta-D-glucosaminide, 0.1 M sodium citrate, 0.25% Triton, pH 5.0.
4. Glycine-EDTA developing solution: 50 mM glycine, 5 mM EDTA, pH 10.5.
Methods: 1. Plates (96 well, Nunc Maxi Sorp) were coated overnight at 4 C with human vitronectin (3 ug/ml) in 50 mM carbonate buffer (pH 9/.6), using 100 KLVwell. Plates were then washed 2X with DPBS and blocked with 2% BSA in DPBS for 2h at room temperature. After additional washes (2X) with DPBS, plates were used for cell attachment assay.
2. 293 (alpha v beta 5) cells were grown in MEM media in presence of 10% fetal calf serum to 90% confluence. Cells were then lifted from dishes with 1X Trypsin/EDTA and washed 3X with serum free MEM. Cells were resuspended in attachment medium (3 X 105 cells/ml).
3. Test compounds were prepared as a series of dilutions at 2X concentrations and added as 50 FVwell. Cell suspension was then added as 50 ml/well. Plates were incubated at 37 C with 55 CO2 for 1 hour to allow attachment.
4. Non-adherent cells were removed by gently washing the plates (3X) with DPBS and then incubated with glucosaminidase substrate solution (100 IlVwell), overnight at room temperature in the dark. To quantitate cell numbers, standard curve of glucosaminidase activity was determined for each experiment by adding samples of cell suspension directly to wells containing the enzyme substrate solution.
5. The next day, the reaction was developed by addition of 185 CLVwell of gylcine/EDTA solution and reading absorbance at 405 nm using a Molecular Devices V-Max plate reader.
Average test absorbance values (4 wells per test samples) were calculated. Then, the number of attached cells at each drug concentration was quantitated versus the standard curve of cells using the Softmax program.

Claims (13)

WHAT IS CLAIMED IS:
1. A method for eliciting an avI3S or dual ctvss3/avss5 antagonizing effect in a mammal which comprises administering to the mammal a therapeutically effective amount of a compound of the formula:
and pharmaceutically acceptable salts thereof, wherein Xis a 5- or 6-membered monocyclic partially or fully saturated ring system containing 0, 1, 2, 3 or 4 heteroatoms selected from N, 0 and S and either unsubstituted or substituted with R1 or R2, or a 9- to 1 0-membered polycyclic ring system, wherein one or more of the rings is partially or fully saturated, containing 0, 1, 2, 3 or 4 heteroatoms selected from N, 0 and S and either unsubstituted or substituted with R1 or R2, wherein R1 and R2 are independently selected from the group consisting of hydrogen, F, Cl, Br, I, Cl-b alkyl, C3-8 cycloalkyl, aryl, aryl C1-8 alkyl, amino, amino C1-8 alkyl, Cl -3 acylamino, C1-3 acylamino C1-8 alkyl, C1-6 alkylamino, C1-6 alkylamino C1-8 alkyl, C1-6 dialkylamino, C1-6 dialkylamino C1-8 alkyl, C14 alkoxy, C14 alkoxy C1-6 alkyl, carboxy, carboxy C1-6 alkyl, C1 3 alkoxycarbonyl, C1-3 alkoxycarbonyl C1-6 alkyl, carboxy C1-6 alkyloxy, hydroxy, and hydroxy Cl -6 alkyl; Yis
where Z is O, NR8, or S; and R8 is defined as R above; R3 and R4 are independently hydrogen, a five or six membered mono or nine or ten membered polycyclic partially or fully saturated ring system containing 0, 1, 2, 3, or 4 heteroatoms selected from nitrogen, oxygen and sulfur, either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, C1-3 alkoxy, C1-5 alkylcarbonyloxy, C1-5 alkoxycarbonyl, C1-5 alkyl, aminoC1-5 alkyl, hydroxycarbonyl, hydroxycarbonylC1-5 alkyl, or hydroxycarbonylC 1-5 alkoxy, -(CH2)n-aryl, wherein n=14 and aryl is defined as a five or six membered unsaturated or partially saturated monocyclic ring system, or nine or ten membered polycyclic ring system wherein at least one of the rings is unsaturated or partially saturated and each of the other rings may be unsaturated, partially saturated or fully saturated, said aryl group containing 0, 1, 2, 3, or 4 heteroatoms selected from nitrogen, oxygen and sulfur, either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, Cl -3 alkoxy, C1-5 alkylcarbonyloxy, C1-5 alkoxycarbonyl, C1-5 alkyl, aminoC 1-5 alkyl, hydroxycarbonyl, hydroxycarbonylC 1-5 alkyl, or hydroxycarbonylCl.5 alkoxy, halogen, hydroxyl, Cl .Salkylcarbonylamino, arylCl-S alkoxy, C1-5 alkoxycarbonyl, aminocarbonyl, C1-5 alkylaminocarbonyl, C 1-5 alkylcarbonyloxy, C3-8 cycloalkyl, oxo, amino, Cl -3 alkylamino, aminoC 1-3 alkyl, arylaminocarbonyl, arylC1-5alkylaminocarbonyl, aminocarbonyl, aminocarbonyl-Cl 4 alkyl, hydroxycarbonyl, hydroxycarbonyl C1-5 alkyl, C1-6alkyl, either unsubstituted or substituted, with one or more groups selected from halogen, hydroxyl, C1-5alkylcarbonylamino, arylC1-5 alkoxy, C1-5 alkoxycarbonyl, aminocarbonyl, C1-5 alkylaminocarbonyl, C1-5 alkylcarbonyloxy, C3-8 cycloalkyl, oxo, amino, Cl -3 alkylamino, amino 3 alkyl, arylaminocarbonyl, arylCl 5alkylaminocarbonyl, aminocarbonyl, aminocarbonyl-C14 alkyl, hydroxycarbonyl, or hydroxycarbonyl C1-5 alkyl, provided that the carbon atom to which R3 and R4 are attached bears only one heteroatom, -(CH2)m C=CH, -(CH2)m C#C-C1-6 alkyl, -(CH2)m C#C-C3-7cycloalkyl, -(CH2)m C#C- aryl, -(CH2)m C#C-C1-6 alkyl aryl, -(CH2)m CH=CH2, (CH2)m CH=CH C1-6 alkyl, -(CH2)m CH=CH-C3 7cycloalkyl, -(CH2)m CH=CH aryl, -(CH2)m CH=CH C1-6 alkyl aryl, -(CH2)m S02C1-6 alkyl, or -(CH2)m So2Cl-6 alkylaryl; R5is hydrogen, fluorine, C1-8 alkyl, hydroxyl, hydroxy C1-6 alkyl, carboxy, carboxy C 1-6 alkyl, C1-6 alkyloxy.
C3-8 cycloalkyl, aryl C1-6 alkyloxy, aryl, aryl C1-6 alkyl, C1-6 alkylcarbonyloxy, amino, C 1-6 alkylamino, amino C1-6 alkyl, Cl -6 alkylamino C1-6 alkyl1 aryl amino, aryl amino C1-6 alkyl, aryl C 1-6 alkylamino, aryl C1-6 alkylamino C1-6 alkyl, aryl carbonyloxy, aryl C1-6 alkylcarbonyloxy, C1-6 dialkylamino, C1-6 dialkylamino C1-6 alkyl, C 1-6 alkylaminocarbonyloxy, C1 8 alkylsulfonylamino, C1-8 alkylsulfonylamino C1-6 alkyl, aryl sulfonylamino C1-6 alkyl, aryl sulfonylamino, aryl C1-6 alkylsulfonylamino, aryl C1-6 alkylsulfonylamino C1-6 alkyl, C1-8 alkyloxycarbonylamino, C1-8 alkyloxycarbonylamino Cl -8 alkyl, aryl C1-8 alkyloxycarbonylamino, aryl oxycarbonylamino, aryl oxycarbonylamino Cl -8 alkyl, aryl C1-8 alkyloxycarbonylamino C1-8 alkyl, C1-8 alkylcarbonylamino, C1-8 alkylcarbonylamino C1-6 alkyl, aryl carbonylamino C1-6 alkyl, aryl carbonylamino, aryl C1-6 alkylcarbonylamino, aryl C1-6 alkylcarbonylamino C1-6 alkyl, C1-8 alkylaminocarbonylamino, aminocarbonylamino, aminocarbonylamino C1-6 alkyl, C1-8 alkylaminocarbonylamino C1-6 alkyl, aryl aminocarbonylamino Cl -6 alkyl, aryl aminocarbonylamino, aryl C1-8 alkylaminocarbonylamino, aryl C1-8 alkylaminocarbonylamino C1-6 alkyl, aminosulfonylamino C1-6 alkyl, aminosulfonylamino, C1-8 alkylaminosulfonylamino, C1-8 alkylaminosulfonylamino C1-6 alkyl, aryl aminosulfonylamino C1-6 alkyl, aryl aminosulfonylamino, aryl C1-8 alkylaminosulfonylamino, aryl C1-8 alkylaminosulfonylamino C1-6 alkyl, C1-6 alkylsulfonyl, C1-6 alkylsulfonyl C1-6alkyl, aryl sulfonyl, aryl sulfonyl C1-6alkyl, aryl alkylsulfonyl, aryl C1-6 alkylsulfonyl, aryl C1-6 alkylsulfonyl C1-6alkyl, C1-6 alkylcarbonyl, C1-6 alkylcarbonyl C1-6 alkyl, aryl carbonyl Cl.6alkyl, aryl carbonyl, aryl C1-6 alkylcarbonyl, aryl C1-6 alkylcarbonyl C1-6alkyl, Cl -6 alkylthiocarbonylamino, C1-6 alkylthiocarbonylamino C1-6 alkyl, aryl thiocarbonylamino C1-6 alkyl, aryl thiocarbonylamino, aryl C1-6 alkylthiocarbonylamino, aryl C1-6 alkylthiocarbonylamino C1-6 alkyl, aminocarbonyl C 1-6 alkyl, aminocarbonyl, C1 8 alkylaminocarbonyl, C1-8 alkylaminocarbonyl C1-6 alkyl, aryl aminocarbonyl C1-6 alkyl, aryl aminocarbonyl, aryl C1-8 alkylaminocarbonyl, aryl C1-8 alkylaminocarbonyl C1-6 alkyl, wherein alkyl groups and aryl groups may be unsubstituted or substituted with one or more substituents selected from R1 and R2; and R6, R7, and R9 are independently hydrogen, C1-8 alkyl, aryl, aryl C1-8 alkyl, hydroxy, C1-8 alkyloxy, aryloxy, aryl C1-6 alkyloxy, C1-8 alkylcarbonyloxy C14 alkyloxy, aryl C1-8 alkylcarbonyloxy C14 alkyloxy, C1-8 alkylaminocarbonylmethyleneoxy, or Cl -8 dialkylaminocarbonylmethyleneoxy, and wherein m and n are integers 0-6.
2. A method for inhibiting a condition selected from the group consisting of restenosis, angiogenesis, diabetic retinopathy, macular degeneration, inflammation, and tumor growth in a mammal which comprises administering to the mammal a therapeutically effective amount of a compound of the formula:
and pharmaceutically acceptable salts thereof, wherein Xis a 5- or 6-membered monocyclic partially or fully saturated ring system containing 0, 1, 2, 3 or 4 heteroatoms selected from N, 0 and S and either unsubstituted or substituted with R1 or R2, or a 9- to l0-membered polycyclic ring system, wherein one or more of the rings is partially or fully saturated, containing 0, 1, 2, 3 or 4 heteroatoms selected from N, 0 and S and either unsubstituted or substituted with R1 or R2, wherein R1 and R2 are independently selected from the group consisting of hydrogen, F, Cl, Br, I, Cl-b alkyl, C3-8 cycloalkyl, aryl, aryl Cl.8 alkyl, amino, amino C1-8 alkyl, C1-3 acylamino, C1-3 acylamino C1-8 alkyl, C1-6 alkylamino, C1-6 alkylamino C1-8 alkyl, Cl -6 dialkylamino, C1-6 dialkylamino C1-8 alkyl, C14 alkoxy, C14 alkoxy C1-6 alkyl, carboxy, carboxy C1-6 alkyl, C1-3 alkoxycarbonyl, C1-3 alkoxycarbonyl C1-6 alkyl, carboxy C1-6 alkyloxy, hydroxy, and hydroxy C1-6 alkyl; Yis
where Z is O, NR8, or S; and R8 is defined as R1 above; R3 and R4 are independently hydrogen, a five or six membered mono or nine or ten membered polycyclic partially or fully saturated ring system containing 0, 1, 2, 3, or 4 heteroatoms selected from nitrogen, oxygen and sulfur, either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, C 1-3 alkoxy, C1-5 alkylcarbonyloxy, C1-5 alkoxycarbonyl, C1-5 alkyl, aminoC1-5 alkyl, hydroxycarbonyl, hydroxycarbonylC 1-5 alkyl, or hydroxycarbonylC 1-5 alkoxy, -(CH2)n-aryl, wherein n=l -4 and aryl is defined as a five or six membered unsaturated or partially saturated monocyclic ring system, or nine or ten membered polycyclic ring system wherein at least one of the rings is unsaturated or partially saturated and each of the other rings may be unsaturated, partially saturated or fully saturated, said aryl group containing 0, 1, 2, 3, or 4 heteroatoms selected from nitrogen, oxygen and sulfur, either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, C1 3 alkoxy, C1-5 alkylcarbonyloxy, C1-5 alkoxycarbonyl, C1 -5 alkyl, aminoC 1-5 alkyl, hydroxycarbonyl, hydroxycarbonylC 1-5 alkyl, or hydroxycarbonylC1-5 alkoxy, halogen, hydroxyl, C1-5alkylcarbonylamino, arylC 1-5 alkoxy, C1-5 alkoxycarbonyl, aminocarbonyl, C1-5 alkylaminocarbonyl, C1-5 alkylcarbonyloxy, C3-8 cycloalkyl, oxo, amino, Cl 3 alkylamino, aminoC 1-3 alkyl, arylaminocarbonyl, arylC1-5alkylaminocarbonyl, aminocarbonyl, aminocarbonyl-C 14 alkyl, hydroxycarbonyl, hydroxycarbonyl C1-5 alkyl, C1 6alkyl, either unsubstituted or substituted, with one or more groups selected from halogen, hydroxyl, C1-5alkylcarbonylamino, arylC1-5 alkoxy, C1-5 alkoxycarbonyl, aminocarbonyl, C1-5 alkylaminocarbonyl, C1-5 alkylcarbonyloxy, C3-8 cycloalkyl, oxo, amino, C1-3 alkylamino, aminoC1-3 alkyl, arylaminocarbonyl, arylC1-5alkylaminocarbonyl, aminocarbonyl, aminocarbonyl-C1-4 alkyl, hydroxycarbonyl, or hydroxycaibonyl C1-5 alkyl, provided that the carbon atom to which R3 and R4 are attached bears only one heteroatom, -(CH2)m C#CH, -(CH2)m C#C-C1-6 alkyl, -(CH2)m C#C-C3-7cycloalkyl, -(CH2)m C=C- aryl, -(CH2)m C-=C-Cl-6 alkyl aryl, -(CH2)m CH=CH2, -(CH2)m CH=CH C1-6 alkyl, -(CH3)m CH=CH-CH3-7cycloalkyl, -(CH2)m CH=CH aryl, -(CH2)m CH=CH C1-6 alkyl aryl, -(CH2)m S02C1-6 alkyl, or -(CH2)m S02C1-6 alkylaryl; R5is hydrogen, fluorine, C1-8 alkyl, hydroxyl, hydroxy C1-6 alkyl, carboxy, carboxy C1-6 alkyl, C1-6 alkyloxy.
C3-8 cycloalkyl, aryl C1-6 alkyloxy, aryl, aryl C1-6 alkyl, C1-6 alkylcarbonyloxy, amino, C1 6 alkylamino, amino C1-6 alkyl, C1-6 alkylamino C1-6 alkyl, aryl amino, aryl amino C1-6 alkyl, aryl C1-6 alkylamino, aryl C1-6 alkylamino C1-6 alkyl, aryl carbonyloxy, aryl C1-6 alkylcarbonyloxy, Cl -6 dialkylamino, C1-6 dialkylamino C1-6 alkyl, C1-6 alkylaminocarbonyloxy, C 1-8 alkylsulfonylamino, C1-8 alkylsulfonylamino C1-6 alkyl, aryl sulfonylamino C1-6 alkyl, aryl sulfonylamino, aryl C1-6 alkylsulfonylamino, aryl C1-6 alkylsulfonylamino C1-6 alkyl, Cl -8 alkyloxycarbonylamino, C1-8 alkyloxycarbonylamino C1-8 alkyl, aryl C1-8 alkyloxycarbonylamino, aryl oxycarbonylamino, aryl oxycarbonylamino C1-8 alkyl, aryl C1-8 alkyloxycarbonylamino C1-8 alkyl, C1-8 alkylcarbonylamino, C1-8 alkylcarbonylamino C 1-6 alkyl, aryl carbonylamino C1-6 alkyl, aryl carbonylamino, aryl C1-6 alkylcarbonylamino, aryl C 1-6 alkylcarbonylamino C 1-6 alkyl, C1-8 alkylaminocarbonylamino, amino carbonylamino, aminocarbonylamino C1-6 alkyl, C1-8 alkylaminocarbonylamino C 1-6 alkyl, aryl aminocarbonylamino C1-6 alkyl, aryl aminocarbonylamino, aryl C1-8 alkylaminocarbonylamino, aryl C1-8 alkylaminocarbonylamino Cl -6 alkyl, aminosulfonylamino C1-6 alkyl, aminosulfonylamino, C1-8 alkylaminosulfonylamino, C1 8 alkylaminosulfonylamino C1-6 alkyl, aryl aminosulfonylamino Cl -6 alkyl, aryl aminosulfonylamino, aryl C 1-8 alkylaminosulfonylamino, aryl C1-8 alkylaminosulfonylamino C1-6 alkyl, C1-6 alkylsulfonyl, C1-6 alkylsulfonyl C1-6alkyl, aryl sulfonyl, aryl sulfonyl C1-6alkyl, aryl alkylsulfonyl, aryl Cl -6 alkylsulfonyl, aryl C1-6 alkylsulfonyl C1 6alkyl, C1-6 alkylcarbonyl, C1-6 alkylcarbonyl C1-6 alkyl, aryl carbonyl Cl.6alkyl, aryl carbonyl, aryl C1-6 alkylcarbonyl, aryl C1-6 alkylcarbonyl C1-6alkyl, C 1-6 alkylthiocarbonylamino, C1-6 alkylthiocarbonylamino C1-6 alkyl, aryl thiocarbonylamino C1-6 alkyl, aryl thiocarbonylamino, aryl C1-6 alkylthiocarbonylamino, aryl C1-6 alkylthiocarbonylamino C1-6 alkyl, aminocarbonyl Cl-6 alkyl, aminocarbonyl, C1-8 alkylaminocarbonyl, C1-8 alkylaminocarbonyl C1-6 alkyl, aryl aminocarbonyl C1-6 alkyl, aryl aminocarbonyl, aryl C1-8 alkylaminocarbonyl, aryl Cl -8 alkylaminocarbonyl C1-6 alkyl, wherein alkyl groups and aryl groups may be unsubstituted or substituted with one or more substituents selected from R1 and R2; and R6, R7, and R9 are independently hydrogen, C1-8 alkyl, aryl, aryl C1-8 alkyl, hydroxy, C1-8 alkyloxy, aryloxy, aryl C1-6 alkyloxy, C1-8 alkylcarbonyloxy C1-4 alkyloxy, aryl C1-8 alkylcarbonyloxy C1-4 alkyloxy, C1-8 alkylaminocarbonylmethyleneoxy, or C1-8 dialkylaminocarbonylmethyleneoxy, and wherein m and n are integers 0-6.
3. A method of Claim 2 wherein the compound has the formula:
and pharmaceutically acceptable salts thereof, wherein Xis
wherein n is 2-4, and n' is 2 or 3, and wherein R1 and R2 are independently selected from the group consisting of hydrogen, F, Cl, Br, I, C1-10 alkyl, C38 cycloalkyl, aryl, aryl C1-8 alkyl, amino, amino C1-8 alkyl, C1-3 acylamino, C1-3 acylamino C1-8 alkyl, C1-6 alkylamino, C1-6 alkylamino C1-8 alkyl, C 1-6 dialkylamino, Cl.6 dialkylamino C1-8 alkyl, C1-4 alkoxy, C14 alkoxy C1-6 alkyl, carboxy, carboxy C1-6 alkyl, C1-3 alkoxycarbonyl, C1 3 alkoxycarbonyl C1-6 alkyl, carboxy C1-6 alkyloxy, hydroxy, and hydroxy C1-6 alkyl.
4. A method of Claim 3 wherein the compound has the formula
and pharmaceutically acceptable salts thereof, wherein Xis
wherein n' is 2 or 3, and Yis
where Z is O, NR8, or S; and R8 is defined as R1.
5. A method of Claim 4 wherein the compound has the formula
and pharmaceutically acceptable salts thereof, wherein Xis
wherein R1 and R2 are independently selected from the group consisting of hydrogen or amino, amino C1-8 alkyl; Yis
R8 is hydrogen or aryl C0-8 alkyl; R3 is hydrogen, a six membered monocyclic partially or fully saturated ring system, either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, C1-3 alkoxy, Cl S alkylcarbonyloxy, C1-5 alkoxycarbonyl, Cl S alkyl, aminoC1-5 alkyl, hydroxycarbonyl, hydroxycarbonylCl 5 alkyl, or hydroxycarbonylC 1-5 alkoxy, -(CH2)n-aryl, wherein n=14 and aryl is defined as a six membered monocyclic unsaturated or partially saturated ring system, either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, Cl -3 alkoxy, C1 S alkylcarbonyloxy, C1-5 alkoxycarbonyl, C1-5 alkyl, aminoC1-5 alkyl, hydroxycarbonyl, hydroxycarbonylC 1-S alkyl, or hydroxycarbonylC 1-5 alkoxy, C35 cycloalkyl, or Cl.6aIkyl, either unsubstituted or substituted, with C3-8 cycloalkyl; R4is hydrogen, -(CH2)n-aryl, wherein n=04 and aryl is defined as a six membered monocyclic unsaturated or partially saturated ring or a nine or ten membered polycyclic ring system wherein at least one of the rings is unsaturated or partially saturated and each of the other rings may be unsaturated, partially saturated or fully saturated, said aryl group either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, C1-3 alkoxy, C1-5 alkylcarbonyloxy, C1-5 alkoxycarbonyl, C1 5 alkyl, aminoCl-S alkyl, hydroxycarbonylC0-5 alkyl, or hydroxycarbonylCl -5 alkoxy, Cl-6alkyl, or -(CH2)0-4 C#CH; R5is hydrogen, aryl sulfonylamino C1-6 alkyl, aryl sulfonylamino, aryl C1-6 alkylsulfonylamino, aryl C1-6 alkylsulfonylamino C1-6 alkyl, C1-8 alkylsulfonylamino, C1-8 alkylsulfonylamino C1-6 alkyl, aryl sulfonylamino C1-6 alkyl, aryl sulfonylamino, aryl C1-6 alkylsulfonylamino, aryl C1-6 alkylsulfonylamino C1-6 alkyl, aminosulfonylamino C1-6 alkyl, aminosulfonylamino, C1-8 alkylaminosulfonylamino, C1-8 alkylaminosulfonylamino Cl -6 alkyl, aryl aminosulfonylamino C1-6 alkyl, aryl aminosulfonylamino, aryl C1-8 alkylaminosulfonylamino, aryl C1-8 alkylaminosulfonylamino C 1-6 alkyl, C1-6 alkylsulfonyl, C1-6 alkylsulfonyl C1-6alkyl, aryl sulfonyl C1-6alkyl, aryl sulfonyl, aryl C1-6 alkylsulfonyl, aryl C1-6 alkylsulfonyl C1-6alkyl, wherein alkyl groups and aryl groups may be unsubstituted or substituted with one or more substituents selected from R1 and R2; and R6 is hydrogen, C1-8 alkyl, or aryl, aryl C1-8 alkyl.
6. A method of Claim S wherein the compound has the formula
and pharmaceutically acceptable salts thereof, wherein X is
Yis
R3 is hydrogen, methyl,
R4 is hydrogen, methyl,
R5 is hydrogen, or NHSO2M)\/ ; and R6 is hydrogen, methyl, ethyl, or t-butyl.
7. A method of Claim 6 wherein the compound is selected from the group consisting of 4-(2-Aminothiazol-4-yl)butanoyl-glycyl-2(S)-phenylsulfonamido- alanine t-butyl ester, 4-(2-Aminothiazol-4-yl)butanoyl-glycyl-2(S)-phenylsulfonamido- alanine, 4-(2-Aminothiazol-4-yl)butanoyl-glycyl-3(R)-(2-phenethyl)- -alanine methyl ester, 4-(2-Aminothiazol-4-yl)butanoyl-glycyl-3(R)-(2-phenethyl)- -alanine, 5-(2-Pyridylamino)pentanoylglycyl-2(S)-phenylsulfonamido- -alanine ethyl ester, 5-(2-Pyridylamino)pentanoylglycyl-2(S)-phenylsulfonamido- -alanine, 4-(2-Amino-pyridin-6-yl)butanoyl-sarcosine-3(R)-[(2-indol-3-yl)ethyl].
-alanine ethyl ester, 4-(2-Aminopyridin-6-yl)butanoyl-sarcosine-3(R)-[(2-indol-3-yl)ethyl] -alanine, 4-(2-Aminopyridin-6-yl)butanoyl-glycyl-2(S)-phenylsulfonamido- p- alanine t-butyl ester, 4-(2-Aminopyridin-6-yl)butanoyl-glycyl-2(S)-phenylsulfonamido-- alanine, 4-(Pyridin-4-yl)butanoyl-sarcosine-3(R)-[2-(indol-3-yl)ethyl]- -alanine ethyl ester, 4-(Pyridin-4-yl)butanoyl-sarcosine-3(R)-[2-(indol-3-yl)ethyl]- -alanine, 4-(2-B ocamino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3 (R)-(2 phenethyl)- -alanine ethyl ester, 4-(2-Amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-(2- phenethyl)- -alanine ethyl ester, 4-(2-Amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-(2- phenethyl) -p-alanine, 4-(2-Boc-amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-[(2- indol-3-yl)ethyl]- -alanine, 4-(2-Amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-[(2-indol 3-yl)ethyl] - -alanine, 4-(2-Bocamino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)- methyl- -alanine ethyl ester, 4-(2-Amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-methyl- alanine ethyl ester, 4-(2-Amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-methyl- alanine, 4-(Pyridin-4-yl)butanoyl-N-(2-phenylethyl)glycyl-3(R)-(2-phenethyl) -alanine ethyl ester, 4-(Pyridin-4-yl)butanoyl-N-(2-phenyl)glycyl-3(R)-(2-phenethyl)- alanine, 4-(2-BOC-Aminopyridin-4-yl)butanoyl-N-(2-phenethyl)glycyl-3(R)methyl- -alanine, 4-(2-Aminopyridin4-yl)butanoyl-N-(2-phenethyl)glycyl-3 (R)-methyl -alanine, 4-(Pyridyloxy)butyrate-N-(2-phenethyl)glycyl-3(R)-2-phenethyl- alanine ethyl ester, 4-(Pyridyloxy)butyrate-N-(2-phenethyl)glycyl-3(R)-2-phenethyl- alanine, 3 -[(N-Methyl)-N-(4-pyridyl)Jaminopropionyl-sarcosine-3(R)-(2- phenethyl)- -alanine ethyl ester, 3-[(N-Methyl)-N-(4-pyridyl)]aminopropionyl-sarcosine-3(R)-(2phenethyl)- -alanine, N- ( N'-3-(4-t-Butoxycarbonyl-1 -piperizinyl)benzoyl)glycyl ) -3(R)- methyl-(3-alanine benzyl ester, N-[N'-[3-(1-Piperazinyl)benzoyl]glycyl]-3(R)-methyl- -alanine, N-[N'-[3-(4-t-Butoxycarbonyl-1-piperazinyl)benzoyl]glycyl]-3(R)-(2 phenethyl)- -alanine methyl ester1 N-[N'-[3-(1 -Piperazinyl)benzoyl]glycyl]-3(R)-(2-phenethyl)- -alanine, N- [N'-[3 -(4-t-Butoxycarbonyl- 1 -piperazinyl)benzoyl]-N'-(2- phenethyl)glycyl]-3(R)-(2-phenethyl)- -alanine methyl ester, N-[N'-[3-(1-Piperazinyl)benzoyl]-N'-(2-phenethyl)glycyl]-33(R)-(2 phenethyl)- -alanine, 4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-yl)butanol-glycyl- -alanine t-butyl ester, 4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-yl)butanoyl-glycyl- alanine, 4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-yl)butanoyl-glycyl-3(S) pyridin-3-yl- -alanine ethyl ester, 4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-yl)butanoyl-glycyl 3(S)pyridin-3-yl- -alanine, Ethyl N-pyridin-4-ylisonipecotyl -N-cyclopropylglycine-3 (S)-ethynyl- -alanine, N-Pyridin-ylisonipecotyl-N-cyclopropylglycine-3(S)-ethynyl- alanine, Ethyl N-pyridin4-ylnipecotyl-N-cyclopropylglycine-3(S)-ethynyl-'3- alanine, N-Pyridin-4-ylnipecotyl-N-cyclopropylglycine-3(S)-ethynyl- - alanine, 4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-5-yl)butanoyl-N-(cyclo propyI)gly-3(S)ethynyl-P-alanine ethyl ester, 4-(1,2,3,4-Tetrahydro-1,8-naphthyridin-5-yl)butanoyl-N-(cyclopropyl)glycyl-3(S)-ethynyl- -alanine, and 3-{2-[5-(1H-Benzoimidazol-2-yl-amino)-pentanoylamino]-acetylamino} 3(S)-pyridin-3-yl- propionic acid, and pharmaceutically acceptable salts thereof.
8. A method of Claim 7 wherein the salt is selected from the group consisting of 4-(2-Aminothiazol-4-yl)butanoyl-glycyl-3(R)-(2-phenethyl)- -alanine trifluoroacetate salt, 5-(2-Pyridylamino)pentanoylglycyl-2(S)-phenylsulfonamido- -alanine trifluoroacetate salt' N-[N'-[3-(1 -Piperazinvl)benzoyl glycyl]-3(R)-methyl -p-alanine trifluoroacetic acid salt, N-[N'-[3-(1-Piperazinyl)benzoyl]glycyl]-3(R)-(2-phenethyl)- -alanine trifluoroacetic acid salt, N-[N'-[3-(1-Piperazinyl)benzoyl]-N'-(2-phenethyl)glycyl]-3(R)-(2phenethyl)- -alanine trifluoroacetic acid salt, and 4-(2-Amino-pyridin-6-yl)butanoyl-N-cyclopropylglycyl-3(R)-(2- phenethyl)- -alanine ethyl ester hydrochloride.
9. The method of Claim 1, wherein the antagonizing effect is an av S antagonizing effect.
10. The method of Claim 2 wherein the condition is diabetic retinopathy or macular degeneration.
11. A composition comprising a carrier suitable for topical ophthamological administration and between about 0.01-5% w/v of a compound of the formula:
and pharmaceutically acceptable salts thereof, wherein Xis a 5- or 6-membered monocyclic partially or fully saturated ring system containing 0, 1, 2, 3 or 4 heteroatoms selected from N, 0 and S and either unsubstituted or substituted with R1 or R2, or a 9- to 10-membered polycyclic ring system, wherein one or more of the rings is partially or fully saturated, containing 0, 1, 2, 3 or 4 heteroatoms selected from N, 0 and S and either unsubstituted or substituted with R1 or R2, wherein R1 and R2 are independently selected from the group consisting of hydrogen, F, Cl, Br, I, C1-10 alkyl, C35 cycloalkyl, aryl, aryl C1-8 alkyl, amino, amino C1-8 alkyl, Cl -3 acylamino, C 1-3 acylamino C1-8 alkyl, C1-6 alkylamino, C1-6 alkylamino C1-8 alkyl, C1-6 dialkylamino, C1-6 dialkylamino C1-8 alkyl, C14 alkoxy, C14 alkoxy C14 alkyl, carboxy, carboxy C1-6 alkyl, C1-3 alkoxycarbonyl, C1-3 alkoxycarbonyl C 1-6 alkyl, carboxy C1-6 alkyloxy, hydroxy, and hydroxy C1-6 alkyl; Yis
where Z is O, NR8, or S; and R8 is defined as R1 above; R3 and R4 are independently hydrogen, a five or six membered mono or nine or ten membered polycyclic partially or fully saturated ring system containing 0, 1, 2, 3, or 4 heteroatoms selected from nitrogen, oxygen and sulfur, either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, C 1-3 alkoxy, C1-5 alkylcarbonyloxy, C1-5 alkoxycarbonyl, C1-5 alkyl, aminoC1-5 alkyl, hydroxycarbonyl, hydroxycarbonylC 1-S alkyl, or hydroxycarbonylC 1-5 alkoxy, -(CH2)n-aryl, wherein n=14 and aryl is defined as a five or six membered unsaturated or partially saturated monocyclic ring system, or nine or ten membered polycyclic ring system wherein at least one of the rings is unsaturated or partially saturated and each of the other rings may be unsaturated, partially saturated or fully saturated, said aryl group containing 0, 1, 2., 3, or 4 heteroatoms selected from nitrogen, oxygen and sulfur, either unsubstituted or substituted, with one or more groups selected from hydroxyl, halogen, cyano, trifluoromethyl, Cl -3 alkoxy, C1-5 alkylcarbonyloxy, C1-5 alkoxycarbonyl, C1-5 alkyl, aminoC 1-5 alkyl, hydroxycarbonyl, hydroxycarbonylC 1-5 alkyl, or hydroxycarbonylC 1-5 alkoxy, halogen, hydroxyl, Cl-S alkylcarbonylamino,
aminoC1-3 alkyl, arylaminocarbonyl, arylC 1 .salkylaminocarbonyl, aminocarbonyl, aminocarbonyl-C1-4 alkyl, hydroxycarbonyl, hydroxycarbonyl C1-5 alkyl, C1 6aLkyl, either unsubstituted or substituted, with one or more groups selected from halogen, hydroxyl, C1-5alkylcarbonylamino, arylC1-5 alkoxy, C1-5 alkoxycarbonyl, aminocarbonyl, C1-5 alkylaminocarbonyl, Cl-5 alkylcarbonyloxy, C3-8 cycloalkyl, oxo, amino, C1-3 alkylamino, aminoC1-3 alkyl, arylaminocarbonyl, arylC1-5alkylaminocarbonyl, aminocarbonyl, aminocarbonyl-C1-4 alkyl, hydroxycarbonyl, or hydroxycarbonyl C1-5 alkyl, provided that the carbon atom to which R3 and R4 are attached bears only one heteroatom, -(CH2)m C=-CH, -(CH2)m C#C-C1-6 alkyl, -(CH2)m C-C-C3 7cycloalkyl, -(CH2)m C--C- aryl, -(CH2)m C#C-C1-6 alkyl aryl, -(CH2)m CH=CH2, -(CH2)m CH=CH C1-6 alkyl, -(CH2)m CH=CH-C3 7cycloalkyl, -(CH2)m CH=CH aryl, -(CH2)m CH=CH C1-6 alkyl aryl, -(CH2)m S02C1-6 alkyl, or -(CH2)m S02C1-6 alkylaryl; R5 is hydrogen, fluorine, C1-8 alkyl, hydroxyl, hydroxy C1-6 alkyl, carboxy, carboxy C1-6 alkyl, C1-6 alkyloxy.
C3-8 cycloalkyl, aryl Cl -6 alkyloxy, aryl, aryl C1-6 alkyl, C 1-6 alkylcarbonyloxy, amino, C1-6 alkylamino, amino C1-6 alkyl, C1-6 alkylamino C1-6 alkyl, aryl amino, aryl amino C1-6 alkyl, aryl C1-6 alkylamino, aryl C 1-6 alkylamino C 1-6 alkyl, aryl carbonyloxy, aryl C1-6 alkylcarbonyloxy, C1-6 dialkylamino, C1-6 dialkylamino C1-6 alkyl, Cl -6 alkylaminocarbonyloxy, C1-8 alkylsulfonylamino, C1-8 alkylsulfonylamino C1-6 alkyl, aryl sulfonylamino C1-6 alkyl, aryl sulfonylamino, aryl C1-6 alkylsulfonylamino, aryl C1-6 alkylsulfonylamino C1-6 alkyl, Cl -8 alkyloxycarbonylamino, C1-8 alkyloxycarbonylamino C1-8 alkyl, aryl C1-8 alkyloxycarbonylamino, aryl oxycarbonylamino, aryl oxycarbonylamino C1-8 alkyl, aryl C1-8 alkyloxycarbonylamino C1-8 alkyl, C 1-8 alkylcarbonylamino, C1-8 alkylcarbonylamino C1-6 alkyl, aryl carbonylamino C1-6 alkyl, aryl carbonylamino, aryl C1-6 alkylcarbonylamino, aryl C1-6 alkylcarbonylamino C1-6 alkyl, C1-8 alkylaminocarbonylamino, aminocarbonylamino, aminocarbonylamino C1-6 alkyl, C1-8 alkylaminocarbonylamino C1-6 alkyl, aryl aminocarbonylamino C1-6 alkyl, aryl aminocarbonylamino, aryl C1-8 alkylaminocarbonylamino, aryl C1-8 alkylaminocarbonylamino C1-6 alkyl, aminosulfonylamino C1-6 alkyl, aminosulfonylamino, C1-8 alkylaminosulfonylamino, C1-8 alkylaminosulfonylamino C1-6 alkyl, aryl aminosulfonylamino Cl -6 alkyl, aryl aminosulfonylamino, aryl C1-8 alkylaminosulfonylamino, aryl C1 8 alkylaminosulfonylamino C1-6 alkyl, C1-6 alkylsulfonyl, C1-6 alkylsulfonyl C1-6alkyl, aryl sulfonyl, aryl sulfonyl C1-6alkyl, aryl alkylsulfonyl, aryl C1-6 alkylsulfonyl, aryl C1-6 alkylsulfonyl C16alkyl, C1-6 alkylcarbonyl, C 1-6 alkylcarbonyl C 1-6 alkyl, aryl carbonyl C1-6alkyl, aryl carbonyl, aryl C1-6 alkylcarbonyl, aryl C1-6 alkylcarbonyl C1-6alkyl, Cl -6 alkylthiocarbonylamino, C1-6 alkylthiocarbonylamino C1-6 alkyl, aryl thiocarbonylamino C1-6 alkyl, aryl thiocarbonylamino, aryl C1-6 alkylthiocarbonylamino, aryl C1-6 alkylthiocarbonylamino C1-6 alkyl, aminocarbonyl C1-6 alkyl, aminocarbonyl, C1-8 alkylaminocarbonyl, C1-8 alkylaminocarbonyl C1-6 alkyl, aryl aminocarbonyl Cl -6 alkyl, aryl aminocarbonyl, aryl C1-8 alkylaminocarbonyl, aryl Cl-8 alkylaminocarbonyl C1-6 alkyl, wherein alkyl groups and aryl groups may be unsubstituted or substituted with one or more substituents selected from R1 and R2; and R6, R7, and R9 are independently hydrogen, C1-8 alkyl, aryl, aryl C1-8 alkyl, hydroxy, C1-8 alkyloxy, aryloxy, aryl C1-6 alkyloxy, C1-8 alkylcarbonyloxy C14 alkyloxy, aryl C1-8 alkylcarbonyloxy C14 alkyloxy, C1-8 alkylaminocarbonylmethyleneoxy, or Cl -8 dialkylaminocarbonylmethyleneoxy, and wherein m and n are integers 0-6.
12. A method for administering a composition of Claim 11 to a patient in need of such compound comprising topically applying to the patient's eye an effective amount of the composition.
13. A method for inhibiting inhibiting diabetic retinopathy or macular degeneration in a patient comprising topically applying to the patient's eye a therapeutically effective amount of the composition of Claim 11.
GB9815165A 1997-07-23 1998-07-13 A method for eliciting an avß5 or dual avß3/avß5 antagonizing effect Withdrawn GB2327609A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US5353797P 1997-07-23 1997-07-23
US5364497P 1997-07-24 1997-07-24
GBGB9800961.6A GB9800961D0 (en) 1998-01-16 1998-01-16 A method for eliciting an µv›5 or dual µv›3/µv›5 antagonizing effect
GBGB9808017.9A GB9808017D0 (en) 1998-04-15 1998-04-15 A method for eliciting an av› or dual av›3/av›5 antagonizing effect

Publications (2)

Publication Number Publication Date
GB9815165D0 GB9815165D0 (en) 1998-09-09
GB2327609A true GB2327609A (en) 1999-02-03

Family

ID=27451741

Family Applications (1)

Application Number Title Priority Date Filing Date
GB9815165A Withdrawn GB2327609A (en) 1997-07-23 1998-07-13 A method for eliciting an avß5 or dual avß3/avß5 antagonizing effect

Country Status (1)

Country Link
GB (1) GB2327609A (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000048996A2 (en) * 1999-02-20 2000-08-24 Merck Patent Gmbh β-ALANINE DERIVATIVES
WO2000066618A1 (en) * 1999-04-28 2000-11-09 Basf Aktiengesellschaft Integrin receptor antagonists
US6750219B1 (en) 1999-08-05 2004-06-15 Meiji Seika Kaisha, Ltd. Ω-amino-α-hydroxycarboxylic acid derivatives having integrin ανβ3 antagonistic activity
US7317019B2 (en) 2003-08-21 2008-01-08 Bristol Myers Squibb Co. N-alkylated diaminopropane derivatives as modulators of chemokine receptor activity
US8071624B2 (en) * 2004-06-24 2011-12-06 Incyte Corporation N-substituted piperidines and their use as pharmaceuticals

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026250A1 (en) * 1996-01-16 1997-07-24 Merck & Co., Inc. Integrin receptor antagonists

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026250A1 (en) * 1996-01-16 1997-07-24 Merck & Co., Inc. Integrin receptor antagonists

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000048996A2 (en) * 1999-02-20 2000-08-24 Merck Patent Gmbh β-ALANINE DERIVATIVES
WO2000048996A3 (en) * 1999-02-20 2000-11-16 Merck Patent Gmbh β-ALANINE DERIVATIVES
US6576637B1 (en) 1999-02-20 2003-06-10 Merck Patent Gmbh β-alanine derivatives
WO2000066618A1 (en) * 1999-04-28 2000-11-09 Basf Aktiengesellschaft Integrin receptor antagonists
US6750219B1 (en) 1999-08-05 2004-06-15 Meiji Seika Kaisha, Ltd. Ω-amino-α-hydroxycarboxylic acid derivatives having integrin ανβ3 antagonistic activity
US7317019B2 (en) 2003-08-21 2008-01-08 Bristol Myers Squibb Co. N-alkylated diaminopropane derivatives as modulators of chemokine receptor activity
US7863333B2 (en) 2003-08-21 2011-01-04 Bristol-Myers Squibb Company N-alkylated diaminopropane derivatives as modulators of chemokine receptor activity
US8071624B2 (en) * 2004-06-24 2011-12-06 Incyte Corporation N-substituted piperidines and their use as pharmaceuticals
US8288417B2 (en) 2004-06-24 2012-10-16 Incyte Corporation N-substituted piperidines and their use as pharmaceuticals

Also Published As

Publication number Publication date
GB9815165D0 (en) 1998-09-09

Similar Documents

Publication Publication Date Title
AU748621B2 (en) Integrin receptor antagonists
CA2277273C (en) Integrin antagonists
AU741769B2 (en) Integrin receptor antagonists
US6066648A (en) Integrin receptor antagonists
AU747784B2 (en) Integrin receptor antagonists
AU736026B2 (en) Integrin receptor antagonists
AU780364B2 (en) Alpha V integrin receptor antagonists
AU713676B2 (en) Integrin receptor antagonists
US6017925A (en) Integrin antagonists
US6358970B1 (en) Integrin receptor antagonists
CA2268940A1 (en) Integrin antagonists
AU1822099A (en) Integrin receptor antagonists
PT1187592E (en) Alpha v integrin receptor antagonists
AU1912899A (en) Integrin receptor antagonists
CA2251017A1 (en) .alpha.v.beta.3 antagonists
GB2327609A (en) A method for eliciting an avß5 or dual avß3/avß5 antagonizing effect
US6294549B1 (en) Method for eliciting an αvβ5 or dual αvβ3/αvβ5 antagonizing effect
US5952306A (en) Integrin receptor antagonists
AU2001290884A1 (en) Alpha V integrin receptor antagonists
WO2002022123A1 (en) Alpha v integrin receptor antagonists

Legal Events

Date Code Title Description
WAP Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1)