Prostaglandin analog for treating osteoporosis

This invention relates to a prostaglandin-bisphosphonate compound of the formula: ##STR1## and its pharmaceutically acceptable salts. The claimed compounds are effective as delivery agents of prostaglandins to treat osteoporosis and related bone diseases. The claimed compounds also simultaneously deliver a bisphosphonate which inhibits bone resorption and delivers prostaglandins which increase bone formation in vivo.

BACKGROUND OF THE INVENTION 
The compounds of the present invention are analogues of the natural 
prostaglandin PGE.sub.2, PGE.sub.1 and PGF.sub.2 alpha useful in the 
treatment of osteoporosis. 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. PGE.sub.2 has the following structure: 
##STR2## 
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. U.S. Pat. No. 4,171,331 teaches 1 
and 2 substituted analogues of certain prostaglandins. Disclosed are trans 
1 and 2 di(loweralkyl)phosphono; 1 and 2 chloro, bromo, and iodo; 1 and 
2-thio; and 1 and 2 amino analogues of PGE.sub.1. U.S. Pat. No. 3,927,197 
discloses the formation of various acid derivatives of prostaglandins such 
as amides, carboxylate-amine salts, and the 
2-decarboxy-2-(2,3,4,5-tetryol-1-yl) derivative. 
Osteoporosis is the most common form of metabolic bone disease and is 
commonly observed in postmemopausal women but also occurs in elderly males 
and females or in young individuals. Commonly, the disease is 
characterized by fractures of the wrist and spine, while femoral fractures 
are the dominant feature of senile osteoporosis. The physical causitive 
factor which creates susceptibility to fracturing is the gradual loss of 
bone or bone minerals such as calcium. Apparently, the normal balance of 
bone resorption activity by the osteoclasts (bone dissolving or resorbing 
cells) and bone formation activity by the osteoblasts (bone forming cells) 
is disrupted by development of the disease so that the cavities created by 
the osteoclasts are not refilled by the osteoblasts. A number of 
pharmaceutical compounds are known in the art which hinder the activity of 
osteoclasts so that bone loss is diminished. For example, bisphosphonates 
as a class are useful in inhibiting bone loss and are therefore important 
in treating diseases associated with bone loss, including osteoporosis. A 
more difficult treatment regime or area has been the effective 
acceleration or stimulation of bone formation to maintain bone growth or 
strengthen weakened bones. 
It is clear, however, that the activity of osteoblasts and osteoclasts is 
coordinated and regulated by a complex mechanism and is affected by a 
variety of hormones and prostaglandins. See Raisz et al., Ann. Rev. 
Physiol., 43:225 (1981); U.S. Pat. No. 4,921,697 which teaches that 
inhibition of prostaglandin production by IFN-gamma is an effective 
treatment for osteoporosis and other bone-resorption diseases since 
prostaglandins have been implicated in bone loss or resorption. The 
literature also suggests that pro staglandins may also play an important 
role in bone formation. See W. Harvey and A. Bennett, "Prostaglandins in 
Bone Resorption" CRC Press, pp. 37 (1988). Osteoblasts are responsible for 
carrying out the bone formation process. It has been established that bone 
formation in vivo in animals is stimulated by systemic injection of 
PGE.sub.2. See Rodan G. J. Cell Biochem. Suppl. 0 (15 Part F), 160 (1991). 
The effects of prostaglandins administered alone has been disclosed in the 
art. Ueno et al., Bone, 6, 79-86, (1985) administered PGE.sub.2 to rapidly 
growing rats at dosages of 1, 3 and 6 mg of PGE.sub.2 /Kg/day. The results 
showed an increase in hard tissue mass in the secondary spongiosa of the 
proximal tibial metaphysis and an increase in the number of trabeculae. 
Jee et al., Bone and Mineral, 15, 33-55 (1991), disclosed that 
subcutaneous injections of PGE.sub.2 over 60, 120, and 180 days produced 
an increased tibial diaphyseal bone mass and elevated bone activity. The 
authors reported that the anabolic effects of PGE.sub.2 increases 
periosteal and corticoendosteal bone mass and sustains the transient 
increase in bone mass with daily administration of PGE.sub.2. It is known 
that very little control is possible over the duration and the 
concentration at which PGs reach the bone cells. It is also known that 
systemic injection or infusion of PGs is an alternative with significant 
drawbacks since the lungs efficiently remove PGs from circulation. See W. 
Harvey and A. Bennett, "Prostaglandins in Bone Resorption" CRC Press, pp. 
37 (1988). 
It is also known that toxicity of prostaglandins due to systemic 
distribution of the administered drug reduces or diminishes the 
pharmaceutical utility of these compounds. Delivery of high doses of 
prostaglandins which would be necessary because of the short half life of 
these compounds may cause unwanted side effects. Ueno et al reported that 
when PGE.sub.2 was administered systemically through subcutaneous 
injections to rats, diarrhea and flushing of the extremities along with 
weight loss occurred at doses of 3 mg/Kg/day or higher. In addition, 
significant decreases in serum phosphate levels of 1 mg of PGE.sub.2 were 
noted. Jee et al reported that long term administration of PGE.sub.2 
administered via subcutaneous injection resulted in soft tissue weight 
increases in adrenal glands, liver, kidneys, and lungs. U.S. Pat. No. 
4,621,100 discloses side effects after oral dosing with PGE.sub.2 
including loose stools, diarrhea, vomiting, infected sclerae, and 
increased serum alkaline phosphatase levels. 
Frost et al. in "Treatment of Osteoporosis by Manipulation of Coherent Bone 
Cell Populations", Clinical Orthopedics and Related Research, 143, 227 
(1979) discloses a theoretical model that suggests it should be possible 
to synchronize the activity and metabolism of bone cells by administering 
bone cell activating agents first and then administering a bone resorption 
inhibiting agent. This proposed model assumes that bone formation 
inhibition does not take place, because no bone resorption inhibiting 
agent is administered during the bone formation phase of the bone 
remodeling unit. EPO App. No. 0 381 296 teaches the use of a kit wherein a 
bone activating period or treatment regime is followed by a bone 
resorption inhibiting regime. Examples of bone activating compounds cited 
in this reference include parathyroid hormone (PTH), inorganic phosphate, 
growth hormone, fluoride, thyroid hormone (e.g. thyroxin), certain vitamin 
D metabolites and prostaglandins (PGE.sub.2 in a dose regime of 10 mg/kg 
per day). See also U.S. Pat. No. 5,118,667. Examples of bone resorption 
inhibiting polyphosphonates include ethane-1-hydroxy 1,1-diphosphonic 
acid, methane diphosphonic acid, pentane-1-hydroxy-1,1-diphosphonic acid, 
methane dichloro dipho sphonic acid, methane hydroxy dipho sphonic acid, 
ethane-1-amino- 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 (administered after 
PGE.sub.2 at a dosage per day of 0.005 mg P/kg), 
pentane-5-amino-1-hydroxy-1,1-diphosphonic acid, and 
hexane-6-amino-1-hydroxy-1,1-diphosphonic acid. Combinations of a 
methylene bisphosphonate coupled to a medicinal compound such as a 
Non-Steroidal Anti-Inflammatory Agent (NS AID) have been disclosed. See 
Japanese Patent Publication No. H2-104593. 
The present invention, on the other hand, provides simultaneous delivery of 
a bone activating agent such as a prostaglandin that is chemically coupled 
to a bone resorption inhibiting compound which selectively delivers the 
bone activating agent to the target area. Upon gradual hydrolysis of the 
novel compound, the hydrolyzed products are able to provide bone 
resorption inhibiting activity (via the bisphosphonates) and bone growth 
or stimulating activity (via PGE.sub.2). The present invention also 
enables more effective delivery of PGE.sub.2 to the target region and 
therefore overcomes the serious side effect disadvantages associated with 
administration of larger quantities of PGE.sub.2 alone. In addition, 
PGE.sub.2 administered systemically has a short half-life. The present 
invention overcomes the disadvantages prevalent in the background art and 
at the same time provides a compound that promotes bone growth and deters 
bone resorption to provide a treatment for osteoporosis and related 
disorders of calcium metabolism. 
SUMMARY OF THE INVENTION 
The claimed invention's primary objective is to use compounds within the 
scope of the invention as chemical delivery agents of prostaglandins. This 
invention claims a novel chemical method for simultaneously delivering a 
bone formation enhancer such as a prostaglandin and a bone resorption 
inhibitor such as an amino bisphosphonate. The invention is a 
prostaglandin-bisphosphonate compound which when administered systemically 
has high affinity for bone cells. The compounds of the invention are then 
hydrolyzed to form a bisphosphonate and a prostaglandin. The invention is 
useful in the prevention and treatment of osteoporosis and has the 
distinct advantage that lower doses of prostaglandins may be administered 
to a mammal or patient in need thereof since the prostaglandin is 
delivered to the site of action before it is metabolized. This method also 
avoids the undesirable side affects associated with higher doses of 
prostaglandins. The invention is also directed to a compound of the 
following formula: 
##STR3## 
and the pharmaceutically acceptable salts thereof wherein: 
EQU [A] 
is 
a dioxygenated cyclopentane moiety of the formula: 
##STR4## 
wherein R is: 
H, 
THP, or 
Si(CH.sub.3).sub.2 tBu; 
R.sup.1 is: 
H, or 
C.sub.1-10 alkyl; 
M is: 
OH, 
OC.sub.1-6 alkyl, 
##STR5## 
wherein R" is H, C.sub.1-10 alkyl, aryl, or benzyl; 
##STR6## 
wherein Z is NH, C(R.sup.1).sub.2, or absent; 
##STR7## 
Y is: 
OR' wherein R' is C.sub.1-6 alkyl; or 
##STR8## 
wherein Q is NR.sup.1, O, or S, 
##STR9## 
wherein Z is HN, C(R.sup.1).sub.2 or absent, or 
##STR10## 
and n is an integer from 1-10. 
This invention is also directed to a method of treating or preventing 
osteoporosis by administering a pharmaceutically effective amount of the 
compound according to claim 1. It is directed to a method of increasing 
the bone fracture healing rate in a mammal exhibiting a bone fracture by 
systemically administering a pharmaceutically effective amount of the 
compound according to claim 1 and to method for enhancing the rate of 
successful bone grafts comprising administering to a mammal in need 
thereof a pharmaceutically effective amount of the compound according to 
claim 1. This invention is advantageously directed to a method of 
delivering a prostaglandin according to claim 1 to a mammalian organism in 
need of treatment thereof via a bisphosphonate delivery agent wherein the 
prostaglandin enhances the rate of bone formation and is thus effective in 
treating osteoporosis, bone fractures, and effective in enhancing the rate 
of successful bone grafts.

DETAILED DESCRIPTION OF THE INVENTION 
This invention comprises a compound that is effective as a chemical 
delivery agent and a compound which is useful in the treatment and 
prevention of osteoporosis and calcium metabolism disorders. The compound 
of the invention may also have dual activity as a bone growth promoter and 
as a bone resorption inhibitor. Prostaglandins of the PGE.sub.2, PGE.sub.1 
and PGF.sub.2 a class or other suitable prostaglandin with a carboxylic 
acid moiety at the 1 position and a hydroxyl group at the 15 position of 
the PG moiety may be reacted with an amino bisphosphonate such as ABP or 
its salts to form the compounds claimed in the instant invention. Any 
known bisphosphonate which has an amine fuctionality capable of coupling 
to a prostaglandin and which targets in vivo to bone may be used in this 
invention as a chemical delivery agent whether or not that particular 
bisphosphonate has bone resorption inhibiting activity. 
The following scheme describes a synthesis of a 
bisphosphonate-prostaglandin compound: 
##STR11## 
1,3-Dicyclohexylcarbodiimide is added to a stirred solution of PGE.sub.2 
(I) or other suitable prostaglandin and N-hyroxysuccinimide in dry 
acetonitrile and stirred at room temperature (25.degree. C.) until thin 
layer chromatography or other suitable analytical method such as HPLC 
indicates that the reaction is complete. The solvent is removed under an 
inert atmosphere (nitrogen) and the residue is dissolved in methylene 
chloride and applied to a small column of silica gel in a pasteur pipette. 
The pipette is then eluted with ethyl acetate to afford the 
hydroxysuccinimide ester (IIa) and a small quantity of dicyclohexylurea. A 
solution of this ester in 1,4-dioxane is added to a stirred solution of a 
suitable bisphosphonate such as ABP in water and 1.0 molar (M) aqueous 
NaOH. After 10 minutes or so the pH of the reaction mixture is adjusted to 
approximately 9 with 1.0M aqueous NaOH, and then 1 hr later the pH is 
adjusted to 7 with 0.1M aqueous HC1. The solution is filtered and the 
filtrate is concentrated to dryness. The residue is then dissolved in 
water and applied to a Varian Bond Elute C.sub.18 pak which is eluted with 
water. When the product begins to elute, the solvent system on the 
C.sub.18 column is changed to acetonitrile/water (50:50). Evaporation of 
fractions containing the product will afford the target amide (III). 
The prostaglandins used in the above scheme can be chosen from the 
PGE.sub.2 class or from the PGF.sub.a class or from any prostaglandin or 
prostaglandin analog which has known bone growth enhancement activity. A 
compound of the general formula depicted below is reacted with DCC to form 
the activated ester V which is then reacted with an 
aminoalkylbisphosphonate to form the coupled amide product. 
##STR12## 
The claimed compounds may be prepared according to Scheme 3: 
##STR13## 
The silyl protected PGE.sub.2 is prepared and reacted with an activated 
carbonyl compound at the C.sup.15 hydroxy to form the activated ester. 
This reactant is then treated with a bisphosphonate such as ABP disodium 
salt to form a prostaglandin-bisphosphonate ester compound that can 
deliver the prostaglandin to the bone cells and is more labile to 
enzymatic hydrolysis. 
The prostaglandins used in the above scheme to produce the amido ester 
derivative may be chosen from the PGE.sub.2 or PGFa class. A compound of 
the general formula depicted below is reacted with oxodiimidazole or 
oxalyl chloride or reactant of the general formula CO(X).sub.2 to form the 
activated ester which is then reacted with an aminoalkylbisphosphonate 
salt to form the coupled amido ester product. 
##STR14## 
Compounds of the instant invention may also be prepared according to the 
following scheme: 
##STR15## 
Protected PGE.sub.2 is reacted with an amide chloride or a bifunctional 
reagent such as 2- (or 3-, or 4-) succinamido-N-oxycarbonylphenylamino 
carbonyl chloride using a base catalyst in THF or in methlene chloride to 
form the activated PGE.sub.2 analog which is further reacted with a 
bisphosphonate such as the disodium salt of ABP in aqueous THF at pH 9-10. 
The resultant bisphosphonate-PG compound is hydrolyzed to remove the 
protecting groups on the cyclopentane moiety to give compound XIV. The 
prostaglandins used in the above scheme can be chosen from the PGE.sub.2 
or PGF.sub.2 a class. A compound of the general formula depicted below is 
reacted with a diactivated ester species to form a reactive intermediate 
which is reacted with a bisphosphonate salt to form the coupled product. 
##STR16## 
Alternatively, the compounds of the instant invention may be prepared 
according to the following scheme: 
##STR17## 
The protected prostaglandin is reacted with carbonyldiimidazole or oxalyl 
chloride to form the 15 hydroxy ester which is further reacted with 
1,3-diaminopropane and 1,3-difloro-4,6-dinitrobenzene to form the 
dinitrophenyl-amino amide-PG analog shown in Scheme 7. The disodium salt 
of ABP acts as a nucleophile and displaces fluorine to form the 
PG-bisphosphonate molecule which is then hydrolyzed to remove the 
remaining protecting groups. Scheme 8 below describes a general synthesis 
wherein the particular prostaglandin used may be from the PGE.sub.2, 
PGE.sub.1 or PGF.sub.2a series. 
##STR18## 
Scheme 9 depicts another method of producing the claimed compounds. 
##STR19## 
The diflouro,dinitro benzene is reacted with a mercaptyl ester to form the 
thioaromatic species which is further reacted with base and oxalyl 
chloride to form the activated aromatic species. This is reacted with 
protected PGE.sub.2 to form the thioaromatic-PGE.sub.2 compound which is 
reacted with a bisphosphonate such as ABP disodium salt and then 
hydrolyzed to give the PGE.sub.2 -bisphosphonate compound which contains 
the aromatic linking moiety. A similar reaction scheme may also be 
performed wherein an NH moiety replaces the thio group to give a compound 
of the formula: 
##STR20## 
This reaction may also be performed on members of the PGE.sub.2, PGE.sub.1 
or PGF.sub.2a class as shown below: 
##STR21## 
The claimed compounds may be prepared as described in Scheme 11 and may be 
further reacted as shown in Scheme 12. 
##STR22## 
Scheme 13 exemplifies production of the claimed compounds. 
##STR23## 
As shown above, DCC is added to a solution of N-(4-carboxybutyl) maleimide 
in a suitable solvent such as dichloromethane that contains 
N-hydroxysuccinimide. The reaction is allowed to proceed for several hours 
and is then purified to afford the activated ester 1. A solution of this 
ester is then added to a stirred solution of a suitable biphosphonate such 
as but not limited to ABP in water and sodium hydroxide. The reaction is 
allowed to proceed for several minutes and then the pH is adjusted to 7 
and then the batch is lyophilized. The resulting powder is then purified 
via a suitable means such as HPLC and the resultant purified powder is 
again lyophilized. Compound 3 or other suitable aminobisphosphonate 
maleimide derivative is produced. In a separate process, a suitable 
prostaglandin derivative, such as PGE.sub.2 or others as disclosed in the 
instant invention, is reacted with DCC and a dithiol compound (such as 
1,3-propanedithiol) or other suitable dinucleophilic agent such as 
3-thio-1-propanol (protected as necessary) to form a compound such as 5. 
The suitable prostaglandin analog such as 5 is then reacted with compound 
3 or other aminobisphosphonate maleimide to form a final product such as 
that depicted in Scheme 13. It is understood that other derivatized 
prostaglandins which have an activated ester group at the C-1 position may 
be reacted with aminoalcohols, thiolalcohols, or dithiols to form 
compounds analogous to 5. For example, Scheme 14 shows the reaction of an 
analog of 5 wherein Q is O, NR.sup.1, or S and the carbon chain may be a 
substituted or unsubstitued chain of 1-10 carbon atoms (n=1-10) with the 
aminobisphosphonate maleimide shown above to form the depicted ester, 
amide or thioester derivative. This compound may be used as an effective 
delivery agent of a prostaglandin. 
##STR24## 
The claimed compounds may be used in treating a variety of calcium 
metabolism disorders including: 
(1) A method of treating or preventing osteoporosis by administering a 
pharmaceutically effective amount of compounds within the scope of the 
present invention. 
(2) A method of increasing the bone fracture healing rate in a mammal 
exhibiting a bone fracture by systemically administering a 
pharmaceutically effective amount of compounds within the scope of the 
present invention. 
(3) A method for enhancing the rate of successful bone grafts comprising 
administering to a mammal in need thereof a pharmaceutically effective 
amount compounds within the scope of the present invention. 
(4) A method of treating periodontal disease or alveolar bone loss by 
administering a pharmaceutically effective amount of compounds within the 
scope of the present invention. 
The bisphosphonates which may be used in the present invention include any 
aminoalkyl bisphosphonate such as alendronate, pamidronate 
(3-amino-1-hydroxypropylidene) bisphosphonic acid disodium salt, 
pamidronic acid, riserdronate 
(1-hydroxy-2-(3-pyridinyl)ethylidene)bisphosphonate, YM 175 
((cycloheptylamino) methylene-bisphosphonic acid, piridronate, 
aminohexanebisphosphonate, tiludronate, BM-210955, CGP-42446, and EB-1053. 
The novel method of delivering prostaglandins via the claimed compounds 
disclosed and claimed in the instant invention to the site at which bone 
growth stimulation is desired requires, in order to enhance bone 
formation, daily delivery of about 0.0001 to about 1 mg of prostaglandin. 
The preferred range to achieve increased bone volume is between 0.1 .mu.g 
and 0.3 .mu.g per day of PGE.sub.2. Cortical bone mass may also be 
increased using a PGE.sub.2 equivalent dose of 0.3 .mu.g per day. The 
quantities delivered via the novel method claimed in the instant invention 
are clearly an improvement over the 3 mg/day necessary to achieve an 
equivalent bone formation effect when a prostaglandin is administered 
systemically. 
The prostaglandins which may be used in the present invention include but 
are not limited to PGE.sub.2, PGE.sub.1, and their analogs and 
PGF.sub.2.alpha. and its analogs. The invention also encompasses 
pharmaceutical compositions containing compounds within the scope of the 
invention as active ingredients and those fillers or other inactive 
ingredients which those skilled in the art recognize as important for the 
safe and effective delivery of the claimed composition to a patient or 
patients in need thereof. 
Protecting groups utilized in the synthesis of compounds within the scope 
of the present invention include, but are not limited to, THP. Other well 
known alcohol protecting groups include benzyl halides, MEM, and 
alkylcarbonylhalides. 
The following examples demonstrate both the syntheses of some of the 
compounds within the scope of the present invention and also demonstrate 
the specific ability of the claimed compounds to target to bone cells in 
vitro and in vivo. The examples show that the uptake of .sup.14 C/.sup.3 H 
dual labeled compound shown below and claimed in the instant invention to 
human bone powder in vitro occurs within one minute in fetal bovine serum. 
About 77% of the .sup.14 C moiety and 53% of the .sup.3 H moiety of the 
compound shown below is taken up by the bone powder. Dissociation of the 
PG moiety from the bisphosphonate from human bone powder in fetal bovine 
serum occurs at a rate of approximately 5%/day at 37.degree. C. Both 
radiolabel experiments and radioimmunoassay experiments confirm release of 
the prostaglandin from the bisphosphonate at the bone cell site. 
In vivo experiments also demonstrate that compounds disclosed and claimed 
in the present invention are delivered to bone. For example, uptake of the 
labeled compound shown below into rat tibiae and femora after a single 
dose was administered intravaneously was demonstrated. The animals used in 
this experiment were sacrificed at 24 hours, 14 and 28 days after the 
compounds claimed in the instant invention were administered. The 
radioactivity of the .sup.14 C and .sup.3 H was measured after 
incineration of the long bones to determine the percentage of compound 
retained in the bone. The examples further show that compounds within the 
scope of the present invention significantly inhibit the production of 
lysylpyridinolines (LP) over certain time periods. High LP levels are 
normally associated with the breakdown of bone collagen. 
The compounds claimed in the instant invention are therefore useful in the 
treatment of diseases or conditions in which bone loss or degradation or 
fracture has occurred. The compounds claimed in the instant invention, as 
the specification discloses and as the schemes and examples demonstrate, 
administered in pure form or in a pharmaceutical composition are effective 
in delivering a bone healing or bone growth enhancing amount of a 
prostaglandin to a patient or organism in need of such treatment. In 
addition, the compounds may also be used as bone growth enhancers and bone 
resorption inhibitors if the particular bisphosphonate used has bone 
resorption inhibiting activity or if the entire compound prior to 
hydrolysis has bone resorption inhibiting activity. 
The term "pharmaceutically acceptable salts" shall mean nontoxic salts of 
the compounds of this invention which are generally prepared by reacting 
the free base with a suitable organic or inorganic acid. Representative 
salts include the following salts: Acetate, benzenesulfonate, benzoate, 
bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, 
camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, 
edetate, edisylate, estolate, esylate, fumarate, glucoheptanate, 
gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, 
hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, 
lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, 
methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, 
oleate, oxalate, pamaote, palmitate, pantothenate, phosphate/diphosphate, 
polygalactouronate, salicylate, stearate, subacetate, succinate, tannate, 
tartrate, teoclate, tosylate, triethiodide, valerate. 
The term "pharmaceutically effective amount" shall mean that amount of a 
drug or pharmaceutical agent that will elicit the biological or medical 
response of a tissue, system or animal that is being sought by a physician 
or veterinarian. 
The term "aryl" shall mean a mono- or polycyclic system s composed of 5- 
and/or 6-membered aromatic tings containing 0, 1, 2, 3, or 4 heteroatoms 
chosen from N, O or S and either unsubstituted or substituted 
independently with R.sup.1 to R.sup.12. The term "alkyl" shall mean 
straight or branched alkane, alkene or alkyne. The term "alkoxy" shall be 
taken to include an alkyl portion where alkyl is as defined above. 
The terms "arylalkyl" and "alkylaryl" shall be taken to include an alkyl 
portion where alkyl is as defined above and to include an aryl portion 
where aryl is as defined above. The C.sub.0-n or C.sub.1-n designation 
where n may be an integer from 1-10 or 2-10 respectively refers to the 
alkyl component of the arylalkyl or alkylaryl unit. 
The term "halogen" shall include fluorine, chlorine, iodine and bromine. 
The term "oxy" shall mean an oxygen (0) atom. The term "oxo" refers to a 
bivalent oxygen atom (.dbd.0). The term "thio" shall mean a sulfur (S) 
atom. 
The site at which bone growth stimulation is desired is meant both the area 
adjacent to a section of bone or group of bones in need of treatment in a 
human or other organism in need thereof or a region inside the bone, 
including the site of a fracture or opening which occurs naturally or is 
intentionally made in the bone or group of bones. 
The term "broken bone" means all types of broken bones such as green stick 
fractures, compound fractures, lateral fractures, pathologic fractures 
resulting from invasive tumors, compression fractures and fractures that 
require surgical procedures for realignment of bones. 
The term "bisphosphonate delivery agent" as recited herein means any known 
bisphosphonate that effectively targets bone and is capable of reacting 
with a prostaglandin as recited herein. The bisphosphonate delivery agents 
include all commercially known bisphosphonates used in the treatment of 
osteoporosis and further includes those specifically recited in this 
disclosure. The above term also includes those bisphosphonates that target 
bone and are safe and effective whether or not the bisphosphonate is 
useful in the treatment of osteoporosis. 
In the schemes and examples below, various reagent symbols have the 
following meanings: 
BOC(Boc): t-butyloxycarbonyl. 
THP: tetrahydropyran 
Pd-C: Palladium on activated carbon catalyst. 
DMF: Dimethylformamide. 
DMSO: Dimethylsulfoxide. 
DCC: 1,3-Dicyclohexylimidazole 
CBZ(CBz): Carbobenzyloxy or benzyloxycarbonyl. 
CH.sub.2 Cl.sub.2 : Methylene chloride. 
CHCl.sub.3 : chlorform. 
CH.sub.3 CN: acetonitrile 
EtOH: ethanol. 
CDI: Carbonyldiimidazole 
MeOH: methanol. 
EtOAc: ethylacetate. 
HOAc: acetic acid. 
EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbo-diimide 
LDA: Lithium diisopropylamide 
THF: tetrahydrofuran 
The compounds of the present invention can be administered in such oral 
forms as tablets, capsules (each of which includes sustained release or 
timed release formulations), pills, powders, granules, elixers, tinctures, 
suspensions, syrups, and emulsions. Likewise, they may be administered in 
intravenous (bolus or infusion), intraperitoneal, subcutaneous, or 
intramusculsar form, all using forms well known to those of ordinary skill 
in the pharmaceutical arts. An effective but non-toxic amount of the 
compound desired can be employed as an anti-osteoporosis agent or as a 
fracture healing agent. 
Compounds of the invention may be administered to patients where prevention 
of osteoporosis or other bone related disorder is desired. 
The dosage regimen utilizing the compounds of the present invention is 
selected in accordance with a variety of factors including type, species, 
age, weight, sex and medical condition of the patient; the severity of the 
condition to be treated; the route of adminstration; the renal and hepatic 
function of the patient; and the particular compound or salt thereof 
employed. An ordinarilly skilled physician or veterinarian can readily 
determine and prescribe the effective amount of the drag required to 
prevent, counter, or arrest the progress of the condition. 
In the methods of the present invention, the compounds herein described in 
detail can form the active ingredient, and are typically administered in 
admixture with suitable pharmaceutical diluents, excipients or carders 
(collectively referred to herein as "carrier" materials) suitably selected 
with respect to the intended form of administration, that is, oral 
tablets, capsules, elixers, syrups and the like, and consistent with 
convention pharmaceutical practices. 
For instance, for oral administration in the form of a tablet or capsule, 
the active drag component can be combined with an oral, nontoxic, 
pharmaceutically acceptable, inert carder such as lactose, starch, 
sucrose, glucose, methyl cellulose, magnesium sterate, dicalcium 
phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral 
administration in liquid form, the oral drag components can be combined 
with any oral, non-toxic, pharmaceutically acceptable inert carder such as 
ethanol, glycerol, water and the like. Moreover, when desired or 
necessary, suitable binders, lubricants, disintegrating agents, 
electrolytes, and coloring agents can also be incorporated into the 
mixture. The present composition may be administered in the form of 
tablets, caplets, gelcaps, capsules, elixirs, syrups, or suspensions. For 
oral administration, the active ingredients may be admixed with a 
pharmaceutically acceptable diluent such as lactose, sucrose, cellulose, 
dicalcium phosphate, calcium sulfate, mannitol, and, in a liquid 
composition, ethyl alcohol. Acceptable emulsifying or suspending agents 
such as PVP, gelatin, natural sugars, corn sweeteners, natural and 
synthetic gums such as acacia, sodium alginate, guar gum, agar, bentonite, 
carboxymethylcellulose sodium, polyethylene glycol and waxes, may also be 
admixed with the active components. Where necessary, lubricants such as 
magnesium stearic acid talc or magnesium stearate, and disintegrators or 
superdisintegrators such as starch, sodium starch glycolate or 
cross-linked PVP may also be included. Electrolytes such as dicalcium 
phosphate, sodium benzoate, sodium acetate and sodium chloride may also be 
used. Disintegrators also include, without limitation, starch methyl 
cellulose, agar, bentonite, xanthan gum and the like. 
The compounds of the present invention can also be administered in the form 
of liposome delivery systems, such as small unilamellar vesicles, large 
unilamellar vesicles and multilamellar vesicles. Liposomes can be formed 
from a variety of phospholipids, such as cholesterol, stearylamine or 
phosphatidylcholines. 
The compounds of the present invention may also be coupled with soluble 
polymers as targetable drug carriers. Such polymers can include 
poly-vinlypyrrolidone, pyran copolymer, 
polyhydroxypropyl-methacrylamide-phenol, 
polyhydroxyethylaspartamide-phenol, or polyethyleneoxide-polylysine 
substituted with palmitoyl residues. Furthermore, the compounds of the 
present invention may be coupled to a class of biodegradable polymers 
useful in achieving controlled release of a drug, for example, polylactic 
acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, 
polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, 
polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or 
amphipathic block copolymers of hydrogels. 
The compounds of the present invention can also be co-administered with 
suitable anti-osteoporosis drags to achieve synergystic effects in the 
treatment of various pathologies. They may also be combined with known 
bisphosphonates or other suitable compounds which are used to treat 
osteoporosis, bone-related disorders, or bone fractures. 
The novel compounds of the present invention were prepared according to the 
procedure of the schemes and examples described in this specification, 
using appropriate materials and are further exemplified by the following 
specific examples. The most preferred compounds of the invention are any 
or all of those specifically set forth in these examples and schemes. 
These compounds are not, however, to be construed as forming the only 
genus that is considered as the invention, and any combination of the 
compounds or their moieties may itself form a genus. The following 
examples further illustrate details for the preparation of the compounds 
of the present invention. Those skilled in the art will readily understand 
that known variations of the conditions and processes of the following 
preparative procedures can be used to prepare these compounds. All 
temperatures are degrees Celcius unless otherwise noted. 
EXAMPLES 
Example 1 
Synthesis of PGE.sub.2 -ABP Sodium Compound 
1,3-Dicyclohexylcarbodiimide (3.6 mg) was added to a stirred solution of 
PGE.sub.2 (I) (3.1 mg) and N-hyroxysuccinimide (3.0 mg) in dry 
acetonitrile (200 .mu.L) and stirred at room temperature (25.degree. C.) 
until thin layer chromatography indicated that the reaction was complete. 
The solvent is removed under an inert atmosphere (nitrogen) and the 
residue was dissolved in methylene chloride and applied to a small column 
of silica gel in a pasteur pipette. The pipette was then eluted with ethyl 
acetate (EtOAc) to afford the hydroxysuccinimide ester (IIa) and a small 
quantity of dicyclohexylurea. (CDCl.sub.3) 5.45-5.7 (2H, m, H-13, 14), 
5.37 (2H, m, s H-5,6), 3.95-4.15 (2H, m, H-11,15), 2.85 (4H, s, 
O.dbd.C(CH.sub.2).sub.2 /CO). A solution of this ester in 1,4-dioxane was 
added to a stirred solution of ABP disodium salt (2.4 mg) in water (150 
.mu.L)and 1.0 molar (M) aqueous NaOH (10 .mu.L). After 10 minutes or so 
the pH of the reaction mixture was adjusted to approximately 9 with 1.0M 
aqueous NaOH, and then 1 hr later the pH was adjusted to 7 with 0.1M 
aqueous HCl. The solution is filtered and the filtrate was concentrated to 
dryness. The residue was then dissolved in water and applied to a Varian 
Bond Elute C.sub.18 pak which was eluted with water. When the product 
began to elute, the solvent system on the C.sub.18 column was changed to 
acetonitrile/water (50:50). Evaporation of fractions containing the 
product afforded the target amide (III) (3.7 mg). (D.sub.2 O) (2H, m, 
H-5,6), 5.1-5.4 (2H, m, H-13,14), 3.9-4.1 (2H, m, H-11,15), 3.0 (2H, t, 
HN--CH.sub.2). 
Example 2 
The identical procedure as described in Example 1 was followed with 
tritiated PGE.sub.2 and .sup.14 C labeled ABP monosodium salt to produce a 
compound with the following structure: 
##STR25## 
This compound was then used in the following in vitro and in vivo 
experiments to exemplify the release of the PGE.sub.2 moiety from bone 
cells after administration of the claimed compound and to exemplify and 
demonstrate that the claimed compound is attached in vivo to bone. 
Example 3 
Synthesis of a PGE.sub.2 -Dithio Compound as depticted in Scheme 13 
Dicyclohexylcarbodiimide (0.2 g) was added to a stirred solution of 
N-(4-carboxybutyl)maleimide (0.12 g) [m.p. 87-89.degree. C. prepared in 
the same way as the procedure in Coleman et al, J. Org. Chem., 1959, 24, 
135] in dichloromethane (10 ml)containing N-hydroxysuccinimide (0.38 g). 
After two hours, the reaction mixture was poured onto a silica gel column 
which was eluted with ethyl acetate affording the active ester (1) (0.086 
g). .sup.1 H NMR [(CD.sub.3).sub.2 CO].delta.6.85(2H, s), 3.50(2H,t), 
2.87(4H,s), 2.72(2H,t ), 1.98(2H,dt). 
A solution of the active ester (1) (12 mg) in 1,4-dioxane (200 .mu.l) was 
addied to a stirred solution of bisphosphonate (ABP) (7 ng) in water (400 
.mu.l) and 1 N sodium hydroxide (25 .mu.l). After 15 minutes the solution 
was adjusted to pH 7 with 0.1 N HCl and then lyophilized. The resulting 
powder was dissolved in water and eluted through two Varian 6 ml C.sub.18 
"bond elute" cartridges with water, collecting the first 4 nl from each 
cartridge. This solution was lyophilized and the resulting colorless 
powder contained the maleimide derivative (3) as well as 
N-hydroxysuccinamide and, perhaps, some unreacted ABP. .sup.1 H NMR 
(D.sub.2 O).delta.6.72(2H,s), 3.40(2H,t), 3.01(2H,t), 2.13(2H,t), 
1.9-1.6(6H,m). 
A solution of PGE.sub.2 (4) (5 mg) in CH.sub.2 Cl.sub.2 (500 .mu.l) was 
stirred under nitrogen and treated with 1,3-propanedithiol (14 .mu.l) and 
dicyclohexylcarbodiimide (8 ng). The reaction was followed by thin layer 
chromatography (t.l.c.) and when complete (.about.4 hours) the reaction 
mixture was poured onto a small silica gel column in a pasteur pipette. 
Elution with deoxygenated ethyl acetate afforded the thiolester (5). This 
was immediately dissolved in methanol (500 .mu.l) and added to a solution 
of (4) in aqueous methanol (1 ml, 1:1 v/v). The solution was allowed to 
stand for 15 minutes, then most of the methanol was evaporated and the 
residual aqueous solution was freeze-dried. The crude product was 
dissolved in water and absorbed onto a Varian 6 ml C-18 bond elute 
cartridge. This was eluted with water (9 ml), 30% MeOH/H.sub.2 O (6 ml), 
then 60% MeON/H.sub.2 O (6 ml). The first 3 ml of the 60% MeOH fraction 
contained all the product (6) obtained as a white powder (4.6 mg) after 
lyophilization. m.p. &gt;260.degree. C. (dec). 
.sup.13 C NMR (D.sub.2 O).delta.(ppm) 215.7(C.dbd.O), 198.2 (C--S), 176.0, 
176.1,172.2 (C--N), 133.8, 129.6, 127.7, 124.5 (HC.dbd.), 71.1(t, Jclp=134 
Hz, C-p), 70.2, 68.4(CH--O), 51.7, 50.6, 37.0(CH), 43.2, 40.6, 37.4, 35.9, 
34.0, 33.4, 30.3, 28.9, 28.4, 27.4, 26.1, 24.8, 23.6, 22.5, 20.8, 20.6, 
19.9(CH.sub.2), 11.3(CH.sub.3). 
Example 4 
The identical procedure as described in Example 3 was followed with 
tritiated PGE.sub.2 and .sup.14 C labeled ABP monosodium salt to produce a 
compound with the following structure: 
##STR26## 
The above compound was used in the same biological experiments as the 
compound IIIa as a delivery agent of labeled prostaglandin. 
Biological Experiments 
Example 5 
Binding of (IIIa) to bone powder 
1 .mu.l of .sup.3 H-PGE.sub.2 /.sup.14 C-ABP (IIIa) (21.64 .mu.Ci of 
.sup.14 C and 19.05 .mu.Ci of .sup.3 H) was placed in 1 ml 100% fetal 
bovine serum to yield a final concentration of 3.5 .mu.M. 200 ml of this 
solution was incubated with 10 mg bone powder for 1, 2, 3 and 5 mins with 
vigorous shaking. The mixture was centrifuged (20 sec), 125 .mu.l aliquot 
was taken from each sample and counted in 10 ml Atomlight in an LKB liquid 
scintillation counter. 125 .mu.l of the radioactive sample was also 
counted at 0 time. The uptake of radioactivity into the bone powder was 
calculated by subtracting the dpms in the medium counted at the times 
indicated above from dpms at 0 time and this number was divided by the 
dpms at 0 time. The data demonstrated that about 76% of the .sup.14 
C-moiety and 53% of the .sup.3 H-moiety were taken up by bone particles 
within 1 min. In a separate experiment, we found that 77% .sup.3 H-ABP was 
taken up by bone in 1 min. 
Example 6 
The .sup.3 H moiety associated with the PGE.sub.2 component of the molecule 
and its release into the medium surrounding the collected bone particles 
was measured over a period of hours to days. The data suggested that 5% 
release occurred per day. 
Dissociation of .sup.3 H-PGE.sub.2 /.sup.14 C-ABP (IIIa) from human bone 
powder 
Dissociation of .sup.3 H-PGE.sub.2 /.sup.14 C-ABP from human bone powder in 
fetal bovine serum at 37.degree. C. was measured by incubating 10 mg of 
human bone powder with 1 .mu.l .sup.3 H-PGE.sub.2 /.sup.14 C-ABP in 1 ml 
for 5 mins. The mixture was centrifuged (20 sec), 100 .mu.l aliquot was 
taken and counted in Atomlight in an LKB liquid scintillation counter. The 
rest of the 900 .mu.l solution was withdrawn, the bone powder was washed 
once with 1 ml phosphate buffered saline, 1 ml fresh fetal bovine serum 
was added and incubated with the bone powder for 15, 24, 39, 48, 59, 79 
and 103 hours in a shaking bath at 37.degree. C. 100 .mu.l aliquots were 
withdrawn at these times and counted in 10 mls Atomlight in an LKB liquid 
scintillation counter. The release of radioactivity from the human bone 
powder into the medium was calculated as follows: dpms from 100 .mu.l of 
the .sup.3 H-PGE.sub.2 /.sup.14 C-ABP at 5 mins were subtracted from dpms 
at 0 time. The resulting dpms reflect radioactivity taken up by bone 
powder. The dpms obtained by counting 100 .mu.l aliquots at each time 
point were then divided by the dpms taken up by bone. 13% of the .sup.3 
H-moiety was released into the medium at 15 hrs and by 103 hours 32.9% of 
the radioactivity was released into the medium. About 5% of the .sup.3 
H-moiety was released per day whereas the dpms of .sup.14 C-moiety in the 
medium were not significantly changed during this time frame. 
Example 7 
Uptake of .sup.3 H-ABP or .sup.3 H-PGE.sub.2 /.sup.14 C-ABP (IIIa) in rat 
tibia and femora 
Both compounds were administered intravenously via the tail vein to 
Sprague-Dawley female rats as a single dose of 28 nmoles of radiolabeled 
compound, equivalent to 0.2 .mu.Ci/animal. .sup.3 H-ABP, which was 
administered to nine rats, is correspondent to 0.1 mg/kg and .sup.3 
H-PGE.sub.2 /.sup.14 C-ABP (IIIa), which was administered to seven rats, 
is correspondent to 0.24 mg/kg. After 1, 14 or 28 days, animals were 
sacrificed by CO.sub.2 and the tibia and femora were dissected weighed and 
then stored at -20.degree. C. The amount of radioactivity incorporated 
into the bone was determined by incineration in a Packard combuster after 
first air drying the bone for three days at ambient temperature. The 
percent of the compound retained in bone at each time point was calculated 
on the basis of the radioactivity, converted to nmoles/gm bone on the 
assumption that the skeleton represents 8 % of the body weight. The 
skeletal retention was expressed as percent administered dose. FIG. 1 
shows the relative percentage of compound IIIa retained in rat tibiae and 
femora versus the bisphosphonate .sup.3 H-alendronate 
(4-amino-1-hydroxybutylidene bisphosphonic acid disodium salt). 
Example 8 
Effect of PGE.sub.2 /ABP (IIIa) on bone resorption estimated by urinary 
excretion of lysypyridinoline in the rat 
4 week old Sprague-Dawley female rats were injected intravenously via the 
tail vein with equimolar weekly doses of ABP (1 mg/kg, n=5), PGE.sub.2 
/ABP (2.4 mg/kg, n=5), PGE.sub.2 (1.4 mg/kg, n=5), or saline (n=4) each. 
Filtered urine was collected after 12 and 26 days by housing individual 
rats in metabolic cages and providing them with food and water ad libitum. 
The overnight collections of urine were centrifuged at 1000.times.g for 10 
minutes to remove any particles and the supernatant fluid was stored at 
-80.degree. C. until analysis. Lysylpyridinoline (LP) was extracted from 
duplicate 1 ml aliquots by acid hydrolysis and subsequent low pressure 
CF-1 chromatography according to the method of Beardsworth et al. (1990). 
LP was further resolved by high pressure liquid chromatography according 
to the method of Uebelhart et al. (1990) and quantitated by comparison 
with an external standard. Urinary creatinine was measured using the 
picric acid colorimetric assay (Pharmacia Diagnostics Inc., Fairfield, 
N.J.). Final results were expressed as pmoles LP per .mu.mole creatinine. 
The results as depicted in FIG. 2 showed that animals treated with 
compound Ilia had significantly lower levels of LP after a 12 day period 
compared to vehicle alone. References which describe the procedures 
utilized in the above examples include: Beardsworth, L. J., Eyre, D. R., 
and Dickson I. R. 1990 Journal of Bone and Mineral Research 5 (7):671-676 
and Uebelhart, D, Gineyts, E, Chapuy, M. C., and Delmas, P. D. 1990 Bone 
and Mineral 8:87-96. 
Example 9 
ABP, PGE.sub.2 /ABP, and PGE.sub.2 effects on bone loss due to limb 
immobilization in the rat 
Male Sprague-Dawley rats weighing 270 grams (10-12 wks) were injected 
subcutaneously on two consecutive days prior to unilateral sciatic 
neurectomy induced hindlimb immobilization with the following doses: 
Vehicle (0.0 mg/kg), ABP (0.5mg/kg), PGE.sub.2 /ABP (1.2 mg/kg), PGE.sub.2 
(0.7 mg/kg). Ten days post-neurectomy femora were removed at necropsy, 
dissected from the musculature, and placed in crucibles for incineration 
at 700.degree. C. for twenty-four hours. Following incineration, the 
femoral ash content was weighed to the nearest 0.1 mg and the femoral ash 
weight differences between the control and immobilized hindlimb were 
calculated. Data represent mean .+-.SEM (n=6). The results showed that in 
this particular experiment there was no statistical difference between 
PGE.sub.2, the labeled compound claimed within the scope of the instant 
invention, and an inert vehicle in preventing bone loss which accompanies 
limb immobilization in the rat. ABP alone used as a positive control was 
effective.