BACKGROUND OF THE INVENTION 
Osteoporosis is a skeletal disorder which is evidenced by an increase in 
fracture incidence resulting from a decrease in bone density. In fact, 
both the bone mineral (calcium phosphate called "hydroxyapatite") and the 
bone matrix (major protein called "collagen") are lost. This condition may 
begin to occur in humans as early as age 30. In general, the process is 
more rapid in postmenopausal women than in men. However, after age 80 
there is no sex difference in the incidence of osteoporosis. In the course 
of 10 to 20 years of bone loss there may be symptoms of back pain and 
X-ray evidence of deformation of the spine. At older ages, the brittleness 
of the bones becomes evident by the ease with which the proximal femur 
("hip") fractures. Osteoporosis is the most common cause of fractures in 
people over age 45. 
Although the cause of osteoporosis is poorly understood, it is believed 
that there is an imbalance between bone production and bone resorption 
(bone break-down). Bone remains a dynamic tissue throughout the life of an 
animal. That is, new bone is continuously being formed and old bone is 
continuously being resorbed. However, in animals suffering from an 
osteoporotic condition, net bone resorption exceeds bone formation. 
A survey indicates that in the United States there may be fifteen to twenty 
million people afflicted with osteoporosis W. A. Peck (Chairman), NIH 
Osteoporosis Consensus Conference, J. Am. Med. Assoc., 10, 252:799-802 
(1984)!. Various types of osteoporosis are designated according to special 
conditions believed to be causative: senile (aging); post-menopausal 
(female loss of estrogenesis); disuse (chronic immobilization); steroid 
(long term steroid treatment as in arthritis); hypercalcemia of 
malignancy. Osteoporosis may also be manifested in dental problems since 
the mandible appears to lose mass more rapidly than any other bone. Thus, 
periodontal disease involving a loosening of the adult teeth may be an 
early sign of osteoporosis. 
The mechanism of bone loss is at present poorly understood. Moreover, the 
present methods of treatment are generally unsatisfactory. These include 
anabolic agents, various drugs containing phosphorous, Vitamin D, calcium 
salts, fluorides and calcitonin. 
Estrogen replacement therapy has been the therapy of choice for 
osteoporosis in post-menopausal women. 
Physical therapy is another method currently used to treat osteoporosis 
since immobilization can cause osteoporosis at any age. Thus, many 
physicians believe that exercise and physical therapy can prevent the 
progression of the disease in elderly patients. However, physical therapy 
can be harmful for patients with fractures and, moreover, over strenuous 
exercise can cause fractures in patients with severe osteoporosis. 
Other treatments include the administration of a fluoride salt such as 
sodium fluoride which has been shown to promote bone growth clinically, 
apparently by stimulating collagen synthesis. However, a serious side 
effect is poorly calcified, irregular bone growth. Another treatment 
involves infusion of calcium and Vitamin D to counteract the deficiency of 
calcium or impaired absorption of calcium which is symptomatic in some 
elderly patients. There is, however, no evidence that a higher intake of 
calcium will prevent osteoporosis or increase bone mass in adults. 
Calcitonin, a 32 amino acid polypeptide hormone, regulates plasma calcium 
by decreasing intestinal calcium absorption, increasing urinary calcium 
excretion, and inhibiting osteoclast-mediated osteolysis (hypocalcemic 
effects). The bone resorption inhibitory activity of calcitonin has proven 
therapeutically useful in the treatment of high turnover metabolic bone 
diseases such as Paget's Disease, hypercalcemia of malignancy, and 
post-menopausal osteoporosis. Although the safety and efficacy of 
calcitonin has been well demonstrated, delivery of the orally-inactive 
peptide, either by parental administration or nasal spray, has limited the 
prophylactic use in asymptomatic individuals. 
DESCRIPTION OF THE INVENTION 
This invention provides a compound of Formula I, which is named {5R*-6R*, 
7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 9-trihydroxy-2, 6, 8, 
12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 
18-hendecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 (5H)-one, and 
which is useful in inhibiting bone resorption, and treating and inhibiting 
osteoporosis. The structure of the compound of Formula I is shown below. 
##STR2## 
As used in describing this invention, 5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 
18S*, 19S*, (13E)!} 4, 7, 19-trihydroxy-2, 6, 8, 12, 12, 14, 16, 
18-heptamethyl-6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19-dodecahydro-1, 
19:5, 9-diepoxybenzocyclooctadecen-3 (5H)-one and the compound of Formula 
I are used interchangeably. 
{5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 
19-Trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 
12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 
(5H)-one is produced by culturing an actinomycete of the genus 
Streptomyces spp., that was designated NRRL 21370, in an aqueous nutrient 
medium. NRRL 21370 has been deposited under the Budapest Treaty with the 
Northern Utilization and Research Division, Agricultural Research Service, 
U.S. Department of Agriculture, Peoria, Ill, U.S.A. 
This invention also provides a compound of Formula I substantially free of 
actinomycete protein, which is useful in inhibiting bone resorption, and 
treating or inhibiting osteoporosis. 
This invention also relates to substantially pure compound of Formula I, 
which is useful in inhibiting bone resorption, and treating or inhibiting 
osteoporosis. Substantially pure is defined as being in excess of 75% 
purity. 
This invention additionally provides a pharmaceutical composition 
comprising a compound of Formula I and a pharmaceutical carrier. 
The stereochemistry designated for the compound of Formula I represents the 
relative stereochemical assignments at the optically active centers. This 
invention covers both absolute configurations of the compound of Formula 
I. 
{5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 
19-Trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 
12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 
(5H)-one can be obtained by aerobic fermentation of NRRL 21370 using 
standard fermentation, isolation, and purification techniques, as 
described in Example 1. The isolation and purification of {5R*-6R*, 7S*, 
8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 19-trihydroxy-2, 6, 8, 12, 
12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 
19-dodecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 (5H)-one is 
provided in Example 2. 
The ability of {5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 
7, 19-trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 
11, 12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 
9-diepoxybenzocyclooctadecen-3 (5H)-one to inhibit bone resorption and 
treat or inhibit osteoporosis was established in three in vitro and one in 
vivo standard pharmacological test procedures. 
The first in vitro standard pharmacological test procedure measures the 
ability of the compound to be evaluated to bind to the calcitonin 
receptor. The following describes the procedure used and the results 
obtained. 
Adult female or male Sprague-Dawley rats (250-350 gm), were euthanized by 
CO.sub.2 asphyxiation. Kidneys were removed and placed in ice cold saline; 
all subsequent procedures were carded out at 4.degree. C. Kidneys were 
decapsulated and coarsely minced with scissors. The resulting fragments 
were homogenized in homogenization buffer (10% w/v: 250 mM sucrose; 20 mM 
Tris-HCl, pH 7.5; 1 mM EDTA; and 0.5 mM Phenylmethylsulfonyl fluoride 
(PMSF) by Polytron homogenization with two 15 seconds bursts in a cold 
room. The homogenate was centrifuged (4.degree. C.) at 1000.times.g for 10 
min. The pellet was discarded and the supernatant was centrifuged 
(4.degree. C.) at 3000.times.g for 10 min. The pellet was discarded and 
the supernatant was centrifuged (4.degree. C.) at 22000.times.g for 15 
min. The pellet was washed 1.times. with 7 ml of membrane buffer (40 mM 
Tris-HCl, pH 7.5; 10 mM MgCl.sub.2 ; 1 mM EGTA; 0.2 mM PMSF) per 2 kidneys 
by resuspension with 5 strokes of a Dounce homogenizer (tight pestle), 
recentrifuged at 22000.times.g for 15 min., and resuspended in 5 ml of 
membrane buffer per 2 kidneys by Dounce homogenization. 
Wallac Printed Filtermat B (for use with 1205 Betaplate.TM.) were soaked in 
0.1% bovine serum albumin in distilled/deionized water for 1 hour at room 
temperature. Binding of radiolabeled .sup.125 I-salmon calcitonin (sCT; 
specific activity about 2000 Ci/mmol; stock diluted to 1 nM prior to use) 
was performed in a Beckman 96 deep-well Polypropylene titer plate in a 
volume of 500 .mu.l. Additions are made in the following order: 
Total binding 
275 .mu.l assay buffer (50 mM tris-HCl, pH 7.5; 10 mM MgCl.sub.2 ; 
1 mM EGTA; 0.2% bovine serum albumin; 0.2 nM PMSF; 
1 mg/ml bacitracin) 
25 .mu.l test compound (or 5 .mu.l of extracts plus 20 .mu.l of assay 
buffer) 
100 .mu.l renal plasma membrane (1:2 dilution from stock) 
Vortex (adapter needed for 96 well format), then add 100 .mu.l labeled sCT 
(1:4 dilution from 1 nM stock) 
Nonspecific binding 
275 .mu.l assay buffer 
25 .mu.l unlabeled sCT 
100 .mu.l renal plasma membrane (1:2 dilution from stock) 
Vortex, then add 
100 .mu.l labeled sCT (1:4 dilution from 1 nM stock) 
Samples were incubated for 1 hour at room temperature. Samples were 
harvested with a cell harvester designed for 96 deep-well titer plate. The 
filtermats were washed with 3 cycles, 4 seconds of ice-cold 0.9% saline. 
The filtermats were dried in 90.degree. C. oven for 20 min., and were then 
bagged, 19 ml of Beta-Scint (LKB) scintillation fluid added, and sealed. 
The filters were counted for a period of 2 minutes per sample utilizing an 
LKB Betaplate Reader. 
The results obtained in this standard pharmacological test procedure showed 
that {5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 
19-trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 
12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 
(5H)-one competitively inhibited the binding of calcitonin to the 
calcitonin receptor with an IC.sub.50 of 0.010 mg/ml). 
The second in vitro standard pharmacological test procedure determines the 
ability of the compound to be evaluated to act as a calcitonin agonist, by 
measuring the levels of cAMP produced in response to the test compound. 
The following briefly describes the procedure used and the results 
obtained. 
cAMP was generated in T-47D cells (a human mammary carcinoma cell line 
available from the American Type Culture Collection, Rockville, Md., USA) 
according to the following procedure. T-47D cells were seeded at 
5.times.10.sup.4 cells per well in a 96 well plate and incubated overnight 
in growth media (RPMI-1640 medium and 10% fetal calf serum) at 37.degree. 
C. in a cell incubator. After the incubation was completed, the growth 
medium was aspirated from the cells in the plate. The cells were washed 
once with 200 .mu.l of prewarmed sample buffer (RPMI-1640 medium; 10% 
fetal calf serum; 20 mM HEPES buffer, pH 7.2, and 10 .mu.M 
3-isobutyl-1-methylxanthine (IBMX). Sample buffer (100 .mu.l) containing 
the compound to be evaluated was added to each well and the plate was 
incubated in a 37.degree. C. water bath for 10 min. The medium was 
aspirated from each well and 200 .mu.l 0.01N HCl were added to the cells. 
After shaking the plate for 10 min, the supernatants were removed and 
stored at -80.degree. C. 
cAMP levels were measured using a scintillation proximity assay (SPA) 
system (Amersham) according to the manufacturer's protocol. cAMP standards 
(range: 0.2 to 12.8 pmoV 100 ul) and samples were diluted in 50 mM acetate 
buffer, pH=5.8 (assay buffer). Fifty ul of each standard and sample were 
pipetted into individual wells, followed by 50 ul of .sup.125 I-cAMP. 
Fifty ul of antiserum was added to all wells with the exception of the 
non-specific binding wells (NSB). Fifty ul of the SPA anti-rabbit reagent 
was added to all wells. The assay plate was sealed and mixed on an orbital 
shaker for 15-20 hours at room temperature The amount of .sup.125 I-cAMP 
bound to fluormicrospheres was determined by counting the plate in a LKB 
betaplate counter for 2 minutes. 
Intracellular cAMP levels in the T-47D cells following salmon calcitonin 
stimulation for 10 rain at 37.degree. C. are as follow: 
______________________________________ 
Calcitonin (nM) 
cAMP (pmol/ml) 
______________________________________ 
0 (Basal) 1 
0.0001 2 
0.001 13 
0.01 105 
0.1 &gt;640 
______________________________________ 
When evaluated in this standard pharmacological test procedure, {5R*-6R*, 
7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 19-trihydroxy-2, 6, 
8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 
19-dodecahydro-1, 19:5, 9-diepoxybenzocyclootadecen-3 (5H)-one had an 
EC.sub.50 of 0.3 .mu.g/ml, demonstrating that this compound was a potent 
calcitonin agonist, and is useful in inhibiting bone resorption. 
The ability of {5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 
7, 19-trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 
11, 12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 
9-diepoxybenzocyclooctadecen-3 (5H)-one to inhibit bone resorption also 
demonstrated in a third in vitro standard pharmacological test procedure. 
Briefly, osteoclasts were isolated from neonatal rat long bones by mincing 
the bones in Medium 199 containing Hank's salts (3 ml per 10 bones from 
each of 5 animals). Osteoclasts were dislodged from the bone fragments by 
aspirating the bone fragments 50 times into and out of a transfer pipette. 
100 .mu.l of the resulting cell suspension from each animal was then 
plated onto each of 3 (per dose) 4.4 mm.sup.2 slices of devitalized bovine 
femoral cortical bone that had been placed in the wells of a 96 well plate 
and pre-wetted with 100 .mu.l of the medium. The osteoclasts were then 
allowed to adhere for 25 minutes at 37.degree. C. The slices, with 
adherent osteoclasts, were then rinsed in 5 ml of medium to remove 
non-adherent cells. The slices were then transferred to Medium 199 
containing Earle's salts with only 0.7 g/L NaHCO.sub.3 (to ensure that the 
medium equilibrates to a pH of 6.8-7.1 in a 5% CO.sub.2 atmosphere) and 
10% heat-inactivated fetal bovine serum. After a 24 h incubation at 
37.degree. C. in a humidified 5% CO.sub.2 /95% air atmosphere, the slices 
were removed from the culture medium and the cells stripped from the bone 
surface using sonication for two 15 second bursts in 0.25M NH.sub.4 OH. 
The excavations (pits) made by the osteoclasts in the surface of the bone 
were visualized by staining with 1% toluidine blue for 3 minutes and bone 
resorption quantified microscopically by counting the number of pits 
excavated per slice. An IC.sub.50 value of 0.8 pg/ml was obtained, 
demonstrating that {5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, 
(13E)!} 4, 7, 19-trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 
8, 9, 10, 11, 12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 
9-diepoxybenzocyclooctadecen-3 (5H)-one has the ability to inhibit bone 
resorption. 
{5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 
19-Trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 
12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 
(5H)-one was also evaluated in an in vivo standard pharmacological test 
procedure that measures the effect of the test compound on serum calcium 
levels. Agents that inhibit bone resorption, such as calcitonin, regulate 
circulating calcium levels by inhibiting osteoclast mediated osteolysis 
and thereby inhibit the release of calcium from bone. The decrease in 
serum calcium levels following drug treatment indicates bone sparing 
activity. Calcitonin acts on specific membrane receptors of the bone 
resorbing osteoclast cells eliciting second messenger events such as 
increased cAMP generation that leads to inhibition of the cell's motility 
and inhibition of its bone resorbing functional activity. If bone turnover 
activity is high, such as in the young growing rat, this rapid inhibition 
in bone resorption is reflected by a transient decrease in circulating 
calcium. The following briefly describes the procedure used and results 
obtained in the in vivo standard pharmacological test procedure in young 
growing rats. Young adult male rats weighing 160-170 g were held for at 
least 72 hours after receipt to acclimatize to colony conditions and then 
were randomly assigned to groups of 5-7 rats/group for treatment. The rats 
were administered vehicle or test compound, and at intervals between 0.5 
and 3 hours after dosing, 1.0 ml of blood was collected from each rat 
under ketamin/acepromazine anesthesia via tail artery or (terminal via) 
cardiac puncture. Serum was evaluated for total calcium using a NOVA 7+7 
blood analyzer. 
The following table summarizes the results for {5R*-6R*, 7S*, 8R*, 9R*, 
12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 19-trihydroxy-2, 6, 8, 12, 12, 14, 
16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 
19-dodecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 (5H)-one (referred 
to as the compound of Formula I) and calcitonin. In this standard 
pharmacological test procedure, all animals were dosed at 0 hours. For 
animals treated with the compound of Formula I, animals were bled 
sequentially. For animals treated with salmon calcitonin and untreated 
normal animals, a different group was used at each time point. 
______________________________________ 
0.5 hours.sup.b 
1.5 hours.sup.b 
3.0 hours.sup.b 
Serum Serum Serum 
Treatment.sup.a 
N Calcium.sup.c 
N Calcium.sup.c 
N Calcium.sup.c 
______________________________________ 
Cmpd Formula I 
8 10.73 8 10.21** 
7 9.99** 
10 mg/kg, i.p. .+-.0.13 .+-.0.17 .+-.0.09 
Cmpd Formula I 
8 10.60 8 10.43* 6 10.18 
30 mg/kg, i.p. .+-.0.05 .+-.0.12 .+-.0.05 
Cmpd Formula I 
8 10.29** 8 10.19** 
8 10.06* 
100 mg/kg, i.p. .+-.0.13 .+-.0.09 .+-.0.10 
Salmon Calcitonin 
6 8.17** 6 7.45** 6 6.65** 
5 IU/rat, s.c. .+-.0.09 .+-.0.07 .+-.0.09 
Normals 6 10.92 6 10.88 6 10.40 
.+-.0.13 .+-.0.10 .+-.0.11 
______________________________________ 
.sup.a All animals were dosed at 0 hours. For animals treated with the 
compound of Formula I, animals were bled sequentially. For animals treate 
with salmon calcitonin and untreated normal animals, a different group wa 
used at each time point. 
.sup.b Time after dosing 
.sup.c Mean .+-. SEM 
*p &lt; 0.05 vs corresponding normals 
**p &lt; 0.01 vs corresponding normals 
The results of the in vitro and in vivo standard pharmacological test 
procedures showed that {5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, 
(13E)!} 4, 7, 19-trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 
8, 9, 10, 11, 12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 
9-diepoxybenzocyclooctadecen-3 (5H)-one bound to the calcitonin receptor, 
acted as a potent agonist at the calcitonin receptor, and significantly 
reduced serum calcium levels demonstrating its ability to inhibit bone 
resorption. Based on these results, {5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 
18S*, 19S*, (13E)!} 4, 7, 19-trihydroxy-2, 6, 8, 12, 12, 14, 16, 
18-heptamethyl-6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19-dodecahydro-1, 
19:5, 9-diepoxybenzocyclooctadecen-3 (5H)-one is useful in treating or 
inhibiting osteoporosis, bone loss secondary to glucocorticoid and other 
drug treatments, and other metabolic bone diseases such as Paget's disease 
and hypercalcemia resulting from malignancy. 
{5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 
19-trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 
12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 
(5H)-one can be formulated neat or with a pharmaceutical carrier. The 
pharmaceutical carrier may be solid or liquid. 
A solid carrier can include one or more substances which may also act as 
flavoring agents, lubricants, solubilizers, suspending agents, fillers, 
glidants, compression aids, binders or tablet-disintegrating agents; it 
can also be an encapsulating material. In powders, the carrier is a finely 
divided solid which is in admixture with the finely divided active 
ingredient. In tablets, the active ingredient is mixed with a carrier 
having the necessary compression properties in suitable proportions and 
compacted in the shape and size desired. The powders and tablets 
preferably contain up to 99% of the active ingredient. Suitable solid 
carriers include, for example, calcium phosphate, magnesium stearate, 
talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl 
cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low 
melting waxes and ion exchange resins. 
Liquid carriers are used in preparing solutions, suspensions, emulsions, 
syrups, elixirs and pressurized compositions. The active ingredient can be 
dissolved or suspended in a pharmaceutically acceptable liquid carrier 
such as water, an organic solvent, a mixture of both or pharmaceutically 
acceptable oils or fats. The liquid carrier can contain other suitable 
pharmaceutical additives such as solubilizers, emulsifiers, buffers, 
preservatives, sweeteners, flavoring agents, suspending agents, thickening 
agents, colors, viscosity regulators, stabilizers or osmo-regulators. 
Suitable examples of liquid carriers for oral and parenteral 
administration include water (partially containing additives as above, 
e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose 
solution), alcohols (including monohydric alcohols and polyhydric 
alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated 
coconut oil and arachis oil). For parenteral administration, the carrier 
can also be an oily ester such as ethyl oleate and isopropyl myristate. 
Sterile liquid carriers are useful in sterile liquid form compositions for 
parenteral administration. The liquid carrier for pressurized compositions 
can be halogenated hydrocarbon or other pharmaceutically acceptable 
propellant. 
Liquid pharmaceutical compositions which are sterile solutions or 
suspensions can be utilized by, for example, intramuscular, 
intraperitoneal or subcutaneous injection. Sterile solutions can also be 
administered intravenously. The compound can also be administered orally 
either in liquid or solid composition form. 
{5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 
19-Trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 
12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 
(5H)-one may be administered rectally in the form of a conventional 
suppository. For administration by intranasal or intrabronchial inhalation 
or insuffiation, {5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 
4, 7, 19-Trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 
10, 11, 12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 
9-diepoxybenzocyclooctadecen-3 (5H)-one may be formulated into an aqueous 
or partially aqueous solution, which can then be utilized in the form of 
an aerosol. {5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 
7, 19-Trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 
11, 12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 
9-diepoxybenzocyclooctadecen-3 (5H)-one may also be administered 
transdermally through the use of a transdermal patch containing the active 
compound and a carrier that is inert to the active compound, is non toxic 
to the skin, and allows delivery of the agent for systemic absorption into 
the blood stream via the skin. The carrier may take any number of forms 
such as creams and ointments, pastes, gels, and occlusive devices. The 
creams and ointments may be viscous liquid or semisolid emulsions of 
either the oil-in-water or water-in-oil type. Pastes comprised of 
absorptive powders dispersed in petroleum or hydrophilic petroleum 
containing the active ingredient may also be suitable. A variety of 
occlusive devices may be used to release the active ingredient into the 
blood stream such as a semipermeable membrane coveting a reservoir 
containing the active ingredient with or without a carrier, or a matrix 
containing the active ingredient. Other occlusive devices are known in the 
literature. 
Preferably, the pharmaceutical composition is in unit dosage form, e.g. as 
tablets or capsules. In such form, the composition is sub-divided in unit 
dose containing appropriate quantities of the active ingredient; the unit 
dosage forms can be packaged compositions, for example, packeted powders, 
vials, ampoules, prefilled syringes or sachets containing liquids. The 
unit dosage form can be, for example, a capsule or tablet itself, or it 
can be the appropriate number of any such compositions in package form. 
The dosage to be used in the treatment must be subjectively determined by 
the attending physician. 
The dosage requirements vary with the particular compositions employed, the 
route of administration, the severity of the symptoms presented and the 
particular subject being treated. Based on the results obtained in the 
standard pharmacological test procedures, projected daily intravenous 
dosages of {5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 
19-trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 
12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 
(5H)-one, would be 0.1-10 mg/kg. Projected daily oral dosages of 
{5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 
19-trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 
12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 
(5H)-one would be 1-25 mg/kg. 
Treatment will generally be initiated with small dosages less than the 
optimum dose of the compound. Thereafter the dosage is increased until the 
optimum effect under the circumstances is reached; precise dosages for 
oral, parenteral, intranasal, intrabronchial, transdermal, or rectal 
administration will be determined by the administering physician based on 
experience with the individual subject treated. In general, {5R*-6R*, 
7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 19-trihydroxy-2, 6, 
8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 
19-dodecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 (5H)-one is most 
desirably administered at a concentration that will generally afford 
effective results without causing any harmful or deleterious side effects.

EXAMPLE 1 
Isolation and Fermentation of NRRL 21370 
Streptomyces spp. NRRL 21370, was isolated from a soil sample collected 
from the side of a highway in North Miami, Fla., and samples thereof have 
been deposited with the ARS Patent Culture Collection at the USDA/ARS 
Northern Regional Research Center. The original soil sample was air dried 
at room temperature and then heat treated at 120.degree. C. for 1 hour. 
The soil sample was applied to Actinomycetes Isolation Agar containing 50 
pg/ml nystatin, 50 .mu.g/ml cyclohexamide, and 10 .mu.g/ml rifampicin. The 
strain was identified to be a member of the genus Streptomyces and 
produces spore chains which are large with spacers and of the type 
Spirales. 
Strain NRRL 21370 was grown and maintained on Starch Casein Agar which 
contains: 
______________________________________ 
Hayashi Soluble Starch (Hayashi Pure Chemical 
10 g/liter 
Industries Ltd. Japan) 
Casein (Sigma) 1 g/liter 
Potassium Phosphate, Dibasic (Mallinckrodt) 
0.5 g/liter 
MgSO.sub.4.7H.sub.2 O (Sigma) 
0.5 g/liter 
Yeast Extract (Difco) 1 g/liter 
______________________________________ 
Grown on this medium the spore mass is gray while the reverse of the 
mycelium is tan to maroon with a brown diffusible pigment. 
The best utilized carbon sources were glucose, rhamnose, fructose, 
raffinose, mannitol, and sucrose; and the less well utilized carbon 
sources were xylose and inositol. Carbon sources arabinose and cellulose 
were not utilized. The microorganism is NaCl tolerant up to and including 
10%. 
NRRL 21370 was grown on Starch Casein Agar demonstrated good growth after 
14 days at 28.degree. C. The plates were then scraped and the mycelia and 
spore mass were placed in a tube containing glass beads plus a storage 
solution of 5% lactose and 10% glycerol. The tubes were then vortexed 
until a turbid solution was obtained. Fifteen milliliters of the turbid 
solution was pipetted into 2-two liter flasks containing 400 mls of the 
seed media which contained: 
______________________________________ 
Glucose 20 g/liter 
Pharmamedia (Trader's Protein) 
25 g/liter 
(NH.sub.4).sub.2 SO.sub.4 
3 g/liter 
ZnSO.sub.4.7H.sub.2 O 0.03 g/liter 
CaCO.sub.3 4 g/liter 
Yeast Extract (Difco) 5 g/liter 
______________________________________ 
These flasks were shaken at 250 RPM at 28.degree. C. for 48 hours. The seed 
material was then transferred to New Brunswick Scientific Micros 30 
fermenters. The fermenter parameters included 20 liters of fermentation 
media, 28.degree. C., 500 RPM, 10 liters air per minute, 5 psi above 
atmospheric, and 2 ml/L Polypropylene glycol 2000 as an antifoaming agent. 
Fermentation was conducted for 6 days followed by harvesting of the entire 
fermentation to provide NRRL 21370. The fermentation medium contained: 
______________________________________ 
Alpha Lactose l0 g/liter 
Soluble Starch 30 g/liter 
Fishmeal (Sigma) l0 g/liter 
CaSO.sub.4.2H.sub.2 O 6 g/liter 
CaCO.sub.3 5 g/liter 
______________________________________ 
EXAMPLE 2 
{5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 
19-Trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 
12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 
(5H)-one 
Procedure A 
The fermentation mixture was filtered through cheese cloth by suction 
filtration. The filtrate was extracted three times with 0.25 v/v ethyl 
acetate. The combined extracts were evaporated under reduced pressure to 
dryness. The mycelium was briefly homogenized and then extracted three 
times with 0.4 v/v of ethyl acetate. The ethyl acetate layers were 
combined and the solvent removed by rotary evaporation. The oily residues 
resulting from the extraction of both the filtrate and mycelium were 
combined and dried on a vacuum pump overnight. 
The crude extract was subjected to countercurrent partition chromatography 
(CPC) fractionation on a high speed countercurrent chromatograph 
containing a "Tripple" coil column (PC, Inc.). A 1:3:3:3 v/v/v/v of 
n-hexane, ethyl acetate, methanol and water was mixed and allowed to 
settle overnight. The lower layer was pumped into the CPC column as the 
stationary phase. The upper layer was used as the mobile phase. After 2 
hours, the lower and upper layer were switched. The CPC run was completed 
after 4 hours. The fractions were tested in the in vitro calcitonin 
receptor binding standard pharmacological procedure (described above) and 
the crude desired product was found to elute from the CPC between 30 and 
33 minutes. The active fractions were pooled and evaporated under reduced 
pressure to dryness. 
The pooled CPC fractions were dissolved in 1 volume of 10% aqueous methanol 
and extracted three times with 1 volume of n-hexane. The desired product 
remained present in the aqueous methanol layer. The aqueous methanol 
layers were combined and evaporated under reduced pressure to dryness. 
The aqueous methanol portion obtained above was subjected to two subsequent 
HPLC fractionation's (Waters HPLC with Millennium software) using the 
following conditions: 
a) 81.5 mg of material dissolved in 5 ml of 1 v/v acetonitrile/water was 
injected into a preparative C18-column (40.times.100 mm, Deltapak, 15 
micron) with a flow rate of 20 ml/minute for each separation run and 
monitored by photodiode array (PDA) at 314 nm. Separation conditions were 
isocratic using 2 volumes of water to 3 volumes of acetontrile over 100 
minutes. The desired material eluted between 75 and 90 minutes. The 
desired fractions were combined and evaporated to under reduced pressure 
to dryness. 
b) 18.3 mg of crude material from the preparative column above dissolved in 
0.08 ml of DMSO was injected into two semipreparative C18-columns in 
series (25.times.100 mm, Nova Pak, 10 micron) with a flow rate of 10 
ml/minute for each separation run and monitored by PDA at 270 nm. 
Separation conditions were isocratic using 1 volume of water to 4 volumes 
of acetonitrile over 27 minutes. The desired fraction eluted between 22 
and 25 minutes. The desired fractions were combined and evaporated under 
reduced pressure dryness. 
The material obtained from semipreparative chromatography was partitioned 
between 1 volume of chloroform and 1 volume of 50% aqueous methanol. The 
desired product remained with the chloroform layer. The chloroform layer 
was evaporated and purified product was precipitated with acetonitrile 
m.p.: greater than 195.degree. C. (dec). The optical rotation of the 
purified compound was determined to be alphaD: -97.4 (c=0.195,acetone). 
The following represent modifications of Procedure A, with procedure B 
being preferred for scale up. 
Procedure B 
The initial crude ethyl acetate extract was obtained as in Procedure A. 
This material was partitioned between 1 volume of n-hexane and 1 volume of 
10% aqueous methanol. The desired product remained with the aqueous 
methanol layer. This material was subjected to CPC fractionation under the 
same conditions as described in Procedure A, and the desired fractions 
were combined and evaporated under reduced pressure. 
This oily residue was then subjected to column chromatography on Sephadex 
LH20 (130 g dry weight) in ethanol (315 ml). The product, identified by a 
characteristic yellow color, was eluted with ethanol. The methanol eluate 
was reduced to a small volume and the desired product was precipitated 
with ethanol. An analytical sample was prepared by crystallization from 
ethanol. 
Procedure C 
The initial crude ethyl acetate extract was obtained as described in 
Procedure A. This material was partitioned between 1 volume n-hexane and 1 
volume of 10% aqueous methanol, and followed by subsequent CPC 
fractionation under the same conditions as described in Procedure A. This 
produced an oily residue as described in Procedure B. 
The resulting material was precipitated with acetonitrile. An analytical 
sample was prepared by crystallization from ethanol. 
The following tables show the proton and carbon magnetic resonances for 
{5R*-6R*, 7S*, 8R*, 9R*, 12S*, 16S*, 18S*, 19S*, (13E)!} 4, 7, 
19-trihydroxy-2, 6, 8, 12, 12, 14, 16, 18-heptamethyl-6, 7, 8, 9, 10, 11, 
12, 15, 16, 17, 18, 19-dodecahydro-1, 19:5, 9-diepoxybenzocyclooctadecen-3 
(5H)-one. The resonance assignments are based on the numbering system in 
the figure below. 
__________________________________________________________________________ 
##STR3## 
H-1 NMR Chemical Shifts 
Proton* 
.delta..sup.# 
Multiplicity.sup.% 
.delta..sup.$ 
Multiplicity.sup.% 
__________________________________________________________________________ 
C-40H 
8.078 
s 8.145 
s 
H-5 4.335 
d, J=10.5 4.512 
d, J=10.5 
H-6 1.680 
tq, J=10.5,6.6 
2.2 obscured 
H-7 3.541 
dt, J=10.5, 4.8 
3.610 
dt, J=10.3, 4.4 
C-7 OH 
3.920 
d, J=4.8 3.820 
d, J=4.4 
H-8 1.857 
qdd, J=6.8, 4.8, 2.6 
2.2 obscured 
H-9 3.505 
ddd, J=10.8, 2.6, 1.1 
3.434 
ddd, J=10.9, 2.0, 1.0 
H-10a 
1.55 
m 1.5 obscured 
H-10b 
1.2 m 1.11 
tdd, J=12.8, 5.3, 1.0 
H-11a 
1.3 m 1.39 
tdd, J=12.8, 11.4, 2.6 
H-11b 
1.3 m 1.27 
tdd, J=12.8, 5.3, 2.4 
H-12 2.322 
tq, J=9.9, 6.6 
2.324 
ddqd, J=11.4, 9.8, 6.5, 2.4 
H-13 4.614 
d, J=9.9 4.687 
d, J=9.8 
H-15a 
2.101 
dm, J=17.2, unresolved 
2.068 
bd, J=17.1 
H-15b 
1.666 
dd, J=17.2, 2.0 
1.5 obscured 
H-16 1.950 
dqdt, J=11.1, 6.5, 4.0, 2.0 
2.2 obscured 
H-17a 
1.65 
m 1.703 
td, J=12.8, 2.0 
H-17b 
1.424 
ddd, J=12.9, 11.1, 3.2 
1.5 obscured 
H-18 2.390 
dqd, J=12.9, 6.7, 3.2 
2.537 
dqd, J=12.8, 6.7, 3.2 
C-19-OH 
6.499 
s 6.610 
s 
H-20 7.165 
s 7.350 
s 
CH.sub.3 -23 
1.817 
s 1.985 
s 
CH.sub.3 -24 
0.866 
d, J=6.6 1.058 
d, J=6.6 
CH.sub.3 -25 
0.923 
d, J=6.8 0.962 
d, J=6.5 
CH.sub.3 -26 
0.845 
d, J=6.6 0.933 
d, J=6.5 
CH.sub.3 -27 
1.585 
bs 1.577 
bs 
CH.sub.3 -28 
0.917 
d, J=6.5 1.034 
d, J=6.9 
CH.sub.3 -29 
0.688 
d, J=6.7 0.752 
d, J=6.7 
__________________________________________________________________________ 
*See Figure for numbering system used 
.sup.# Solution in Acetone-d6; Chemical shifts are reported relative to 
TMS and were 
measured relative to internal Acetone-d5 = 2.02 ppm 
.sup.% s = singlet, d = doublet, t = triplet, q = quartet, b = broad, m = 
multiplet; Coupling 
constants, J, are in Hz and are the average of all multiplets in which 
they are measured. 
.sup.$ Solution in Benzene-d6: Acetone-d6 1:1; Chemical shifts are 
reported relative to TMS 
and were measured relative to internal Benzene-d5 = 7.27 ppm 
C-13 NMR Chemical Shifts 
Carbon No.* 
Chemical Shift.sup.# 
Chemical Shift.sup.$ 
APT results.sup.% 
__________________________________________________________________________ 
C-1 168.60 168.67 np-sp.sup.2 -C 
C-2 119.14 119.25 np-sp.sup.2 -C 
C-3 182.13 182.26 np-sp.sup.2 -C 
C-4 146.85 146.83 np-sp.sup.2 -C 
C-4a 130.22 130.30 np-sp.sup.2 -C 
C-5 77.73 77.82 CH 
C-6 38.17 38.28 CH 
C-7 78.72 78.67 CH 
C-8 40.85 40.83 CH 
C-9 76.32 76.44 CH 
C-10 33.46 33.42 CH.sub.2 
C-11 35.87 35.79 CH.sub.2 
C-12 33.62 33.65 CH 
C-13 129.88 129.88 sp.sup.2 -CH 
C-14 132.12 132.01 np-sp.sup.2 -C 
C-15 46.09 46.02 CH.sub.2 
C-16 26.50 26.45 CH 
C-17 39.75 39.67 CH.sub.2 
C-18 41.47 41.46 CH 
C-19 104.26 104.35 np-sp.sup.3 -C 
C-20 141.33 141.60 sp.sup.2 -CH 
C-20a 110.99 110.94 np-sp.sup.2 -C 
C-23 7.62 7.93 CH.sub.3 
C-24 13.29 13.49 CH.sub.3 
C-25 19.92 19.87 CH.sub.3 
C-26 22.70 22.90 CH.sub.3 
C-27 19.72 20.01 CH.sub.3 
C-28 7.18 7.32 CH.sub.3 
C-29 12.66 12.84 CH.sub.3 
__________________________________________________________________________ 
*See Figure for numbering system used 
.sup.# Solution in Acetoned6; Chemical shifts are reported relative to TM 
and were measured relative to internal Acetoned6 methyl carbon = 29.8 ppm 
.sup.% APT = attached proton test; np = nonprotonated 
.sup.$ Solution in Benzened6: Acetoned6 1:1; Chemical shifts are reported 
relative to TMS and were measured relative to internal Acetoned6 methyl 
carbon = 29.8 ppm 
Mass spectra were run on a Finnigan MAT 8230 and were obtained in the 
ei(electron impact) mode at 70 eV. HRMS (high resolution mass spectra) 
were performed using PKF (perfluorokerosene) as an internal standard. MS 
m/z (rel intensity) 486(M.sup.+, 100), 468(M-28, 86), 220(M-266, 96), 
193(M-293, 80); HRMS m/z 486.297879(M.sup.+ calcd for C.sub.29 H.sub.42 
O.sub.6 486.297879).