Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
INTEGRIN ANTAGONISTS
FIELD OF THE INVENTION
The present invention provides novel compounds and
derivatives thereof, their synthesis, and their use as vitronectin receptor
ligands. More particularly, the compounds of the present invention are
avP3 antagonists, avP5 antagonists or dual av(33/ av(35 antagonists
useful for inhibiting bone resorption, treating and preventing
osteoporosis, and inhibiting vascular restenosis, diabetic retinopathy,
macular degeneration, angiogenesis, atherosclerosis, inflammation,
viral disease, and tumor growth.
BACKGROUND OF THE INVENTION
This invention relates to compounds for inhibiting bone
resorption that is mediated by the action of a class of cells known as
osteoclasts.
Osteoclasts are multinucleated cells of up to 400 m in
diameter that resorb mineralized tissue, chiefly calcium carbonate and
calcium phosphate, in vertebrates. They are actively motile cells that
migrate along the surface of bone. They can bind to bone, secrete
necessary acids and proteases and thereby.cause the actual resorption of
mineralized tissue from the bone.
More specifically, osteoclasts are believed to exist in at least
two physiological states. In the secretory state, osteoclasts are flat,
attach to the bone matrix via a tight attachment zone (sealing zone),
become highly polarized, form a ruffled border, and secrete lysosomal
enzymes and protons to resorb bone. The adhesion of osteoclasts to bone
surfaces is an important initial step in bone resorption. In the
migratory or motile state, the osteoclasts migrate across bone matrix
and do not take part in resorption until they attach again to bone.
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Integrins are transmembrane, heterodimeric,
glycoproteins which interact with extracellular matrix and are involved
in osteoclast attachment, activation and migration. The most abundant
integrin in osteoclasts (rat, chicken, mouse and human) is the
vitronectin receptor, or av(33, thought to interact in bone with matrix
proteins that contain the RGD sequence. Antibodies to av(33 block bone
resorption in vitro indicating that this integrin plays a key role in the
resorptive process. There is increasing evidence to suggest that av(33
ligands can be used effectively to inhibit osteoclast mediated bone
resoption in vivo in mammals.
The current major bone diseases of public concern are
osteoporosis, hypercalcemia of malignancy, osteopenia due to bone
metastases, periodontal disease, hyperparathyroidism, periarticular
erosions in rheumatoid arthritis, Paget's disease, immobilization-
induced osteopenia, and glucocorticoid treatment.
All these conditions are characterized by bone loss,
resulting from an imbalance between bone resorption (breakdown) and
bone formation, which continues throughout life at the rate of about 14%
per year on the average. However, the rate of bone turnover differs from
site to site, for example, it is higher in the trabecular bone of the
vertebrae and the alveolar bone in the jaws than in the cortices of the
long bones. The potential for bone loss is directly related to turnover and
can amount to over 5% per year in vertebrae immediately following
menopause, a condition which leads to increased fracture risk.
There are currently 20 million people with detectable
fractures of the vertebrae due to osteoporosis in the United States. In
addition, there are 250,000 hip fractures per year attributed to
osteoporosis. This clinical situation is associated with a 12% mortality
rate within the first two years, while 30% of the patients require nursing
home care after the fracture.
Individuals suffering from all the conditions listed above
would benefit from treatment with agents which inhibit bone resorption.
Additionally, av(i3 ligands have been found to be useful in
treating and/or inhibiting restenosis (recurrence of stenosis after
corrective surgery on the heart valve), atherosclerosis, diabetic
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retinopathy, macular degeneration and angiogenesis (formation of new
blood vessels), and inhibiting viral disease. Moreover, it has been
postulated that the growth of tumors depends on an adequate blood
supply, which in turn is dependent on the growth of new vessels into the
tumor; thus, inhibition of angiogenesis can cause tumor regression in
animal models. (See, Harrison's Princinles of Internal Medicine, 12th
ed., 1991). avP3 antagonists, which inhibit angiogenesis, are therefore
useful in the treatment of cancer for inhibiting tumor growth. (See e.g.,
Brooks et al., Cell, 79:1157-1164 (1994)).
Moreover, compounds of this invention can also inhibit
neovascularization by acting as antagonists of the integrin receptor
av(35. A monoclonal antibody for av(35 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 avP5 are useful for treating and
preventing macular degeneration, diabetic retinopathy, and tumor
growth.
In addition, certain compounds of this invention antagonize
both the av(33 and av(35 receptors. These compounds, referred to as
"dual av(33/av(35 antagonists," are useful for inhibiting bone resorption,
treating and preventing osteoporosis, and inhibiting vascular
restenosis, diabetic retinopathy, macular degeneration, angiogenesis,
atherosclerosis, inflammation and tumor growth.
It is an object of the present invention to identify compounds
which bind to the av(33 receptor, av(35 receptor or both the av(33 and av(35
receptors.
It is a further object of the invention to identify compounds
which act as antagonists of the av(33 receptor. It is another object of the
invention to identify avP3 antagonist compounds which are useful
agents for inhibiting: bone resorption mediated by osteoclast cells,
restenosis, atherosclerosis, inflammation, diabetic retinopathy,
macular degeneration and angiogenesis in animals, preferably
mammals, especially humans. Still another object of the invention is to
identify av(33 antagonists which cause tumor regression and/or inhibit
tumor growth in animals.
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A further object of the invention is to identify av(33
antagonists useful for preventing or treating osteoporosis. An
additional object of the invention is to identify av(33 antagonists useful for
treating cancer.
It has now been found that the compounds of the present
invention, av(33 ligands, are useful for inhibiting bone resorption in
mammals. Thus, the compounds of the present invention are useful for
preventing or reducing the incidence of osteoporosis. Additionally, the
avP3 ligands of the present invention are also useful for treating and/or
inhibiting restenosis, diabetic retinopathy, macular degeneration, viral
disease, atherosclerosis and/or angiogenesis in mammals.
SUMMARY OF THE INVENTION
The present invention provides av(33 antagonist compounds
of the formula
X-Y-Z-Aryl-A-B
wherein:
Aryl is a 6-membered aromatic ring containing 0, 1, 2 or 3 nitrogen
atoms and either unsubstituted or substituted with R8 and R9;
X is selected from
NR2 NR' NR2
~~ tNR2R3, -NR1 R2, NR1R3, -NR1-C-NR3R4,
NR1 NR2
11 25 -aryl-NR1R2, -aryI-C-NR2R3, -aryl-NR1- C-NR3R'
a 5- or 6-membered monocyclic aromatic or nonaromatic ring
system containing 0, 1, 2, 3 or 4 heteroatoms selected from N, 0
or S wherein the 5- or 6-membered ring system is either
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unsubstituted or substituted with R1, R2, R3 and R4, or
a 9- to 14-membered polycyclic ring system, wherein one or more
of the rings is aromatic, and wherein the polycyclic ring system
contains 0, 1, 2, 3 or 4 heteroatoms selected from N, 0 or S, and
wherein the polycyclic ring system is either unsubstituted or
substituted with Rl, R2, R3 and R4 ;
Y is selected from
CO-g alkylene,
C3-10 cycloalkyl,
CO-g alkylene-NR10-CO-CO-8 alkylene,
CO-8 alkylene-CONR10-CO-8 alkylene,
CO-g alkylene-O-CO-g alkylene,
CO-g alkylene-NR10-Cp-8 alkylene,
CO-g alkylene-S(O)0-2-CO-8 alkylene,
CO-g alkylene-S02-NR10-CO-8 alkylene,
CO-g alkylene-NR10-S02-CO-8 alkylene,
CO-8 alkylene-CO-CO-g alkylene,
(CH2)0-6 aryl(CH2)0-6,
(CH2)0-6 aryl-CO-(CH2)0-6,
(CH2)0-6 aryl-C0-NR10-(CH2)0-6,
(CH2)0-6 ary1NR10C0(CH2)0-6, or
OR'
I
(CHz)o-sCH (CH2)o-a=
,
Z and A are each independently selected from
-5-
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O
(CH2)m, (CH2)mO(CH2)m (CH2)mNR11(CH2)n,(CH2)mNR111CNR12(CH2)n
0 11 11 11O O
(CH2)mCNR (CH2)n,(CH2)mNR C(CH2)n,(CH2)m~(CH2)n,
S
(CH2J(CH2)n, (CH2)mSO2(CH2)n, (CH2)mS(CH2)n,
(CH2)mSO(CH2)n, (CH2)mSO2NR11(CH2)n,
(CH2)mNR11SO2(CH2)n, (CH2)mCR11=CR12(CH2)n, or
(CH2)mC=C- (CH2)n,
where m and n are each independently an integer from 0 to 6;
B is
O
11
C_R1s
R6 R~
p
where p is an integer from 1 to 3;
R1, R2, R3, R4, R5, R8, Rg, R10, R,11 and R12 are each independently
selected from
hydrogen,
halogen,
C1-10 alkyl,
aryl CO-8 alkyl,
amino CO-8 alkyl,
C1_3 acylamino CO-8 alkyl,
C1-6 alkylamino CO-8 alkyl,
-6-
r- T
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C1-6 dialkylamino Cp-g alkyl,
aryl CO-6 alkylamino CO-6 alkyl,
C1-4 alkoxyamino Cp-8 alkyl,
hydroxy C 1-6 alkylamino CO-8 alkyl,
C1-4 alkoxy CO-6 alkyl,
hydroxycarbonyl C0-6 alkyl,
C1-4 alkoxycarbonyl C0-6 alkyl,
hydroxycarbonyl C0-6 alkyloxy,
hydroxy C1-6 alkylamino CO-6 alkyl or
hydroxy CO-6 alkyl;
R6 is selected from
hydrogen,
fluorine,
C 1-g alkyl,
hydroxyl,
hydroxy C 1-6 alkyl,
carboxy CO-6 alkyl,
C 1-6 alkyloxy,
C1-6 alkylcarbonyl,
aryl CO-6 alkylcarbonyl,
C1-6 alkylcarbonyloxy,
aryl CO-6 alkylcarbonyloxy,
C 1-6 alkylaminocarbonyloxy,
C3-8 cycloalkyl,
aryl CO-6 alkyl,
CO-6 alkylamino CO-6 alkyl,
CO-6 dialkylamino CO-6 alkyl,
C 1_g alkylsulfonylamino C0-6 alkyl,
aryl CO-6 alkylsulfonylamino CO-6 alkyl,
C1-8 alkyloxycarbonylamino C0-8 alkyl,
aryl CO-8 alkyloxycarbonylamino Cp-g alkyl,
Cl-8 alkylcarbonylamino C0-6 alkyl,
aryl C0-6 alkylcarbonylamino C0-6 alkyl,
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Cp-g alkylaminocarbonylamino CO-6 alkyl,
aryl C0-8 alkylaminocarbonylamino Cp-6 alkyl,
C0-8 alkylaminosulfonylamino C0-6 alkyl,
aryl C0-8 alkylaminosulfonylamino Cp-6 alkyl,
C1-6 alkylsulfonyl Cp-6 alkyl,
aryl Cp-6 alkylsulfonyl C0-6 alkyl,
C1-6 alkylcarbonyl Cp-6 alkyl,
aryl C0-6 alkylcarbonyl C0-6 alkyl,
C 1-6 alkylthiocarbonylamino Cp-6 alkyl, or
aryl C0-6 alkylthiocarbonylamino Cp-6 alkyl;
wherein the alkyl or N atoms may be unsubstituted or
substituted with R5;
R7 is selected from
C7-20 polycyclyl Cp-8 alkylsulfonylamino C0-6 alkyl;
C7-20 polycyclyl Cp-g alkylcarbonylamino C0-6 alkyl;
C7-20 polycyclyl Cp-8 alkylaminosulfonylamino CO-6 alkyl;
C7-20 polycyclyl Cp-8 alkylaminocarbonylamino Cp-6 alkyl or
C7-20 polycyclyl C0-8 alkyloxycarbonylamino Cp-6 alkyl;
wherein the polycyclyl may be unsubstituted or substituted with
R14, R15, R16 and R17; and wherein any of the alkyl groups may
be unsubstituted or subsituted with R14 and R15.
,
R13 is selected from
hydroxy,
C 1-8 alkyloxy,
aryl C0-6 alkyloxy,
C1-8 alkylcarbonyloxy C1-4 alkyloxy,
aryl C 1-g alkylcarbonyloxy C 1-4 alkyloxy,
C 1-6 dialkylaminocarbonylmethyloxy,
aryl C 1-6 dialkylaminocarbonylmethyloxy or
an L- or D-amino acid joined by an amide linkage and
wherein the carboxylic acid moiety of said amino acid
is as the free acid or is esterified by C1-6 alkyl; and
-8-
____
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R14, R15, R16 and R17 are each independently selected from
hydrogen, halogen, Cl-lo alkyl, C3-8 cycloalkyl, oxo, aryl,
aryl C1-8 alkyl, amino, amino Cl-8 alkyl, C1-3 acylamino,
C1-3 acylamino C1-8 alkyl, Cl-6 alkylamino, C1-6 alkylamino-
C1-8 alkyl, C1-6 dialkylamino, C1-6 dialkylamino Cl-8 alkyl,
C 1-4 alkoxy, C 1-4 alkoxy C 1-6 alkyl, hydroxycarbonyl,
hydroxycarbonyl C 1-6 alkyl, C 1-3 alkoxycarbonyl,
C1-3 alkoxycarbonyl C1-6 alkyl, hydroxycarbonyl-
C1-6 alkyloxy, hydroxy, hydroxy C1-6 alkyl, Cl-6 alkyloxy-
C 1-6 alkyl, nitro, cyano, trifluoromethyl, trifluoromethoxy,
trifluoroethoxy, C1-8 alkyl-S(O)q, C1-8 alkylaminocarbonyl,
C 1-8 dialkylaminocarbonyl, C 1-8 alkyloxycarbonylamino,
C1-8 alkylaminocarbonyloxy or C1-8alkylsulfonylamino;
and the pharmaceutically acceptable salts thereof;
provided that the compound is not 4-[2-(1,2,3,4-Tetrahydro-1,8-
naphthyridin-7-yl)ethyl]benzoyl-2(S)[1(S)10-camphorsulfonylamino] (3-
alanine ethyl ester (1-14) or 4-[2-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-
yl)ethyl]benzoyl-2(S)-[1(S)10-camphorsulfonylamino] (3-alanine (1-15).
In one embodiment of the invention is the compound
wherein
Aryl is a 6-membered aromatic ring containing 0, 1 or 2 nitrogen atoms
wherein Aryl is unsubstituted or substituted with R8 and R9;
X is selected from
NR1 NR2
-NR'R2, -C-NR2R3, -NR1-C-NR3R4
a 5- or 6-membered monocyclic aromatic or nonaromatic ring
system containing 0, 1 or 2 heteroatoms selected from N, 0
or S wherein the 5- or 6-membered ring system is either
-9-
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unsubstituted or substituted with R1 and R2, or
a 9- to 14-membered fused polycyclic ring system, wherein one or
more of the rings is aromatic, and wherein the polycyclic ring
system contains 0, 1, 2 or 3 heteroatoms selected from N. 0 or S,
and wherein the polycyclic ring system is either unsubstituted or
substituted with Rl and R2;
Y is selected from the group consisting of
CO-g alkylene,
C3-10 cycloalkyl,
CO-8 alkylene-NR10-CO-CO-8 alkylene,
CO-8 alkylene-CONR10-CO-8 alkylene,
CO-8 alkylene-O-CO-g alkylene,
CO-8 alkylene-NR10-CO-8 alkylene,
CO-8 alkylene-S(O)0-2-CO-8 alkylene,
CO-8 alkylene-S02-NR10-CO-8 alkylene,
CO-g alkylene-NR10-S02-CO-8 alkylene,
CO-8 alkylene-CO-CO-g alkylene,
(CH2)0-6 aryl(CH2)0-6,
(CH2)0-6 aryl-CO-(CH2)0-6,
(CH2)0-6 aryl-CO-NH-(CH2)0-6, or
OR1
I
(CH2)0-8CH(CH2)0-8
Z and A are each independently selected from
-10-
r T
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(CH2)m, (CH2)mO(CH2)n, (CH2)mNR11(CH2)n,
O O 11 I
(CH2)mCNR11(CH2)n,(CH2)mNR11C(CH2)n,(CH2)mC(CH2)n,
-1 1{
(CH2)mC(CH2)n, (CH2)mS02(CH2)n, (CH2)mS(CH2)n,
(CH2)mSO(CH2)n, (CH2)mSO2NR11(CH2)n,
(CH2)mNR1iSO2(CH2)n, (CH2)mCR11=CR12(CH2)n or
(CH2)mC =C - (CH2)n,
where m and n are each independently an integer from 0 to 4;
R1, R2, R3, R4, R5, R8, R9, R10, R,11 and R12 are each independently
selected from
hydrogen,
halogen,
C1-10 alkyl,
aryl CO-g alkyl,
amino CO-8 alkyl,
C1-3 acylamino CO-8 alkyl,
C1-6 alkylamino CO-g alkyl,
C1-6 dialkylamino CO-8 alkyl,
aryl C0-6 alkylamino CO-6 alkyl,
C1-4 alkoxyamino CO-g alkyl,
hydroxy C 1-g alkylamino CO-g alkyl,
C1-4 alkoxy C0-6 alkyl, or
C1-4 alkoxycarbonyl CO-6 alkyl;
R6 is selected from
hydrogen,
C 1-g alkyl or
aryl CO-g alkyl;
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R7 is selected from
C7-15 polycyclyl C0-6 alkylsulfonylamino CO-6 alkyl or
C7-15 polycyclyl C0-6 alkylcarbonylamino CO-6 alkyl;
wherein the polycyclyl may be unsubstituted or substituted with
R14~ R15, R16 and R17; and wherein any of the alkyl groups may
be unsubstituted or subsituted with R14 and R15;
and all other variables are as defined above;
and the pharmaceutically acceptable salts thereof;
provided that the compound is not 4-[2-(1,2,3,4-Tetrahydro-1,8-
naphthyridin-7-yl)ethyl]benzoyl-2(S)[1(S)10-camphorsulfonylamino] (3-
alanine ethyl ester (1-14) or 4-[2-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-
yl)ethyl]benzoyl-2(S)-[1(S)10-camphorsulfonylamino] (3-alanine (1-15).
In a class of the invention is the compound
wherein
Aryl is a phenyl or pyridyl ring wherein the phenyl or pyridyl ring is
unsubstituted or substituted with R8;
X is
a 9- to 14-membered fused polycyclic ring system, wherein one or
more of the rings is aromatic, and wherein the polycyclic ring
system contains 0, 1, 2 or 3 heteroatoms selected from N, 0 or S,
and wherein the polycyclic ring system is either unsubstituted or
substituted with Rl and R2;
Y is selected from
CO-6 alkylene,
C0-6 alkylene-NR10-CO-CO-6 alkylene,
C0-6 alkylene-CONR10-CO-6 alkylene,
C0-6 alkylene-O-CO-6 alkylene,
CO-6 alkylene-NR10-CO-6 alkylene,
C0-6 alkylene-S(O)0-2-CO-6 alkylene,
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C0-6 alkylene-S02-NR10-CO-6 alkylene, or
C0-6 alkylene-aryl-CO-g alkylene;
Z is selected from
O
(CH2)m, (CH2)mO(CH2)n or (CH2)mNR11,C(CH2)n
A is
0
11
(CH2)mCNR11(CH2)n =
B is
0
11
C-R13
R6 R7
R1, R2, R3, R4, R5, R8, R9, R10, R11 and R12 are each independently
selected from
hydrogen,
halogen,
C1-10 alkyl,
aryl Cp-g alkyl,
C 1-4 alkoxy CO-6 alkyl, or
C1-4 alkoxycarbonyl CO-6 alkyl;
R6 is hydrogen;
R7 is selected from
-13-
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C7-10 polycyclyl C0-6 alkylsulfonylamino CO-6 alkyl or
C7-10 polycyclyl CO-6 alkylcarbonylamino C0-6 alkyl;
wherein the polycyclyl may be unsubstituted or substituted with
R14, R15, R16 and R17;
R13 is selected from
hydroxy or
C 1-8 alkyloxy;
R14P R15, R16 and R17 are each independently selected from
hydrogen, halogen, Cl-6 alkyl, C3-7 cycloalkyl, oxo, amino,
amino Cl-8 alkyl, Cl-3 acylamino, Cl-3 acylamino Cl-8 alkyl,
C 1-6 alkylamino, C 1-6 alkylamino C 1-g alkyl,
C 1-6 dialkylamino, C 1-6 dialkylamino C 1-8 alkyl,
C 1-4 alkoxy, C 1-4 alkoxy C 1-6 alkyl, hydroxycarbonyl,
hydroxycarbonyl C 1-6 alkyl, C 1-3 alkoxycarbonyl,
C 1-3 alkoxycarbonyl C 1-6 alkyl, hydroxycarbonyl-
C 1-6 alkyloxy, hydroxy, hydroxy C l-6 alkyl, C l-6 alkyloxy-
Cl-6 alkyl, nitro, cyano, trifluoromethyl, trifluoromethoxy,
trifluoroethoxy, Cl-8 alkylsulfonyl, C1-8 alkylaminocarbonyl,
Cl-8 alkyloxycarbonylamino, C1-8 alkylaminocarbonyloxy or
C 1-8alkylsulfonylamino;
and all other variables are as defined above;
and the pharmaceutically acceptable salts thereof;
provided that the compound is not 4-[2-(1,2,3,4-Tetrahydro-1,8-
naphthyridin-7-yl)ethyl]benzoyl-2(S)[1(S)10-camphorsulfonylamino] {3-
alanine ethyl ester (1-14) or 4-[2-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-
yl)ethyl]benzoyl-2(S)-[1(S)10-camphorsulfonylamino] P-alanine (1-15).
In a subclass of the invention is the compound of the
formula
-14-
~._
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RB
O
X-Y-Z ~~ N R13
~ H H R7
wherein X is selected from the group consisting of
R' R' i
(5R2
~ CN N
or
N H H H H
Yis selected from
C0-6 alkylene,
CO-6 alkylene-O-CO-6 alkylene or
CO-6 alkylene-1vR10-CO-6 alkylene;
Z is selected from
(CH2)m or (CH2)m O-(CH2)n ;
m and n are each independently an integer from 0 to 6;
R14, R15, R,16 and R17 are each independently selected from
hydrogen, halogen, C1-6 alkyl, oxo or hydroxy;
and all other variables are as defined above;
and the pharmaceutically acceptable salts thereof;
provided that the compound is not 4-[2-(1,2,3,4-Tetrahydro-1,8-
naphthyridin-7-yl)ethyl]benzoyl-2(S)[1(S)10-camphorsulfonylamino] (3-
alanine ethyl ester (1-14) or 4-[2-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7=-
yl)ethyl]benzoyl-2(S)-[1(S)10-camphorsulfonylamino] P-alanine (1-15).
Illustrative of the invention is the compound of the formula:
-15
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RS
O O
X-Y-Z ~ ~ N R' 3
H H H-SO2-(CH2)q R1a
wherein
q is an integer from 0 to 2;
R18 is selected from
or
and all other variables are as defined previously;
and the pharmaceutically acceptable salts thereof;
provided that the compound is not 4-[2-(1,2,3,4-Tetrahydro-1,8-
naphthyridin-7-yl)ethyl]benzoyl-2(S)[1(S)10-camphorsulfonylamino] (3-
alanine ethyl ester (1-14) or 4-[2-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-
yl)ethyl]benzoyl-2(S)-[1(S)10-camphorsulfonylamino] (3-alanine (1-15).
Exemplifying the invention is the compound selected from
-16-
T.._... . _ i
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O
CO2H
N O
:
N N~ H H H' S02
/
O
~CO2H
H N ;'
N N H H~ N'S
H O2 or
/
O
N I-/CO2H
H N H H'''N'S
N H 02
and the pharmaceutically acceptable salts thereof.
An illustration of the invention is the compound of the
formula
O
CO2H
H
N H ,S
H 02 O
and the pharmaceutically acceptable salts thereof.
Exemplifying the invention is a pharmaceutical
composition comprising any of the compounds described above and a
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pharmaceutically acceptable carrier. An example of the invention is a
pharmaceutical composition made by combining any of the compounds
described above and a pharmaceutically acceptable carrier. Another
illustration of the invention is a process for making a pharmaceutical
composition comprising combining any of the compounds described
above and a pharmaceutically acceptable carrier.
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 bone
resorption, osteoporosis, restenosis, diabetic retinopathy, macular
degeneration, angiogenesis, atherosclerosis, inflammation, viral
disease, cancer and tumor growth. More preferably, the condition is
selected from osteoporosis and cancer. Most preferably, the condition is
osteoporosis.
More specifically exemplifying the invention is a method of
eliciting a vitronectin antagonizing effect in a mammal in need thereof,
comprising administering to the mammal a therapeutically effective
amount of any of the compounds or any of the pharmaceutical
compositions described above. Preferably, the vitronectin antagonizing
effect is an av(33 antagonizing effect; more specifically the av(33
antagonizing effect is selected from inhibition of bone resorption,
inhibition of restenosis, inhibition of atherosclerosis, inhibition of
angiogenesis, inhibition of diabetic retinopathy, inhibition of macular
degeneration, inhibition of inflammation, inhibition of viral disease, or
inhibition of tumor growth. Most preferably, the av(33 antagonizing
effect is inhibition of bone resorption. Alternatively, the vitronectin
antagonizing effect is an av(35 antagonizing effect or a dual avp3/av(35
antagonizing effect. Examples of av(35 antagonizing effects are
inhibition of: restenosis, atherosclerosis, angiogenesis, diabetic
retinopathy, macular degeneration, inflammation, or tumor growth.
Examples of dual av(33/avP5 antagonizing effects are inhibition of bone
resorption, restenosis, atherosclerosis, angiogenesis, diabetic
-18-
1
.T.._ _.~__ .
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retinopathy, macular degeneration, inflammation, viral disease, or
tumor growth.
Additional examples of the invention are methods of
inhibiting bone resorption and of treating and/or preventing osteoporosis
in a mammal in need thereof, comprising administering to the
mammal a therapeutically effective amount of any of the compounds or
any of the pharmaceutical compositions decribed above.
Additional illustrations of the invention are methods of
inhibiting tumor growth and of treating and/or preventing cancer in a
mammal in need thereof, comprising administering to the mammal a
therapeutically effective amount of any of the compounds or any of the
pharmaceutical compositions decribed above.
More specifically exemplifying the invention is any of the
compositions described above, further comprising a therapeutically
effective amount of a second bone resorption inhibitor; preferably, the
second bone resorption inhibitor is alendronate.
More specifically illustrating the invention is any of the
methods of treating and/or preventing osteoporosis and/or of inhibiting
bone resoption described above, wherein the compound is administered
in combination with a second bone resorption inhibitor; preferably, the
second bone resorption inhibitor is alendronate.
Additional illustrations of the invention are methods of
treating hypercalcemia of malignancy, osteopenia due to bone
metastases, periodontal disease, hyperparathyroidism, periarticular
erosions in rheumatoid arthritis, Paget's disease, immobilization-
induced osteopenia, and glucocorticoid treatment in a mammal in need
thereof, comprising administering to the mammal a therapeutically
effective amount of any of the compounds or any of the pharmaceutical
compositions described above.
More particularly exemplifying the invention is the use of
any of the compounds described above in the preparation of a
medicament for the treatment and/or prevention of osteoporosis in a
mammal in need thereof. Still further exemplifying the invention is the
use of any of the compounds described above in the preparation of a
medicament for the treatment and/or prevention of: bone resorption,
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tumor growth, cancer, restenosis, artherosclerosis, inflammation, viral
disease, diabetic retinopathy, macular degeneration and/or
angiogenesis.
Another illustration of the invention is a drug which is
useful for treating and/or preventing osteoporosis in a mammal in need
thereof, the effective ingredient of the said drug being any of the
compounds descibed above. More specifically illustrating the invention
is a drug which is useful for treating and/or preventing: bone
resorption, tumor growth, cancer, restenosis, artherosclerosis,
inflammation, viral disease, diabetic retinopathy, macular degeneration
and/or angiogenesis in a mammal in need thereof, the effective
ingredient of the said drug being any of the compounds described above.
Additional illustrations of the invention are methods of
treating tumor growth in a mammal in need thereof, comprising
administering to the mammal a therapeutically effective amount of a
compound described above and one or more agents known to be cytotoxic
or antiproliferative, e.g., Taxol'+'and doxorubicin. Further illustrative are
methods of treating tumor growth wherein the compounds of the
present invention are administered in conjunction with radiation
therapy.
Further illustrative are methods of inhibiting angiogenesis
comprising administering a compound as described above in
combination with a VEGF (a vascular endothethial growth factor)
inhibitor compound. Such combinations are useful for treating disease
states such as macular degeneration, diabetic retinopathy, and cancer.
DETAILED DESCRIPTION OF THE INVENTION
Representative compounds of the present invention are
av03 antagonists which display submicromolar affinity for the human
avP3 receptor. Compounds of this invention are therefore useful for
treating mammals suffering from a condition caused or mediated by the
av(33 receptor, who are in need of such therapy. Pharmacologically
effective amounts of the compounds, including pharamaceutically
acceptable salts thereof, are administered to the mammal, to inhibit: the
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activity of mammalian osteoclasts, restenosis, tumor growth,
artherosclerosis, inflammation, macular degeneration, diabetic
retinopathy and angiogenesis.
The compounds of the present invention are administered
in dosages effective to antagonize the av(33 receptor where such
treatment is needed, as, for example, in the prevention or treatment of
osteoporosis or cancer. For use in medicine, the salts of the compounds
of this invention refer to non-toxic "pharmaceutically acceptable salts."
Other salts may, however, be useful in the preparation of the compounds
according to the invention or of their pharmaceutically acceptable salts.
Salts encompassed within the term "pharmaceutically acceptable salts"
refer to non-toxic salts of the compounds of this invention which are
generally prepared by reacting the free base with a suitable organic or
inorganic acid. Representative salts include the following:
Acetate, Benzenesulfonate, Benzoate, Bicarbonate,
Bisulfate, Bitartrate, Borate, Bromide, Calcium, Camsylate, Carbonate,
Chloride, Clavulanate, Citrate, Dihydrochloride, Edetate, Edisylate,
Estolate, Esylate, Fumarate, Gluceptate, Gluconate, Glutamate,
Glycollylarsanilate, Hexylresorcinate, Hydrabamine, Hydrobromide,
Hydrochloride, Hydroxynaphthoate, Iodide, Isothionate, Lactate,
Lactobionate, Laurate, Malate, Maleate, Mandelate, Mesylate,
Methylbromide, Methylnitrate, Methylsulfate, Mucate, Napsylate,
Nitrate, N-methylglucamine ammonium salt, Oleate, Oxalate, Pamoate
(Embonate), Palmitate, Pantothenate, Phosphate/diphosphate,
Polygalacturonate, Salicylate, Stearate, Sulfate, Subacetate, Succinate,
Tannate, Tartrate, Teoclate, Tosylate, Triethiodide and Valerate.
Furthermore, where the compounds of the invention carry an acidic
moiety, suitable pharmaceutically acceptable salts thereof may include
alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal
salts, e.g., calcium or magnesium salts; and salts formed with suitable
organic ligands, e.g., quaternary ammonium salts.
The compounds of the present invention, may have chiral
centers and occur as racemates, racemic mixtures and as individual
diastereomers, or enantiomers with all isomeric forms being included
in the present invention. Therefore, where a compound is chiral, the
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separate enantiomers, substantially free of the other, are included
within the scope of the invention; further included are all mixtures of
the two enantiomers. Also included within the scope of the invention
are polymorphs and hydrates of the compounds of the instant invention.
The present invention includes within its scope prodrugs of
the compounds of this invention. In general, such prodrugs will be
functional derivatives of the compounds of this invention which are
readily convertible in vivo into the required compound. Thus, in the
methods of treatment of the present invention, the term "administering"
shall encompass the treatment of the various conditions described with
the compound specifically disclosed or with a compound which may not
be specifically disclosed, but which converts to the specified compound in
vivo after administration to the patient. Conventional procedures for the
selection and preparation of suitable prodrug derivatives are described,
for example, in "Design of Prodrugs," ed. H. Bundgaard, Elsevier, 1985.
Metabolites of these compounds include active species produced upon
introduction of compounds of this invention into the biological milieu.
The term "therapeutically effective amount" shall mean
that amount of a drug or pharmaceutical agent that will elicit the
biological or medical response of a tissue, system, animal or human
that is being sought by a researcher or clinician.
The term "bone resorption," as used herein, refers to the
process by which osteoclasts degrade bone.
The term "alkyl" shall mean straight, branched or
monocyclic alkanes, alkenes or alkynes of the specified number of
carbon atoms. Preferably, the term "alkyl" refers to straight or branced
chain alkanes of C1-10 carbon atoms, or any number within this range
(i.e., methyl, ethyl, 1-propyl, 2-propyl, n-butyl, s-butyl, t-butyl, etc.).
The term "alkenyl" shall mean straight or branched chain
alkenes of two to ten total carbon atoms, or any number within this
range.
The term "alkynyl" shall mean straight or branched chain
alkynes of two to ten total carbon atoms, or any number within this
range.
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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 terms "polycyclic" or "polycyclyl," as used herein, refer
to unsubstituted or substituted fused or bridged polycyclic systems
containing from 7 to 20 carbon atoms and which can contain one or
more degrees of unsaturation. Preferably, the term "polycyclyl" refers to
unsubstituted or substituted fused or bridged bi- or tri-cyclic systems
containing from 7-15 carbon atoms and which can contain one or two
degrees of unsaturation. More preferably, the term "polycyclyl" refers to
unsubstituted or substituted fused or bridged bi- or tri-cyclic systems
containing from 7-10 carbon atoms and which can contain one or two
degrees of unsaturation. Examples of prefered polycyclyl
systems include, but are not limited to, decaline, camphor, adamantyl
and norbornyl.
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, 0, or S. Examples of cycloheteroalkyl
groups include, but are not limited to piperidinyl, pyrrolidinyl,
azetidinyl, morpholinyl, piperazinyl.
The term "alkoxy," as used herein, refers to straight or
branched chain alkoxides of the number of carbon atoms specified (e.g.,
Cl-b alkoxy), or any number within this range (i.e., methoxy, ethoxy,
etc.).
The term "alkylene" shall include both straight and
branched chain alkylenes (e.g., -CH2-, -CH(CH3)-, -CH(CH3)-CH2-, etc.).
The term "aryl," as used herein, refers to a monocyclic or
polycyclic system composed of 5- and 6-membered rings, such that the
system comprises at least one fully unsaturated (i.e., aromatic) ring,
wherein the rings contain 0, 1, 2, 3 or 4 heteroatoms chosen from N, 0 or
S, and either unsubstituted or substituted with one or more groups
independently selected from hydrogen, halogen, C 1-10 alkyl, C3-8
cycloalkyl, aryl, aryl C 1-8 alkyl, amino, amino C 1-8 alkyl, C 1-3
acylamino, C1-3 acylamino C1-8 alkyl, C1-6 alkylamino, C1-6 alkylamino
C 1-8 alkyl, C 1-6 dialkylamino, C 1-6 dialkylamino-C 1-8 alkyl, C 1-4 alkoxy,
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C 1-4 alkoxy C 1-6 alkyl, hydroxycarbonyl, hydroxycarbonyl C 1-6 alkyl, C 1-
alkoxycarbonyl, Cl-3 alkoxycarbonyl C 1-6 alkyl, hydroxycarbonyl Cl-6
alkyloxy, hydroxy, hydroxy Cl-6 alkyl, cyano, trifluoromethyl, oxo or C 1-
5 alkylcarbonyloxy. Examples of aryl include, but are not limited to,
5 phenyl, naphthyl, pyridyl, pyrazinyl, pyrimidinyl, imidazolyl,
benzimidazolyl, indolyl, thienyl, furyl, dihydrobenzofuryl, benzo(1,3)
dioxolane, oxazolyl, isoxazolyl and thiazolyl, which are either
unsubstituted or substituted with one or more groups independently
selected from hydrogen, halogen, C1-10 alkyl, C3-8 cycloalkyl, aryl, aryl
C l-g alkyl, amino, amino C 1-g alkyl, C 1-3 acylamino, C 1-3 acylamino
Cl-8 alkyl, C 1-6 alkylamino, C l-6 alkylamino-C 1-g alkyl, C 1-6
dialkylamino, C1-6 dialkylamino C1-8 alkyl, C1-4 alkoxy, C1-4 alkoxy Cl-
6 alkyl, hydroxycarbonyl, hydroxycarbonyl C1-6 alkyl, Cl-5
alkoxycarbonyl, C 1-3 alkoxycarbonyl C 1-6 alkyl, hydroxycarbonyl C 1-6
alkyloxy, hydroxy, hydroxy C l-6 alkyl, cyano, trifluoromethyl, oxo or C 1-
5 alkylcarbonyloxy. Preferably, the aryl group is unsubstituted, mono-,
di-, tri- or tetra-substituted with one to four of the above-named
substituents; more preferably, the aryl group is unsubstituted, mono-,
di- or tri-substituted with one to three of the above-named substituents;
most preferably, the aryl group is unsubstituted, mono- or di-substituted
with one to two of the above-named substituents.
Whenever the term "alkyl" or "aryl" or either of their prefix
roots appear in a name of a substituent (e.g., aryl CO-g alkyl) it shall be
interpreted as including those limitations given above for "alkyl" and
"aryl." Designated numbers of carbon atoms (e.g., C1-10) shall refer
independently to the number of carbon atoms in an alkyl or cyclic alkyl
moiety or to the alkyl portion of a larger substituent in which alkyl
appears as its prefix root.
The terms "arylalkyl" and "alkylaryl" include an alkyl
portion where alkyl is as defined above and to include an aryl portion
where aryl is as defined above. The CO-m or Cl-m designation where m
may be an integer from 1-10 or 2-10 respectively refers to the alkyl
component of the arylalkyl or alkylaryl unit. Examples of arylalkyl
include, but are not limited to, benzyl, fluorobenzyl, chlorobenzyl,
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phenylethyl, phenylpropyl, fluorophenylethyl, chlorophenylethyl,
thienylmethyl, thienylethyl, and thienylpropyl. Examples of alkylaryl
include, but are not limited to, toluene, ethylbenzene, propylbenzene,
methylpyridine, ethylpyridine, propylpyridine and butylpyridine.
When substituent R1, R2, R3, R4, R5, R6, R7, R8, R9, R10,
R,11, R12, R13 or R14 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 m, q, r or s is zero, then the group modified by the
variable is not present; for example, when s is zero, the group "-(CH2)s
C=CH" is "-C=CH".
The term "halogen" shall include iodine, bromine, chlorine
and fluorine.
The term "oxy" means an oxygen (0) atom. The term "thio"
means a sulfur (S) atom. The term "oxo" shall mean a bivalent oxygen
atom (=0).
The term "L- or D-amino acids" means naturally occurring
L- or D-amino acids, for example, those naturally occurring L-amino
acids present in humans, e.g. protein amino acids,, including L-
alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-
glutamine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-
leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,
L-threonine, L-tryptophan, L-tyrosine, and L-valine, and those naturally
occurring D-amino acids which are non-protein amino acids, such as
those found, for example, in antibiotic substances produced by bacteria
and fungi, including D-valine, D-asparagine, D-glutamate, D-ornithine,
D-phenylalanine, D-leucine, D-cysteine, and D-aspartate. (see Zubay
"BIOCHEMISTRY" Addison-Wesley Publishing Company, Inc.
(Reading, MA) 1983 pp. 867-870 and Stryer "BIOCHEMISTRY" W.H.
Freeman and Company (New York, NY) 3rd Edition 1988 pp. 16-21).
The term "substituted" shall be deemed to include multiple
degrees of substitution by a named substitutent. Where multiple
substituent moieties are disclosed or claimed, the substituted compound
can be independently substituted by one or more of the disclosed or
claimed substituent moieties, singly or plurally.
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Under standard nonmenclature 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 Cl-5 alkylcarbonylamino C1-6 alkyl
substituent is equivalent to
0
11
-C1-6 alkyl-NH-C-C1_5 alkyl.
The present invention is also directed to combinations of the
compounds of the present invention with one or more agents useful in
the prevention or treatment of osteoporosis. For example, the
compounds of the instant invention may be effectively administered in
combination with effective amounts of other agents 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 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. It will be understood that the scope of combinations of the
compounds of this invention with other agents useful for treating (xvP3
related conditions includes in principle any combination with any
pharmaceutical composition useful for treating osteoporosis.
Further exemplifying the invention are compositions
further comprising an active ingredient selected from the group
consisting of
a.) an organic bisphosphonate or a pharmaceutically-
acceptable salt or ester thereof,
b.) an estrogen receptor modulator, and
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c.) a cytotoxic/antiproliferative agent, and mixtures thereof.
Nonlimiting examples of such bisphosphonates include
alendronate, etidronate, pamidronate, risedronate, and
pharmaceutically acceptable salts and esters thereof. A particularly
preferred bisphosphonate is alendronate, especially alendronate
monosodium trihydrate.
Nonlimiting examples of estrogen receptor modulators
include estrogen, progesterin, estradiol, raloxifene, and tamoxifene.
Nonlimiting examples of cytotoxic/antiproliferative agents
are Taxo~and doxorubicin.
As used herein, the term "composition" is intended to
encompass a product comprising the specified ingredients in the
specified amounts, as well as any product which results, directly or
indirectly, from combination of the specified ingredients in the specified
amounts.
The compounds of the present invention can be
administered in such oral dosage forms as tablets, capsules (each of
which includes sustained release or timed release formulations), pills,
powders, granules, elixers, tinctures, suspensions, syrups and
emulsions. Likewise, they may also be administered in intravenous
(bolus or infusion), intraperitoneal, topical (e.g., ocular eyedrop),
subcutaneous, intramuscular or transdermal (e.g., patch) form, all
using forms well known to those of ordinary skill in the pharmaceutical
arts. An effective but non-toxic amount of the compound desired can be
employed as an avP3 inhibitor.
The dosage regimen utilizing-the compounds of the present
invention is selected in accordance with a variety of factors including
type, species, age, weight, sex and medical condition of the patient; the
severity of the condition to be treated; the route of administration; the
renal and hepatic function of the patient; and the particular compound
or salt thereof employed. An ordinarily skilled physician, veterinarian
or clinician can readily determine and prescribe the effective amount of
the drug required to prevent, counter or arrest the progress of the
condition.
* trade-mark
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Oral dosages of the present invention, when used for the
indicated effects, will range between about 0.01 mg per kg of body weight
per day (mg/kg/day) to about 100 mg/kg/day, preferably 0.01 to 10
mg/kg/day, and most preferably 0.1 to 5.0 mg/kg/day. For oral
administration, the compositions are preferably provided in the form of
tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0,
50.0, 100
and 500 milligrams of the active ingredient for the symptomatic
adjustment of the dosage to the patient to be treated. A medicament
typically contains from about 0.01 mg to about 500 mg of the active
ingredient, preferably, from about 1 mg to about 100 mg of active
ingredient. Intravenously, the most preferred doses will range from
about 0.1 to about 10 mg/kg/minute during a constant rate infusion.
Advantageously, compounds of the present invention may be
administered in a single daily dose, or the total daily dosage may be
administered in divided doses of two, three or four times daily.
Furthermore, preferred compounds for the present invention can be
administered in intranasal form via topical use of suitable intranasal
vehicles, or via transdermal routes, using those forms of transdermal
skin patches well known to those of ordinary skill in the art. To be
administered in the form of a transdermal delivery system, the dosage
administration will, of course, be continuous rather than intermittant
throughout the dosage regimen.
In the methods of the present invention, the compounds
herein described in detail can form the active ingredient, and are
typically administered in admixture with suitable pharmaceutical
diluents, excipients or carriers (collectively referred to herein as
'carrier' materials) suitably selected with respect to the intended form of
administration, that is, oral tablets, capsules, elixirs, syrups and the
like, and consistent with conventional pharmaceutical practices.
For instance, for oral administration in the form of a tablet
or capsule, the active drug component can be combined with an oral,
non-toxic, pharmaceutically acceptable, inert carrier such as lactose,
starch, sucrose, glucose, methyl cellulose, magnesium stearate,
dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like;
for oral administration in liquid form, the oral drug components can be
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combined with any oral, non-toxic, pharmaceutically acceptable inert
carrier such as ethanol, glycerol, water and the like. Moreover, when
desired or necessary, suitable binders, lubricants, disintegrating agents
and coloring agents can also be incorporated into the mixture. Suitable
binders include starch, gelatin, natural sugars such as glucose or beta-
lactose, corn sweeteners, natural and synthetic gums such as acacia,
tragacanth or sodium alginate, carboxymethylcellulose, polyethylene
glycol, waxes and the like. Lubricants used in these dosage forms
include sodium oleate, sodium stearate, magnesium stearate, sodium
benzoate, sodium acetate, sodium chloride and the like. Disintegrators
include, without limitation, starch, methyl cellulose, agar, bentonite,
xanthan gum and the like.
The compounds of the present invention can also be
administered in the form of liposome delivery systems, such as small
unilamellar vesicles, large unilamellar vesicles and multilamellar
vesicles. Liposomes can be formed from a variety of phospholipids, such
as cholesterol, stearylamine or phosphatidylcholines.
Compounds of the present invention may also be delivered
by the use of monoclonal antibodies as individual carriers to which the
compound molecules are coupled. The compounds of the present
invention may also be coupled with soluble polymers as targetable drug
carriers. Such polymers can include polyvinylpyrrolidone, pyran
copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-
ethylaspartamide-phenol, or polyethyleneoxide-polylysine substituted
with palmitoyl residues. Furthermore, the compounds of the present
invention may be coupled to a class of biodegradable polymers useful in
achieving controlled release of a drug, for example, polylactic acid,
polyglycolic acid, copolymers of polyactic and polyglycolic acid,
polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,
polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or
amphipathic block copolymers of hydrogels.
In the schemes and examples below, various reagent
symbols and abbreviations have the following meanings:
AcOH (HOAc): Acetic acid.
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BH3 = DMS: Borane = dimethylsulfide.
BOC(Boc): t-Butyloxycarbonyl.
BOP: Benzotriazol- 1-yloxytris(dimethylamino)-
phosphonium hexafluorophosphate.
CBZ(Cbz): Carbobenzyloxy or benzyloxycarbonyl.
CDI: Carbonyldiimidazole.
CH2C12: Methylene chloride.
CHC13: Chloroform.
DEAD: Diethyl azodicarboxylate.
DIAD: Diisopropyl azodicarboxylate.
DIBAH or
DIBAL-H: Diisobutylaluminum hydride.
DIPEA: Diisopropylethylamine.
DMAP: 4-Dimethylaminopyridine.
DME: 1,2-Dimethoxyethane.
DMF: Dimethylformamide.
DMSO: Dimethylsulfoxide.
DPFN: 3,5-Dimethyl-l-pyrazolylformamidine nitrate.
EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide.
Et: Ethyl.
EtOAc: Ethyl acetate.
EtOH: Ethanol.
HOBT: 1-Hydroxybenzotriazole.
LDA: Lithium diisopropylamide.
MeOH: Methanol.
NEt3: Triethylamine.
NMM: N-methylmorpholine.
PCA=HC1: Pyrazole carboxamidine hydrochloride.
Pd/C: Palladium on activated carbon catalyst.
Pd(OAc)2: Palladium(II) acetate.
Ph: Phenyl.
pTSA: p-Toluene sulfonic acid.
TEA: Triethylamine.
TFA: Trifluoroacetic acid.
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THF: Tetrahydrofuran.
TLC: Thin Layer Chromatography.
TMEDA: N,N,N',N'-Tetramethylethylenediamine.
TMS: Trimethylsilyl.
TsCl: Tosyl chloride.
The novel compounds of the present invention were
prepared according to the procedure of the following schemes and
examples, using appropriate materials and are further exemplified by
the following specific 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.
The following Schemes and Examples describe procedures
for making representative compounds of the present invention.
Moreover, by utilizing the procedures described in detail in PCT
International Application Publication Nos. W095/32710, published 7
December 1995, and W095/17397, published 29 June 1995, in conjunction
with the disclosure contained herein, one of ordinary skill in the art can
readily prepare additional compounds of the present invention claimed
herein.
More specifically, procedures for preparing the N-terminus
of the compounds of the present invention are described in WO 95/32710.
Additionally, for a general review describing the synthesis of P-alanines
which can be utilized as the C-terminus of the compounds of the present
invention, see Cole, D.C., Recent Stereoselective Synthetic Approaches
to PAmino Acids, Tetrahedron, 1994, 50, 9517-9582; Juaristi, E, et al.,
Enantioselective Synthesis of 6-Amino Acids, Aldrichemica Acta, 1994,
27, 3. In particular, synthesis of the 3-methyl (3-alanine is taught in
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Duggan, M.F. et al., J. Med. Chem., 1995, 38, 3332-3341; the 3-ethynyl (3-
alanine is taught in Zablocki, J.A., et al., J. Med. Chem., 1995, 38, 2378-
2394; the 3-pyrid-3-yl 0-alanine is taught in Rico, J.G. et al., J. Org.
Chem., 1993, 58, 7948-7951; and the 2-amino and 2-toslylamino P-
alanines are taught in Xue, C-B, et al., Biorg. Med. Chem. Letts., 1996,
6, 339-344.
Scheme 1
C02H SOC12 C02Et
H2N HCI=H2N
EtOH H,.
NHCBZ NHCBZ
1-2
BOC2O, NEt3, CH3CN
BOCNC02Et 10% Pd/C gOCN
H H~.= CO2Et
H H~'NHCBZ
NH2 H2
2
1=4 1-3
10(+) camphorsulfonyl chloride
NMM, CH2C12
CO2Et HCI(g)
BOCH H, EtOAc
NHSO2 O HCI=H2H N~C02Et
1-5 NHSO2 O
1-6
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Scheme 1 (continued)
OH k
+ OEt
1-7 1-8 O
Pd(OAc)2,
CH3CN, 100 C
O
OEt
O
1-9
O
cl H L-proline,
EtOH, reflux
N NH2
1-10
N N
OEt
1-11 O
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Scheme 1 (continued)
1-11
10% Pd/C, ethanol, H2
HN ~ 2, R= Et
OR ~ 6N HCI, 50 C
1-13,R-H
O
BOP
DMF 1S
NMM
H
N
H NH C02iAo
O N HS02 1-14, R = Et
D NaOH, C2H5OH
Ethyl 2(S)-Na-Cbz-2.3-diaminopropionate hydrochloride (1-2)
1-1 (5 g, 21 mmol; Bachem) was dissolved in 100 mL EtOH
and cooled to 0 C. SOC12 (9.2 mL, 126 mmol) was added followed by
removal of the cooling bath. After 6 hours, the reaction was
concentrated to provide 1=2 as a white solid.
1H NMR (300 MHz, CD3OD) S 7.35 (m, 5H), 5.14 (s, 2H), 4.44 (m, 1H), 4.22
(q, J=7Hz, 2H), 3.43 (m, 1H), 3.20 (m, 1H), 1.25 (t, J=7Hz, 3H).
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Ethy12(S)-Nu-Cbz-Np-Boc-2.3-diaminopropionate (1-3)
,1-2 (2 g, 6.6 mmol) was dissolved in 60 mL CH3CN. NEt3 (1
mL, 7.2 mmol) was added followed by BOC20 (1.58 g, 7.3 mmol). After
two hours, the reaction was concentrated, diluted with EtOAc, washed
with sat. NaHCO3, 10% KHSO4 and brine, dried (MgSO4), filtered and
concentrated to provide .1- as a clear oil.
TLC Rf 0.87 (silica, 80% EtOAc/hexanes).
1H NMR (300 MHz, CDC13) 8 7.35 (s, 5H), 5.75 (bs, 1H), 5.12 (s, 2H), 4.81
(bs, 1H), 4.39 (m, 1H), 4.19 (m, 2H), 3.56 (m, 2H), 1.42 (s, 9H), 1.29 (q,
J=7Hz, 3H).
Ethy12(S)-NR-Boc-2.3-diaminopropionate (1-4)
1=3 (2.4 g, 6.6 mmol) with 10% Pd/C (240 mg) in EtOAc (35
mL) was stirred under a H2 atmosphere for 20 hours. The reaction was
filtered through a Celite*pad and concentrated to provide 1=4 as a clear
oil.
TLC Rf 0.13 (silica, 80% EtOAc/hexanes).
1H NMR (300 MHz, CDC13) 5 5.00 (bs, 1H), 4.19 (m, 2H), 3.55 (m, 2H), 3.25
(m, 1H), 1.44 (s, 9H), 1.29 (q, J=7Hz, 3H).
Ethyl-2(S)-Na-(1(S)10-camphorsulfonylamino)-Np-Boc-2, 3-diamino-
ro,pionate (1-5)
Amine 14 (760 mg, 3.27 mmol) was dissolved in 35 mL
CH2C12 and cooled to 0 C. NMM (755 mL, 6.87 mmol) and 10(+)
camphorsulfonyl chloride (1.23 g, 4.9 mmol; Aldrich) were added. After
stirring at 0 C for one hour, the reaction was concentrated, then diluted
with EtOAc, washed with H20, sat. NaHCO3, 10% KHSO4 and brine,
dried (MgSO4), and concentrated to an oil. Flash chromatography
(silica, 25-40% EtOAc/hexanes) provided 1-5 as a clear oil.
TLC Rf 0.66 (silica, 50% Et0Aclhexanes).
1H-NMR (300 MHz, CDC13) 5 6.37 (d, J=8Hz, 1H), 4.99 (bt, 1H), 4.32 (m,
1H), 4.23 (q, J=8Hz, 2H), 3.56 (m, 3H), 3.0 (d, J=15 Hz, 1H), 2.4 (m, 1H),
2.05 (m, 4H), 1.43 (s, 9H), 1.30 (t, J=7 Hz, 3H), 1.00 (s, 3H), 0.91 (s, 3H).
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Ethyl-2(S)-Na-(1(S)10-camphorsulfonylamino)-2,3-diaminopro-
pionate hydrochloride (1-6)
Ester 1;5 (900 mg, 2.18 mmol) was dissolved in 15 mL EtOAc
and cooled to 0 C. HCl (g) was bubbled through the reaction mixture for
15 minutes. The reaction was removed from the cooling bath and
purged with Ar (g) for 20 minutes followed by concentration to provide 1-
6 as a foamly solid.
TLC Rf 0.05 (silica, 20% MeOH/EtOAc).
1H-NMR (300 MHz, CDC13): 8 4.75 (m, 1H), 4.26 (q, J=7Hz, 2H), 3.50 (m,
4H), 2.40 (m, 3H), 1.98 (m, 4H), 1.30 (t, J=7Hz, 3H), 1.04 (s, 3H), 0.91 (s,
3H).
Ethy14-(3-oxo-butyl)benzoate (1-9)
3-Buten-2-ol 1=7 (2.15 mL, 25 mmol; Aldrich), ethyl 4-
iodobenzoate 1-8 (5.5 g, 20 mmol, Aldrich) and NEt3 (3.5 mL, 25 mmol)
were combined in CH3CN (6 mL) under argon in a pressure tube.
Pd(OAc)2 (19 mg, 80 umol) was added and the reaction heated at 100 C
for 3 hours. The reaction was cooled, then diluted with ether and
washed with H20, 10% KHSO4, sat. NaHCO3, and brine, dried (MgSO4),
and concentrated to furnish 1- as a brown solid.
TLC Rf 0.23 (silica, 30% EtOAc/hexanes).
Ethv14-f2-(1.8-Naphthvridin-7-vl)ethvllbenzoate (1-11)
An ethanol solution (70 mL) of 1-10 (1.75 g, 14.3 mmol; JOC,
1983, 48, 3401-3408),19 (3.15 g, 14.3 mmol, and L-proline (0.8 g, 7.0
mmol) was refluxed for 16 hours. The reaction was concentrated to
dryness. Flash chromatography (silica, 60%-80% EtOAc/hexane) gave 1-
11 as a yellow solid.
Rf 0.21 (silica, 3/1 EtOAc/hexane).
1H NMR (300 MHz, CDC13) 8 9.1 (m, 1H), 8.18 (j, J=8 Hz, 1H), 8.08 (d, J=8
Hz, 1H), 7.95 (d, J=8 Hz, 2H), 7.48 (m, 1H), 7.30 (dd, J=8 Hz, 2H), 4.35 (q,
J=7Hz, 2H), 3.35 (m, 4H), 1.38 (t, J=7Hz, 3H).
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Ethyl 4-[2-(1,2,3,4-tetrahydro-1,8-naphthyridin-7-yl)ethylbenzoate
(1-12)
A mixture of 1-1 (645 mg, 2.1 mmol), 10% Pd/C (65 mg), and ethanol (10
mL) was stirred under a hydrogen atmosphere for 18 hour. Filtration
through a Celite*pad followed by concentration provided crude 1-12.
Trituration of the solid with 1:1 ether/hexanes furnished ,1-12 as a light
yellow solid.
TLC Rf 0.75 (silica, 70% EtOAc/hexanes).
1H NMR (300 MHz, CDC13) 5 7.94 (d, J=8Hz, 2H), 7.26 (d, J=8 Hz, 2H),
7.03 (d, J=7 Hz, 1H), 6.28 (d, J=7 Hz, 1H), 4.81 (s, 1H), 4.35 (q, J=7 Hz,
2H),
3.40 (m, 2H), 3.03 (m, 2H), 2.84 (m, 2H), 2.69 (t, J=6 Hz, 2H), 1.93 (t, J=6
Hz, 2H), 1.38 (t, J=7 Hz, 3H).
4-f 2-(1.2.3.4-Tetrahydro-1.8-nanhthvridin-7-vl)ethvlbenzoic acid (1-13)
The ester 1-12 (680 mg, 2.1 mmol) in 6N HCI (10 mL) was
heated to 50 C for 18 hours. Concentration provided 1-13 as a light yellow
solid.
1H NMR (300 MHz, CD3OD) S 7.93 (d, J=8 Hz, 2H), 7.52 (d, J=8 Hz, 1H),
7.31 (d, J=8 Hz, 2H), 5.54 (d, J=8 Hz, 1), 3.48 (t, J-5 Hz, 2H), 3.03 (m, 4H),
2.79 (t, J=6 Hz, 2H), 1.93 (t, J=6 Hz, 2H).
4-[2-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-yl)ethyl]benzoyl-2(S)[ 1(S)10-
camphorsulfonylaminol (i-alanine ethyl ester (1-14)
-13 (200 mg, 0.627 mmol), amine 1& (240 mg, 0.69 mmol),
NMM (345 mL, 3.13 mmol) and BOP reagent (332 mg, 0.75 mmol) were
combined in 5 mL CH3CN. After stirring -overnight, the reaction was
concentrated, then diluted with EtOAc, washed with H20, sat. NaHCO3
and brine, dried (MgSO4), filtered and concentrated. Flash
chromatography (silica, EtOAc) provided 1-1 as an off-white foamy
solid.
TLC Rf 0.13 (silica, EtOAc).
1H NMR (300 MHz, CDC13) 5 7.70 (d, J=8Hz, 2H), 7.25 (d, J=8Hz, 2H), 7.03
(d, J=7Hz, 1H), 6.72 (t, J=5Hz, 1H), 6.5 (bm, 1H), 6.28 (d, J=7Hz, 1H), 4.79
(s, 1H), 4.42 (bs, 1H), 4.25 (q, J=7Hz, 2H), 4.04 (m, 1H), 3.85 (m, 1H), 3.55
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(d, J=15Hz, 1H), 3.41 (m, 2H), 3.00 (m, 3H), 2.82 (t, J=4Hz, 2H), 2.69 (t,
J=6Hz, 2H), 2.04 (m, 8H), 1.58 (bs, 3H), 1.31 (t, J=7Hz, 3H), 1.00 (s, 3H),
0.90 (s, 3H).
4-[2-(1,2,3,4-Tetrahydro-1,8-naphthyridin-7-yl)ethyl]benzoyl-
2(S)-fl(S)10-camphorsulfonvlaminol (3-alanine (1-15)
1-14 (250 mg, 0.409 mmol) was dissolved in 4 mL EtOH, 1M
NaOH (1.02 mL, 1.02 mmol) was added and the reaction mixture was
stirred for two hours. The reaction mixture was neutralized with 1N
HCl and then concentrated to a foamy solid. Flash chromatography
(silica, 18:10:1:1 EtOAc/EtOH/NH4OHlH20) provided 1-15 as a slightly
yellow solid.
TLC Rf 0.49 (silica, 12:10:1:1 EtOAc/EtOH/NH4OH/H20).
1H NMR (400 MHz, DMSO) 8 8.48 (bt, 1H), 7.72 (d, J=8Hz, 2H), 7.55 (bs,
1H), 7.28 (d, J=8Hz, 2H), 7.02 (d, J=7Hz, 1H), 6.37 (s, 1H), 6.26 (d, J=7Hz,
1H), 4.13 (s, 1H), 3.54 (m, 3H), 3.37 (m, 2H), 2.94 (m, 3H), 2.73 (t, J=7Hz,
2H), 2.6 (t, J=6Hz, 2H), 2.3 (m, 3H), 2.02 (m, 1H), 1.89 (m, 2H), 1.75 (m,
2H), 1.49 (m, 1H), 1.37 (m, 1H), 1.05 (m, 1H), 0.95 (s, 3H), 0.66 (s, 3H).
Structurally related analogs of 1-15 can be prepared from
commercially available starting materials utilizing the chemistry
shown in Scheme 1. Several such representative compounds are
examplified as compounds 1-4 depicted in Table 1. Compounds 1 and 2
are readily available through the chemistry illustrated in Scheme 1 by
using 2(R)-Na-Cbz-2,3-diaminopropionic acid 5(Bachem) or (1R)-(-)10-
camphorsulfonyl chloride 6(Aldrich), respectively. Compounds 3 and
are attained by starting with the appropriate alcohols, 1-
adamantanemethanol 7 (Aldrich) or (1R)-(-)-Nopol 8 (Aldrich),
respectively. These alcohols can be converted to their corresponding
sulfonyl chlorides via the standard chemistry shown in Scheme 2.
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Scheme 2
HO TsO
TsCI, pyridine
=-,j - '~
2-1 2-2
NaHCO3, CH3 COSH,
DMF
HS CH3COS
,, \ f CH3OH, K2CO3
2-4 2-3
Ci2,
H20 CIS02
2-5
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Table 1
Starting
Compound Material
O
A NC02H ~H N C02H
N H HN ' S 2 HNHCBZ
H 02 1 ~
0
CO2H
N O
N N~ H H H, SO2 O
2 02
Ci,S
.~.
O
N C02H HO
H ,
H
N N~ \ I H H-S 0
2 7
O
N HO
"XC02H
H H H-S j
UNN,_
02
4 \
,,~ 8
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SCHEME 3
H2N~~ CO2H
O H NH2
3-1
S02C1 NaOH, dioxane
H20
H2N~~ CO2H
O H H ~ SO
2
3-2
1. Br2, NaOH,
H20
2. HCI
H2N CO2H
/ =''
H S02
3-3
HCI
EtOH
HCI=H2N \,. C02CH2CH3
H H' S02
3-4
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SCHEME 3 (Cont'd)
~ CO2CH2CH3
N
H2N 3 5a
H2,
10% Pd/C
EtOH
H2N "3-5
6N HCI
HCI=H2NfN
C02H
3-6
HCI=H2N WCO2CH2CH3 EDC, HOBT,
H HNSO2C6H41 NMM, DMF
3-4
H2N N O2S
~ H H NH
N CO2CH2CH3
O
3-7
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SCHEME 3 (Cont'd) ~
/ I
\
H2N N H H O NH
N CO2CH2CH3
O
3-7
6N HCI
6 0 C /
\ I
H2N N 02S
N H NH
C02H
3-8 O
(CH3Sn)2, Pd(PPH3)4,
dioxane, 90 C Sn(CH3)3
H2N N I \ 02S
N H 1
NH
CO2H
3-9 0
1251
\/I
H2N N H H 021
C02H
O
3-10
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N-(4-Iodo-phenvlsulfonvlamino)-L-asparagine (3-2)
To a stirred solution of acid 3_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 H20, was added followed by the removal of the
cooling bath. After 2.0 h, the reaction mixture was concentrated. The
residue was dissolved in H20 (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 3=2 as a white solid.
1H NMR (300 MHz, D20) S 7.86 (d, 2H, J=8Hz), 7.48 (d, 2H, J=8Hz) 3.70
(m, 1H), 2.39 (m, 2H).
2(S)-(4-Iodo-phenylsulfonvlamino)-(3-alanine (3-3)
To a stirred solution of NaOH (7.14 g, 181.8 mmol) and H20
(40 ml) at 0 C was added Br2 (1.30 ml, 24.9 mmol) dropwise over a ten
minute period. After -5 minutes, acid 3!2 (9.9 g, 24.9 mmol), NaOH (2.00
g, 49.8 mmol) and H20 (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 3-3 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).
Ethyl 2(S)-(4-iodo-phenvlsulfonylamino)-D-alanine-hvdrochloride (3-4)
HCl gas was rapidly bubbled through a suspension of acid
-3 (4.0 g, 10.81 mmol) in EtOH (50 ml) at 0 C for 10 minutes. The cooling
bath was removed and the reaction was heated to 60 C. After 18 h, the
reaction was concentrated to provide ester 3=4 as a white solid.
1H NMR (300 MHz, CD3OD) 8 7.98 (d, 2H, J=8Hz), 7.63 (d, 2H, J=8Hz),
4.25 (q, 1H, J=5Hz), 3.92 (m, 2H), 3.33 (m, 1H), 3.06 (m, 1H), 1.01 (t, 3H,
J=7Hz).
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Ethv14-f2-(2-Aminopvridin-6-vl)ethvllbenzoate (3-5)
A mixture of ester 3-5a (700 mg, 2.63 mmol), (for
preparation, see: Scheme 29 of PCT International Application
Publication No. WO 95/327 10, 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 3-5 as
a brown oil.
TLC Rf = 0.23 (silica, 40% EtOAc/hexanes)
1H NMR (300 MHz, CDC13) S 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-f2-(2-AminQpvridin-6-vl)ethvllbenzoic acid hydrochloride (3-6)
A suspension of ester -5 (625 mg, 2.31 mmol) in 6N HCI
(12 ml) was heated to 60 C. After -20 h, the reaction was concentrated to
give acid L-6 as a tan solid.
1H NMR (300 MHz, CD30D) 8 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).
Ethy14-[2-(2-Aminopyridin-6-yl)ethyl]benzoyl-2(S)-(4-iado-
phenvlsulfonylamino)-D-alanine (3-7)
A solution of acid 3-6 (400 mg, 1.43 mmol), amine 3a
(686 mg, 1.57 mmol), EDC (358 mg, 1.86 mmol), HOBT (252 mg, 1.86
mmol), NMM (632 1, 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 to 5% isopropanol/EtOAc) provided
amide Qr_7 as a white solid.
TLC Rf = 0.4 (silica, 10% isopropanol/EtOAc)
1H NMR (300 MHz, CD30D) S 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).
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4-[2-(2-Aminopyridin-6-yl)ethyl]benzoyl-2( S)-(4-iodophenyl-
su1 onylamino)-o-alanine (3-8)
A solution of ester 3-7 (200 mg, 0.3213 mmol) and 6N HCl (30
ml) was heated to 60 C. After -20 h, the reaction mixture was
concentrated. Flash chromatography (silica, 20:20:1:1 EtOAc/EtOH/
NH40H/H20) provided acid 3-$ as a white solid.
TLC Rf = 0.45 (silica, 20:20:1:1 EtOAc/EtOH/NH4OH/H20)
1H NMR (400 MHz, DMSO) S 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-
phenvlsulfonvlamino-D-alanine (3-9)
A solution of iodide 3-$ (70 mg, 0.1178 mmol), (CH3Sn)2
(49 l, 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 pM 100A , 40 x 100 mm; 95:5 iE 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 9 as a white solid.
1H NMR, (400 MHz, DMSO) S 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)ethyllbenzoyl-2(S)-4-125iodo-
enylsulfonylaminq;f3-alanine (3-10)
An iodobead (Pierce) was added to a shipping vial of 5 mCi
of Na1251 (Amersham, IMS30) and stirred for five minutes at room
temperature. A solution of 0.1 mg of la in 0.05 mL of 10% H2SO4/MeOH
was made and immediately added to the Na125Uiodobead 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
[Vydaepeptide-protein C-18 column, 4.6 x 250 mm, linear gradient of
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10% acetonitrile (0.1% (TFA):H20 (0.1% TFA) to 90% acetonitrile (0.1%
TFA):H20 (0.1% TFA) over 30 minutes, 1 mL/min]. The retention time
of -1( 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 -,QI , which coeluted on HPLC
analysis with an authentic sample of 3-$.
Instrumentation: Analytical and preparative HPLC was
carried out using a Waters 600E Powerline Multi Solvent Delivery
System with 0.1 mL heads with a Rheodyne 7125 injector and a Waters
990 Photodiode Array Detector with a Gilson FC203 Microfraction
collector. For analytical and preparative HPLC a Vydac peptide-protein
C-18 column, 4.6 x 250 mm was used with a C-18 Brownlee modular
guard column. The acetonitrile used for the HPLC analyses was Fisher
Optima grade. The HPLC radiodetector used was a Beckman 170
Radioisotope detector. A VydatC-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 W/Vis Diode Array Spectrophotometer.
Sample radioactivities were determined in a Packard A5530 gamma
counter.
The test procedures employed to measure av(33 binding and
the bone resorption inhibiting activity of the compounds of the present
invention are described below. In addition, the activity of the compounds
of the present invention for treating cancer and/or inhibiting tumor
growth is confirmed utilizing the nude mouse tumor xenograft assay
described in Kohl et al., PNAS 91 (1994) 9141-45.
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BONE RESORPTION-PIT ASSAY
When osteoclasts engage in bone resorption, they will
literally cause the formation of pits in the surface of bone that they are
acting upon. Therefore, when testing compounds for their ability to
inhibit osteoclasts, it is useful to measure the ability of osteoclasts to
excavate these resorption pits when the inhibiting compound is present.
Consecutive 200 micron thick cross sections from a six mm
cylinder of bovine femur diaphysis were cut with a low speed diamond
saw (Isomet, Beuler, Ltd., Lake Bluff, Il). Bone slices were pooled,
placed in a 10% ethanol solution and refrigerated until further use.
Prior to experimentation, bone slices were ultrasonicated
twice, 20 minutes each in H20. Cleaned slices were placed in 96 well
plates such that two control lanes and one lane for each drug dosage are
available. Each lane represents either triplicate or quadruplicate
cultures. The bone slices in 96 well plates were sterilized by UV
irradiation. Prior to incubation with osteoclasts, the bone slices were
hydrated by the addition of 0.1 ml Medium 199, pH 6.9 containing 15%
fetal bovine serum and 1% penicillin/streptomycin.
Osteoclasts were isolated from the long bones of 1 to 3 day
old rat pups (Sprague-Dawley) by modifications of Chambers et al., (J .
Cell. Science, 66:383-399). The resulting suspension (0.75 mUbone) was
gently triturated 90-120 times using a wide bore transfer pipet. The
cellular population was separated from bone fragments by a cell
strainer with a 100 micron nylon mesh. 100 l of the cell suspension
was placed onto each bone slice. Test compounds were then added at the
desired experimental concentrations. -
Bone slices exposed to osteoclasts for 20-24 hrs were
processed for staining. Tissue culture media was removed from each
bone slice. Each well was washed with 200 l of H20, and the bone slices
were then fixed for 20 minutes in 2.5% glutaraldehyde, 0.1 M cacodylate,
pH 7.4. After fixation, any remaining cellular debris was removed by 2
min. ultrasonication in the presence of 0.25 M NH4OH followed by 2 X 15
min ultrasonication in H20. The bone slices were immediately stained
for 6-8 min with filtered 1% toluidine blue and 1% borax.
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After the bone slices have dried, resorption pits were
counted in test and control slices. Resorption pits were viewed in a
Microphot Fx (Nikonn fluorescence microscope using a polarizing Nikor*
IGS filter cube. Test dosage results were compared with controls and
resulting IC50 values were determined for each compound tested.
The appropriateness of extrapolating data from this assay
to utility and use in mammalian (including human) disease states is
supported by the teaching found in Sato, M., et al., Journal of Bone and
Mineral Research, Vol. 5, No. 1, 1990. That article teaches that certain
bisphosphonates have been used clinically and appear to be effective in
the treatment of Paget's disease, hypercalcemia of malignancy,
osteolytic lesions produced by bone metastases, and bone loss due to
immobilization or sex hormone deficiency. These same
bisphosphonates are then tested in the resorption pit assay described
above to confirm a correlation between their known utility and positive
performance in the assay.
EIB ASSAY
Duong A al., J. Bone Miner. Res., 8:S 378, describe a system
for expressing the human integrin av(33. It has been suggested that the
integrin stimulates attachment of osteoclasts to bone matrix, since
antibodies against the integrin, or RGD-containing molecules, such as
echistatin (European Publication 382 451), can effectively block bone
resorption.
Reaction Mixture:
1. 175 1 TBS buffer (50 mM Tris=HCl pH 7.2, 150 mM NaCI,
1% BSA, 1 mM CaC12, 1 mM MgC12).
2. 25 l cell extract (dilute with 100 mM octylglucoside buffer to
give 2000 cpm/25 l).
3. 1251-echistatin (25 l/50,000 cpm) (see EP 382 451).
4. 25 l buffer (total binding) or unlabeled echistatin (non-
specific binding).
* trade-mark
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The reaction mixture was then incubated for 1 h at room
temp. The unbound and the bound av(33 were separated by filtration
using a Skatron Cell Harvester. The filters (prewet in 1.5% poly-
ethyleneimine for 10 mins) were then washed with the wash buffer (50
mM Tris HCl, 1mM 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. 3-10 (specific activity 500-1000 Ci/mmole)
10. test compound
11. Purified integrin receptor: av(33 was purified from 293 cells
overexpressing av(33 (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.
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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 l of binding buffer and 20 ml of 50-OG
buffer. 5 l (-30 ng/ l) of purified receptor was added to the
beads in suspension with stirring at room temperature for 30
minutes. The mixture was then centrifuged at 2,500 rpm in a
Beckman GPR Benchtop centrifuge for 10 minutes at 4 C. The
pellets were then resuspended in 50 l of binding buffer and 25
l of 50-OG buffer.
3. Reaction
The following were sequentially added into Optiplate in
corresponding wells:
(i) Receptor/beads mixture (75 l)
(ii) 25 l of each of the following: compound to be tested, binding
buffer for total binding or 3_8 for non-specific
binding (final concentration 1 M )
(iii) 3-10 in binding buffer (25 l, final concentration 40 pM)
(iv) Binding buffer (125 l)
(v) Each plate was sealed with plate sealer from PACKARD and
incubated overnight with rocking at 4 C
4. Plates were counted using PACKARD TOPCOUNT
5. % inhibition was calculated as follows:
A = total counts
B = nonspecific counts
C = sample counts
% inhibition = [{(A-B)-(C-B)}/(A-B)]/(A-B) x 100
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OCFORM ASSAY
Osteoblast-like cells (1.8 cells), originally derived from
mouse calvaria, were plated in CORNING 24 well tissue culture plates
in a MEM medium containing ribo- and deoxyribonucleosides, 10% fetal
bovine serum and penicillin-streptomycin. Cells were seeded at
40,000/well in the morning. In the afternoon, bone marrow cells were
prepared from six week old male Balb/C mice as follows:
Mice were sacrificed, tibiae removed and placed in the
above medium. The ends were cut off and the marrow was flushed out
of the cavity into a tube with a 1 mL syringe with a 27.5 gauge needle.
The marrow was suspended by pipetting up and down. The suspension
was passed through >100 m nylon cell strainer. The resulting
suspension was centrifuged at 350 x g for seven minutes. The pellet was
resuspended, and a sample was diluted in 2% acetic acid to lyse the red
cells. The remaining cells were counted in a hemacytometer. The cells
were pelleted and resuspended at 1 x 106 cells/mL. 50 L was added to
each well of 1.8 cells to yield 50,000 cells/well and 1,25-dihydroxy-vitamin
D3(D3) was added to each well to a final concentration of 10 nM. The
cultures were incubated at 37 C in a humidified, 5% CO2 atmosphere.
After 48 h, the medium was changed. 72 h after the addition of bone
marrow, test compounds were added with fresh medium containing D3
to quadruplicate wells. Compounds were added again after 48 h with
fresh medium containing D3. After an additional 48 h the medium was
removed, cells were fixed with 10% formaldehyde in phosphate buffered
saline for 10 minutes at room temperature, followed by a 1-2 minute
treatment with ethanol: acetone (1:1) and air dried. The cells were then
stained for tartrate resistant acid phosphatase as follows:
The cells were stained for 10-15 minutes at room
temperature with 50 mM acetate buffer, pH 5.0 containing 30 mM
sodium tartrate, 0.3 mg/mL Fast Red Violet LB Salt and 0.1 mg/mL
Naphthol AS -MX phosphate. After staining, the plates were washed
extensively with deionized water and air dried. The number of
multinucleated, positive staining cells were counted in each well.
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EXAMPLE OF A PHARMACEUTICAL FORMULATION
As a specific embodiment of an oral composition, 100 mg of
compound 1(shown in Table 1) is formulated with sufficient finely
divided lactose to provide a total amount of 580 to 590 mg to fill a size 0
hard gel capsule.
While the invention has been described and illustrated in
reference to certain preferred embodiments thereof, those skilled in the
art will appreciate that various changes, modifications and
substitutions can be made therein without departing from the spirit and
scope of the invention. For example, effective dosages other than the
preferred doses as set forth hereinabove may be applicable as a
consequence of variations in the responsiveness of the mammal being
treated for severity of bone disorders caused by resorption, or for other
indications for the compounds of the invention indicated above.
Likewise, the specific pharmacological responses observed may vary
according to and depending upon the particular active compound
selected or whether there are present pharmaceutical carriers, as well
as the type of formulation and mode of administration employed, and
such expected variations or differences in the results are contemplated
in accordance with the objects and practices of the present invention. It
is intended, therefore, that the invention be limited only by the scope of
the claims which follow and that such claims be interpreted as broadly
as is reasonable.
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