Osteoporosis compounds

This invention relates to prostaglandin agonists, methods of using such prostaglandin agonists, pharmaceutical compositions containing such prostaglandin agonists and kits containing such prostaglandin agonists. The prostaglandin agonists are useful for the treatment of bone disorders including osteoporosis.

BACKGROUND OF INVENTION 
This invention relates to prostaglandin agonists, pharmaceutical 
compositions containing such agonists and the use of such agonists to 
prevent bone loss or restore or augment bone mass and to enhance bone 
healing including the treatment of conditions which present with low bone 
mass and/or bone defects in vertebrates, and particularly mammals, 
including humans. 
Osteoporosis is a systemic skeletal disease, characterized by low bone mass 
and deterioration of bone tissue, with a consequent increase in bone 
fragility and susceptibility to fracture. In the U.S., the condition 
affects more than 25 million people and causes more than 1.3 million 
fractures each year, including 500,000 spine, 250,000 hip and 240,000 
wrist fractures annually. Hip fractures are the most serious consequence 
of osteoporosis, with 5-20% of patients dying within one year, and over 
50% of survivors being incapacitated. 
The elderly are at greatest risk of osteoporosis, and the problem is 
therefore predicted to increase significantly with the aging of the 
population. Worldwide fracture incidence is forecasted to increase 
three-fold over the next 60 years, and one study estimated that there will 
be 4.5 million hip fractures worldwide in 2050. 
Women are at greater risk of osteoporosis than men. Women experience a 
sharp acceleration of bone loss during the five years following menopause. 
Other factors that increase the risk include smoking, alcohol abuse, a 
sedentary lifestyle and low calcium intake. 
There are currently two main types of pharmaceutical therapy for the 
treatment of osteoporosis. The first is the use of anti-resorptive 
compounds to reduce the resorption of bone tissue. 
Estrogen is an example of an anti-resorptive agent. It is known that 
estrogen reduces fractures. In addition, Black, et al. in EP 0605193A1 
report that estrogen, particularly when taken orally, lowers plasma levels 
of LDL and raises those of the beneficial high density lipoproteins 
(HDL's). However, estrogen failed to restore bone back to young adult 
levels in the established osteoporotic skeleton. Furthermore, long-term 
estrogen therapy, however, has been implicated in a variety of disorders, 
including an increase in the risk of uterine cancer, endometrial cancer 
and possibly breast cancer, causing many women to avoid this treatment. 
The significant undesirable effects associated with estrogen therapy 
support the need to develop alternative therapies for osteoporosis that 
have the desirable effect on serum LDL but do not cause undesirable 
effects. 
A second type of pharmaceutical therapy for the treatment of osteoporosis 
is the use of anabolic agents to promote bone formation and increase bone 
mass. This class of agents is expected to restore bone to the established 
osteoporotic skeleton. 
U.S. Pat. No. 4,112,236, incorporated herein by reference, discloses 
certain interphenylene 8-aza-9-dioxothia-11,12-secoprostaglandins for the 
treatment of patients with renal impairment. 
Certain prostagladin agonists are disclosed in GB 1478281, GB1479156 and 
U.S. Pat. Nos. 4,175,203, 4,055,596, 4,175,203, 3,987,091 and 3,991,106 as 
being useful as, for example, renal vasodilators. U.S. Pat. Nos. 
4,175,203, 4,055,596, 4,175,203, 3,987,091 and 3,991,106 are each 
incorporated herein by reference. 
U.S. Pat. No. 4,033,996, which is incorporated herein by reference, 
discloses certain 8-aza-9-oxo(and dioxo)-thia-11,12-secoprostaglandins 
which are useful as renal vasodilators, for the prevention of thrombus 
formation, to induce growth hormone release, and as regulators of the 
immune response. 
French Pat. No. 897,566 discloses certain amino acid derivatives for the 
treatment of neurological, mental or cardiovascular disease. 
J. Org. Chem. 26; 1961; 1437 discloses 
N-acetyl-N-benzyl-p-aminophenylmercaptoacetic acid. 
Certain 11,12-secoprostaglandins are disclosed in U.S. Pat. Nos. 3,991,087 
and 4,066,692, each of which is incorporated herein by reference. 
In addition to osteoporosis, approximately, 20-25 million women and an 
increasing number of men have detectable vertebral fractures as a 
consequence of reduced bone mass, with an additional 250,000 hip fractures 
reported yearly in America alone. The latter case is associated with a 12% 
mortality rate within the first two years and with a 30% rate of patients 
requiring nursing home care after the fracture. While this is already 
significant, the economic and medical consequences of convalescence due to 
slow or imperfect healing of these bone fractures is expected to increase, 
due to the aging of the general population. 
Estrogens have been shown (Bolander et al., 38th Annual Meeting Orthopedic 
Research Society, 1992) to improve the quality of the healing of 
appendicular fractures. Therefore, estrogen replacement therapy might 
appear to be a method for the treatment of fracture repair. However, 
patient compliance with estrogen therapy is relatively poor due to its 
side effects, including the resumption of menses, mastodynia, an increased 
risk of uterine cancer, an increased perceived risk of breast cancer, and 
the concomitant use of progestins. In addition, men are likely to object 
to the use of estrogen treatment. Clearly the need exists for a therapy 
which would be beneficial to patients who have suffered debilitating bone 
fractures and which would increase patient compliance. 
Although there are a variety of osteoporosis therapies there is a 
continuing need and a continuing search in this field of art for 
alternative osteoporosis therapies. In addition, there is a need for bone 
fracture healing therapies. Also, there is a need for therapy which can 
promote bone re-growth into skeletal areas where defects exist such as 
defects caused or produced by, for example, tumors in bone. Further, there 
is a need for therapy which can promote bone re-growth into skeletal areas 
where bone grafts are indicated. 
SUMMARY OF THE INVENTION 
This invention is directed to a compound of Formula I 
##STR1## 
a prodrug thereof or a pharmaceutically acceptable salt of said compound 
or said prodrug, wherein: 
B is N or C(Q.sup.1), where Q.sup.1 is H or (C.sub.1 -C.sub.3)alkyl; 
L is n-propylenyl-X-- or CH.sub.2 -metaphenylene-CH.sub.2, wherein X is 
furanyl, thienyl, thiazolyl or tetrahydrofuranyl, said CH.sub.2 
-metaphenylene-CH.sub.2 or X being optionally mono-, di- or 
tri-substituted on aromatic carbon independently with one to three chloro, 
fluoro, methoxy, difluoromethoxy, trifluoromethoxy, trifluoromethyl or 
methyl; 
R is carboxyl, (C.sub.1 -C.sub.6)alkoxycarbonyl, tetrazolyl, 
5-oxo-1,2,4-thiadiazolyl; 5-oxo-1,2,4-oxadiazolyl, (C.sub.1 
-C.sub.4)alkylsulfonylcarbamoyl or phenylsulfonylcarbamoyl; 
R.sup.1 is H, methyl, ethyl or propyl; 
R.sup.2 is H or (C.sub.2 -C.sub.5) alkanoyl; 
R.sup.3 is independently H, fluoro or methyl; 
R.sup.4 is H, (C.sub.1 -C.sub.7) alkyl, or R.sup.4 and R.sup.1 are taken 
together to form a 5-9 membered carbocyclic ring, said alkyl being 
optionally monounsaturated and optionally mono-, di- or tri-substituted 
independently with one to three fluoro, chloro, methoxy, difluoromethoxy, 
trifluoromethoxy, trifluoromethyl or methyl; 
R.sup.5 is (C.sub.1 -C.sub.6)alkylsulfonyl, (C.sub.3 
-C.sub.7)cycloalkylsulfonyl, (C.sub.3 -C.sub.7)cycloalkyl(C.sub.1 
-C.sub.6)alkylsulfonyl, (C.sub.1 -C.sub.6)alkylcarbonyl, (C.sub.3 
-C.sub.7)cycloalkylcarbonyl, (C.sub.3 -C.sub.7)cycloalkyl(C.sub.1 
-C.sub.6)alkylcarbonyl, G-sulfonyl or G-carbonyl, said (C.sub.1 
-C.sub.6)alkylsulfonyl, (C.sub.3 -C.sub.7)cycloalkylsulfonyl, (C.sub.3 
-C.sub.7)cycloalkyl(C.sub.1 -C.sub.6)alkylsulfonyl, (C.sub.1 
-C.sub.6)alkylcarbonyl, (C.sub.3 -C.sub.7)cycloalkylcarbonyl, (C.sub.3 
-C.sub.7)cycloalkyl(C.sub.1 -C.sub.6)alkylcarbonyl optionally mono-, di- 
or tri-substituted on carbon independently with hydroxy, fluoro, chloro, 
methoxy, difluoromethoxy, trifluoromethoxy, trifluoromethyl or methyl; 
Z is methylene, ethylene, propylene or ethenylene; 
G is Ar, Ar.sup.1 -V-Ar.sup.2, Ar-(C.sub.1 -C.sub.6)alkylene, 
Ar-CONH-(C.sub.1 -C.sub.6)alkylene, R.sup.12 R.sup.13 -amino, oxy(C.sub.1 
-C.sub.6)alkylene, amino substituted with Ar, or amino substituted with 
Ar(C.sub.1 -C.sub.4)alkylene and R.sup.11, wherein R.sup.11 is H or 
(C.sub.1 -C.sub.8)alkyl, R.sup.12 and R.sup.13 may be taken separately and 
are independently selected from H and (C.sub.1 -C.sub.8)alkyl, or R.sup.12 
and R.sup.13 are taken together with the nitrogen atom to which they are 
attached to form a five- or six-membered azacycloalkyl, said azacycloalkyl 
optionally containing an oxygen atom and optionally substituted with up to 
two oxo, hydroxy, (C.sub.1 -C.sub.4)alkyl, fluoro or chloro; 
Ar is a partially saturated or fully unsaturated five to eight membered 
ring optionally having one to four heteroatoms selected independently from 
oxygen, sulfur and nitrogen, or a bicyclic ring consisting of two fused 
independently partially saturated, fully saturated or fully unsaturated 
five or six membered rings, taken independently, optionally having one to 
four heteroatoms selected independently from nitrogen, sulfur and oxygen, 
or a tricyclic ring consisting of three fused independently partially 
saturated, fully saturated or fully unsaturated five or six membered 
rings, optionally having one to four heteroatoms selected independently 
from nitrogen, sulfur and oxygen, said partially or fully saturated ring, 
bicyclic ring or tricyclic ring optionally having one or two oxo groups 
substituted on carbon or one or two oxo groups substituted on sulfur; or 
Ar is a fully saturated five to seven membered ring having one or two 
heteroatoms selected independently from oxygen, sulfur and nitrogen; 
Ar.sup.1 and Ar.sup.2 are each independently a partially saturated, fully 
saturated or fully unsaturated five to eight membered ring optionally 
having one to four heteroatoms selected independently from oxygen, sulfur 
and nitrogen, or a bicyclic ring consisting of two fused independently 
partially saturated, fully saturated or fully unsaturated five or six 
membered rings, taken independently, optionally having one to four 
heteroatoms selected independently from nitrogen, sulfur and oxygen, or a 
tricyclic ring consisting of three fused independently partially 
saturated, fully saturated or fully unsaturated five or six membered 
rings, taken independently, optionally having one to four heteroatoms 
selected independently from nitrogen, sulfur and oxygen, said partially or 
fully saturated ring, bicyclic ring or tricyclic ring optionally having 
one or two oxo groups substituted on carbon or one or two oxo groups 
substituted on sulfur; 
said Ar, Ar.sup.1 and Ar.sup.2 moieties are optionally substituted on 
carbon or nitrogen, on one ring if the moiety is monocyclic, on one or 
both rings if the moiety is bicyclic, or on one, two or three rings if the 
moiety is tricyclic, with up to three substituents per moiety, 
independently selected from R.sup.14, R.sup.15 and R.sup.16 wherein 
R.sup.14, R.sup.15 and R.sup.16 are independently hydroxy, nitro, halo, 
carboxy, (C.sub.1 -C.sub.7)alkoxy, (C.sub.1 -C.sub.4)alkoxy(C.sub.1 
-C.sub.4)alkyl, (C.sub.1 -C.sub.4)alkoxycarbonyl, (C.sub.1 -C.sub.7)alkyl, 
(C.sub.2 -C.sub.7)alkenyl, (C.sub.2 -C.sub.7)alkynyl, (C.sub.3 
-C.sub.7)cycloalkyl, (C.sub.3 -C.sub.7)cycloalkyl(C.sub.1 -C.sub.4)alkyl, 
(C.sub.3 -C.sub.7)cycloalkyl(C.sub.1 -C.sub.4)alkanoyl, formyl, (C.sub.1 
-C.sub.8)alkanoyl, (C.sub.1 -C.sub.6)alkanoyl(C.sub.1 -C.sub.6)alkyl, 
(C.sub.1 -C.sub.4)alkanoylamino, (C.sub.1 -C.sub.4)alkoxycarbonylamino, 
hydroxysulfonyl, aminocarbonylamino or mono-N-, di-N,N-, di-N,N'- or 
tri-N,N,N'-(C.sub.1 -C.sub.4)alkyl substituted aminocarbonylamino, 
sulfonamido, (C.sub.1 -C.sub.4)alkylsulfonamido, amino, mono-N- or 
di-N,N-(C.sub.1 -C.sub.4)alkylamino, carbamoyl, mono-N- or di-N,N-(C.sub.1 
-C.sub.4)alkylcarbamoyl, cyano, thiol, (C.sub.1 -C.sub.6)alkylthio, 
(C.sub.1 -C.sub.6)alkylsulfinyl, (C.sub.1 -C.sub.4)alkylsulfonyl or 
mono-N- or di-N,N-(C.sub.1 -C.sub.4)alkylaminosulfinyl; and 
V is a bond, thio(C.sub.1 -C.sub.4)alkylene, (C.sub.1 
-C.sub.4)alkylenethio, (C.sub.1 -C.sub.4)alkyleneoxy, oxy(C.sub.1 
-C.sub.4)alkylene or (C.sub.1 -C.sub.3)alkylene optionally mono- or 
di-substituted, when V is not a bond, independently with hydroxy or 
fluoro. 
A preferred group of compounds of Formula I, designated Group A, comprises 
those compounds of claim 1, prodrugs thereof and pharmaceutically 
acceptable salts of said compounds and said prodrugs, wherein B is N; R is 
carboxyl, (C.sub.1 -C.sub.6)alkoxycarbonyl or tetrazolyl; Z is ethylenyl; 
R.sup.1 and R.sup.2 are each H; and L is CH.sub.2 -metaphenylene-CH.sub.2. 
A preferred group of compounds within Group A comprises those compounds, 
prodrugs thereof and pharmaceutically acceptable salts of said compounds 
and said prodrugs, wherein R.sup.5 is (C.sub.1 -C.sub.3)alkylsulfonyl or 
(C.sub.3 -C.sub.7)cycloalkylsulfonyl. 
Another preferred group of compounds within Group A comprises those 
compounds, prodrugs thereof and pharmaceutically acceptable salts of said 
compounds and said prodrugs, wherein R.sup.5 is G-sulfonyl and G is 
phenyl, imidazolyl, pyridyl, pyrazolyl or pyrimidyl optionally mono-, di- 
or tri-substituted on carbon or nitrogen with chloro, fluoro, methoxy, 
difluoromethoxy, trifluoromethoxy, trifluoromethyl or methyl. 
Another preferred group of compounds within Group A comprises those 
compounds, prodrugs thereof and pharmaceutically acceptable salts of said 
compounds and said prodrugs, wherein R.sup.5 is (C.sub.1 
-C.sub.6)alkylcarbonyl optionally mono-, di- or tri-substituted with 
hydroxy or fluoro. 
Another preferred group of compounds of Formula I, designated Group B, 
comprises those compounds, prodrugs thereof and pharmaceutically 
acceptable salts of said compounds and said prodrugs, wherein B is N; R is 
carboxyl, (C.sub.1 -C.sub.6)alkoxycarbonyl or tetrazolyl; Z is ethylenyl; 
R.sup.1 and R.sup.2 are each H; and L is n-propylenyl-X--. 
A preferred group of compounds within Group B comprises those compounds, 
prodrugs thereof and pharmaceutically acceptable salts of said compounds 
and said prodrugs, wherein R.sup.5 is (C.sub.1 -C.sub.6)alkylsulfonyl or 
(C.sub.3 -C.sub.7)cycloalkylsulfonyl. 
Another preferred group of compounds within Group B comprises those 
compounds, prodrugs thereof and pharmaceutically acceptable salts of said 
compounds and said prodrugs, wherein R.sup.5 is G-sulfonyl and G is 
phenyl, imidazolyl, pyridyl, pyrazolyl or pyrimidyl optionally mono-, di- 
or tri-substituted on carbon or nitrogen with chloro, fluoro, methoxy, 
difluoromethoxy, trifluoromethoxy, trifluoromethyl or methyl. 
Another preferred group of compounds within Group B comprises those 
compounds; prodrugs thereof and pharmaceutically acceptable salts of said 
compounds and said prodrugs, wherein R.sup.5 is (C.sub.1 
-C.sub.6)alkylcarbonyl optionally mono-, di- or tri-substituted with 
hydroxy or fluoro. 
This invention is also directed to methods of using compounds of Formula 
II, 
##STR2## 
prodrugs thereof and pharmaceutically acceptable salts of said compounds 
and said prodrugs, wherein: 
A is ethylene, n-propylene, .alpha.-methylethylene, .beta.-methylethylene, 
.alpha.,.alpha.-dimethylethylene, .beta.,.beta.-dimethylethylene or 
oxymethylene; 
B is N or C(Q); 
R is carboxy, (C.sub.1 -C.sub.10) alkoxycarbonyl, carbamoyl, mono-N- or 
di-N,N-(C.sub.1 -C.sub.4)alkyl-substituted carbamoyl or --COOY wherein Y 
is 1-succinimidoethyl, 1-pivaloyloxyethyl, 2-acetamidoethyl, 
diloweralkylaminoloweralkyl, or carbazoyl; 
R.sup.1 is H, methyl, ethyl or propyl; 
R.sup.2 is H or (C.sub.2 -C.sub.5) alkanoyl; 
R.sup.3 is independently H or methyl; 
R.sup.4 is H, (C.sub.1 -C.sub.4) alkyl, vinyl or 2,2,2-trifluoroethyl; or 
R.sup.4 and R.sup.1 are taken together to form a 5-9 membered carbocyclic 
ring; 
R.sup.5, when B is C(Q), is formyl, acetyl, pivaloyl, propionyl, acryloyl, 
hydroxyacetyl, 3-hydroxypropionyl, hydroxymethyl, 1-hydroxyethyl, 
1,2-dihydroxyethyl, 1,3-dihydroxypropyl or 1-hydroxyl-1-methylethyl; 
R.sup.5, when B is N, is formyl, acetyl, propionyl, acryloyl, 
hydroxyacetyl, 2,2,2-trifluoroethyl or R.sup.6 SOy; 
Z is methylene, ethylene, n-propylene, tetramethylene, vinylene or 
ethynylene; 
Q is H, chloro, bromo, methyl, phenyl or substituted phenyl; 
R.sup.6 is methyl, ethyl, propyl or isopropyl; and 
y is 1 or 2; , a prodrug thereof or a pharmaceutically acceptable salt of 
said compound or said prodrug as described below. 
This invention is also directed to methods of using compounds of Formula 
III, 
##STR3## 
prodrugs thereof and pharmaceutically acceptable salts of said compounds 
and said prodrugs wherein: 
R.sup.7 is (C.sub.1 -C.sub.4) alkyl; 
R.sup.8 is carboxy or (C.sub.1 -C.sub.10) alkoxycarbonyl; 
R.sup.9 is H or methyl; 
R.sup.10 is independently H or methyl; 
R.sup.11 is (C.sub.3 -C.sub.6) alkyl; or R.sup.11 and R.sup.9 are taken 
together to form a 5-9 membered carbocyclic ring; 
G is (CH.sub.2).sub.n, oxymethylene or vinylene; 
X is (CH.sub.2).sub.m ; 
L is ethylene, vinylene or ethynylene; 
n is 0 or 2; and 
m is 1, 3 or 4; provided that the sum of chain-forming elements in G and X 
is limited to either 3 or 4, a prodrug thereof or a pharmaceutically 
acceptable salt of said compound or said prodrug as described below. 
This invention is also directed to methods for treating vertebrates, e.g., 
a mammal, having a condition which presents with low bone mass comprising 
administering to said vertebrate, e.g., a mammal, having a condition which 
presents with low bone mass, a therapeutically effective amount of a 
compound of Formulas I, II or III above a prodrug thereof or a 
pharmaceutically acceptable salt of said compound or said prodrug. 
Preferably post-menopausal women and men over the age of 60 are treated. 
Also included are individuals regardless of age who have significantly 
reduced bone mass, i.e., greater than or equal to 1.5 standard deviations 
below young normal levels. 
Yet another aspect of this invention is directed to methods for treating 
osteoporosis, bone fractures, osteotomy, bone loss associated with 
periodontitis, or prosthetic ingrowth in a vertebrate, e.g., a mammal 
(including a human being), comprising administering to said vertebrate, 
e.g., a mammal suffering from osteoporosis, bone fracture, osteotomy, bone 
loss associated with periodontitis, or prosthetic ingrowth an 
osteoporosis, bone fracture, osteotomy, bone loss associated with 
periodontitis, or prosthetic ingrowth treating amount of a compound of 
Formulas I, II or III, a prodrug thereof or a pharmaceutically acceptable 
salt of said compound or said prodrug. 
Yet another aspect of this invention is directed to methods for treating 
osteoporosis in a vertebrate, e.g., a mammal (including a human being), 
comprising administering to said vertebrate, e.g., a mammal suffering from 
osteoporosis, an osteoporosis treating amount of a Formula I, II or III 
compound, a prodrug thereof or a pharmaceutically acceptable salt of said 
compound or said prodrug. 
Yet another aspect of this invention is directed to methods for treating 
osteotomy in a vertebrate, e.g., a mammal (including a human being), 
comprising administering to said vertebrate, e.g. a mammal having 
undergone an osteotomy, a bone restoration treating amount of a Formula I, 
II or III compound, a prodrug thereof or a pharmaceutically acceptable 
salt of said compound or said prodrug, wherein a bone restoration treating 
amount is an amount of said Formula I compound sufficient to restore bone 
in areas containing bone defects due to said osteotomy. In one aspect the 
Formula I, II or III compound, prodrug thereof or pharmaceutically 
acceptable salt of said compound or said prodrug is applied locally to a 
site of osteotomy. 
Yet another aspect of this invention is directed to methods for treating 
alveolar or mandibular bone loss in a vertebrate, e.g., a mammal 
(including a human being), comprising administering to said vertebrate, 
e.g., a mammal suffering from an alveolar or mandibular bone loss, an 
alveolar or mandibular bone loss treating amount of a Formula I, II or III 
compound, a prodrug thereof or a pharmaceutically acceptable salt of said 
compound or said prodrug. 
Yet another aspect of this invention is directed to methods for treating 
bone loss associated with periodontitis in a vertebrate, e.g., a mammal 
(including a human being), comprising administering to said vertebrate, 
e.g., mammal suffering from bone loss associated with periodontitis, a 
bone loss associated with periodontitis treating amount of a Formula I, II 
or III compound, a prodrug thereof or a pharmaceutically acceptable salt 
of said compound or said prodrug. 
Yet another aspect of this invention is directed to methods for treating 
childhood idiopathic bone loss in a child comprising administering to said 
child suffering from childhood idiopathic bone loss, a childhood 
idiopathic bone loss treating amount of a Formula I, II or III compound, a 
prodrug thereof or a pharmaceutically acceptable salt of said compound or 
said prodrug. 
Yet another aspect of this invention is directed to methods for treating 
"secondary osteoporosis", which includes glucocorticoid-induced 
osteoporosis, hyperthyroidism-induced osteoporosis, immobilization-induced 
osteoporosis, heparin-induced osteoporosis or immunosuppressive-induced 
osteoporosis in a vertebrate, e.g., a mammal (including a human being), by 
administering to said vertebrate, e.g., a mammal suffering from "secondary 
osteoporosis," a "secondary osteoporosis" treating amount of a Formula I, 
II or III compound, a prodrug thereof or a pharmaceutically acceptable 
salt of said compound or said prodrug. 
Yet another aspect of this invention is directed to methods for treating 
glucocorticoid-induced osteoporosis in a vertebrate, e.g., a mammal 
(including a human being), comprising administering to said vertebrate, 
e.g., a mammal suffering from glucocorticoid-induced osteoporosis, a 
glucocorticoid-induced osteoporosis treating amount of a Formula I, II or 
III compound, a prodrug thereof or a pharmaceutically acceptable salt of 
said compound or said prodrug. 
Yet another aspect of this invention is directed to methods for treating 
hyperthyroidism-induced osteoporosis in a vertebrate, e.g., a mammal 
(including a human being), comprising administering to said vertebrate, 
e.g., a mammal suffering from hyperthyroidism-induced osteoporosis, a 
hyperthyroidism-induced osteoporosis treating amount of a Formula I, II or 
III compound, a prodrug thereof or a pharmaceutically acceptable salt of 
said compound or said prodrug. 
Yet another aspect of this invention is directed to methods for treating 
immobilization-induced osteoporosis in a vertebrate, e.g., a mammal 
(including a human being), comprising administering to said vertebrate, 
e.g., a mammal suffering from immobilization-induced osteoporosis, an 
immobilization-induced osteoporosis treating amount of a Formula I, II or 
III compound, a prodrug thereof or a pharmaceutically acceptable salt of 
said compound or said prodrug. 
Yet another aspect of this invention is directed to methods for treating 
heparin-induced osteoporosis in a vertebrate, e.g., a mammal (including a 
human being), comprising administering to said vertebrate, e.g., a mammal 
suffering from heparin-induced osteoporosis, a heparin-induced 
osteoporosis treating amount of a Formula I, II or III compound, a prodrug 
thereof or a pharmaceutically acceptable salt of said compound or said 
prodrug. 
Yet another aspect of this invention is directed to methods for treating 
immunosuppressive-induced osteoporosis in a vertebrate, e.g., a mammal 
(including a human being), comprising administering to said vertebrate, 
e.g., a mammal suffering from immunosuppressive-induced osteoporosis, an 
immunosuppressive-induced osteoporosis treating amount of a Formula I, II 
or III compound, a prodrug thereof or a pharmaceutically acceptable salt 
of said compound or said prodrug. 
Yet another aspect of this invention is directed to methods for treating a 
bone fracture in a vertebrate, e.g., a mammal-(including a human being), 
comprising administering to said vertebrate, e.g., a mammal suffering from 
a bone fracture, a bone fracture treating amount of a Formula I, II or III 
compound, a prodrug thereof or a pharmaceutically acceptable salt of said 
compound or said prodrug. In one aspect of this invention for treating a 
bone fracture the Formula I, II or III compound, prodrug thereof or 
pharmaceutically acceptable salt of said compound or said prodrug is 
applied locally to the site of bone fracture. In another aspect of this 
invention the Formula I, II or III compound, prodrug thereof or 
pharmaceutically acceptable salt of said compound or said prodrug is 
administered systemically. 
Yet another aspect of this invention is directed to methods for enhancing 
bone healing following facial reconstruction, maxillary reconstruction or 
mandibular reconstruction in a vertebrate, e.g., a mammal (including a 
human being), comprising administering to said vertebrate, e.g., a mammal 
which has undergone facial reconstruction maxillary reconstruction or 
mandibular reconstruction, a bone enhancing amount of a Formula I, II or 
III compound, a prodrug thereof or a pharmaceutically acceptable salt of 
said compound or said prodrug. In one aspect of this method the Formula I, 
II or III compound, prodrug thereof or pharmaceutically acceptable salt of 
said compound or said prodrug is applied locally to the site of bone 
reconstruction. 
Yet another aspect of this invention is directed to methods for treating 
prosthetic ingrowth in a vertebrate such as promoting bone ingrowth into a 
bone prosthesis in, e.g., a mammal (including a human being), comprising 
administering to said vertebrate, e.g., a mammal suffering from prosthetic 
ingrowth, a prosthetic ingrowth treating amount of a Formula I, II or III 
compound, a prodrug thereof or a pharmaceutically acceptable salt of said 
compound or said prodrug. 
Yet another aspect of this invention is directed to methods for inducing 
vertebral synostosis in a vertebrate, e.g., a mammal (including a human 
being), comprising administering to said vertebrate, e.g., a mammal 
undergoing surgery for vertebral synostosis, a therapeutically effective 
amount of a Formula I, II or III compound, a prodrug thereof or a 
pharmaceutically acceptable salt of said compound or said prodrug. 
Yet another aspect of this invention is directed to methods for enhancing 
long bone extension in a vertebrate, e.g., a mammal (including a human 
being), comprising administering to said vertebrate, e.g., a mammal 
suffering from an insufficiently sized long bone, a long bone enhancing 
amount of a Formula I, II or III compound, a prodrug thereof or a 
pharmaceutically acceptable salt of said compound or said prodrug. 
Yet another aspect of this invention is directed to methods for 
strengthening a bone graft in a vertebrate, e.g., a mammal (including a 
human being), comprising administering to said vertebrate, e.g., a mammal 
in receipt of a bone graft, a bone graft strengthening amount of a Formula 
I, II or III compound, a prodrug thereof or a pharmaceutically acceptable 
salt of said compound or said prodrug. Additionally, a compound of Formula 
I, II or III, a prodrug thereof or a pharmaceutically acceptable salt of 
said compound or said prodrug can be used as an alternative to bone graft 
surgery. In one aspect of this method the Formula I, II or III compound, a 
prodrug thereof or a pharmaceutically acceptable salt of said compound or 
said prodrug is applied locally to the site of the bone graft. In another 
aspect of this method, a Formula I, II or III compound, a prodrug thereof 
or a pharamceutically acceptable salt of said compound or said prodrug is 
applied directly to the bone by injection or direct application to the 
bone surface. 
A preferred dosage is about 0.001 to 100 mg/kg/day of the Formula I, II or 
III compound, prodrug thereof or pharmaceutically acceptable salt of said 
compound or said prodrug. An especially preferred dosage is about 0.01 to 
10 mg/kg/day of the Formula I, II or III compound, prodrug thereof or 
pharmaceutically acceptable salt of said compound or said prodrug. 
This invention is also directed to pharmaceutical compositions which 
comprise a therapeutically effective amount of a compound of Formula I, a 
prodrug thereof or a pharmaceutically acceptable salt of said compound or 
said prodrug and a pharmaceutically acceptable carrier or diluent. 
This invention is also directed to pharmaceutical compositions for the 
augmentation of bone mass which comprise a bone mass augmenting amount of 
a compound of Formula I, a prodrug thereof or a pharmaceutically 
acceptable salt of said compound or said prodrug and a pharmaceutically 
acceptable carrier or diluent. 
This invention is also directed to pharmaceutical compositions for the 
treatment of a condition which presents with low bone mass in a 
vertebrate, e.g., a mammal (including a human being), which comprise a low 
bone mass condition treating amount of a compound of Formula I, a prodrug 
thereof or a pharmaceutically acceptable salt of said compound or said 
prodrug and a pharmaceutically acceptable carrier or diluent. 
This invention is also directed to pharmaceutical compositions for the 
treatment of osteoporosis, bone fractures, osteotomy, bone loss associated 
with periodontitis, or prosthetic ingrowth in a vertebrate, e.g., a mammal 
(including a human being), which comprises a therapeutically effective 
amount of a compound of Formula I, a prodrug thereof or a pharmaceutically 
acceptable salt of said compound or said prodrug and a pharmaceutically 
acceptable carrier or diluent. 
This invention is also directed to pharmaceutical compositions for the 
treatment of "secondary osteoporosis," which includes 
glucocorticoid-induced osteoporosis, hyperthyroidism-induced osteoporosis, 
immobilization-induced osteoporosis, heparin-induced osteoporosis or 
immunosuppressive-induced osteoporosis in a vertebrate, e.g., a mammal 
(including a human being), which comprise a "secondary osteoporosis" 
treating amount of a compound of Formula I, a prodrug thereof or a 
pharmaceutically acceptable salt of said compound or said prodrug and a 
pharmaceutically acceptable carrier or diluent. 
This invention is also directed to pharmaceutical compositions for the 
treatment of osteoporosis in a vertebrate, e.g., a mammal (including a 
human being), which comprise an osteoporosis treating amount of a compound 
of the Formula I, a prodrug thereof or a pharmaceutically acceptable salt 
of said compound or said prodrug and a pharmaceutically acceptable carrier 
or diluent. 
This invention is also directed to pharmaceutical compositions for 
enhancing bone fracture healing in a vertebrate, e.g., a mammal (including 
a human being), which comprise a bone fracture treating amount of a 
compound of the Formula I a prodrug thereof or a pharmaceutically 
acceptable salt of said compound or said prodrug and a pharmaceutically 
acceptable carrier or diluent. 
This invention is also directed to pharmaceutical compositions for the 
treatment of osteotomy in a vertebrate, e.g., a mammal (including a human 
being), comprising administering to said vertebrate, e.g. a mammal having 
undergone an osteotomy, a bone restoration treating amount of a Formula I 
compound, a prodrug thereof, or a pharmaceutically acceptable salt of said 
compound or said prodrug, wherein a bone restoration treating amount is an 
amount of said Formula I compound, prodrug thereof or pharmaceutically 
acceptable salt of said compound or said prodrug sufficient to restore 
bone in areas containing bone defects due to said osteotomy. In one aspect 
the Formula I compound, prodrug thereof or pharmaceutically acceptable 
salt of said compound or said prodrug is applied locally to an osteotomy 
site. 
This invention is also directed to pharmaceutical compositions facilitating 
bone healing after an osteotomy in a vertebrate, e.g., a mammal (including 
a human being), comprising administering to said vertebrate, e.g., a 
mammal having undergone an osteotomy, a bone healing amount of a Formula I 
compound, a prodrug thereof or a pharmaceutically acceptable salt of said 
compound or said prodrug. In one aspect, the Formula I compound, prodrug 
or pharmaceutically acceptable salt of said compound or said prodrug is 
applied locally to an osteotomy site. 
This invention is also directed to pharmaceutical compositions for the 
treatment of alveolar or mandibular bone loss in a vertebrate, e.g., a 
mammal (including a human being), which comprise an alveolar or mandibular 
bone loss treating amount of a compound of the Formula I, a prodrug 
thereof or a pharmaceutically acceptable salt of said compound or said 
prodrug and a pharmaceutically acceptable carrier or diluent. 
This invention is also directed to pharmaceutical compositions for the 
treatment of childhood idiopathic bone loss in a child which comprise a 
childhood idiopathic bone loss treating amount of a compound of the 
Formula I, a prodrug thereof or a pharmaceutically acceptable salt of said 
compound or said prodrug and a pharmaceutically acceptable carrier or 
diluent. 
This invention is also directed to pharmaceutical compositions for the 
augmentation of bone healing following facial reconstruction, maxillary 
reconstruction or mandibular reconstruction in a vertebrate, e.g., a 
mammal (including a human being), which comprise a bone healing amount of 
a compound of the Formula I, a prodrug thereof or a pharmaceutically 
acceptable salt of said compound or said prodrug and a pharmaceutically 
acceptable carrier or diluent. 
This invention is also directed to pharmaceutical compositions for the 
treatment of bone loss associated with periodontitis in a vertebrate, 
e.g., a mammal (including a human being), which comprise a bone loss 
associated with periodontitis treating amount of a compound of the Formula 
I, a prodrug thereof or a pharmaceutically acceptable salt of said 
compound or said prodrug and a pharmaceutically acceptable carrier or 
diluent. 
This invention is also directed to pharmaceutical compositions for the 
treatment of prosthetic ingrowth in a vertebrate, e.g., a mammal 
(including a human being), which comprise a prosthetic ingrowth treating 
amount of a compound of the Formula I, a prodrug thereof or a 
pharmaceutically acceptable salt of said compound or said prodrug and a 
pharmaceutically acceptable carrier or diluent 
This invention is also directed to pharmaceutical compositions for inducing 
vertebral synostosis in a vertebrate, e.g., a mammal (including a human 
being), which comprise a therapeutically effective amount of a compound of 
the Formula I, a prodrug thereof or a pharmaceutically acceptable salt of 
said compound or said prodrug and a pharmaceutically acceptable carrier or 
diluent. 
This invention is also directed to pharmaceutical compositions for 
enhancing bone union in a long bone extension procedure in a vertebrate, 
e.g., a mammal (including a human being), which comprise a bone mass 
augmentation treating amount of a compound of the Formula I, a prodrug 
thereof or a pharmaceutically acceptable salt of said compound or said 
prodrug and a pharmaceutically acceptable carrier or diluent. 
This invention is also directed to pharmaceutical compositions for the 
treatment of glucocorticoid-induced osteoporosis in a vertebrate, e.g., a 
mammal (including a human being), which comprise a glucocorticoid-induced 
osteoporosis treating amount of a compound of the Formula I, a prodrug 
thereof or a pharmaceutically acceptable salt of said compound or said 
prodrug and a pharmaceutically acceptable carrier or diluent. 
This invention is also directed to pharmaceutical compositions for the 
treatment of hyperthyroidism-induced osteoporosis in a vertebrate, e.g., a 
mammal (including a human being), which comprise a hyperthyroidism-induced 
osteoporosis treating amount of a compound of the Formula I, prodrug 
thereof or a pharmaceutically acceptable salt of said compound or said 
prodrug and a pharmaceutically acceptable carrier or diluent. 
This invention is also directed to pharmaceutical compositions for the 
treatment of immobilization-induced osteoporosis in a vertebrate, e.g., a 
mammal (including a human being), which comprise an immobilization-induced 
osteoporosis treating amount of a compound of the Formula I, a prodrug 
thereof or a pharmaceutically acceptable salt of said compound or said 
prodrug and a pharmaceutically acceptable carrier or diluent. 
This invention is also directed to pharmaceutical compositions for the 
treatment of heparin-induced osteoporosis in a vertebrate, e.g., a mammal 
(including a human being) which comprise a heparin-induced osteoporosis 
treating amount of a compound of the Formula I, a prodrug thereof or a 
pharmaceutically acceptable salt of said compound or said prodrug and a 
pharmaceutically acceptable carrier or diluent. 
This invention is also directed to pharmaceutical compositions for the 
treatment of immunosuppressive-induced osteoporosis in a vertebrate, e.g., 
a mammal (including a human being) which comprise an 
immunosuppressive-induced osteoporosis treating amount of a compound of 
the Formula I, a prodrug thereof or a pharmaceutically acceptable salt of 
said compound or said prodrug and a pharmaceutically acceptable carrier or 
diluent. 
Yet another aspect of this invention is directed to combinations of the 
Formula I, II or III compounds a prodrug thereof or a pharmaceutically 
acceptable salt of said compound or said prodrug and other compounds as 
described below. 
Yet another aspect of this invention is directed to pharmaceutical 
compositions comprising a compound of Formula I, a prodrug thereof or a 
pharmaceutically acceptable salt of said compound or said prodrug and an 
anti-resorptive agent, a prodrug thereof and a pharmaceutically acceptable 
salt of said agent or said prodrug and for the use of such compositions 
for the treatment or prevention of conditions which present with low bone 
mass, including osteoporosis in a vertebrates, e.g., mammals (e.g., 
humans, particularly women) or the use of such compositions for other bone 
mass augmenting uses. 
The combinations of this invention comprise a therapeutically effective 
amount of a first compound, said first compound being a Formula I, II or 
III compound, a prodrug thereof or a pharmaceutically acceptable salt of 
said compound or said prodrug; and a therapeutically effective amount of a 
second compound, a prodrug of said second compound or a pharmaceutically 
acceptable salt of said second compound or said prodrug, said second 
compound being an anti-resorptive agent such as an estrogen 
agonist/antagonist or a bisphosphonate. 
Another aspect of this invention is methods for treating vertebrates, e.g., 
mammals which present with low bone mass comprising administering to said 
vertebrate, e.g., a mammal having a condition which presents with low bone 
mass 
a. an amount of a first compound, said first compound being a Formula I, II 
or III compound, a prodrug thereof or a pharmaceutically acceptable salt 
of said compound or said prodrug; and 
b. an amount of a second compound, a prodrug thereof or a pharmaceutically 
acceptable salt of said second compound or said prodrug, said second 
compound being an anti-resorptive agent such as an estrogen 
agonist/antagonist or a bisphosphonate. 
Such compositions and methods may also be used for other bone mass 
augmenting uses. 
A preferred aspect of this method is wherein the condition which presents 
with low bone mass is osteoporosis. 
Another preferred aspect of this method is wherein the first compound and 
the second compound are administered substantially simultaneously. 
Another preferred aspect of this method is wherein the first compound is 
administered for a period of from about one week to about five years. 
An especially preferred aspect of this method is wherein the first compound 
is administered for a period of from about one week to about three years. 
Optionally the administration of the first compound is followed by 
administration of the second compound wherein the second compound is an 
estrogen agonist/antagonist for a period of from about three months to 
about three years without the administration of the first compound during 
the second period of from about three months to about three years. 
Alternatively, the administration of the first compound is followed by 
administration of the second compound wherein the second compound is an 
estrogen agonist/antagonist for a period greater than about three years 
without the administration of the first compound during the greater than 
about three year period. 
Another aspect of this invention is a kit comprising: 
a. an amount of a Formula I, II or III compound, a prodrug thereof or a 
pharmaceutically acceptable salt of said compound or said prodrug and a 
pharmaceutically acceptable carrier or diluent in a first unit dosage 
form; 
b. an amount of an anti-resorptive agent such as an estrogen 
agonist/antagonist or a bisphosphonate, a prodrug thereof or a 
pharmaceutically acceptable salt of said agent or said prodrug and a 
pharmaceutically acceptable carrier or diluent in a second unit dosage 
form; and 
c. container means for containing said first and second dosage forms. 
Yet another aspect of this invention is directed to pharmaceutical 
compositions including a compound of Formula I, II or Ill, a prodrug 
thereof or a pharmaceutically acceptable salt of said compound or said 
prodrug and another bone anabolic agent (although the other bone anabolic 
agent may be a different Formula I, II or III compound) and for the use of 
such compositions for the treatment of conditions which present with low 
bone mass, including osteoporosis in vertebrates, e.g., mammals (e.g., 
humans, particularly women) or the use of such compositions for other bone 
mass augmenting uses. 
The compositions comprise a therapeutically effective amount of a first 
compound, said first compound being a Formula I, II or III compound, a 
prodrug thereof or a pharmaceutically acceptable salt of said compound or 
said prodrug; and a therapeutically effective amount of a second compound, 
said second compound being another bone anabolic agent, a prodrug thereof 
or a pharmaceutically acceptable salt of said agent or said prodrug. 
Another aspect of this invention is methods for treating vertebrates, e.g., 
mammals which present with low bone mass comprising administering to said 
vertebrate, e.g., a mammal having a condition which presents with low bone 
mass 
a. an amount of a first compound, said first compound being a Formula I, II 
or III compound, a prodrug thereof or a pharmaceutically acceptable salt 
of said compound or said prodrug; and 
b. an amount of a second compound, said second compound being another bone 
anabolic agent, a prodrug thereof or a pharmaceutically acceptable salt of 
said agent or said prodrug. 
Such compositions and methods may also be used for other bone mass 
augmenting uses. 
A preferred aspect of this method is wherein the condition which presents 
with low bone mass is osteoporosis. 
Another preferred aspect of this method is wherein the first compound and 
the second compound are administered substantially simultaneously. 
Another aspect of this invention is a kit comprising: 
a. an amount of a Formula I, II or III compound, a prodrug thereof or a 
pharmaceutically acceptable salt of said compound or said prodrug and a 
pharmaceutically acceptable carrier or diluent in a first unit dosage 
form; 
b. an amount of a second compound, said second compound being another bone 
anabolic agent, a prodrug thereof or a pharmaceutically acceptable salt of 
said agent or said prodrug and a pharmaceutically acceptable carrier or 
diluent in a second unit dosage form; and 
c. container means for containing said first and second dosage forms. 
Where used in any of the above methods, kits and compositions, certain bone 
anabolic agents, estrogen agonists/antagonists and bisphosphonates are 
preferred or especially preferred. 
Preferred bone anabolic agents include IGF-1, bone morphogenetic protein 
(BMP), prostaglandins, prostaglandin agonists/antagonists, sodium 
fluoride, parathyroid hormone (PTH), active fragments of parathyroid 
hormone, parathyroid hormone related peptides and active fragments and 
analogues of parathyroid hormone related peptides, growth hormones or 
growth hormone secretagogues and the pharmaceutically acceptable salts 
thereof. 
Preferred estrogen agonists/antagonists include droloxifene, raloxifene, 
tamoxifen; 4-hydroxy-tamoxifen; toremifene; centchroman;levormeloxifene; 
idoxifene; 
6-(4-hydroxy-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)benzyl]-naphthalen-2-ol; 
{4-[2-(2-aza-bicyclo[2.2.1]hept-2-yl)-ethoxy]-phenyl}-[6-hydroxy-2-(4-hydro 
xy-phenyl)-benzo[b]thiophen-3-yl]-methanone; 
3-(4-(1,2-diphenyl-but-1-enyl)-phenyl)-acrylic acid; 
2-(4-methoxy-phenyl)-3-[4-(2-piperidin-1-yl-ethoxy)-phenoxy]benzo[b]thiophe 
n-6-ol; 
cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetr 
ahydro-naphthalene-2-ol; 
(-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro 
naphthalene-2-ol; 
cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaph 
thalene-2-ol; 
cis-1-[6'-pyrrolodinoethoxy-3'-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahyd 
ronaphthalene; 
1-(4'-pyrrolidinoethoxyphenyl)-2-(4"-fluorophenyl)-6-hydroxy-1,2,3,4-tetrah 
ydroisoquinoline; 
cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetr 
ahydro-naphthalene-2-ol; 
1-(4'-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoqui 
noline; and the pharmaceutically acceptable salts thereof. 
Especially preferred estrogen agonists/antagonists include droloxifene; 
3-(4-(1,2-diphenyl-but-1-enyl)-phenyl)-acrylic acid; 
2-(4-methoxy-phenyl)-3-[4-(2-piperidin-1-yl-ethoxy)-phenoxy]benzo[b]thiophe 
n-6-ol; 
cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetr 
ahydro-naphthalene-2-ol; 
(-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro 
-naphthalene-2-ol; 
cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaph 
thalene-2-ol; 
cis-1-[6'-pyrrolodinoethoxy-3'-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahyd 
ro-naphthalene; 
1-(4'-pyrrolidinoethoxyphenyl)-2-(4"-fluorophenyl)-6-hydroxy-1,2,3,4-tetrah 
ydroisoquinoline; 
cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetr 
ahydro-naphthalene-2-ol; 
1-(4'-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoqui 
noline; and the pharmaceutically acceptable salts thereof. 
Preferred bisphosphonates include, tiludronic acid, alendronic acid, 
zoledronic acid, ibandronic acid, risedronic acid, etidronic acid, 
clodronic acid, pamidronic acid and their pharmaceutically acceptable 
salts. 
It will be recognized that prodrugs and pharmaceutically acceptable salts 
may be formed from the compounds used as the second compounds in the 
combinations of this invention. All of such prodrugs and pharmaceutically 
acceptable salts so formed are within the scope of this invention. 
Particularly preferred salt forms include droloxifene citrate, raloxifene 
hydrochloride, tamoxifen citrate and toremifen citrate. 
The phrase "condition(s) which presents with low bone mass" refers to a 
condition where the level of bone mass is below the age specific normal as 
defined in standards by the World Health Organization "Assessment of 
Fracture Risk and its Application to Screening for Postmenopausal 
Osteoporosis (1994). Report of a World Health Organization Study Group. 
World Health Organization Technical Series 843". Included in "condition(s) 
which presents with low bone mass" are primary and secondary osteoporosis. 
Secondary osteoporosis includes glucocorticoid-induced osteoporosis, 
hyperthyroidism-induced osteoporosis, immobilization-induced osteoporosis, 
heparin-induced osteoporosis and immunosuppressive-induced osteoporosis. 
Also included is periodontal disease, alveolar bone loss, osteotomy bone 
loss and childhood idiopathic bone loss. The phrase "condition(s) which 
presents with low bone mass" also includes long term complications of 
osteoporosis such as curvature of the spine, loss of height and prosthetic 
surgery. 
The phrase "condition which presents with low bone mass" also refers to 
said vertebrate, e.g., a mammal known to have a significantly higher than 
average chance of developing such diseases as are described above 
including osteoporosis (e.g., post-menopausal women, men over the age of 
60). 
Other bone mass augmenting or enhancing uses include bone restoration, 
increasing the bone fracture healing rate, enhancing the rate of 
successful bone grafts, bone healing following facial reconstruction or 
maxillary reconstruction or mandibular reconstruction, prosthetic 
ingrowth, vertebral synostosis or long bone extension. 
The compounds and compositions of this invention may also be used in 
conjunction with orthopedic devices such as spinal fusion cages, spinal 
fusion hardware, internal and external bone fixation devices, screws and 
pins. 
Those skilled in the art will recognize that the term bone mass actually 
refers to bone mass per unit area which is sometimes (although not 
strictly correctly) referred to as bone mineral density. 
The term "treating", "treat" or "treatment" as used herein includes 
preventative (e.g., prophylactic) and palliative and curative treatment. 
By "pharmaceutically acceptable" it is meant the carrier or diluent, 
diluent, excipients, and/or salt must be compatible with the other 
ingredients of the formulation, and not deleterious to the recipient 
thereof. 
The expression "prodrug" refers to compounds that are drug precursors which 
following administration, release the drug in vivo via some chemical or 
physiological process (e.g., a prodrug on being brought to the 
physiological pH or through enzyme action is converted to the desired drug 
form). Exemplary prodrugs upon cleavage release the corresponding free 
acid, and such hydrolyzable ester-forming residues of the Formula I, II or 
III compounds include but are not limited to substituents wherein the R 
moiety in Formula I or II and the R.sup.8 moiety in Formula III is 
independently carboxyl and the free hydrogen is replaced by (C.sub.1 
-C.sub.4)alkyl, (C.sub.2 -C.sub.7)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl 
having from 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having 
from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 
carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 
1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, 
N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 
1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 
3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N-(C.sub.1 
-C.sub.2)alkylamino(C.sub.2 -C.sub.3)alkyl (such as b-dimethylaminoethyl), 
carbamoyl-(C.sub.1 -C.sub.2)alkyl, N,N-di(C.sub.1 
-C.sub.2)alkylcarbamoyl-(C.sub.1 -C.sub.2)alkyl and piperidino-, 
pyrrolidino- or morpholino(C.sub.2 -C.sub.3)alkyl. 
Exemplary five to six membered aromatic rings optionally having one or two 
heteroatoms selected independently from oxygen, nitrogen and sulfur are 
phenyl, furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, 
pyrazolyl, isoxazolyl, isothiazolyl, pyridyl, pyridiazinyl, pyrimidinyl 
and pyrazinyl. 
Exemplary partially saturated, fully saturated or fully unsaturated five to 
eight membered rings optionally having one to four heteroatoms selected 
independently from oxygen, sulfur and nitrogen (i.e., Ar, Ar.sup.1 and 
Ar.sup.2) are cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and phenyl. 
Further exemplary five membered rings are furyl, thienyl, 2H-pyrrolyl, 
3H-pyrrolyl, pyrrolyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrrolidinyl, 
1,3-dioxolanyl, oxazolyl, thiazolyl, imidazolyl, 2H-imidazolyl, 
2-imidazolinyl, imidazolidinyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, 
isoxazolyl, isothiazolyl, 1,2-dithiolyl, 1,3-dithiolyl, 3H-1,2-oxathiolyl, 
1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 
1,3,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-thiadiazolyl, 
1,2,3,4-oxatriazolyl, 1,2,3,5-oxatriazolyl, 3H-1,2,3-dioxazolyl, 
1,2,4-dioxazolyl, 1,3,2-dioxazolyl, 1,3,4-dioxazolyl, 
5H-1,2,5-oxathiazolyl and 1,3-oxathiolyl. 
Further exemplary six membered rings are 2H-pyranyl, 4H-pyranyl, pyridyl, 
piperidinyl, 1,2-dioxinyl, 1,3-dioxinyl, 1,4-dioxanyl, morpholinyl, 
1,4-dithianyl, thiomorpholinyl, pyridazinyl, pyrimidinyl, pyrazinyl, 
piperazinyl, 1,3,5-triazinyl, 1,2,4-triazinyl, 1,2,3-triazinyl, 
1,3,5-trithianyl, 4H-1,2-oxazinyl, 2H-1,3-oxazinyl, 6H-1,3-oxazinyl, 
6H-1,2-oxazinyl, 1,4-oxazinyl, 2H-1,2-oxazinyl, 4H-1,4-oxazinyl, 
1,2,5-oxathiazinyl, 1,4-oxazinyl, o-isoxazinyl, p-isoxazinyl, 
1,2,5-oxathiazinyl, 1,2,6-oxathiazinyl, 1,4,2-oxadiazinyl and 
1,3,5,2-oxadiazinyl. 
Further exemplary seven membered rings are azepinyl, oxepinyl, thiepinyl 
and 1,2,4-diazepinyl. 
Further exemplary eight membered rings are cyclooctyl, cyclooctenyl and 
cyclooctadienyl. 
Exemplary bicyclic rings consisting of two fused independently partially 
saturated, fully saturated or fully unsaturated five and/or six membered 
rings, taken independently, optionally having one to four heteroatoms 
selected independently from nitrogen, sulfur and oxygen are indolizinyl, 
indolyl, isoindolyl, 3H-indolyl, 1H-isoindolyl, indolinyl, 
cyclopenta(b)pyridinyl, pyrano(3,4-b)pyrrolyl, benzofuryl, isobenzofuryl, 
benzo(b)thienyl, benzo(c)thienyl, 1H-indazolyl, indoxazinyl, benzoxazolyl, 
anthranilyl, benzimidazolyl, benzthiazolyl, purinyl, 4H-quinolizinyl, 
quinolinyl, isoquinotinyl, cinnolinyl, phthalazinyl, quinazolinyl, 
quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, indenyl, isoindenyl, 
naphthyl, tetralinyl, decalinyl, 2H-1-benzopyranyl, 1,4-benzodioxan, 
pyrido(3,4-b)-pyridinyl, pyrido(3,2-b)-pyridinyl, pyrido(4,3-b)-pyridinyl, 
2H-1,3-benzoxazinyl, 2H-1,4-benzoxazinyl, 1H-2,3-benzoxazinyl, 
4H-3,1-benzoxazinyl, 2H-1,2-benzoxazinyl and 4H-1,4-benzoxazinyl. 
Exemplary tricyclic rings consisting of three fused independently partially 
saturated, fully saturated or fully unsaturated five and/or six membered 
rings, taken independently, optionally having one to four heteroatoms 
selected independently from nitrogen, sulfur and oxygen are indacenyl, 
biphenylenyl, acenaphthylenyl, fluorenyl, phenalenyl, phenanthrenyl, 
anthracenyl, naphthothienyl, thianthrenyl, xanthenyl, phenoxathiinyl, 
carbazolyl, carbolinyl, phenanthridinyl, acridinyl, perimidinyl, 
phenanthrolinyl, phenazinyl, phenothiazinyl and phenoxazinyl. It will be 
understood that the fully saturated and all partially unsaturated forms of 
these rings are within the scope of this invention. Further, it will be 
understood that the heteroatom or heteroatoms in the heterocyclic rings 
can be substituted at any non-bridgehead position within said ring. 
By alkylene is meant saturated hydrocarbon (straight chain or branched) 
wherein a hydrogen atom is removed from each of the terminal carbons. 
Exemplary of such groups (assuming the designated length encompases the 
particular example) are methylene, ethylene, propylene, butylene, 
pentylene, hexylene and heptylene. 
By alkenylene is meant a hydrocarbon containing monounsaturation in the 
form of one double bond wherein said hydrocarbon is a straight chain or 
branched and wherein a hydrogen atom is removed from each of the terminal 
carbons. Exemplary of such groups (assuming the designated length 
encompasses the particular example) are ethenylene (or vinylene), 
propenylene, butenylene, pentenylene, hexenylene and heptenylene. 
By alkynylene is meant a hydrocarbon containing di-unsaturation in the form 
of one triple bond wherein said hydrocarbon is a straight chain or 
branched and wherein a hydrogen atom is removed from each of the terminal 
carbons. Exemplary of such groups (assuming the designated length 
encompasses the particular example) are ethynylene, propynylene, 
butynylene, pentynylene, hexynylene and heptynylene. 
By halo is meant chloro, bromo, iodo, or fluoro. 
By alkyl is meant a straight chain saturated hydrocarbon or branched 
saturated hydrocarbon. Exemplary of such alkyl groups (assuming the 
designated length encompasses the particular example) are methyl, ethyl, 
propyl, isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, 
neopentyl, tertiary pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 
hexyl, isohexyl, heptyl and octyl. 
By alkoxy is meant a straight chain saturated alkyl or branched saturated 
alkyl bonded through an oxy. Exemplary of such alkoxy groups (assuming the 
designated length encompasses the particular example) are methoxy, ethoxy, 
propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy, 
isopentoxy, neopentoxy, tertiary pentoxy, hexoxy, isohexoxy, heptoxy and 
octoxy. 
As used herein, the term mono-N- or di-N,N-(C.sub.1 -C.sub.x)alkyl . . . 
refers to the (C.sub.1 -C.sub.x)alkyl moiety taken independently when it 
is di-N,N-(C.sub.1 -C.sub.x)alkyl . . . (x refers to integers and is taken 
independently when two (C.sub.1 -C.sub.x)alkyl groups are present, e.g., 
methylethylamino is within the scope of di-N,N-(C.sub.1 -C.sub.x)alkyl). 
It is to be understood that if a carbocyclic or heterocyclic moiety may be 
bonded or otherwise attached to a designated substrate, through differing 
ring atoms without denoting a specific point of attachment, then all 
possible points are intended, whether through a carbon atom or, for 
example, a trivalent nitrogen atom For example, the term "pyridyl" means 
2-, 3-, or 4-pyridyl, the term "thienyl" means 2-, or 3-thienyl, and so 
forth. 
The expression "pharmaceutically acceptable salt" refers to nontoxic 
anionic salts containing anions such as (but not limited to) chloride, 
bromide, iodide, sulfate, bisulfate, phosphate, acetate, maleate, 
fumarate, oxalate, lactate, tartrate, citrate, gluconate, methanesulfonate 
and 4-toluene-sulfonate. The expression also refers to nontoxic cationic 
salts such as (but not limited to) sodium, potassium, calcium, magnesium, 
ammonium or protonated benzathine (N,N'-dibenzylethylenediamine), choline, 
ethanolamine, diethanolamine, ethylenediamine, meglamine 
(N-methyl-glucamine), benethamine (N-benzylphenethylamine), piperazine or 
tromethamine (2-amino-2-hydroxymethyl-1,3-propanediol). 
As used herein, the expressions "reaction inert solvent" and "inert 
solvent" refers to a solvent which does not interact with starting 
materials, reagents, intermediates or products in a manner which adversely 
affects the yield of the desired product. 
The chemist of ordinary skill will recognize that certain compounds of this 
invention will contain one or more atoms which may be in a particular 
stereochemical or geometric configuration, giving rise to stereoisomers 
such as enantiomers and diastereomers; and configurational isomers such as 
cis and trans olefins and cis and trans substitution patterns on saturated 
alicyclic rings. All such isomers and mixtures thereof are included in 
this invention. 
Hydrates and solvates of the compounds of this invention are also included. 
The methods and compounds of this invention result in bone formation 
resulting in decreased fracture rates. This invention makes a significant 
contribution to the art by providing compounds and methods that increase 
bone formation resulting in prevention, retardation, and/or regression of 
osteoporosis and related bone disorders. 
Other features and advantages will be apparent from the specification and 
claims which describe the invention. 
DETAILED DESCRIPTION OF THE INVENTION 
In general the compounds of this invention can be made by processes which 
include processes well known in the chemical arts. 
Compounds of the formula I 
##STR4## 
wherein B, L, R, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and Z are as 
defined above are prepared using methods analagous to the methods 
disclosed in U.S. Pat. Nos. 3,987,091; 3,991,087; 4,033,996; and 
4,066,692, each of which is incorporated herein by reference. 
Compounds of the formula II 
##STR5## 
wherein A is ethylene, n-propylene, .alpha.-methylethylene, 
.beta.-methylethylene, .alpha.,.alpha.-dimethylethylene, 
.beta.,.beta.-dimethylethylene or oxymethylene; 
B is N; 
R is carboxy, (C.sub.1 -C.sub.10)alkanoyloxy, carbamoyl or mono-N- or 
di-N,N-(C.sub.1 -C.sub.4)alkyl-substituted carbamoyl; 
R.sup.1 is H; 
R.sup.2 is H or (C.sub.2 -C.sub.5) alkanoyl; 
R.sup.3 is independently H or methyl; 
R.sup.4 is H, (C.sub.1 -C.sub.4) alkyl, vinyl or 2,2,2-trifluoroethyl; 
R.sup.5 is formyl, acetyl, propionyl, acryloyl, hydroxyacetyl or 
2,2,2-trifluoroethyl; and 
Z is ethylene, vinylene or ethynylene are prepared as described in U.S. 
Pat. No. 3,987,091, which is incorporated herein by reference. 
Compounds of the formula II 
##STR6## 
wherein A is ethylene, n-propylene, .alpha.-methylethylene, 
.beta.-methylethylene, .alpha.,.alpha.-dimethylethylene, 
.beta.,.beta.-dimethylethylene or oxymethylene; 
B is C(Q); 
R is carboxy, (C.sub.1 -C.sub.10)alkanoyloxy, carbamoyl or mono-N- or 
di-N,N-(C.sub.1 -C.sub.4)alkyl-substituted carbamoyl; 
R.sup.1 is hydrogen or methyl; 
R.sup.2 is H or (C.sub.2 -C.sub.5)alkanoyl; 
R.sup.3 is independently H or methyl; 
R.sup.4 is H, (C.sub.1 -C.sub.4) alkyl, vinyl or 2,2,2-trifluoroethyl; or 
R.sup.4 and R.sup.1 are taken together to form a 5-9 membered carbocyclic 
ring; 
R.sup.5 is acetyl; 
Q is chloro, bromo, methyl, phenyl or substituted phenyl; and 
Z is ethylene are prepared as disclosed in U.S. Pat. No. 3,991,087, which 
is incorporated herein by reference. 
Compounds of the formula II 
##STR7## 
wherein A is ethylene, n-propylene, .alpha.-methylethylene, 
.beta.-methylethylene, .alpha.,.alpha.-dimethylethylene, 
.beta.,.beta.-dimethylethylene or oxymethylene; 
B is N; 
R is carboxy, (C.sub.1 -C.sub.10) alkoxycarbonyl, carbamoyl, mono-N- or 
di-N,N-(C.sub.1 -C.sub.4)alkyl-substituted carbamoyl or --COOY wherein Y 
is 1-succinimidoethyl, 1-pivaloyloxyethyl, 2-acetamidoethyl or 
diloweralkylaminoloweralkyl; 
R.sup.1 is H, methyl, ethyl or propyl; 
R.sup.2 is H or (C.sub.2 -C.sub.5) alkanoyl; 
R.sup.3 is independently H or methyl; 
R.sup.4 is H, (C.sub.1 -C.sub.4) alkyl, vinyl or 2,2,2-trifluoroethyl; or 
R.sup.4 and R.sup.1 are taken together to form a 5-9 membered carbocyclic 
ring; 
R.sup.5 is R.sup.6 SOy; 
Z is ethylene, vinylene or ethynylene; 
R.sup.6 is methyl, ethyl, propyl or isopropyl; and 
y is 1 or 2 are prepared as disclosed in U.S. Pat. No. 4,033,996, which is 
incorporated herein by reference. 
Compounds of the formula II 
##STR8## 
wherein A is ethylene, n-propylene, .alpha.-methylethylene, 
.beta.-methylethylene, .alpha.,.alpha.-dimethylethylene, 
.beta.,.beta.-dimethylethylene or oxymethylene; 
B is C(Q); 
R is carboxy, (C.sub.1 -C.sub.10) alkoxycarbonyl, carbamoyl, mono-N- or 
di-N,N-(C.sub.1 -C.sub.4)alkyl-substituted carbamoyl or --COOY wherein Y 
is 1-succinimidoethyl, 1-pivaloyloxyethyl, 2-acetamidoethyl; 
diloweralkylaminoloweralkyl; or carbazoyl; 
R.sup.1 is H or methyl; 
R.sup.2 is H or (C.sub.2 -C.sub.5) alkanoyl; 
R.sup.3 is independently H or methyl; 
R.sup.4 is H, (C.sub.1 -C.sub.4) alkyl, vinyl or 2,2,2-trifluoroethyl; or 
R.sup.4 and R.sup.1 are taken together to form a 5-9 membered carbocyclic 
ring; 
R.sup.5, when B is C(Q), is formyl, acetyl, pivaloyl, propionyl, acryloyl, 
hydroxyacetyl, 3-hydroxypropionyl, hydroxymethyl, 1-hydroxyethyl, 
1,2-dihydroxyethyl, 1,3-dihydroxypropyl or 1-hydroxyl-1-methylethyl; 
Q is H; and 
Z is methylene, ethylene, n-propylene, tetramethylene, vinylene or 
ethynylene are prepared as disclosed in U.S. Pat. No. 4,066,692, which is 
incorporated herein by reference. 
Compounds of the formula III 
##STR9## 
wherein R.sup.7 is (C.sub.1 -C.sub.4) alkyl; 
R.sup.8 is carboxy or (C.sub.1 -C.sub.10) alkoxycarbonyl; 
R.sup.9 is H or methyl; 
R.sup.10 is independently H or methyl; 
R.sup.11 is (C.sub.3 -C.sub.6) alkyl; or 
R.sup.11 and R.sup.9 are taken together to form a 5-9 membered carbocyclic 
ring; 
G is (CH.sub.2).sub.n, oxymethylene or vinylene; 
X is (CH.sub.2).sub.m ; 
L is ethylene, vinylene or ethynylene; 
n is 0 or 2; and 
M is 1, 3 or 4; provided that the sum of chain-forming elements in G and X 
is limited to either 3 or 4 are prepared as disclosed in U.S. Pat. No. 
4,112,236, which is incorporated herein by reference. 
Compounds of the formula NH.sub.2 --L--R can be prepared according to 
methods well known to those skilled in the art, e.g., as set forth in U.S. 
Pat. No. 3,987,091 or as set forth in Preparations 2, 2a, 4, 5 and 7 
below. Compounds of the formula R.sup.5 --NH--L--R are prepared according 
to methods well known to those skilled in the art, e.g., see Preparations 
3, 6 and 8-11. Particularly compounds of the formula R.sup.5 --NH--L--R 
are prepared by reacting the corresponding amine with the appropriate 
carboxylic acid anhydride, carboxylic acid chloride or sulfonyl chloride 
in the presence of a mild base such as triethylamine, N,N-diisopropylamine 
or pyridine or by reacting the amine with a carboxylic acid in the 
presence of a suitable activating agent, to obtain the corresponding amido 
or sulfonamido compounds. Suitable activating agents are 
dicyclohexylcarbodiimide or carbonyl diimidazole. 
It will be recognized that the compounds of Formula I of this invention and 
the compounds of Formulas II and III used in the methods of this invention 
can exist in radiolabelled form, i.e., said compounds may contain one or 
more atoms containing an atomic mass or mass number different from the 
atomic mass or mass number ordinarily found in nature. Radioisotopes of 
hydrogen, carbon, phosphorous, fluorine and chlorine include .sup.3 H, 
.sup.14 C, .sup.32 P, .sup.35 S, .sup.18 F and .sup.36 CI, respectively. 
Compounds of Formula I of this invention and compounds of Formulas II and 
III used in the methods of this invention which contain such radioisotopes 
and/or other radioisotopes of other atoms are within the scope of this 
invention. Tritiated, i.e., .sup.3 H, and carbon-14, i.e., .sup.14 C, 
radioisotopes are particularly preferred for their ease of preparation and 
detectability. Radiolabelled compounds of Formula I of this invention and 
radiolabelled compounds of Formulas II and III used in the methods of this 
invention can generally be prepared according to methods well known to 
those skilled in the art. Conveniently, such radiolabelled compounds can 
be prepared by carrying out the procedures disclosed herein or referenced 
hereby by substituting a readily available radiolabelled reagent for a 
non-radiolabelled reagent. 
Those skilled in the art will recognize that anti-resorptive agents (for 
example progestins, polyphosphonates, bisphosphonate(s), estrogen 
agonists/antagonists, estrogen, estrogeniprogestin combinations, 
Premarin.RTM., estrone, estriol or 17.alpha.- or 17.beta.-ethynyl 
estradiol) may be used in conjunction with the compounds of this 
invention. 
Exemplary progestins are available from commercial sources and include: 
algestone acetophenide, altrenogest, amadinone acetate, anagestone 
acetate, chlormadinone acetate, cingestol, clogestone acetate, 
clomegestone acetate, delmadinone acetate, desogestrel, dimethisterone, 
dydrogesterone, ethynerone, ethynodiol diacetate, etonogestrel, 
flurogestone acetate, gestaclone, gestodene, gestonorone caproate, 
gestrinone, haloprogesterone, hydroxyprogesterone caproate, 
levonorgestrel, lynestrenol, medrogestone, medroxyprogesterone acetate, 
melengestrol acetate, methynodiol diacetate, norethindrone, norethindrone 
acetate, norethynodrel, norgestimate, norgestomet, norgestrel, oxogestone 
phenpropionate, progesterone, quingestanol acetate, quingestrone, and 
tigestol. 
Preferred progestins are medroxyprogestrone, norethindrone and 
norethynodrel. 
Exemplary bone resorption inhibiting polyphosphonates include 
polyphosphonates of the type disclosed in U.S. Pat. No. 3,683,080, the 
disclosure of which is incorporated herein by reference. Preferred 
polyphosphonates are geminal diphosphonates (also referred to as 
bis-phosphonates). Tiludronate disodium is an especially preferred 
polyphosphonate. Ibandronic acid is an especially preferred 
polyphosphonate. Alendronate is an especially preferred polyphosphonate. 
Other preferred polyphosphonates are 
6-amino-1-hydroxy-hexylidene-bisphosphonic acid and 
1-hydroxy-3(methylpentylamino)-propylidene-bisphosphonic acid. The 
polyphosphonates may be administered in the form of the acid, or of a 
soluble alkali metal salt or alkaline earth metal salt. Hydrolyzable 
esters of the polyphosphonates are likewise included. Specific examples 
include ethane-1-hydroxy 1,1-diphosphonic acid, methane diphosphonic acid, 
pentane-1-hydroxy-1,1-diphosphonic acid, methane dichloro diphosphonic 
acid, methane hydroxy diphosphonic acid, ethane-1-amino-1,1-diphosphonic 
acid, ethane-2-amino-1,1-diphosphonic acid, 
propane-3-amino-1-hydroxy-1,1-diphosphonic acid, 
propane-N,N-dimethyl-3-amino-1-hydroxy-1,1-diphosphonic acid, 
propane-3,3-dimethyl-3-amino-1-hydroxy-1,1-diphosphonic acid, phenyl amino 
methane diphosphonic acid, N,N-dimethylamino methane diphosphonic acid, 
N(2-hydroxyethyl) amino methane diphosphonic acid, 
butane-4-amino-1-hydroxy-1,1-diphosphonic acid, 
pentane-5-amino-1-hydroxy-1,1-diphosphonic acid, 
hexane-6-amino-1-hydroxy-1,1-diphosphonic acid and pharmaceutically 
acceptable esters and salts thereof. 
In particular, the compounds of this invention may be combined with a 
mammalian estrogen agonist/antagonist. Any estrogen agonist/antagonist may 
be used as the second compound of this invention. The term estrogen 
agonist/antagonist refers to compounds which bind with the estrogen 
receptor, inhibit bone turnover or prevent bone loss. In particular, 
estrogen agonists are herein defined as chemical compounds capable of 
binding to the estrogen receptor sites in mammalian tissue, and mimicking 
the actions of estrogen in one or more tissue. Estrogen antagonists are 
herein defined as chemical compounds capable of binding to the estrogen 
receptor sites in mammalian tissue, and blocking the actions of estrogen 
in one or more tissues. Such activities are readily determined by those 
skilled in the art according to standard assays including estrogen 
receptor binding assays, standard bone histomorphometric and densitometer 
methods, and Eriksen E. F. et al., Bone Histomorphometry, Raven Press, New 
York, 1994, pages 1-74; Grier S. J. et. al., The Use of Dual-Energy X-Ray 
Absorptiometry In Animals, Inv. Radiol., 1996, 31(1):50-62; Wahner H. W. 
and Fogelman I., The Evaluation of Osteoporosis: Dual Energy X-Ray 
Absorptiometry in Clinical Practice., Martin Dunitz Ltd., London 1994, 
pages 1-296). A variety of these compounds are described and referenced 
below. 
A preferred estrogen agonist/antagonist is droloxifene: (phenol, 
3-[1-[4[2-(dimethylamino)ethoxy]phenyl]-2-phenyl-1-butenyl]-, (E)-) and 
related compounds which are disclosed in U.S. Pat. No. 5,047,431, the 
disclosure of which is incorporated herein by reference. 
Another preferred estrogen agonist/antagonist is tamoxifen: 
(ethanamine,2[-4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethyl, (Z)-2-, 
2-hydroxy-1,2,3-propanetricarboxylate(1:1)) and related compounds which 
are disclosed in U.S. Pat. No. 4,536,516, the disclosure of which is 
incorporated herein by reference. 
Another related compound is 4-hydroxy tamoxifen which is disclosed in U.S. 
Pat. No. 4,623,660, the disclosure of which is incorporated herein by 
reference. 
A preferred estrogen agonist/antagonist is raloxifene: (methanone, 
[6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl][4-[2-(1-piperidinyl)etho 
xy]phenyl]hydrochloride) which is disclosed in U.S. Pat. No. 4,418,068, the 
disclosure of which is incorporated herein by reference. 
Another preferred estrogen agonist/antagonist is toremifene: (ethanamine, 
2-[4-(4-chloro-1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethyl-, (Z-, 
2-hydroxy-1,2,3-propanetricarboxylate (1:1) which is disclosed in U.S. 
Pat. No. 4,996,225, the disclosure of which is incorporated herein by 
reference. 
Another preferred estrogen agonist/antagonist is 
3-(4-(1,2-diphenyl-but-1-enyl)-phenyl)-acrylic acid, which is disclosed in 
Willson et al., Endocrinology, 1997, 138, 3901-3911. 
Another preferred estrogen agonist/antagonist is centchroman: 
1-[2-[[4-(-methoxy-2,2, 
dimethyl-3-phenyl-chroman4-yl)-phenoxy]-ethyl]-pyrrolidine, which is 
disclosed in U.S. Pat. No. 3,822,287, the disclosure of which is 
incorporated herein by reference. Also preferred is levormeloxifene. 
Another preferred estrogen agonist/antagonist is idoxifene: 
(E)-1-(2-(4-(1-(4-iodo-phenyl)-2-phenyl-but-1-enyl)-phenoxy)-ethyl)-pyrrol 
idinone, which is disclosed in U.S. Pat. No. 4,839,155, the disclosure of 
which is incorporated herein by reference. 
Another preferred estrogen agonist/antagonist is 
6-(4-hydroxy-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-benzyl]-naphthalen-2-o 
l which is disclosed in U.S. Pat. No. 5,484,795, the disclosure of which is 
incorporated herein by reference. 
Another preferred estrogen agonist/antagonist is 
2-(4-methoxy-phenyl)-3-(4-(2-piperidin-1-yl-ethoxy)-phenoxy)-benzo[b]thiop 
hen-6-ol which is disclosed in U.S. Pat. No. 5,488,058, the disclosure of 
which is incorporated herein by reference. 
Another preferred estrogen agonist/antagonist is 
{4-[2-(2-aza-bicyclo[2.2.1]hept-2-yl)-ethoxy]-phenyl}-[6-hydroxy-2-(4-hydr 
oxy-phenyl)benzo[b]thiophen-3-yl]-methanone which is disclosed, along with 
methods of preparation, in PCT publication no. WO 95/10513 assigned to 
Pfizer Inc. 
Other preferred estrogen agonist/antagonists include compounds as described 
in commonly assigned U.S. Pat. No. 5,552,412, the disclosure of which is 
incorporated herein by reference. Especially preferred compounds described 
therein are: 
cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetr 
ahydro-naphthalene-2-ol; 
(-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro 
-naphthalene-2-ol; 
cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-nap 
hthalene-2-ol; 
cis-1-[6'-pyrrolodinoethoxy-3'-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahyd 
ronaphthalene; 
1-(4'-pyrrolidinoethoxyphenyl)-2-(4"-fluorophenyl)-6-hydroxy-1,2,3,4-tetrah 
ydroisoquinoline; 
cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetr 
ahydro-naphthalene-2-ol; and 
1-(4'-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoqui 
noline. 
Other estrogen agonist/antagonists are described in U.S. Pat. No. 4,133,814 
(the disclosure of which is incorporated herein by reference). U.S. Pat. 
No. 4,133,814 discloses derivatives of 2-phenyl-3-aroyl-benzothiophene and 
2-phenyl-3-aroylbenzothiophene-1-oxide. 
Those skilled in the art will recognize that other bone anabolic agents, 
also referred to as bone mass augmenting agents, may be used in 
conjunction with the compounds of this invention. A bone mass augmenting 
agent is a compound that augments bone mass to a level which is above the 
bone fracture threshold as detailed in the World Health Organization Study 
World Health Organization, "Assessment of Fracture Risk and its 
Application to Screening for Postmenopausal Osteoporosis (1994). Report of 
a WHO Study Group. World Health Organization Technical Series 843." 
Any prostaglandin, or prostaglandin agonist/antagonist may be used as the 
second compound in certain aspects of this invention. This includes 
utilizing two different compounds of Formula I, II or III of this 
invention. Those skilled in the art will recognize that IGF-1, sodium 
fluoride, bone morphogenetic proteins (BMPs), parathyroid hormone (PTH), 
active fragments of parathyroid hormone, growth hormone or growth hormone 
secretagogues may also be used. The following paragraphs describe 
exemplary second compounds of this invention in greater detail. 
Any prostaglandin may be used as the second compound in certain aspects of 
this invention. The term prostaglandin refers to compounds which are 
analogs of the natural prostaglandins PGD.sub.1, PGD.sub.2, PGE.sub.2, 
PGE.sub.1 and PGF.sub.2 which are useful in the treatment of osteoporosis. 
These compounds bind to the prostaglandin receptors. Such binding is 
readily determined by those skilled in the art according to standard 
assays (e.g., An S. et al., Cloning and Expression of the EP.sub.2 Subtype 
of Human Receptors for Prostaglandin E.sub.2, Biochemical and Biophysical 
Research Communications, 1993, 197(1):263-270). 
Prostaglandins are alicyclic compounds related to the basic compound 
prostanoic acid. The carbon atoms of the basic prostaglandin are numbered 
sequentially from the carboxylic carbon atom through the cyclopentyl ring 
to the terminal carbon atom on the adjacent side chain. Normally the 
adjacent side chains are in the trans orientation. The presence of an oxo 
group at C-9 of the cyclopentyl moiety is indicative of a prostaglandin 
within the E class while PGE.sub.2 contains a trans unsaturated double 
bond at the C.sub.13 -C.sub.14 and a cis double bond at the C.sub.5 
-C.sub.6 position. 
A variety of prostaglandins are described and referenced below. However, 
other prostaglandins will be known to those skilled in the art. Exemplary 
prostaglandins are disclosed in U.S. Pat. Nos. 4,171,331 and 3,927,197, 
the disclosures of each of which are incorporated herein by reference. 
Norrdin et al., The Role of Prostaglandins in Bone In Vivo, Prostaglandins 
Leukotriene Essential Fatty Acids 41,139-150, 1990 is a review of bone 
anabolic prostaglandins. 
Any prostaglandin agonistlantagonist may be used as the second compound in 
certain aspects of this invention. The term prostaglandin 
agonist/antagonist refers to compounds which bind to prostaglandin 
receptors (e.g., An S. et al., Cloning and Expression of the EP.sub.2 
Subtype of Human Receptors for Prostaglandin E.sub.2, Biochemical and 
Biophysical Research Communications, 1993, 197(1):263-270) and mimic the 
action of prostaglandin in vivo (e.g., stimulate bone formation and 
increase bone mass). Such actions are readily determined by those skilled 
in the art according to standard assays. Eriksen E. F. et al., Bone 
Histomorphometry, Raven Press, New York, 1994, pages 1-74; Grier S. J. et. 
al., The Use of Dual-Energy X-Ray Absorptiometry In Animals, Inv. Radiol., 
1996, 31(1):50-62; Wahner H. W. and Fogelman I., The Evaluation of 
Osteoporosis: Dual Energy X-Ray Absorptiometry in Clinical Practice., 
Martin Dunitz Ltd., London 1994, pages 1-296. A variety of these compounds 
are described and referenced below. However, other prostaglandin 
agonists/antagonists will be known to those skilled in the art. Exemplary 
prostaglandin agonists/antagonists are disclosed as follows. 
Commonly assigned U.S. Pat. No. 3,932,389, the disclosure of which is 
incorporated herein by reference, discloses 
2-descarboxy-2-(tetrazol-5-yl)-11-desoxy-15-substituted-omega-pentanorpros 
taglandins useful for bone formation activity. 
Commonly assigned U.S. Pat. No. 4,018,892, the disclosure of which is 
incorporated herein by reference, discloses 
16-aryl-13,14-dihydro-PGE.sub.2 p-biphenyl esters useful for bone 
formation activity. 
Commonly assigned U.S. Pat. No. 4,219,483, the disclosure of which is 
incorporated herein by reference, discloses 2,3,6-substituted-4-pyrones 
useful for bone formation activity. 
Commonly assigned U.S. Pat. No. 4,132,847, the disclosure of which is 
incorporated herein by reference, discloses 2,3,6-substituted-4-pyrones 
useful for bone formation activity. 
U.S. Pat. No. 4,000,309, the disclosure of which is incorporated herein by 
reference, discloses 16-aryl-13,14-dihydro-PGE.sub.2 p-biphenyl esters 
useful for bone formation activity. 
U.S. Pat. No. 3,982,016, the disclosure of which is incorporated herein by 
reference, discloses 16-aryl-13,14-dihydro-PGE.sub.2 p-biphenyl esters 
useful for bone formation activity. 
U.S. Pat. No. 4,621,100, the disclosure of which is incorporated herein by 
reference, discloses substituted cyclopentanes useful for bone formation 
activity. 
U.S. Pat. No. 5,216,183, the disclosure of which is incorporated herein by 
reference, discloses cyclopentanones useful for bone formation activity. 
Sodium fluoride may be used as the second compound in certain aspects of 
this invention. The term sodium fluoride refers to sodium fluoride in all 
its forms (e.g., slow release sodium fluoride, sustained release sodium 
fluoride). Sustained release sodium fluoride is disclosed in U.S. Pat. No. 
4,904,478, the disclosure of which is incorporated herein by reference. 
The activity of sodium fluoride is readily determined by those skilled in 
the art according to biological protocols (e.g., see Eriksen E. F. et al., 
Bone Histomorphometry, Raven Press, New York, 1994, pages 1-74; Grier S. 
J. et. al., The Use of Dual-Energy X-Ray Absorptiometry In Animals, Inv. 
Radiol., 1996, 31(1):50-62; Wahner H. W. and Fogelman I., The Evaluation 
of Osteoporosis: Dual Energy X-Ray Absorptiometry in Clinical Practice., 
Martin Dunitz Ltd., London 1994, pages 1-296). 
Bone morphogenetic protein may be used as the second compound in certain 
aspects of this invention (e.g., see Ono, et al., Promotion of the 
Osteogenetic Activity of Recombinant Human Bone Morphogenetic Protein by 
Prostaglandin E1, Bone, 1996,19(6), 581-588). 
Any parathyroid hormone (PTH) may be used as the second compound in certain 
aspects of this invention. The term parathyroid hormone refers to 
parathyroid hormone, fragments or metabolites thereof and structural 
analogs thereof which can stimulate bone formation and increase bone mass. 
Also included are parathyroid hormone related peptides and active 
fragments and analogs of parathyroid related peptides (see PCT publication 
no. WO 94/01460). Such bone anabolic functional activity is readily 
determined by those skilled in the art according to standard assays (e.g., 
see Eriksen E. F. et al., Bone Histomorphometry, Raven Press, New York, 
1994, pages 1-74; Grier S. J. et al., The Use of Dual-Energy X-Ray 
Absorptiometry In Animals, Inv. Radiol., 1996, 31(1):50-62; Wahner H. W. 
and Fogelman I., The Evaluation of Osteoporosis: Dual Energy X-Ray 
Absorptiometry in Clinical Practice., Martin Dunitz Ltd., London 1994, 
pages 1-296). A variety of these compounds are described and referenced 
below. However, other parathyroid hormones will be known to those skilled 
in the art. Exemplary parathyroid hormones are disclosed in the following 
references. 
"Human Parathyroid Peptide Treatment of Vertebral Osteoporosis", 
Osteoporosis Int., 3, (Supp 1):199-203. 
"PTH 1-34 Treatment of Osteoporosis with Added Hormone Replacement Therapy: 
Biochemical, Kinetic and Histological Responses" Osteoporosis Int. 
1:162-170. 
Any growth hormone or growth hormone secretagogue may be used as the second 
compound in certain aspects of this invention. The term growth hormone 
secretagogue refers to a compound which stimulates the release of growth 
hormone or mimics the action of growth hormone (e.g., increases bone 
formation leading to increased bone mass). Such actions are readily 
determined by those skilled in the art according to standard assays well 
known to those of skill in the art. A variety of these compounds are 
disclosed in the following published PCT patent applications: WO 95/14666; 
WO 95/13069; WO 94/19367; WO 94/13696; and WO 95/34311. However, other 
growth hormones or growth hormone secretagogues will be known to those 
skilled in the art. 
In particular a preferred growth hormone secretagogue is 
N-[1(R)-[1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperidin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide:MK-677. 
Other preferred growth hormone secretagogues include: 
2-amino-N-[2-(3a-(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazo-[4 
,3-c]pyridin-5-yl)-1-(R)-benzyloxymethyl-2-oxo-ethyl]-isobutyramide or its 
L-tartaric acid salt; 
2-amino-N-{1-(R)-benzyloxymethyl-2-[3a-(R)-(4-fluoro-benzyl)-2-methyl-3-oxo 
-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl]-2-oxo-ethyl}isobutyram 
ide; 
2-amino-N-[2-(3a-(R)-benzyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyr 
idin-5-yl)-1-(R)benzyloxymethyl-2-oxo-ethyl]isobutyramide; and 
2-amino-N-{1-(2,4-difluoro-benzyloxymethyl)-2-oxo-2-[3-oxo-3a-pyridin-2-ylm 
ethyl-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyrid 
in-5-yl]-ethyl}-2-methyl-propionamide. 
Some of the preparation methods useful for the preparation of the compounds 
described herein may require protection of remote functionality (e.g., 
primary amine, secondary amine, carboxyl in Formula I, II and III 
precursors). The need for such protection will vary depending on the 
nature of the remote functionality and the conditions of the preparation 
methods. The need for such protection is readily determined by one skilled 
in the art. The use of such protection/deprotection methods is also within 
the skill in the art. For a general description of protecting groups and 
their use, see T. W. Greene, Protective Groups in Organic Synthesis, John 
Wiley & Sons, New York, 1991. 
The starting materials and reagents for the above described compounds are 
also readily available or can be easily synthesized by those skilled in 
the art using conventional methods of organic synthesis. For example, many 
of the compounds used herein are related to, or are derived from, 
compounds found in nature, in which there is a large scientific interest 
and commercial need, and accordingly many such compounds are commercially 
available or are reported in the literature or are easily prepared from 
other commonly available substances by methods which are reported in the 
literature. Such compounds include, for example, prostaglandins. 
It will be recognized by persons skilled in the art that some of the 
compounds of this invention have at least one asymmetric carbon atom and 
therefore are enantiomers or diastereomers. Diasteromeric mixtures can be 
separated into their individual diastereomers on the basis of their 
physico-chemical differences by methods known per se, for example, by 
chromatography and/or fractional crystallization. Enantiomers can be 
separated by converting the enantiomeric mixture into a diasteromeric 
mixture by reaction with an appropriate optically active compound (e.g., 
alcohol), separating the diastereomers and converting (e.g., hydrolyzing, 
including both chemical hydrolysis methods and microbial lipase hydrolysis 
methods, e.g., enzyme catalyzed hydrolysis) the individual diastereomers 
to the corresponding pure enantiomers. All such isomers, including 
diastereomers, enantiomers and mixtures thereof are considered as part of 
this invention. Also, some of the compounds of this invention are 
atropisomers (e.g., substituted biaryls) and are considered as part of 
this invention. 
Many of the compounds of this invention, including the compounds of 
Formulas I and II, the anti-resorptive agents, bone anabolic agents, 
prostaglandin agonists/antagonists, parathyroid hormones, growth hormones 
and growth hormone secretagogues, and prodrugs thereof, are acidic and 
they form a salt with a pharmaceutically acceptable cation. Some of the 
compounds of this invention, including the compounds of Formulas I and II, 
the anti-resorptive agents, bone anabolic agents, prostaglandin 
agonists/antagonists, parathyroid hormones, growth hormones and growth 
hormone secretagogues, and prodrugs thereof, are basic and they form a 
salt with a pharmaceutically acceptable anion. All such salts are within 
the scope of this invention and they can be prepared by conventional 
methods. For example, they can be prepared simply by contacting the acidic 
and basic entities, usually in a stoichiometric ratio, in either an 
aqueous, non-aqueous or partially aqueous medium, as appropriate. The 
salts are recovered either by filtration, by precipitation with a 
non-solvent followed by filtration, by evaporation of the solvent, or, in 
the case of aqueous solutions, by lyophilization, as appropriate. 
In addition, when the compounds of this invention, including the compounds 
of Formulas I and II, the anti-resorptive agents, bone anabolic agents, 
prostaglandin agonists/antagonists, parathyroid hormones, growth hormones 
and growth hormone secretagogues, form hydrates or solvates they are also 
within the scope of the invention. 
In addition, all prodrugs of the compounds of this invention, including the 
compounds of Formulas I, II and III, the anti-resorptive agents, bone 
anabolic agents, prostaglandin agonists/antagonists, parathyroid hormones, 
growth hormones and growth hormone secretagogues, are within the scope of 
this invention. Further, many of the prodrugs of the compounds of this 
invention, including the compounds of Formulas I, II and III, the 
anti-resorptive agents, bone anabolic agents, prostaglandin 
agonists/antagonists, parathyroid hormones, growth hormones and growth 
hormone secretagogues can be converted to their pharmaceutically 
acceptable salts by methods described above for the compounds of this 
invention. 
The compounds of this invention are all adapted to therapeutic use as 
agents that stimulate bone formation and increase bone mass in a 
vertebrates, e.g., mammals, and particularly humans. Since bone formation 
is closely related to the development of osteoporosis and bone related 
disorders, these compounds, by virtue of their action on bone, prevent, 
arrest and/or regress osteoporosis. 
The utility of the compounds of the present invention as medical agents in 
the treatment of conditions which present with low bone mass (e.g., 
osteoporosis) in a vertebrates, e.g., mammals (e.g. humans, particularly 
the female) is demonstrated by the activity of the compounds of this 
invention in conventional assays, including an in vivo assay, a receptor 
binding assay, a cyclic AMP assay and the fracture healing assay (all of 
which are described below). The in vivo assay (with appropriate 
modifications within the skill in the art) may be used to determine the 
activity of other anabolic agents as well as the prostaglandin agonists of 
this invention. The estrogen agonist/antagonist protocol may be used to 
determine the activity of estrogen agonists/antagonists in particular and 
also other anti-resorptive agents (with appropriate modifications within 
the skill in the art). The combination and sequential treatment protocol 
described below is useful for demonstrating the utility of the 
combinations of the anabolic agents (e.g., the compounds of this 
invention) and anti-resorptive agents (e.g., estrogen 
agonists/antagonists) described herein. Such assays also provide a means 
whereby the activities of the compounds of this invention (or the other 
anabolic agents and anti-resorptive agents described herein) can be 
compared to each other and with the activities of other known compounds. 
The results of these comparisons are useful for determining dosage levels 
in a vertebrates, e.g., mammals, including humans, for the treatment of 
such diseases. 
Anabolic Agent In Vivo Assay 
The activity of anabolic bone agents in stimulating bone formation and 
increasing bone mass can be tested in intact male or female rats, sex 
hormone deficient male (orchidectomy) or female (ovariectomy) rats. 
Male or female rats at different ages (such as 3 months of age) can be used 
in the study. The rats are either intact or castrated (ovariectomized or 
orchidectomized), and subcutaneously injected or gavaged with 
prostaglandin agonists at different doses (such as 1, 3, or 10 mg/kg/day) 
for 30 days. In the castrated rats, treatment is started at the next day 
after surgery (for the purpose of preventing bone loss) or at the time 
bone loss has already occured (for the purpose of restoring bone mass). 
During the study, all rats are allowed free access to water and a pelleted 
commercial diet (Teklad Rodent Diet #8064, Harlan Teklad, Madison, Wis.) 
containing 1.46% calcium, 0.99% phosphorus and 4.96 IU/g of Vitamin 
D.sub.3. All rats are given subcutaneous injections of 10 mg/kg calcein on 
days 12 and 2 before sacrifice. The rats are sacrificed. The following 
endpoints are determined: 
Femoral Bone Mineral Measurements: 
The right femur from each rat is removed at autopsy and scanned using dual 
energy X-ray absorptiometry (DXA, QDR 1000/W, Hologic Inc., Waltham, 
Mass.) equipped with "Regional High Resolution Scan" software (Hologic 
Inc., Waltham, Mass.). The scan field size is 5.08.times.1.902 cm, 
resolution is 0.0254.times.0.0127 cm and scan speed is 7.25 mm/second. The 
femoral scan images are analyzed and bone area, bone mineral content 
(BMC), and bone mineral density (BMD) of whole femora (WF), distal femoral 
metaphyses (DFM), femoral shaft (FS), and proximal femora (PF) are 
determined. 
Tibial Bone Histomorphometric Analyses: 
The right tibia is removed at autopsy, dissected free of muscle, and cut 
into three parts. The proximal tibia and the tibial shaft are fixed in 70% 
ethanol, dehydrated in graded concentrations of ethanol, defatted in 
acetone, then embedded in methyl methacrylate (Eastman Organic Chemicals, 
Rochester, N.Y.). 
Frontal sections of proximal tibial metaphyses at 4 and 10 .mu.m thickness 
are cut using a Reichert-Jung Polycut S microtome. The 4 .mu.m sections 
are stained with modified Masson's Trichrome stain while the 10 .mu.m 
sections remained unstained. One 4 .mu.m and one 10 .mu.m sections from 
each rat are used for cancellous bone histomorphometry. 
Cross sections of tibial shaft at 10 .mu.m thickness are cut using a 
Reichert-Jung Polycut S microtome. These sections are used for cortical 
bone histomorphometric analysis. 
Cancellous bone histomorphometry: A Bioquant OS/2 histomorphometry system 
(R&M Biometrics, Inc., Nashville, Tenn.) is used for the static and 
dynamic histomorphometric measurements of the secondary spongiosa of the 
proximal tibial metaphyses between 1.2 and 3.6 mm distal to the growth 
plate-epiphyseal junction. The first 1.2 mm of the tibial metaphyseal 
region needs to be omitted in order to restrict measurements to the 
secondary spongiosa. The 4 .mu.m sections are used to determine indices 
related to bone volume, bone structure, and bone resorption, while the 10 
.mu.m sections are used to determine indices related to bone formation and 
bone turnover. 
I) Measurements and calculations related to trabecular bone volume and 
structure: (1) Total metaphyseal area (TV, mm.sup.2): metaphyseal area 
between 1.2 and 3.6 mm distal to the growth plate-epiphyseal junction. (2) 
Trabecular bone area (BV, mm.sup.2): total area of trabeculae within TV. 
(3) Trabecular bone perimeter (BS, mm): the length of total perimeter of 
trabeculae. (4) Trabecular bone volume (BV/TV, %): BV/TV.times.100. (5) 
Trabecular bone number (TBN, #/mm): 1.199/2.times.BS/TV. (6) Trabecular 
bone thickness (TBT, .mu.m): (2000/1.199).times.(BV/BS). (7) Trabecular 
bone separation (TBS, .mu.m): (2000.times.1.199).times.(TV-BV). 
II) Measurements and calculations related to bone resorption: (1) 
Osteoclast number (OCN, #): total number of osteoclast within total 
metaphyseal area. (2) Osteoclast perimeter (OCP, mm): length of trabecular 
perimeter covered by osteoclast. (3) Osteoclast number/mm (OCN/mm, #/mm): 
OCN/BS. (4) Percent osteoclast perimeter (%OCP, %): OCP/BS.times.100. 
III) Measurements and calculations related to bone formation and turnover: 
(1) Single-calcein labeled perimeter (SLS, mm): total length of trabecular 
perimeter labeled with one calcein label. (2) Double-calcein labeled 
perimeter (DLS, mm): total length of trabecular perimeter labeled with two 
calcein labels. (3) Inter-labeled width (ILW, .mu.m): average distance 
between two calcein labels. (4) Percent mineralizing perimeter (PMS, %): 
(SLS/2+DLS)/BS.times.100. (5) Mineral apposition rate (MAR, .mu.m/day): 
ILW/label interval. (6) Bone formation rate/surface ref. (BFR/BS, 
.mu.m.sup.2 /d/.mu.m): (SLS/2+DLS).times.MAR/BS. (7) Bone turnover rate 
(BTR, %/y): (SLS/2+DLS).times.MAR/BV.times.100. 
Cortical bone histomorphometry: A Bioquant OS/2 histomorphometry system 
(R&M Biometrics, Inc., Nashville, Tenn.) is used for the static and 
dynamic histomorphometric measurements of tibial shaft cortical bone. 
Total tissue area, marrow cavity area, periosteal perimeter, endocortical 
perimeter, single labeled perimeter, double labeled perimeter, and 
interlabeled width on both periosteal and endocortical surface are 
measured, and cortical bone area (total tissue area-marrow cavity area), 
percent cortical bone area (cortical area/total tissue area.times.100), 
percent marrow area (marrow cavity area/total tissue area.times.100), 
periosteal and endocortical percent labeled perimeter [(single labeled 
perimeter/2+double labeled perimeter)/total perimeter.times.100], mineral 
apposition rate (interlabeled width/intervals), and bone formation rate 
[mineral apposition rate.times.[(single labeled perimeter/2+double labeled 
perimeter)/total perimeter] are calculated. 
Statistics 
Statistics can be calculated using StatView 4.0 packages (Abacus Concepts, 
Inc., Berkeley, Calif.). The analysis of variance (ANOVA) test followed by 
Fisher's PLSD (Stat View, Abacus Concepts Inc., 1918 Bonita Ave, Berkeley, 
Calif. 94704-1014) are used to compare the differences between groups. 
Determination of CAMP Elevation in 293-S Cell Lines Stably Overexpressing 
Recombinant Human EP.sub.2 and EP.sub.4 Receptors 
cDNAs representing the complete open reading frames of the human EP.sub.2 
and EP.sub.4 receptors are generated by reverse transcriptase polymerase 
chain reaction using oligonucleotide primers based on published sequences 
(1, 2) and RNA from primary human kidney cells (EP.sub.2) or primary human 
lung cells (EP.sub.4) as templates. cDNAs are cloned into the multiple 
cloning site of pcDNA3 (Invitrogen Corporation, 3985B Sorrento Valley 
Blvd., San Diego, Calif. 92121) and used to transfect 293-S human 
embryonic kidney cells via calcium phosphate co-precipitation. 
G418-resistant colonies are expanded and tested for specific [.sup.3 
H]PGE.sub.2 binding. Transfectants demonstrating high levels of specific 
[.sup.3 H]PGE.sub.2 binding are further characterized by Scatchard 
analysis to determine Bmax and Kds for PGE.sub.2. The lines selected for 
compound screening have approximately 338,400 receptors per cell and a 
Kd=12 nM for PGE.sub.2 (EP.sub.2), and approximately 256,400 receptors per 
cell and a Kd=2.9 nM for PGE.sub.2 (EP.sub.4). Constituitive expression of 
both receptors in parental 293-S cells is negligible. Cells are maintained 
in RPMI supplemented with fetal bovine serum (10% final) and G418 (700 
ug/ml final). 
cAMP responses in the 293-S/EP.sub.2 and 293-S/EP.sub.4 lines are 
determined by detaching cells from culture flasks in 1 ml of Ca++ and Mg++ 
deficient PBS via vigorous pounding, adding serum-free RPMI to a final 
concentration of 1.times.10.sup.6 cells/ml, and adding 
3-isobutyl-1-methylxanthine (IBMX) to a final concentration of 1 mM. One 
milliliter of cell suspension is immediately aliquoted into individual 2 
ml screwcap microcentrifuge and incubated for 10 minutes, uncovered, at 
37.degree. C., 5% CO.sub.2, 95% relative humdity. The compound to be 
tested is then added to cells at 1:100 dilutions such that final DMSO or 
ethanol concentrations is 1%. Immediately after adding compound, the tubes 
are covered, mixed by inverting two times, and incubated at 37.degree. C. 
for 12 minutes. Samples are then lysed by incubation at 100.degree. C. for 
10 minutes and immediately cooled on ice for 5 minutes. Cellular debris is 
pelleted by centrifugation at 1000.times. g for 5 minutes, and cleared 
lysates are transferred to fresh tubes. cAMP concentrations are determined 
using a commercially available cAMP radioimmunoassay kit RIA (NEK-033, 
DuPont/NEN Research Products, 549 Albany St., Boston, Mass. 02118) after 
diluting cleared lysates 1:10 in cAMP RIA assay buffer (included in kit). 
Typically, one treats cells with 6-8 concentrations of the compound to be 
tested in 1 log increments. EC50 calculations are performed on a 
calculator using linear regression analysis on the linear portion of the 
dose response curves. 
References 
1. Regan, J. W. Bailey, T. J. Pepperl, D. J. Pierce, K. L. Bogardus, A. M. 
Donello, J. E. Fairbairn, C. E. Kedzie, K. M. Woodward, D. F. and Gil, D. 
W. 1994 Cloning of a Novel Human Prostaglandin Receptorwith 
Characteristics of the Pharmaclogically Defined EP.sub.2 Subtype. Mol. 
Pharmacology 46:213-220. 
2. Bastien, L., Sawyer, N., Grygorczyk, R., Metters, K., and Adam, M. 1994 
Cloning, Functional Expression, and Characterization of the Human 
Prostaglandin E2 Receptor EP2 Subtype. J. Biol. Chem. Vol 
269,16:11873-11877. 
Assay for Binding to Prostaglandin E.sub.2 Receptors 
Membrane Preparation: All operations are performed at 4.degree. C. 
Transfected cells expressing prostaglandin E.sub.2 type 1 receptors 
(EP.sub.1), type 2 (EP.sub.2), type 3 (EP.sub.3) or type 4 (EP.sub.4) 
receptors are harvested and suspended to 2 million cells per ml in Buffer 
A [50 mM Tris-HCl (pH 7.4), 10 mM MgCl.sub.2, 1 mM EDTA, 1 mM Pefabloc 
peptide, (Boehringer Mannheim Corp., Indianapolis, Ind.), 10 uM 
Phosporamidon peptide, (Sigma, St. Louis, Mo.), 1 uM pepstatin A peptide, 
(Sigma, St. Louis, Mo.), 10 uM elastatinal peptide, (Sigma, St. Louis, 
Mo.), 100 uM antipain peptide, (Sigma, St. Louis, Mo.)]. The cells are 
lysed by sonification with a Branson Sonifier (Model #250, Branson 
Ultrasonics Corporation, Danbury, Conn.) in 2 fifteen second bursts. 
Unlysed cells and debris are removed by centrifugation at 100.times. g for 
10 min. Membranes are then harvested by centrifugation at 45,000.times. g 
for 30 minutes. Pelleted membranes are resuspended to 3-10 mg protein per 
ml, protein concentration being determined of the method of Bradford 
[Bradford, M., Anal. Biochem., 72, 248 (1976)]. Resuspended membranes are 
then stored frozen at -80.degree. C. until use. 
Binding Assay: Frozen membranes prepared as above are thawed and diluted to 
1 mg protein per ml in Buffer A above. One volume of membrane preparation 
is combined with 0.05 volume test compound or buffer and one volume of 3 
nM .sup.3 H-prostaglandin E.sub.2 (#TRK 431, Amersham, Arlington Heights, 
Ill.) in Buffer A. The mixture (205 .mu.L total volume) is incubated for 1 
hour at 25.degree. C. The membranes are then recovered by filtration 
through type GF/C glass fiber filters (#1205-401, Wallac, Gaithersburg, 
Md.) using a Tomtec harvester (Model Mach II/96, Tomtec, Orange, Conn.). 
The membranes with bound .sup.3 H-prostaglandin E.sub.2 are trapped by the 
filter, while the buffer and unbound .sup.3 H-prostaglandin E.sub.2 pass 
through the filter into waste. Each sample is then washed 3 times with 3 
ml of [50 mM Tris-HCl (pH 7.4), 10 mM MgCl.sub.2, 1 mM EDTA]. The filters 
are then dried by heating in a microwave oven. To determine the amount of 
.sup.3 H-prostaglandin bound to the membranes, the dried filters are 
placed into plastic bags with scintillation fluid and counted in a LKB 
1205 Betaplate reader (Wallac, Gaithersburg, Md.). IC50s are determined 
from the concentration of test compound required to displace 50% of the 
specifically bound .sup.3 H-prostaglandin E.sub.2. 
The full length EP.sub.1 receptor is made as disclosed in Funk et al., 
Journal of Biological Chemistry, 1993, 268, 26767-26772. The full length 
EP.sub.2 receptor is made as disclosed in Regan et al., Molecular 
Pharmacology, 1994, 46, 213-220. The full length EP.sub.3 receptor is made 
as disclosed in Regan et al., British Journal of Pharmacology, 1994, 112, 
377-385. The full length EP.sub.4 receptor is made as disclosed in 
Bastien, Journal of Biological Chemistry, 1994, 269,11873-11877. These 
full length receptors are used to prepare 293S cells expressing the 
EP.sub.1, EP.sub.2, EP.sub.3 and EP.sub.4 receptors. 
293S cells expressing either the human EP.sub.1, EP.sub.2, EP.sub.3 or 
EP.sub.4 prostaglandin E.sub.2 receptors are generated according to 
methods known to those skilled in the art. Typically, PCR (polymerase 
chain reaction) primers corresponding to the 5' and 3' ends of the 
published full length receptor are made according to the well known 
methods disclosed above and are used in an RT-PCR reaction using the total 
RNA from human kidney (for EP.sub.1), human lung (for EP.sub.2) or human 
lung (for EP.sub.3) as a source. PCR products are cloned by the TA 
overhang method into pCR2.1 (Invitrogen, Carlsbad, Calif.) and identity of 
the cloned receptor is confirmed by DNA sequencing. 
293S cells (Mayo, Dept. of Biochemistry, Northwestern Univ.) are 
transfected with the cloned receptor in pcDNA3 by electroporation. Stable 
cell lines expressing the receptor are established following selection of 
transfected cells with G418. 
Clonal cell lines expressing the maximal number of receptors are chosen 
following a whole cell .sup.3 H-PGE.sub.2 binding assay using unlabeled 
PGE.sub.2 as a competitor. 
Fracture Healing Assays 
Assay for Effects on Fracture Healing After Systemic Administration 
Fracture Technique: Sprage-Dawley rats at 3 months of age are anesthetized 
with Ketamine. A 1 cm incision is made on the anteromedial aspect of the 
proximal part of the right tibia or femur. The following describes the 
tibial surgical technique. The incision is carried through to the bone, 
and a 1 mm hole is drilled 4 mm proximal to the distal aspect of the 
tibial tuberosity 2 mm medial to the anterior ridge. Intramedullary 
nailing is performed with a 0.8 mm stainless steel tube (maximum load 36.3 
N, maximum stiffness 61.8 N/mm, tested under the same conditions as the 
bones). No reaming of the medullary canal is performed. A standardized 
closed fracture is produced 2 mm above the tibiofibular junction by 
three-point bending using specially designed adjustable forceps with blunt 
jaws. To minimize soft tissue damage, care is taken not to displace the 
fracture. The skin is closed with monofilament nylon sutures. The 
operation is performed under sterile conditions. Radiographs of all 
fractures are taken immediately after nailing, and rats with fractures 
outside the specified diaphyseal area or with displaced nails are 
excluded. The remaining animals are divided randomly into the following 
groups with 10-12 animals per each subgroup per time point for testing the 
fracture healing. The first group receives daily gavage of vehicle (water: 
100% Ethnanol=95:5) at 1 ml/rat, while the others receive daily gavage 
from 0.01 to 100 mg/kg/day of the compound to be tested (1 ml/rat) for 10, 
20, 40 and 80 days. 
At 10, 20, 40 and 80 days, 10-12 rats from each group are anesthetized with 
Ketamine and sacrificed by exsanguination. Both tibiofibular bones are 
removed by dissection and all soft tissue is stripped. Bones from 5-6 rats 
for each group are stored in 70% ethanol for histological analysis, and 
bones from another 5-6 rats for each group are stored in a buffered 
Ringers solution (+4.degree. C., pH 7.4) for radiographs and biomechanical 
testing which is performed. 
Histological Analysis: The methods for histologic analysis of fractured 
bone have been previously published by Mosekilde and Bak (The Effects of 
Growth Hormone on Fracture Healing in Rats: A Histological Description. 
Bone, 14:19-27, 1993). Briefly, the fracture side is sawed 8 mm to each 
side of the fracture line, embedded undecalcified in methymethacrylate, 
and cut frontals sections on a Reichert-Jung Polycut microtome in 8 .mu.m 
thick. Masson-Trichrome stained mid-frontal sections (including both tibia 
and fibula) are used for visualization of the cellullar and tissue 
response to fracture healing with and without treatment. Sirius red 
stained sections are used to demonstrate the characterisitics of the 
callus structure and to differentiate between woven bone and lamellar bone 
at the fracture site. The following measurements are performed: (1) 
fracture gap--measured as the shortest distance between the cortical bone 
ends in the fracture, (2) callus length and callus diameter, (3) total 
bone volume area of callus, (4) bony tissue per tissue area inside the 
callus area, (5) fibrous tissue in the callus, and (6) cartilage area in 
the callus. 
Biomechanical Analysis: The methods for biomechanical analysis have been 
previously published by Bak and Andreassen (The Effects of Aging on 
Fracture Healing in Rats. Calcif Tissue Int 45:292-297, 1989). Briefly, 
radiographs of all fractures are taken prior to the biomechanical test. 
The mechanical properties of the healing fractures are analyzed by a 
destructive three- or four-point bending procedure. Maximum load, 
stiffness, energy at maximum load, deflection at maximum load, and maximum 
stress are determined. 
Assay for Effects on Fracture Healing After Local Administration 
Fracture Technique: Female or male beagle dogs at approximately 2 years of 
age are used under anesthesia in the study. Transverse radial fractures 
are produced by slow continuous loading in three-point bending as 
described by Lenehan et al. (Lenehan, T. M.; Balligand, M.; Nunamaker, D. 
M.; Wood, F. E.: Effects of EHDP on Fracture Healing in Dogs. J Orthop Res 
3:499-507; 1985). A wire is pulled through the fracture site to ensure 
complete anatomical disruption of the bone. Thereafter, local delivery of 
prostaglandin agonists to the fracture site is achieved by slow release of 
compound delivered by slow release pellets or by administration of the 
compounds in a suitable formulation such as a paste gel solution or 
suspension for 10, 15, or 20 weeks. 
Histological Analysis: The methods for histologic analysis of fractured 
bone have been previously published by Peter et al. (Peter, C. P.; Cook, 
W. O.; Nunamaker, D. M.; Provost, M. T.; Seedor, J. G.; Rodan, G. A. 
Effects of alendronate on fracture healing and bone remodeling in dogs. J. 
Orthop. Res. 14:74-70, 1996) and Mosekilde and Bak (The Effects of Growth 
Hormone on Fracture Healing in Rats: A Histological Description. Bone, 
14:19-27, 1993). Briefly, after sacrifice, the fracture side is sawed 3 cm 
to each side of the fracture line, embedded undecalcified in 
methymethacrylate, and cut on a Reichert-Jung Polycut microtome in 8 .mu.m 
thick of frontal sections. Masson-Trichrome stained mid-frontal sections 
(including both tibia and fibula) are used for visualization of the 
cellullar and tissue response to fracture healing with and without 
treatment. Sirius red stained sections are used to demonstrate the 
characterisitics of the callus structure and to differentiate between 
woven bone and lamellar bone at the fracture site. The following 
measurements are performed: (1) fracture gap--measured as the shortest 
distance between the cortical bone ends in the fracture, (2) callus length 
and callus diameter, (3) total bone volume area of callus, (4) bony tissue 
per tissue area inside the callus area, (5) fibrous tissue in the callus, 
(6) cartilage area in the callus. 
Biomechanical Analysis: The methods for biomechanical analysis have been 
previously published by Bak and Andreassen (The Effects of Aging on 
Fracture Healing in Rats. Calcif Tissue Int 45:292-2971 1989) and Peter et 
al. (Peter, C. P.; Cook, W. O.; Nunamaker, D. M.; Provost, M. T.; Seedor, 
J. G.; Rodan, G. A. Effects of Alendronate On Fracture Healing And Bone 
Remodeling In Dogs. J. Orthop. Res. 14:74-70, 1996). Briefly, radiographs 
of all fractures are taken prior to the biomechanical test. The mechanical 
properties of the healing fractures are analyzed by a destructive three- 
or four-point bending procedures. Maximum load, stiffness, energy at 
maximum load, deflection at maximum load, and maximum stress are 
determined. 
Estrogen Agonist/Antagonist Protocol 
Estrogen agonist/antagonists are a class of compounds which inhibit bone 
turnover and prevent estrogen-deficiency induced bone loss. The 
ovariectomized rat bone loss model has been widely used as a model of 
postmenopausal bone loss. Using this model, one can test the efficacy of 
the estrogen agonist/antagonist compounds in preventing bone loss and 
inhibiting bone resorption. 
Sprague-Dawley female rats (Charles River, Wilmington, Mass.) at different 
ages (such as 5 months of age) are used in these studies. The rats are 
singly housed in 20 cm.times.32 cm.times.20 cm cages during the 
experimental period. All rats are allowed free access to water and a 
pelleted commercial diet (Agway ProLab 3000, Agway County Food, Inc., 
Syracuse, N.Y.) containing 0.97% calcium, 0.85% phosphorus, and 1.05 IU/g 
of Vitamin D.sub.3 
A group of rats (8 to 10) are sham-operated and treated p.o. with vehicle 
(10% ethanol and 90% saline, 1 ml/day), while the remaining rats are 
bilaterally ovariectomized (OVX) and treated with either vehicle (p.o.), 
17.beta.-estradiol (Sigma, E-8876, E.sub.2, 30 .mu.g/kg, daily 
subcutaneous injection), or estrogen agonist/antagonists (such as 
droloxifene at 5, 10, or 20 mg/kg, daily p.o.) for a certain period (such 
as 4 weeks). All rats are given subcutaneous injections of 10 mg/kg 
calcein (fluorochrome bone marker) 12 and 2 days before being sacrificed 
in order to examine the dynamic changes in bone tissue. After 4 weeks of 
treatment, the rats are sacrificed and autopsied. The following endpoints 
are determined: 
Body Weight Gain: Body weight at autopsy minus body weight at surgery. 
Uterine Weight and Histology: The uterus is removed from each rat during 
autopsy, and weighed immediately. Thereafter, the uterus is processed for 
histologic measurements such as uterine cross-sectional tissue area, 
stromal thickness, and luminal epithelial thickness. 
Total Serum Cholesterol: Blood is obtained by cardiac puncture and allowed 
to clot at 4.degree. C., and then centrifuged at 2,000 g for 10 min. Serum 
samples are analyzed for total serum cholesterol using a high performance 
cholesterol calorimetric assay (Boehringer Mannheim Biochemicals, 
Indianapolis, Ind.). 
Femoral Bone Mineral Measurements: The right femur from each rat is removed 
at autopsy and scanned using dual energy X-ray absorptiometry (DEXA, QDR 
1000/W, Hologic Inc., Waltham, Mass.) equipped with "Regional High 
Resolution Scan" software (Hologic Inc., Waltham, Mass.). The scan field 
size is 5.08.times.1.902 cm, resolution is 0.0254.times.0.0127 cm and scan 
speed is 7.25 mm/second. The femoral scan images are analyzed and bone 
area, bone mineral content (BMC), and bone mineral density (BMD) of whole 
femora (WF), distal femoral metaphyses (DFM), femoral shaft (FS), and 
proximal femora (PF) is determined. 
Proximal Tibial Metaphyseal Cancellous Bone Histomorphometric Analyses: The 
right tibia is removed at autopsy, dissected free of muscle, and cut into 
three parts. The proximal tibia is fixed in 70% ethanol, dehydrated in 
graded concentrations of ethanol, defatted in acetone, then embedded in 
methyl methacrylate (Eastman Organic Chemicals, Rochester, N.Y.). Frontal 
sections of proximal tibial metaphyses at 4 and 10 .mu.m thickness are cut 
using a Reichert-Jung Polycut S microtome. One 4 .mu.m and one 10 .mu.m 
sections from each rat are used for cancellous bone histomorphometry. The 
4 .mu.m sections are stained with modified Masson's Trichrome stain while 
the 10 .mu.m sections remained unstained. 
A Bioquant OS/2 histomorphometry system (R&M Biometrics, Inc., Nashville, 
Tenn.) is used for the static and dynamic histomorphometric measurements 
of the secondary spongiosa of the proximal tibial metaphyses between 1.2 
and 3.6 mm distal to the growth plate-epiphyseal junction. The first 1.2 
mm of the tibial metaphyseal region is omitted in order to restrict 
measurements to the secondary spongiosa. The 4 .mu.m sections are used to 
determine indices related to bone volume, bone structure, and bone 
resorption, while the 10 .mu.m sections are used to determine indices 
related to bone formation and bone turnover. 
I. Measurements and calculations related to trabecular bone volume and 
structure: 
1. Total metaphyseal area (TV, mm.sup.2): metaphyseal area between 1.2 and 
3.6 mm distal to the growth plate-epiphyseal junction. 
2. Trabecular bone area (BV, mm.sup.2): total area of trabeculae within TV. 
3. Trabecular bone perimeter (BS, mm): the length of total perimeter of 
trabeculae. 
4. Trabecular bone volume (BV/TV, %): BV/TV.times.100. 
5. Trabecular bone number (TBN, #/mm): 1.199/2.times.BS/TV. 
6. Trabecular bone thickness (TBT, .mu.m): (2000/1.199).times.(BV/BS). 
7. Trabecular bone separation (TBS, .mu.m): 
(2000.times.1.199).times.(TV-BV). 
II. Measurements and calculations related to bone resorption: 
1. Osteoclast number (OCN, #): total number of osteoclast within total 
metaphyseal area. 
2. Osteoclast perimeter (OCP, mm): length of trabecular perimeter covered 
by osteoclast. 
3. Osteoclast number/mm (OCN/mm, #/mm): OCN/BS. 
4. Percent osteoclast perimeter (%OCP, %): OCP/BS.times.100. 
III. Measurements and calculations related to bone formation and turnover: 
1. Single-calcein labeled perimeter (SLS, mm): total length of trabecular 
perimeter labeled with one calcein label. 
2. Double-calcein labeled perimeter (DLS, mm): total length of trabecular 
perimeter labeled with two calcein labels. 
3. Inter-labeled width (ILW, .mu.m): average distance between two calcein 
labels. 
4. Percent mineralizing perimeter (PMS, %): (SLS/2+DLS)/BS.times.100. 
5. Mineral apposition rate (MAR, .mu.m/day): ILW/label interval. 
6. Bone formation rate/surface ref. (BFR/BS, .mu.m.sup.2 /d/.mu.m): 
(SLS/2+DLS).times.MAR/BS. 
7. Bone turnover rate (BTR, %/y): (SLS/2+DLS).times.MAR/BV.times.100. 
Statistics 
Statistics are calculated using StatView 4.0 packages (Abacus Concepts, 
Inc., Berkeley, Calif.). The analysis of variance (ANOVA) test followed by 
Fisher's PLSD (Stat View, Abacus Concepts Inc. 1918 Bonita Ave, Berkeley, 
Calif. 94704-1014) is used to compare the differences between groups. 
Combination and Sequential Treatment Protocol 
The following protocols can of course be varied by those skilled in the 
art. For example, intact male or female rats, sex hormone deficient male 
(orchidectomy) or female (ovariectomy) rats may be used. In addition, male 
or female rats at different ages (such as 12 months of age) can be used in 
the studies. The rats can be either intact or castrated (ovariectomized or 
orchidectomized), and administered with anabolic agents such as the 
compounds of this invention at different doses (such as 1, 3 or 6 
mg/kg/day) for a certain period (such as two weeks to two months), and 
followed by administration of an anti-resorptive agent such as droloxifene 
at different doses (such as 1,5,10 mg/kg/day) for a certain period (such 
as two weeks to two months), or a combination treatment with both anabolic 
agent and anti-resorptive agent at different doses for a certain period 
(such as two weeks to two months). In the castrated rats, treatment can be 
started on the next day after surgery (for the purpose of preventing bone 
loss) or at the time bone loss has already occurred (for the purpose of 
restoring bone mass). 
The rats are sacrificed under ketamine anesthesia. The following endpoints 
are determined: 
Femoral bone mineral measurements are performed as described above in the 
estrogen agonist/antagonist protocol. 
Lumbar Vertebral Bone Mineral Measurements: Dual energy X-ray 
absorptiometry (QDR 1000/W, Hologic, Inc., Waltham, Mass.) equipped with a 
"Regional High Resolution Scan" software (Hologic, Inc., Waltham, Mass.) 
is used to determined the bone area, bone mineral content (BMC), and bone 
mineral density (BMD) of whole lumbar spine and each of the six lumbar 
vertebrae (LV1-6) in the anesthetized rats. The rats are anesthetized by 
injection (i.p.) of 1 ml/kg of a mixture of ketamine/rompun (ratio of 4 to 
3), and then placed on a rat platform. The scan field sized is 6.times.1.9 
cm, resolution is 0.0254.times.0.0127 cm, and scan speed is 7.25 mm/sec. 
The whole lumbar spine scan image is obtained and analyzed. Bone area 
(BA), and bone mineral content (BMC) is determined, and bone mineral 
density is calculated (MBC divided by BA) for the whole lumbar spine and 
each of the six lumbar vertebrae (LV1-6). 
Proximal tibial metaphyseal cancellous bone histomorphometric analyses are 
performed as described above for in the estrogen agonistlantagonist 
protocol. 
Measurements and calculations related to trabecular bone volume and 
structure are performed as described above in the estrogen 
agonist/antagonist protocol. Further, measurements and calculations 
related to bone resorption are also performed as described above in the 
estrogen agonist/antagonist protocol. Still further, measurements and 
calculations related to bone formation and turnover are performed as 
described above in the estrogen agonist/antagonist protocol. Further 
still, the data obtained is analyzed using the statistical manipulations 
described above in the estrogen agonist/antagonist protocol. 
Kidney Regeneration Assay 
The role of an prostaglandin selective agonist in kidney regeneration is 
investigated by the ability of Prostaglandin E.sub.2 (PGE.sub.2) or a 
selective prostaglandin agonist to induce the expression of Bone 
Morphogenetic Protein 7 (BMP-7) in wild type 293S cells and in 293S cells 
transfected with EP.sub.2. 
Methods: 293S and EP.sub.2 293S cells are grown in Dulbecco's Modified 
Egale medium (DMEM, Gibco, BRL; Gaithersburg, Md.). One day prior to 
treatment with PGE.sub.2 or an prostaglandin agonist, cells are plated at 
a density of 1.5.times.10.sup.6 cells/10 cm dish. Generally about 16 to 24 
hours later the cell monolayer is washed once with OptiMEM (Gico, BRL) 
followed by the addition of 10 ml OptiMEM/dish in the presence and absense 
of vehicle (DMSO), PGE.sub.2 (10.sup.-6 M) or an prostaglandin selective 
agonist (10.sup.-6 M). Cells are harvested and RNA is extracted at 8, 16 
and 24 hours. Northern blot analysis of total (20 mg/lane) is carried out 
by probing the blots with .sup.32 P-labeled BMP-7 probe. The blots are 
normalized for RNA loading by hybridization with .sup.32 P-labeled 18s 
ribosomal RNA probe. PGE.sub.2 and prostaglandin selective agonists induce 
the expression of BMP-7 in the EP.sub.2 293S cells in a time dependent 
manner. Such induction of expression is generally not observed in the 
parental cell line. Given the known role of BMP-7 in kidney regeneration 
and the ability of an prostaglandin agonist to induce BMP-7 expression in 
293S kidney cells in a time and receptor specific manner indicates a role 
for prostaglandin agonist in kidney regeneration. 
Administration of the compounds of this invention can be via any method 
which delivers a compound of this invention systemically and/or locally 
(e.g., at the site of the bone fracture, osteotomy, or orthopedic 
surgery). These methods include oral routes, parenteral, intraduodenal 
routes, etc. Generally, the compounds of this invention are administered 
orally, but parenteral administration (e.g., intravenous, intramuscular, 
transdermal, subcutaneous, rectal or intramedullary) may be utilized, for 
example, where oral administration is inappropriate for the target or 
where the patient is unable to ingest the drug. 
The compounds are used for the treatment and promotion of healing of bone 
fractures and osteotomies by the local application (e.g., to the sites of 
bone fractures of osteotomies) of the compounds of this invention or 
compositions thereof. The compounds of this invention are applied to the 
sites of bone fractures or osteotomies, for example, either by injection 
of the compound in a suitable solvent (e.g., an oily solvent such as 
arachis oil) to the cartilage growth plate or, in cases of open surgery, 
by local application thereto of such compounds in a suitable carrier or 
diluent such as bone-wax, demineralized bone powder, polymeric bone 
cements, bone sealants, etc. Alternatively, local application can be 
achieved by applying a solution or dispersion of the compound in a 
suitable carrier or diluent onto the surface of, or incorporating it into 
solid or semi-solid implants conventionally used in orthopedic surgery, 
such as dacron-mesh, gel-foam and kiel bone, or prostheses. 
The compounds of this invention may also be applied locally to the site of 
the fracture or osteotomy in a suitable carrier or diluent in combination 
with one or more of the anabolic agents or bone anti-resorptive agents 
described above. 
Such combinations within the scope of this invention can be co-administered 
simultaneously or sequentially in any order, or a single pharmaceutical 
composition comprising a Formula I compound, a prodrug thereof or a 
pharmaceutical salt of said compound or said prodrug as described above 
and a second compound as described above in a pharmaceutically acceptable 
carrier or diluent can be administered. 
For example, a bone anabolic agent can be used in this invention alone or 
in combination with an anti-resorptive agent for three months to three 
years, followed by an anti-resorptive agent alone for three months to 
three years, with optional repeat of the full treatment cycle. 
Alternatively, for example, the bone anabolic agent can be used alone or 
in combination with an anti-resorptive agent for three months to three 
years, followed by an anti-resorptive agent alone for the remainder of the 
patient's life. For example, in one preferred mode of administration, a 
Formula I, II or III compound, or a prodrug thereof or a pharmaceutically 
acceptable salt of said compound or said prodrug as described above may be 
administered once daily and a second compound as described above (e.g., 
estrogen agonist/antagonist) may be administered daily in single or 
multiple doses. Alternatively, for example, in another preferred mode of 
administration the two compounds may be administered sequentially wherein 
the Formula I, II or III compound, prodrug thereof or pharmaceutically 
acceptable salt of said compound or said prodrug as described above may be 
administered once daily for a period of time sufficient to augment bone 
mass to a level which is above the bone fracture threshold (World Health 
Organization Study "Assessment of Fracture Risk and its Application to 
Screening for Postmenopausal Osteoporosis (1994). Report of a World Health 
Organization Study Group. World Health Organization Technical Series 843") 
followed by administration of a second compound, as described above (e.g., 
estrogen agonist/antagonist), daily in single or multiple doses. It is 
preferred that the first compound as described above is administered once 
daily in a rapid delivery form such as oral delivery. 
In any event, the amount and timing of compounds administered will, of 
course, be dependent on the subject being treated, on the severity of the 
affliction, on the manner of administration and on the judgment of the 
prescribing physician. Thus, because of patient to patient variability, 
the dosages given below are a guideline and the physician may titrate 
doses of the drug to achieve the treatment (e.g. bone mass augmentation) 
that the physician considers appropriate for the patient. In considering 
the degree of treatment desired, the physician must balance a variety of 
factors such as bone mass starting level, age of the patient, presence of 
preexisting disease, as well as presence of other diseases (e.g., 
cardiovascular disease). 
In general an amount of a compound of this invention is used that is 
sufficient to augment bone mass to a level which is above the bone 
fracture threshold (as detailed in the World Health Organization Study 
previously cited herein). 
In general an effective dosage for the anabolic agents used in this 
invention described above is in the range of 0.001 to 100 mg/kg/day, 
preferably 0.01 to 50 mg/kg/day. 
The following paragraphs provide preferred dosage ranges for various 
anti-resorptive agents. 
The amount of the anti-resorptive agent to be used is determined by its 
activity as a bone loss inhibiting agent. This activity is determined by 
means of the pharmacokinetics of an individual compound and its minimal 
versus maximal effective dose in inhibition of bone loss using a protocol 
such as described above (e.g., Estrogen Agonist/Antagonist Protocol). 
In general, an effective dosage for an anti-resorptive agent is about 0.001 
mg/kg/day to about 20 mg/kg/day. 
In general, an effective dosage for progestins is about 0.1 to 10 mg per 
day; the preferred dose is about 0.25 to 5 mg per day. 
In general, an effective dosage for polyphosphonates is determined by its 
potency as a bone resorption inhibiting agent according to standard 
assays. 
Ranges for the daily administration of some polyphosphonates are about 
0.001 mg/kg/day to about 20 mg/kg/day. 
In general an effective dosage for the treatment of this invention, for 
example the bone resorption treatment of this invention, for the estrogen 
agonists/antagonists of this invention is in the range of 0.01 to 200 
mg/kg/day, preferably 0.5 to 100 mg/kg/day. 
In particular, an effective dosage for droloxifene is in the range of 0.1 
to 40 mg/kg/day, preferably 0.1 to 5 mg/kg/day. 
In particular, an effective dosage for raloxifene is in the range of 0.1 to 
100 mg/kg/day, preferably 0.1 to 10 mg/kg/day. 
In particular, an effective dosage for tamoxifen is in the range of 0.1 to 
100 mg/kg/day, preferably 0.1 to 5 mg/kg/day. 
In particular, an effective dosage for 
2-(4-methoxy-phenyl)-3-[4-(2-piperidin-1-yl-ethoxy)-phenoxy]-benzo[b]thiop 
hen-6-ol is 0.001 to 1 mg/kg/day. 
In particular, an effective dosage for 
cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetr 
ahydro-naphthalene-2-ol; 
(-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro 
-naphthalene-2-ol; 
cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-nap 
hthalene-2-ol; 
cis-1-[6'-pyrrolodinoethoxy-3'-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahyd 
ronaphthalene; 
1-(4'-pyrrolidinoethoxyphenyl)-2-(4"-fluorophenyl)-6-hydroxy-1,2,3,4-tetrah 
ydroisoquinoline; 
cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8tetra 
hydro-naphthalene-2-ol; or 
1-(4'-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoqui 
noline 
is in the range of 0.0001 to 100 mg/kg/day, preferably 0.001 to 10 
mg/kg/day. 
In particular, an effective dosage for 4-hydroxy tamoxifen is in the range 
of 0.0001 to 100 mg/kg/day, preferably 0.001 to 10 mg/kg/day. 
The compounds of the present invention are generally administered in the 
form of a pharmaceutical composition comprising at least one of the 
compounds of this invention together with a pharmaceutically acceptable 
vehicle or diluent. Thus, the compounds of this invention can be 
administered individually or together, locally or systemically, in any 
conventional oral, parenteral, rectal or transdermal dosage form. 
For oral administration a pharmaceutical composition can take the form of 
solutions, suspensions, tablets, pills, capsules, powders, and the like. 
Tablets containing various excipients such as sodium citrate, calcium 
carbonate and calcium phosphate are employed along with various 
disintegrants such as starch and preferably potato or tapioca starch and 
certain complex silicates, together with binding agents such as 
polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, 
lubricating agents such as magnesium stearate, sodium lauryl sulfate and 
talc are often very useful for tabletting purposes. Solid compositions of 
a similar type are also employed as fillers in soft and hard-filled 
gelatin capsules; preferred materials in this connection also include 
lactose or milk sugar as well as high molecular weight polyethylene 
glycols. When aqueous suspensions and/or elixirs are desired for oral 
administration, the compounds of this invention can be combined with 
various sweetening agents, flavoring agents, coloring agents, emulsifying 
agents and/or suspending agents, as well as such diluents as water, 
ethanol, propylene glycol, glycerin and various like combinations thereof. 
For purposes of parenteral administration, solutions in sesame or peanut 
oil or in aqueous propylene glycol can be employed, as well as sterile 
aqueous solutions of the corresponding water-soluble salts. Such aqueous 
solutions may be suitably buffered, if necessary, and the liquid diluent 
first rendered isotonic with sufficient saline or glucose. These aqueous 
solutions are especially suitable for intravenous, intramuscular, 
subcutaneous and intraperitoneal injection purposes. In this connection, 
the sterile aqueous media employed are all readily obtainable by standard 
techniques well-known to those skilled in the art. 
For purposes of transdermal (e.g., topical) administration, dilute sterile, 
aqueous or partially aqueous solutions (usually in about 0.1% to 5% 
concentration), otherwise similar to the above parenteral solutions, are 
prepared. 
Methods of preparing various pharmaceutical compositions with a certain 
amount of active ingredient are known, or will be apparent in light of 
this disclosure, to those skilled in this art. For examples of methods of 
preparing pharmaceutical compositions, see Remington's Pharmaceutical 
Sciences, Mack Publishing Company, Easter, Pa., 15th Edition (1975). 
Pharmaceutical compositions according to the invention may contain 0.1%-95% 
of the compound(s) of this invention, preferably 1%-70%. In any event, the 
composition or formulation to be administered will contain a quantity of a 
compound(s) of this invention in an amount effective to treat the 
disease/condition of the subject being treated, e.g., a bone disorder. 
Since the present invention has an aspect that relates to the augmentation 
and maintenance of bone mass by treatment with a combination of active 
ingredients which may be administered separately, the invention also 
relates to combining separate pharmaceutical compositions in kit form. The 
kit comprises two separate pharmaceutical compositions: a compound of 
Formula I, II or III, a prodrug thereof or a pharmaceutically acceptable 
salt of said compound or said prodrug and a second compound as described 
above. The kit comprises container means for containing the separate 
compositions such as a divided bottle or a divided foil packet, however, 
the separate compositions may also be contained within a single, undivided 
container. Typically the kit comprises directions for the administration 
of the separate components. The kit form is particularly advantageous when 
the separate components are preferably administered in different dosage 
forms (e.g., oral and parenteral), are administered at different dosage 
intervals, or when titration of the individual components of the 
combination is desired by the prescribing physician. 
An example of such a kit is a so-called blister pack. Blister packs are 
well known in the packaging industry and are being widely used for the 
packaging of pharmaceutical unit dosage forms (tablets, capsules, and the 
like). Blister packs generally consist of a sheet of relatively stiff 
material covered with a foil of a preferably transparent plastic material. 
During the packaging process recesses are formed in the plastic foil. The 
recesses have the size and shape of the tablets or capsules to be packed. 
Next, the tablets or capsules are placed in the recesses and the sheet of 
relatively stiff material is sealed against the plastic foil at the face 
of the foil which is opposite from the direction in which the recesses 
were formed. As a result, the tablets or capsules are sealed in the 
recesses between the plastic foil and the sheet. Preferably the strength 
of the sheet is such that the tablets or capsules can be removed from the 
blister pack by manually applying pressure on the recesses whereby an 
opening is formed in the sheet at the place of the recess. The tablet or 
capsule can then be removed via said opening. 
It may be desirable to provide a memory aid on the kit, e.g., in the form 
of numbers next to the tablets or capsules whereby the numbers correspond 
with the days of the regimen which the dosage form so specified should be 
ingested. Another example of such a memory aid is a calendar printed on 
the card e.g., as follows "First Week, Monday, Tuesday, . . . etc. . . . 
Second Week, Monday, Tuesday, . . . " etc. Other variations of memory aids 
will be readily apparent. A "daily dose" can be a single tablet or capsule 
or several tablets or capsules to be taken on a given day. Also, a daily 
dose of a Formula I, II or III compound, a prodrug thereof or a 
pharmaceutically acceptable salt of said compound or said prodrug can 
consist of one tablet or capsule while a daily dose of the second compound 
can consist of several tablets or capsules and vice versa. The memory aid 
should reflect this. 
In another specific embodiment of the invention, a dispenser designed to 
dispense the daily doses one at a time in the order of their intended use 
is provided. Preferably, the dispenser is equipped with a memory-aid, so 
as to further facilitate compliance with the regimen. An example of such a 
memory-aid is a mechanical counter which indicates the number of daily 
doses that has been dispensed. Another example of such a memory-aid is a 
battery-powered micro-chip memory coupled with a liquid crystal readout, 
or audible reminder signal which, for example, reads out the date that the 
last daily dose has been taken and/or reminds one when the next dose is to 
be taken. 
The compounds of this invention either alone or in combination with each 
other or other compounds generally will be administered in a convenient 
formulation. The following formulation examples only are illustrative and 
are not intended to limit the scope of the present invention. 
In the formulations which follow, "active ingredient" means a compound or 
compounds of this invention. 
Formulation 1: Gelatin Capsules 
Hard gelatin capsules are prepared using the following: 
______________________________________ 
Ingredient Quantity (mg/capsule) 
______________________________________ 
Active ingredient 0.25-100 
Starch NF 0-650 
Starch flowable powder 
0-50 
Silicone fluid 350 centistokes 
0-15 
______________________________________ 
A tablet formulation is prepared using the ingredients below: 
Formulation 2: Tablets 
______________________________________ 
Ingredient Quantity (mg/tablet) 
______________________________________ 
Active ingredient 
0.25-100 
Cellulose, microcrystalline 
200-650 
Silicone dioxide, fumed 
10-650 
Stearate acid 5-15 
______________________________________ 
The components are blended and compressed to form tablets. 
Alternatively, tablets each containing 0.25-100 mg of active ingredients 
are made up as follows: 
Formulation 3: Tablets 
______________________________________ 
Ingredient Quantity (mg/tablet) 
______________________________________ 
Active ingredient 0.25-100 
Starch 45 
Cellulose, microcrystalline 
35 
Polyvinylpyrrolidine (as 10% solution in water) 
4 
Sodium carboxymethyl cellulose 
4.5 
Magnesium stearate 0.5 
Talc 1 
______________________________________ 
The active ingredients, starch, and cellulose are passed through a No. 45 
mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone 
is mixed with the resultant powders which are then passed through a No. 14 
mesh U.S. sieve. The granules so produced are dried at 
50.degree.-60.degree. C. and passed through a No. 18 mesh U.S. sieve. The 
sodium carboxymethyl starch, magnesium stearate, and talc, previously 
passed through a No. 60 U.S. sieve, are then added to the granules which, 
after mixing, are compressed on a tablet machine to yield tablets. 
Suspensions each containing 0.25-100 mg of active ingredient per 5 ml dose 
are made as follows: 
Formulation 4: Suspensions 
______________________________________ 
Ingredient Quantity (mg/5 ml) 
______________________________________ 
Active ingredient 0.25-100 mg 
Sodium carboxymethyl cellulose 
50 mg 
Syrup 1.25 mg 
Benzoic acid solution 0.10 mL 
Flavor q.v. 
Color q.v. 
Purified Water to 5 mL 
______________________________________ 
The active ingredient is passed through a No. 45 mesh U.S. sieve and mixed 
with the sodium carboxymethyl cellulose and syrup to form smooth paste. 
The benzoic acid solution, flavor, and color are diluted with some of the 
water and added, with stirring. Sufficient water is then added to produce 
the required volume. 
An aerosol solution is prepared containing the following ingredients: 
Formulation 5: Aerosol 
______________________________________ 
Ingredient Quantity (% by weight) 
______________________________________ 
Active ingredient 0.25 
Ethanol 25.75 
Propellant 22 (Chlorodifluoromethane) 
70.00 
______________________________________ 
The active ingredient is mixed with ethanol and the mixture added to a 
portion of the propellant, cooled to 30.degree. C., and transferred to a 
filling device. The required amount is then fed to a stainless steel 
container and diluted with the remaining propellant. The valve units are 
then fitted to the container. 
Suppositories are prepared as follows: 
Formulation 6: Suppositories 
______________________________________ 
Ingredient Quantity (mg/suppository) 
______________________________________ 
Active ingredient 
250 
Saturated fatty acid glycerides 
2,000 
______________________________________ 
The active ingredient is passed through a No. 60 mesh U.S. sieve and 
suspended in the saturated fatty acid glycerides previously melted using 
the minimal necessary heat. The mixture is then poured into a suppository 
mold of nominal 2 g capacity and allowed to cool. 
An intravenous formulation is prepared as follows: 
Formulation 7: Intravenous Solution 
______________________________________ 
Ingredient Quantity 
______________________________________ 
Active ingredient 20 mg 
Isotonic saline 1,000 mL 
______________________________________ 
The solution of the above ingredients is intravenously administered to a 
patient at a rate of about 1 mL per minute. 
The active ingredient above may also be a combination of agents. 
The abbreviations "Me", "Et", "tPr", "Tf", "Bu", "Ph", "EDC" and "Ac", 
where used herein, define the terms "methyl," "ethyl", "isopropyl", 
"triflyl", "butyl", "phenyl", 
"1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride" and 
"acetyl", respectively. 
General Experimental Procedures 
Unless otherwise specified, all reactions were performed under an inert 
atmosphere such as nitrogen (N.sub.2). 
NMR spectra were recorded on a Varian XL-300 (Varian Co., Palo Alto, 
Calif.), a Bruker AM-300 spectrometer (Bruker Co., Billerica, Mass.) or a 
Varian Unity 400 at about 23.degree. C. at 300 or 400 MHz for proton and 
75.4 MHz for carbon nuclei. Chemical shifts are expressed in parts per 
million downfield from trimethylsilane. The peak shapes are denoted as 
follows: s, singlet; d, doublet; t, triplet, q, quartet; m, multiplet; 
bs=broad singlet. Resonances designated as exchangeable did not appear in 
a separate NMR experiment where the sample was shaken with several drops 
of D.sub.2 O in the same solvent. Atmospheric pressure chemical ionization 
(APCI) mass spectra were obtained on a Fisons Platform II Spectrometer. 
Chemical ionization mass spectra were obtained on a Hewlett-Packard 5989 
instrument (Hewlett-Packard Co., Palo Alto, Calif.) (ammonia ionization, 
PBMS). Where the intensity of chlorine or bromine-containing ions are 
described the expected intensity ratio was observed (approximately 3:1 for 
.sup.35 Cl/.sup.37 Cl-containing ions) and 1:1 for .sup.79 Br/.sup.81 
Br-containing ions) and the intensity of only the lower mass ion is given. 
Column chromatography was performed with either Baker Silica Gel (40 .mu.m) 
(J. T. Baker, Phillipsburg, N.J.) or Silica Gel 60 (EM Sciences, 
Gibbstown, N.J.) in glass columns under low nitrogen pressure. Radial 
Chromatography was performed using a Chromatotron.RTM. (model 7924T, 
Harrison Research). Medium pressure chromatography was performed on a 
Flash 40 Biotage System (Biotage Inc, Dyax Corp., Charlottesville, Va.). 
Unless otherwise specified, reagents were used as obtained from commercial 
sources. Dimethylformamide, 2-propanol, acetonitrile, methanol, 
tetrahydrofuran, and dichloromethane, when used as reaction solvents, were 
the anhydrous grade supplied by Aldrich Chemical Company (Milwaukee, 
Wis.). The terms "concentrated" and "coevaporated" refer to removal of 
solvent at water aspirator pressure on a rotary evaporator with a bath 
temperature of less than 45.degree. C. Reactions conducted at 
"0-20.degree. C." or "0-25.degree. C." were conducted with initial cooling 
of the vessel in an insulated ice bath which was allowed to warm to room 
temperature over several hours. The abbreviation "min" and "h" stand for 
"minutes" and "hours" respectively.

EXAMPLE 1 
7-((4-Hydroxy-nonyl)-methanesulfonyl-amino)-heptanoic acid 
The title compound was prepared as described in U.S. Pat. No. 4,033,996. 
.sup.1 H NMR (CDCl.sub.3) .delta. 3.62-3.60 (m, 1H), 3.19-3.10 (m, 4H), 
2.80 (s, 3H), 2.33 (m, 2H), 1.81-1.69 (m, 1H), 1.67-1.55 (m, 5H), 
1.52-1.26 (m, 15H), 0.87 (t, 3H); MS 364 (M.sup.+ -1). 
EXAMPLE 2 
8-Acetyl-12-hydroxy-heptadecanoic acid 
The title compound was prepared as described in U.S. Pat. No. 4,018,802. 
.sup.1 H NMR (CDCl.sub.3) .delta. 3.60-3.52 (m, 1H), 2.43-2.38 (m, 1H), 
2.34-2.30 (m, 2H), 2.10 (s, 3H), 1.62-1.53 (m, 4H), 1.40-1.23 (m, 22H), 
0.87 (t, 3H); MS 327 (M.sup.+ -1). 
EXAMPLE 3 
(3-(((4-Hydroxy-nonyl)-methanesulfonyl-amino)-methyl)-phenoxy)-acetic acid 
The title compound was prepared from 
[3-(methanesulfonylamino-methyl)phenoxy]-acetic acid methyl ester 
(Preparation 10) and acetic acid 1-(3-chloropropyl)-hexyl ester using the 
alkylation procedure described in U.S. Pat. No. 4,033,996. .sup.1 H NMR 
(CDCl.sub.3) .delta. 7.31-7.26 (m, 1 H), 7.02-6.84 (m, 3H), 4.68 (s, 2H), 
4.35 (s, 2H), 3.91-3.89 (m, 1 H), 3.20-3.12 (m, 2H), 2.83 (s, 3H), 
1.94-1.92 (m, 1H), 1.79-1.24 (m, 12H), 0.87 (t, 3H); MS 400 (M.sup.+ -1). 
PREATION 1 
Pyridine-3-sulfonyl chloride hydrochloride 
A mixture of pyridine-3-sulfonic acid (15.0 g), phosphorous pentachloride 
(24.0 g) and phosphorous oxychloride (30 mL) was heated to 120.degree. C. 
for 16 h. The reaction was cooled to room temperature, and the resulting 
suspension was saturated with HCl (g). A white precipitate was collected, 
washed with CHCl.sub.3, and dried in vacuo to afford the title compound 
(15.6 g). .sup.1 H NMR (400 MHz, DMSO) .delta. 8.98 (s, 1H), 8.85 (d, 1H), 
8.66 (d, 1H), 8.02 (t, 1H). 
PREATION 2 
5-(3-Amino-propyl)-thiophene-2-carboxylic acid methyl ester 
Step A 
5-(3-tert-Butoxycarbonylamino-prop-1-ynyl)-thiophene-2-carboxylic acid 
methyl ester. A mixture of prop-2-ynyl-carbamic acid tert-butyl ester 
(1.67 g, 0.011 mmol), 5-bromo-thiophene-2-carboxylic acid methyl ester 
(2.50 g, 0.011 mmol), tetrakistriphenylphosphine(0) palladium (0.622 g, 
0.0538 mmol), Cul (0.102 g, 0.538 mmol) and triethylamine (1.57 mL, 0.011 
mmol) in 50 mL acetonitrile was heated at reflux for 16 h. The reaction 
was cooled to room temperature, diluted with 75 mL EtOAc, washed with 5.5% 
HCl, water and brine, dried over MgSO.sub.4, filtered and concentrated in 
vacuo to an oil. The product was purified via flash chromatography (9:1 to 
4:1 hexanes:EtOAc) to afford the title compound as an oil (2.06 g). MS 313 
(M+18). 
Step B 
5-(3-tert-Butoxycarbonylamino-propyl)-thiophene-2-carboxylic acid methyl 
ester. A solution of 
5-(3-tert-butoxycarbonylamino-prop-1-ynyl)-thiophene-2-carboxylic acid 
methyl ester (2.06 g) and 10% Pd/C (1.03 g) in 50 mL MeOH was placed in a 
Parr shaker and was hydrogenated at 50 psi H.sub.2 for 16 h. The reaction 
was filtered through Celite.RTM. with the aid of MeOH and the filtrate was 
concentrated in vacuo to afford the title compound of Step B as a solid 
(1.93 g). MS 317 (M+18). 
Step C 
5-(3-Amino-propyl)-thiophene-2-carboxylic acid methyl ester. A solution of 
5-(3-tert-butoxycarbonylamino-propyl)-thiophene-2-carboxylic acid methyl 
ester (0.118 g, 0.5 mmol) in 50 mL MeOH was cooled to 0.degree. C. and was 
saturated with HCl (g). The reaction was stirred at room temperature for 
90 minutes. The solution was concentrated to a solid which was partitioned 
between EtOAc and saturated NaHCO.sub.3. The layers were separated, and 
the organic solution was washed with brine, dried over MgSO.sub.4, 
filtered and concentrated in vacuo to afford the title compound as an oil 
(399 mg). MS 200 (M+1). 
PREATION 2a 
5-(3-Amino-propyl)-furan-2-carboxylic acid methyl ester hydrochloride salt 
Preparation 2a was prepared from the appropriate starting materials in an 
analogous manner to Preparation 2 with the following exceptions. The 
hydrogenation performed in Step B was carried out for 5.5 h. In Step C, 
the reaction was stirred for 16 h at room temperature and was concentrated 
in vacuo to provide the title compound as the hydrochloride salt. 
PREATION 3 
5-(3-Methanesulfonylamino-propyl)-furan-2-carboxylic acid methyl ester 
To a solution of 5-(3-amino-propyl)-furan-2-carboxylic acid methyl ester 
hydrochloride salt (see Preparation 2a) (150 mg, 0.683 mmol), and 
triethylamine (0.313 mL, 2.25 mmol) in CH.sub.2 Cl.sub.2 (15 mL) at 
0.degree. C. was added methanesulfonyl chloride (86 mg, 0.75 mmol). The 
reaction was stirred at room temperature for 18 h. The organic solution 
was washed consecutively with dilute HCl, water, saturated NaHCO.sub.3, 
and brine. The organic solution was dried over MgSO.sub.4, filtered, and 
concentrated to provide the title sulfonamide (156 mg). MS 262 (M+1). 
PREATION 4 
5-(3-Amino-Propyl)-thiophene-2-carboxylic acid tert-butyl ester 
Step A 
Prop-2-ynyl-carbamic acid benzyl ester. To a solution of propargylamine 
(6.4 g, 71.2 mmol) in pyridine (100 mL) was added benzylchloroformate 
(13.37 g, 78.2 mmol) in 100 mL CH.sub.2 Cl.sub.2 over 0.5 h. The reaction 
was stirred for 16 h and the volatiles were removed in vacuo. The residue 
was dissolved in EtOAc and the organic solution was washed with water 
(2.times.). The organic solution was washed with dilute aqueous HCl 
followed by saturated NaHCO.sub.3. The organic solution was dried over 
MgSO.sub.4, filtered, and concentrated in vacuo to provide the title 
compound of Step A (4.43 g). 
Step B 
5-(3-Benzyloxycarbonylamino-prop-1-ynyl)-thiophene-2-carboxylic acid 
tert-butyl ester. The title compound of Step B was prepared from 
prop-2-ynyl-carbamic acid benzyl ester and 5-bromo-thiophene-2-carboxylic 
acid tert-butyl ester in an analagous manner to Step A of Preparation 2. 
Step C 
5-(3-Amino-propyl)-thiophene-2-carboxylic acid tert-butyl ester. To a 
solution of 
5-(3-benzyloxycarbonylamino-prop-1-ynyl)-thiophene-2-carboxylic acid 
tert-butyl ester (1.0 g, 2.69 mmol) in 15 mL MeOH and 2.69 mL 1N HCl (aq) 
was added Pd(OH).sub.2. The mixture was placed on a Parr shaker and was 
hydrogenated under 45 psi H.sub.2 for 16 h. The mixture was filtered 
through Celite.RTM., the catalyst was replaced, and the reaction was 
shaken for another 6 h. The mixture was filtered through Celite.RTM. and 
concentrated in vacuo. The residue was chased with CCl.sub.4 and was 
triturated with Et.sub.2 O. The product was isolated as a solid (360 mg). 
PREATION 5 
5-(3-Amino-propyl)-tetrahydrofuran-2-carboxylic acid methyl ester 
hydrochloride salt 
Step A 
5-(3-tert-Butoxycarbonylamino-prop-1-ynyl)-furan-2-carboxylic acid methyl 
ester. The title compound of Step A was prepared from 
5-bromo-furan-2-carboxylic acid methyl ester and prop-2-ynyl-carbamic acid 
tert-butyl ester as described in Step A of Preparation 2. 
Step B 
5-(3-tert-Butoxycarbonylamino-propyl)-tetrahydrofuran-2-carboxylic acid 
methyl ester and 5-(3-tert-Butoxycarbonylamino-propyl)-furan-2-carboxylic 
acid methyl ester. To a solution of 
5-(3-tert-butoxycarbonylamino-prop-1-ynyl)-furan-2-carboxylic acid methyl 
ester (1.69 g) in MeOH (50 mL) was added 10% palladium on carbon (850 mg) 
and the mixture was hydrogenated on a Parr shaker at 50 psi for 18 h. The 
catalyst was removed via filtration through Celite.RTM. and the volatiles 
were concentrated in vacuo. Flash chromatography (hexanes:EtOAc 4:1) 
provided 5-(3-tert-butoxycarbonylamino-propyl)-furan-2-carboxylic acid 
methyl ester (422 mg, MS 284 M+) followed by 
5-(3-tert-butoxycarbonylamino-propyl)-tetrahydrofuran-2-carboxylic acid 
methyl ester (903 mg). 
Step C 
5-(3-Amino-propyl)-tetrahydrofuran-2-carboxylic acid methyl ester 
hydrochloride salt. The title compound was prepared from 
5-(3-tert-butoxycarbonylaminopropyl)-tetrahydrofuran-2-carboxylic acid 
methyl ester following the procedure described in Step C for Preparation 
2a. 
PREATION 6 
5-(3-Methanesulfonylamino-propyl)-thiophene-2-carboxylic acid methyl ester 
Step A 
5-(3-Methanesulfonylamino-prop-1-ynyl)-thiophene-2-carboxylic acid methyl 
ester. To a solution of 5-bromo-thiophene-2-carboxylic acid methyl ester 
(1.66 g, 8.0 mmol), N-prop-2-ynyl-methanesulfonamide (1.09 g, 8.2 mmol), 
Et.sub.3 N (1.7 mL, 12.1 mmol), and CH.sub.3 CN (30 mL) was added 
Pd(PPh.sub.3).sub.4 (462 mg, 0.4 mmol) followed by Cul (76 mg, 0.4 mmol). 
The reaction was heated at reflux for 24 h and was cooled to room 
temperature. The volatiles were removed in vacuo and the residue was 
purified via flash chromatography (20% EtOAc in hexanes to 33% EtOAc in 
hexanes) to yield 
5-(3-methanesulfonylamino-prop-1-ynyl)-thiophene-2-carboxylic acid methyl 
ester as a pale yellow solid (1.1 g). .sup.1 H NMR (300 MHz, CDCl.sub.3) 
.delta. 7.64 (d, 1H), 7.14 (d, 1H), 4.60 (m, 1H), 4.22 (d, 2H), 3.88 (s, 
3H), 3.10 (s, 3H); MS 274 (M+1). 
Step B: Hydrogenation 
A solution of 5-(3-methanesulfonylamino-prop-1-ynyl)-thiophene-2-carboxylic 
acid methyl ester (3.0 g, 10.9 mmol) in EtOAc (100 mL) and MeOH (50 mL) 
was hydrogenated with 10% palladium on carbon (680 mg) at 50 psi for 7 h. 
The solution was filtered through a pad of Celite.RTM. with the aid of 
MeOH and was concentrated in vacuo to provide the title compound as an 
off-white solid (2.95 g). .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta. 7.62 
(d, 1H), 7.23 (d, 1H), 4.29 (m, 1H), 3.85 (s, 3H), 3.18 (q, 2H), 2.93 (m, 
5H), 1.96 (m, 2H). 
PREATION 7 
(3-Aminomethyl-phenyl)-acetic acid methyl ester hydrochloride 
Step A 
(3-Cyano-phenyl)-acetic acid methyl ester. Nitrogen was bubbled through a 
mixture of (3-bromo-phenyl)-acetic acid methyl ester (26.12 g, 114.02 
mmol), Zn(CN).sub.2 (8.37 g, 71.3 mmol), and DMF (300 mL) for about 5 
minutes followed by addition of tetrakistriphenylphosphine(0) palladium 
(5.0 g, 4.3 mmol). The mixture was heated for 1.5 h at 90.degree. C. and 
was cooled to room temperature. Aqueous 2N NH.sub.4 OH was added and the 
product was extracted into EtOAc (3.times.). The organic solution was 
washed with 2N NH.sub.4 OH (1.times.) followed by brine (2.times.). The 
organic solution was dried (MgSO.sub.4), filtered, and concentrated in 
vacuo. Purification by medium pressure chromatography (9:1 hexanes:EtOAc) 
provided the title compound of Step A as an oil (18.06 g). .sup.1 H NMR 
(400 MHz, CDCl.sub.3) .delta. 7.57-7.41 (m, 4H), 3.706 (s, 3H), 3.703 (s, 
2H). 
Step B 
(3-Aminomethyl-phenyl)-acetic acid methyl ester hydrochloride. To a 
solution of (3-cyano-phenyl)-acetic acid methyl ester (18.06 g, 103 mmol) 
in MeOH (150 mL) and 4M HCl in dioxane (30.0 mL, 120 mmol) was added 10% 
palladium on carbon (2 g). The mixture was placed on a Parr shaker and was 
hydrogenated at 50 psi hydrogen for 24 h. The catalyst was removed via 
filtration through Celite.RTM. and the organic solution was concentrated 
in vacuo. The resulting solid was stirred in EtOAc and filtered to provide 
the title compound as a white solid (20.42 g). .sup.1 H NMR (400 MHz, 
CD.sub.3 OD) .delta. 7.42-7.32 (m, 4H), 4.09 (s, 2H), 3.69 (s, 2H), 3.67 
(s, 3H); MS 180 (M+1). mg). 
PREATION 8 
{3-[(Pyridine-3-sulfonylamino)-methyl]-phenyl}-acetic acid methyl ester 
To a solution of (3-aminomethyl-phenyl)-acetic acid methyl ester 
hydrochloride (0.56 g) and diisopropylamine (2.2 mL) in 10 mL 
dichloromethane was added pyridine-3-sulfonyl chloride (0.601 g, 2.83 
mmol) and the reaction was stirred at room temperature for 16 h. Aqueous 
1N HCl was added and the solution was extracted with CH.sub.2 Cl.sub.2. 
The organic solution was washed with saturated NaHCO.sub.3, dried over 
MgSO.sub.4, filtered and concentrated in vacuo to afford the title 
compound. Purification via flash chromatography on silica gel (2:1 
hexanes: EtOAc) afforded the title compound as a white solid. .sup.1 H NMR 
(400 MHz, CDCl.sub.3) .delta. 8.91 (s, 1H), 8.71 (d, 1H), 8.04 (d, 1H), 
7.37 (m, 1H), 7.05-7.24 (m, 4H), 5.87 (bs, 1H), 4.14 (s, 2H), 3.62 (s, 
3H), 3.52 (s, 2H). 
PREATION 9 
{3-[(Propane-1-sulfonylamino)-methyl]-phenyl}-acetic acid methyl ester 
Following the procedure described in Preparation 8, 
(3-aminomethyl-phenyl)-acetic acid methyl ester hydrochloride (603 mg, 
2.80 mmol) was converted to the title sulfonamide by reaction with 
1-propanesulfonyl chloride (0.375 mL) and N,N-diisopropylethylamine (2.0 
mL) in dichloroethane (10 mL). .sup.1 H NMR (400 MHz, CDCl.sub.3) .delta. 
7.35-7.22 (m, 4H), 4.60 (bs, 1H), 4.29 (s, 2H), 3.70 (s, 3H), 3.63 (s, 
2H), 2.92 (t, 2H), 1.79 (m, 2H), 0.99 (t, 3H). 
PREATION 10 
[3-(Methanesulfonylamino-methyl)-phenoxy]-acetic acid methyl ester 
Step A 
N-(3-Methoxy-benzyl)-methanesulfonamide. Methanesulfonyl chloride (4.170 g, 
36.4 mmol) was added to a solution of 3-methoxybenzylamine (5.000 g, 36.4 
mmol) and triethylamine (3.946 g, 39.0 mmol) in THF (100 mL) at room 
temperature. The mixture was stirred for 18 h and the insolubles were 
removed by filtration. The organic solution was concentrated to a yellow 
oil which was purified by flash chromatography (6:4 hexanes:EtOAc to 1:1 
hexanes:EtOAc) to yield N-(3-methoxy-benzyl)-methanesulfonamide (7.431 g). 
.sup.1 H NMR (400 MHz, CDCl.sub.3) .delta. 7.26 (m, 1H), 6.92-6.82 (m, 
3H), 4.62 (m, 1H), 4.28 (d, 2H), 3.80 (s, 3H), 2.87 (s, 3H); MS 214 (M-1). 
Step B 
N-(3-Hydroxy-benzyl)-methanesulfonamide. A solution of BBr.sub.3 (1.0 M in 
CH.sub.2 Cl.sub.2, 111 mL, 111 mmol) was slowly added to a solution of 
N-(3-methoxy-benzyl)-methanesulfonamide (12.000 g, 55.7 mmol) in CH.sub.2 
Cl.sub.2 (200 mL) 0.degree. C. The reaction was warmed to room temperature 
and was stirred for 4 h. Methanol (100 mL) was cautiously added and the 
solution was concentrated in vacuo. Flash chromatography (1:1 
hexanes:EtOAc) provided N-(3-hydroxy-benzyl)-methanesulfonamide (11.50 g). 
.sup.1 H NMR (400 MHz, CDCl.sub.3) .delta. 7.20 (m, 1H), 6.84 (m, 2H), 
6.77 (m, 1H), 4.83 (bs, 1H), 4.24 (s, 2H), 2.86 (s, 3H); MS 201 (M+). 
Step C 
A mixture of N-(3-hydroxy-benzyl)-methanesulfonamide (6.000 g, 29.82 mmol), 
methyl bromoacetate (4.562 g, 29.82 mmol), K.sub.2 CO.sub.3 (4.121 g, 
29.82 mmol), and acetone (250 mL) was stirred at room temperature for 68 
h. The solids were removed by filtration and the solution was concentrated 
in vacuo. Purification by flash chromatography (1:1 hexanes:EtOAc) 
provided the title compound of Preparation 10 (5.637 g). .sup.1 H NMR (400 
MHz, CDCl.sub.3) .delta. 7.25 (m, 1H), 6.96 (m, 1H), 6.89 (s, 1H), 6.82 
(m, 1H), 4.63 (m, 3H), 4.28 (m, 2H), 3.80 (s, 3H), 2.86 (s, 3H); MS 274 
(M+1). 
PREATION 11 
2-(3-Methanesulfonylamino-propyl)-thiazole-4-carboxylic acid ethyl ester 
Step A 
4-Methanesulfonylamino-butyric acid ethyl ester. Methanesulfonyl chloride 
(4.10 g, 35.8 mmol) was added to a suspension of ethyl 4-aminobutyrate 
hydrochloride (6.00 g, 35.8 mmol) and Et.sub.3 N (10.8 mL, 77.4 mmol) in 
THF (230 mL). The resulting suspension was stirred at room temperature for 
43 h. The reaction mixture was filtered and the filtrate was concentrated. 
Flash chromatography (1:1 EtOAc:hexanes to EtOAc) afforded the title 
compound of Step A (7.08 g). .sup.1 H NMR (400 MHz, CDCl.sub.3) .delta. 
4.51 (s, 1H), 4.12 (q, 2H), 3.18 (q, 2H), 2.94 (s, 3H), 2.40 (t, 2H), 
1.85-1.92 (m, 2H), 1.24 (t, 3H); MS 210 (M.sup.+ +1). 
Step B 
4-Methanesulfonylamino-butyramide. A solution of 
4-methanesulfonylamino-butyric acid ethyl ester (7.08 g, 33.8 mmol) in 
concentrated NH.sub.4 OH (200 mL) was stirred at room temperature for 66 
h. The reaction mixture was concentrated to afford the title compound of 
Step B as a white solid (6.16 g). The product was used in the next step 
without further purification. .sup.1 H NMR (400 MHz, CDCl.sub.3) .delta. 
3.30 (s, 3H), 3.05-3.09 (m, 2H), 2.91 (s, 3H), 2.24-2.30 (m, 2H), 
1.80-1.85 (m, 2H); MS 181 (M.sup.+ +1). 
Step C 
4-Methanesulfonylamino-thiobutyramide. A suspension of 
4-methanesulfonylamino-butyramide (0.50 g, 2.8 mmol) and Lawesson's 
reagent (0.56 g, 1.4 mmol) in THF (50 mL) was stirred at room temperature 
for 45 minutes. During this time all of the solid dissolved. The solution 
was concentrated and purified by flash chromatography (79:1 EtOAc:MeOH) to 
afford the title compound of Step C (0.41 g); .sup.1 H NMR (400 MHz, 
CDCl.sub.3) .delta. 3.29 (s, 3H), 3.07-3.11 (m, 2H), 2.91 (s, 3H), 
2.62-2.66 (m, 2H), 1.93-1.99 (m, 2H); MS 197 (M.sup.+ +1). 
Step D 
2-(3-Methanesulfonylamino-propyl)-thiazole-4-carboxylic acid ethyl ester. A 
solution of 4-methanesulfonylamino-thiobutyramide (0.35 g, 1.8 mmol) and 
ethyl bromopyruvate (0.37 g, 1.9 mmol) in EtOH (50 mL) was stirred at room 
temperature for 17 h. Additional ethyl bromopyruvate (0.05 g, 0.26 mmol) 
was added and the reaction mixture was stirred at room temperature for 5.5 
h. The reaction mixture was concentrated and was purified by flash 
chromatography (79:1 to 19:1 EtOAc:MeOH) to afford the title compound 
(0.47 g). .sup.1 H NMR (400 MHz, CDCl.sub.3) .delta. 8.05 (s, 1H), 4.40 
(q, 2H), 3.24 (t, 2H), 3.17 (t, 2H), 2.96 (s, 3H), 2.10 (t, 2H), 1.39 (t, 
3H); MS 293 (M.sup.+ +1).