Biologically pure culture of Cytospora useful as angiotensin II antagonist

An organism of the Cytospora species produces substituted hexahydrobenzopyran compounds of the Formulas A, B, or C which are useful as angiotensin II antagonists: ##STR1##

BACKGROUND OF THE INVENTION 
The renin-angiotensin system (RAS) plays a central role in the regulation 
of normal blood pressure and seems to be critically involved in 
hypertension development and maintenance as well as congestive heart 
failure. Angiotensin II (A II), is an octapeptide hormone produced mainly 
in the blood during the cleavage of angiotensin I by angiotensin 
converting enzyme (ACE) localized on the endothelium of blood vessels of 
lung, kidney, and many other organs. It is the end product of the 
renin-angiotensin system (RAS) and is a powerful arterial vasoconstrictor 
that exerts its action by interacting with specific receptors present on 
cell membranes. One of the possible modes of controlling the RAS is 
angiotensin II receptor antagonism. Thus, new angiotensin II receptor 
antagonists and methods of producing them are highly desirable. 
SUMMARY OF THE INVENTION 
It has now been found that a novel microorganism which may be isolated from 
a bark of a tree produces an aerobic fermentation of an assimilable carbon 
source in a suitable nutrient medium, three novel hexahydropyran 
compounds, Compounds A, B, and C (of formulas): 
##STR2## 
which are useful as angiotensin II receptor antagonists. The novel 
compounds are described and claimed in concurrently filed copending 
application Ser. No. 07/961,957. The organism a yet unidentifiable species 
of the fungal genus Cytospora, has been deposited under the Budapest 
Treaty with the American Type Culture Collection and has been assigned 
accession number ATCC 74091. 
DETAILED DESCRIPTION OF THE INVENTION 
Strain Isolation 
The producing organism of the present invention was isolated from a sample 
of bark of yellow birch Betula alleganiensis, collected in Randolph 
County, W. Va. To isolate this organism, bark discs were removed from the 
tree with a leather punch. Discs were approximately 1 cm in diameter and 
0.3-1.0 cm thick and included an entire bark cross-section along with the 
vascular cambium, and sometimes a veneer of the outer xylem. Discs were 
soaked in 95% ethanol for 1 minute, 66% household bleach (aqueous 3.3% 
NaClO solution) for 5 minutes, and rinsed with 95% ethanol again for 0.5 
minute prior to application to isolation media. Bark discs were applied 
(outer side down) to an agar medium (BBL Laboratories Mycosel Agar, 
Cockeysville, Md., consisting of phytone 10 g, glucose 10 g cycloheximide 
0.4 g, chloramphenicol 50 mg; in 1 L distilled water) in 100 mm diameter 
quartered plastic petri dishes. Petri dishes were incubated at 24.degree. 
C., and inspected for up to one month for the development of fungal 
colonies on bark discs and the agar. Hyphal fragments of colonies emerging 
from bark discs were transferred to slants of malt yeast extract agar. 
Colonial Morphology in Agar Culture 
Colonies on oatmeal agar (Difco Laboratories), 14 days, 20.degree. C., 12 
hour photoperiod (12/12 light/dark): Attaining 66-70 mm in diameter, with 
little development of aerial mycelium, appressed to submerged, pruinose or 
slightly floccose around inoculation point, dull, hyaline to pale olive 
when young, Buffy Olive, Light Yellowish Olive, soon dark olive to dark 
olivaceous black, Olive, Dark Greenish Olive, Deep Olive, Dark Grayish 
Olive, Dark Olive (capitalized color names from Ridgway, R., 1912, Color 
Standards and Nomenclature, Washington, D.C.) with mottled pigment 
development over outer third of colony, with scattered stromatic 
conidiomatal pustules at first white, but soon pale grayish olive to 
olivaceous brown, margin submerged, irregular. 
Colonies on Emerson's YpSS (Difco Laboratories), 14 days, 20.degree. C., 12 
hour photoperiod: Attaining 35-45 mm in diameter, dull, aerial mycelium 
pruinose to downy, with scattered stromata, hyaline at margin but soon 
pale olive to olivaceous gray, Dark Olive-Buff, Citrine Drab, finally dark 
olive, Dark Greenish Olive, Dark Grayish Olive, margin minutely feathery 
or fimbriate. Colonies on malt-yeast extract agar (Difco Laboratories), 14 
days, 20.degree. C., 12 hour photoperiod: Attaining 70-75 mm in diameter, 
shiny to moist, aerial mycelium appressed or minutely floccose around 
inoculation point, hyaline to greenish yellow, Yellowish Citrine, 
Serpentine Green, Dull Citrine, soon grayish olive to dark olive, Deep 
Grayish Olive, Dark Grayish Olive, Dark Olive, with scattered stromatic 
pustules in older portions, developing long irregular plumose branches 
with submerged, irregular margins. 
Conidiomata scattered across colony surface, up to 1.5 mm wide, up to 2.5 
mm tall, tough, stromatic, with base embedded in agar, pulvinate or 
hemispherical when young, becoming short cylindrical in age, with rounded 
apex, sometimes with subapical constrictions, with surface minutely 
pubescent, dehiscing irregularly in age to yield a gelatinous spore mass 
or spore horns, with outer cortex a textura intricata, golden to dark 
brown, containing a irregularly convoluted conidial chamber, which 
overlies or surrounds an inner cortex, with inner cortex a textura 
intricata to textura angularis, dark brown to black. Conidiophores 
arranged in a dense palisade layer, lining the interal stomatic cavity, 
arising from broad, short cylindrical to subglobose hyphae, branching at 
3-5 levels, acropleurogenous or not, thin-walled, hyaline, smooth 
Conidiogenous cells enteroblastic, phialidic, cylindrical to awl-shaped, 
tapered to a narrow apex, 5-12.times.1-3 .mu.m, with minute pore at 
conidiogenous locus. Conidia 3-5.times.1-1.5 .mu.m cylindrical, narrowly 
ellipsoidal, or allantoid, exuded from conidiomata in a golden brown to 
yellowish olive gelatinous mass or in spore horns. Hyphae septate, 
branching hyaline to blackish brown, occasionally incrusted in age, up to 
12 .mu.m in diameter. 
This strain can be assigned to genus Cytospora (Coelomycetes) because of 
its combination of the following characteristics: tough stromatic 
conidiomata; the production of conidiophores in a well-defined palisade 
layer within a convoluted, multilocular chamber, highly branched 
conidiophores; enteroblastic conidiogenesis; small; hyaline, more-or-less 
allantoid conidia; and dark mycelial colors. Members of the genus 
Cytospora are typical inhabitants of stems of woody plants. Several 
species are associated with canker and dieback diseases of woody stems. 
Cytospora species are often the conidial states in the life cycles of 
Valsa and Leucostoma species (Ascomycetes, Diaporthales), but no sexual 
state of this strain observed in culture. The genus is in need of 
extensive revisionary studies. About 400 species have been named and 
nearly all species description are based on anatomical characteristics as 
they occur fruiting on host tissues (Sutton, B. C. (1980) The 
Coelomycetes, Commonwealth Mycological Ins. Kew; Spielman, L. J. (1985) "A 
Monograph of Valsa on Hardwoods in North America", Can. J. of Botany, 63: 
1355-1378). The stromatic tissues, conidiophores, and conidia of ATCC 
74091 are similar to many of the common Cystospora anamorphs of Valsa 
species described by Spielman, (1985). Because no workable identification 
system currently exists for these fungi in culture, and because this 
strain was derived from vegetative growth from bark rather than from 
spores produced in an identifiable fruiting structure on a host, it is 
impossible to determine the name of this Cytospora species. 
Use of Cytospora sp. in the Production of the Hexahydrobenzopyran Compounds 
Fermentation Conditions 
Compounds of formulas A, B, and C are produced during the aerobic 
fermentation of suitable aqueous nutrient media under controlled 
conditions via inoculation with a culture of the organism ATCC 74091. The 
media contain sources of assimilable carbon, nitrogen, and inorganic 
salts. 
In general, carbohydrates (for example, glucose, fructose, maltose, xylose, 
and the like) can be used either alone or in combination as sources of 
assimilable carbon in the nutrient medium. The exact quantity of the 
carbohydrate source or sources utilized in the medium depends in part upon 
the other ingredients of the medium but, in general, the amount of 
carbohydrate usually varies between about 2% to 5% by weight of the 
medium. These carbon sources may be used individually or combined in the 
medium. 
Generally, many proteinaceous materials may be used as nitrogen sources for 
the fermentation process. Suitable nitrogen sources include, for example, 
yeast hydrolysates, primary yeast, soybean meal, cottonseed flour, 
hydrolysates of casein, cornsteep liquor, distiller's solubles or tomato 
paste and the like. The sources of nitrogen, either alone or in 
combination, are used in amounts preferably ranging from about 0.05% to 
0.2% by weight of the aqueous medium. 
Among the nutrient inorganic salts which can be incorporated in the culture 
media are the customary salts capable of yielding sodium, potassium, 
ammonium, calcium, phosphate, sulfate, chloride, carbonate, and the like 
ions. Also included are trace elements such as cobalt, manganese, copper, 
molybdenun, zinc, boron, iron and magnesium. 
It should be noted that the nutrient media described herein are merely 
illustrative of the wide variety of media which may be employed, and are 
not intended to be limiting. 
The fermentation is carried out at temperatures ranging from about 
25.degree. C. to 35.degree. C.; however, for optimum results it is 
preferably to conduct the fermentation at temperatures of from about 
25.degree. C. to 32.degree. C. The pH of the nutrient media for growing 
the ATCC 74091 culture and producing Compounds A, B, and C can vary from 
about 6 to 8. 
Although these three compounds are produced by both surface and submerged 
culture, it is preferred to carry out the fermentation in the submerged 
state. 
A small scale fermentation is conveniently carried out by inoculating a 
suitable nutrient medium with the culture and, after transfer to a 
production medium, permitting the fermentation to proceed at a constant 
temperature of about 25.degree. C. on a shaker for up to 25 days. 
The fermentation is initiated in a sterilized flask of medium via one or 
more stages of seed development. The nutrient medium for the seed stage 
may be any suitable combination of carbon and nitrogen sources, however, 
the preferred carbon source is glucose or hydrolyzed starch. The seed 
flask is shaken in a constant temperature chamber at about 28.degree. C. 
for 2-4 days, or until growth is satisfactory, and some of the resulting 
growth is used to inoculate either a second stage seed or the production 
medium. Intermediate stage seed flasks, when used, are developed in 
essentially the same manner; that is, part of the contents of the flask 
from the last seed stage are used to inoculate the production medium. The 
inoculated flask are shaken at a constant temperature for several days, 
and at the end of the incubation period the contents of the flasks are 
recovered by precipitation with a suitable alcohol such as isopropanol. 
For large scale work, it is preferable to conduct the fermentation in 
suitable tanks provided with an agitator and a means of aerating the 
fermentation medium. According to this method, the nutrient medium is made 
up in the tank and sterilized by heating at temperatures of up to about 
121.degree. C. Upon cooling, the sterilized medium is inoculated with a 
previously grown seed of the producing culture, and the fermentation is 
permitted to proceed for a period of time as, for example, from 2 to 4 
days while agitating and/or aerating the nutrient medium and maintaining 
the temperature at about 28.degree. C. This method of producing Compounds 
A, B, and C is particularly suited for the preparation of large 
quantities. 
The product is recovered from the fermentation broth by extraction with 
equal volumes of solvents, generally methyl ethyl ketone, n-butanol and 
methanol. The extract is generally filtered to remove mycelia and then 
evaporated to dryness and reconstituted in solvent combinations such as 
1:1 methylene chloride/methanol and then isolated by several stage 
fractionation by thin layer chromatography (TLC) with methylene 
chloride/methanol and final purification and isolation on preparative HPLC 
and collecting bioactive fractions. 
The substituted hexahydrobenzopyran compounds of the formulas A, B, or C, 
which are angiotensin II antagonists and thus useful in the treatment of 
hypertension and congestive heart failure are also adapted to be used as 
ocular antihypertensives. 
Additionally, the novel compounds may be used in pharmaceutically 
acceptable compositions both as the sole therapeutically active ingredient 
and in combination with diuretics and other antihypertensive agents, 
including beta blockers, angiotensin converting enzyme inhibitors, calcium 
channel blockers or a combination thereof. 
The compounds also have central nervous system (CNS) activity. They are 
useful in the treatment of cognitive dysfunctions including Alzheimer's 
disease, amnesia and senile dementia. These compounds also have anxiolytic 
and antidepressant properties and are therefore, adapted to be employed 
for the relief of symptoms of anxiety and tension and in the treatment of 
patients with depressed or dysphoric mental states. 
In addition, these compounds exhibit antidopaminergic properties and thus 
may be used to treat disorders that involve dopamine dysfunction such as 
schizophrenia. The compounds are especially useful in the treatment of 
these conditions in patients who are also hypertensive or have a 
congestive heart failure condition. 
Angiotensin II (AII) is a powerful arterial vasoconstrictor, and it exerts 
its action by interacting with specific receptors present on cell 
membranes. The compounds produced by Cytospora sp. are competitive 
antagonists of AII at receptor sites. The usefulness of compounds as AII 
antagonists may be determined from the results of certain receptor binding 
assays. The initial assay is that using rat adrenal glands and the results 
of this assay are described herein. 
Receptor Assay using Rat Adrenal Gland Preparation 
Adrenal membranes for the receptor assay were readied first by placing 
cleaned, whole adrenal glands in microcentrifuge tubes (20 glands per 
tube), and freezing and storing under liquid nitrogen. As needed, a 
microcentrifuge tube of adrenal glands was homogenized in 20 ml 50 mM tris 
(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) pH 7.7 and 
centrifuged at 20,000 rpm for 15 minutes at 4.degree. C. The pellet was 
then resuspended in a buffer [120 mM NaCl, 10 mM Na.sub.2 HPO.sub.4, 5 mM 
Na.sub.2 EDTA and 0.1 mM phenylmethanesulfonyl fluoride (PMSF)] and 
centrifuged at 20,000 rpm for 15 minutes at 4.degree. C. The last step was 
then repeated. The resulting pellet was resuspended in the same buffer and 
a 0.5 ml aliquot dispensed per microcentrifuge tube and frozen. 
For the assay, the adrenal membrane preparation in one vial was resuspended 
in 30 ml of 100 mM Tris-HCl pH 7.4 with 5 mM MgCl.sub.2, 0.2% bovine serum 
albumin and 0.2 mg/ml bacitracin. 200 microliters of this preparation was 
added to each tube containing 45 microliters of .sup.125 I-Angiotensin II 
to which were added 5 microliters of sample and 10 microliters of 
2-butyl-4-chloro-1-((2'-(1H-tetrazol-5-yl)-1,1'-biphenyl)-4-yl)methyl)1H-i 
midazole-5-methanol monohydrochloride as known inhibitor of Site 1 and 
1-(3-methyl-4-methoxyphenyl)methyl-5-diphenylacetyl-4,5,6,7-tetrahydro-1H- 
imidazo(4,5-c)pyridine-6-carboxylic acid known inhibitor of Site 2. The 
tubes were then vortexed and incubated at 37.degree. C. for 90 minutes. 
After the incubation period, the membranes were collected, the reaction 
mixture aspirated, 3-4 milliliters of 50 mM Tris-HCl pH 7.4 with 0.9% NaCl 
passed through the system, the mixture then filtered and the filter 
deposited in minivials for counting. The vials are counted with a gamma 
counter for one minute and percent inhibition determined from counts/min 
(CPM) as follows: 
##EQU1## 
The IC.sub.50 is determined by plotting percent inhibition v. 
concentration of compound tested. It was found that Compound A has an 
IC.sub.50 of 0.5-1 .mu.g/ml or 1-2 .mu.M at site 2. 
On obtaining positive results with rat adrenal gland assay, assays using 
rabbit aorta and bovine adrenal glands may be employed for further 
determinations. 
Receptor Binding Assay Using Rabbit Aortae Membrane Preparation 
Three frozen rabbit aortae (obtained from Pel-Freeze Biologicals) were 
suspended in 5 mM Tris-0.25M sucrose, pH 7.4 buffer (50 mL) homogenized, 
and then centifuged. The mixture was filtered through a cheesecloth and 
the supernatant was centrifuged for 30 minutes at 20,000 rpm at 4.degree. 
C. The pellet thus obtained was resuspended in 30 mL of 50 mM Tris-5 mM 
MgCl.sub.2 buffer containing 0.2% Bovine Serum Albumin and 0.2 mg/mL 
Bacitracin and the suspension was used for 100 assay tubes. Samples tested 
for screening were done in duplicate. To the membrane preparation (0.25 
mL) there was added .sup.125 I-Sar.sup.1 Ile.sup.8 - angiotensin II 
[obtained from New England Nuclear] (10 mL; 20,000 cpm) with or without 
the test sample and the mixture was incubated at 37.degree. C. for 90 
minutes. The mixture was then diluted with ice-cold 50 mM Tris-0.9% NaCl, 
pH 7.4 (4 mL) and filtered through a glass fiber filter (GF/B Whatman 2.4" 
diameter). The filter was soaked in scintillation cocktail (10 mL) and 
counted for radioactivity using 2660 Tricarb liquid scintillation counter 
[Packard Instruments]. The inhibitory concentration (IC.sub.50) of 
potential AII antagonist which gives 50% displacement of the total 
specifically bound .sup.125 I-Sar.sup.1 Ile.sup.8 -angiotensin II is 
presented as a measure of the efficacy of such compounds as AII 
antagonists. Compound A was found to have IC.sub.50 of only 8 .mu.g/ml (at 
site 1). 
Receptor Assay Using Bovine Aorta Preparation 
Bovine aorta assay may be carried out in the following manner. Weighed 
tissue (0.1 g is needed for 100 assay tubes) is suspended in Tris HCl (50 
mM), pH 7.7 buffer and homogenized. The homogenate is centrifuged at 
20,000 rpm for 15 minutes. Supernatant is discarded and pellets 
resuspended in buffer [Na.sub.2 HPO.sub.4 (10 mM)-NaCl (120 mM)-disodium 
EDTA (5 mM) containing phenylmethane sulfonyl fluoride (PMSF) (0.1 mM)]. 
To the membrane preparation (0.5 mL) there is added .sup.3 H-angiotensin 
II (50 mM) (10 mL) with or without the test sample and the mixture is 
incubated at 37.degree. C. for 1 hour. The mixture is then diluted with 
Tris buffer (4 mL) and filtered through a glass fiber filter (GF/B Whatman 
2.4" diameter). The filter is soaked in scintillation cocktail (10 mL) and 
counted for radioactivity using Packard 2660 Tricarb liquid scintillation 
counter. The inhibitory concentration (IC.sub.50) of potential AII 
antagonist which gives 50% displacement of the total specifically bound 
.sup.3 H-angiotensin II was presented as a measure of the efficacy of such 
compounds as AII antagonists. 
In the management of hypertension and the clinical conditions described 
herein, the hexahydropyran compounds may be utilized in compositions such 
as tablets, capsules or elixirs for oral administration, suppositories for 
rectal administration, sterile solutions or suspensions for parenteral or 
intramuscular administration, and the like. The compounds may be 
administered to patients (animals and human) in need of such treatment in 
dosages that will provide optimal pharmaceutical efficacy. Although the 
dose will vary from patient to patient depending upon the nature and 
severity of disease, the patient's weight, special diets then being 
followed by a patient, concurrent medication, and other factors which 
those skilled in the art will recognize, the dosage range will generally 
be about 1 to 1000 mg per patient per day which can be administered in 
single or multiple doses. Preferably, the dosage range will be about 2.5 
to 250 mg per patient per day; more preferably about 2.5 to 75 mg per 
patient per day. 
The hexahydropyran compounds can also be administered in combination with 
other antihypertensives and/or diuretics and/or angiotensin converting 
enzyme inhibitors and/or calcium channel blockers. For example, the 
compounds of this invention can be given in combination with such 
compounds as amiloride, atenolol, bendroflumethiazide, chlorothalidone, 
chlorothiazide, clonidine, cryptenamine acetates and cryptenamine 
tannates, deserpidine, diazoxide, guanethidene sulfate, hydralazine 
hydrochloride, hydrochlorothiazide, metolazone, metoprolol tartate, 
methylclothiazide, methyldopa, methyldopate hydrochloride, minoxidil, 
pargyline hydrochloride, polythiazide, prazosin, propranolol, rauwolfia 
serpentina, rescinnamine, reserpine, sodium nitroprusside, spironolactone, 
timolol maleate, trichlormethiazide, trimethophan camsylate, benzthiazide, 
quinethazone, ticrynafan, triamterene, acetazolamide, aminophylline, 
cyclothiazide, ethacrynic acid, furosemide, merethoxylline procaine, 
sodium ethacrynate, captopril, delapril hydrochloride, enalapril, 
enalaprilat, fosinopril sodium, lisinopril, pentopril, quinapril 
hydrochloride, ramapril, teprotide, zofenopril calcium, diflunisal, 
diltiazem, felodipine, nicardipine, nifedipine, niludipine, nimodipine, 
nisoldipine, nitrendipine, and the like, as well as admixtures and 
combinations thereof. 
Typically, the individual daily dosages for these combinations can range 
from about one-fifth of the minimally recommended clinical dosages to the 
maximum recommended levels for the entities when they are given singly. 
To illustrate these combinations, one of the hexahydropyran angiotensin II 
antagonists, effective clinically in the 2.5-250 milligrams per day range, 
can be effectively combined at levels at the 0.5-250 milligrams per day 
range with the following compounds at the indicated per day dose range: 
hydrochlorothiazide (15-200 mg), chlorothiazide (125-2000 mg), ethacrynic 
acid (15-200 mg), amiloride (5-20 mg), furosemide (5-80 mg), propranolol 
(20-480 mg), timolol maleate (5-60 mg), methyldopa (65-2000 mg), 
felodipine (5-60 mg), nifedipine (5-60 mg), and nitrendipine (5-60 mg). In 
addition, triple drug combinations of hydrochlorothiazide (15-200 mg) plus 
amiloride (5-20 mg) plus hexahydrobenzopyran angiotensin II antagonist 
(3-200 mg) or hydrochlorothiazide (15-200 mg) plus timolol maleate (5-60) 
plus a hexahydropyran angiotensin II antagonist (0.5-250 mg) or 
hydrochlorothiazide (15-200 mg) and nifedipine (5-60 mg) plus a 
hexahydropyran angiotensin II antagonist (0.5-250 mg) are effective 
combinations to control blood pressure in hypertensive patients. 
Naturally, these dose ranges can be adjusted on a unit basis as necessary 
to permit divided daily dosage and, as noted above, the dose will vary 
depending on the nature and severity of the disease, weight of patient, 
special diets and other factors. 
Typically, these combinations can be formulated into pharmaceutical 
compositions as discussed below. 
About 1 to 100 mg of compound or mixture of Compounds A, B, or C is 
compounded with a physiologically acceptable vehicle, carrier, excipient, 
binder, preservative, stabilizer, flavor, etc., in a unit dosage form as 
called for by accepted pharmaceutical practice. The amount of active 
substance in these compositions or preparations is such that a suitable 
dosage in the range indicated is obtained. 
Illustrative of the adjuvants which can be incorporated in tablets, 
capsules and the like are the following: a binder such as gum tragacanth, 
acacia, corn starch or gelatin; an excipient such as microcrystalline 
cellulose; a disintegrating agent such as corn starch, pregelatinized 
starch, alginic acid and the like; a lubricant such as magnesium stearate; 
a sweetening agent such as sucrose, lactose or saccharin; a flavoring 
agent such as peppermint, oil of wintergreen or cherry. When the dosage 
unit form is a capsule, it may contain, in addition to materials of the 
above type, a liquid carrier such as fatty oil. Various other materials 
may be present as coatings or to otherwise modify the physical form of the 
dosage unit. For instance, tablets may be coated with shellac, sugar or 
both. A syrup or elixir may contain the active compound, sucrose as a 
sweetening agent, methyl and propyl parabens as preservatives, a dye and a 
flavoring such as cherry or orange flavor. 
Sterile compositions for injection can be formulated according to 
conventional pharmaceutical practice by dissolving or suspending the 
active substance in a vehicle such as water for injection, a naturally 
occuring vegetable oil like sesame oil, coconut oil, peanut oil, 
cottonseed oil, etc., or a synthetic fatty vehicle like ethyl oleate or 
the like. Buffers, preservatives, antioxidants and the like can be 
incorporated as required. 
The hexahydrobenzopyran compounds produced by the organism of this 
invention are also useful to treat elevated intraocular pressure and can 
be administered to patients in need of such treatment with typical 
pharmaceutical formulations such as tablets, capsules, injectables, as 
well as topical ocular formulations in the form of solutions, ointments, 
inserts, gels and the like. Pharmaceutical formulations prepared to treat 
intraocular pressure would typically contain about 0.1% to 15% by weight, 
and preferably 0.5% to 2.0% by weight of a compound of this invention. 
The compounds produced by the organism of the invention are useful also in 
the management of acute and chronic congestive heart failure, in the 
treatment of secondary hyperaldosteronism, primary and secondary pulmonary 
hypertension, renal failure such as diabetic nephropathy, 
glomerulonephritis, scleroderma, and the like, renal vascular 
hypertension, left ventricular dysfunction, diabetic retinopathy, and in 
the management of vascular disorders such as migraine or Raynaud's 
disease. 
The useful central nervous system (CNS) activities of the novel 
hexahydrobenzopyran compounds are demonstrated and exemplified by the 
ensuing assays. 
Cognitive Function Assay 
The efficacy of these compounds to enhance cognitive function can be 
demonstrated in a rat passive avoidance assay in which cholinomimetics 
such as physostigmine and nootropic agents are known to be active. In this 
assay, rats are trained to inhibit their natural tendency to enter dark 
areas. The test apparatus used consists of two chambers, one of which is 
brightly illuminated and the other is dark. Rats are placed in the 
illuminated chamber and the elapsed time it takes for them to enter the 
darkened chamber is recorded. On entering the dark chamber, they receive a 
brief electric shock to the feet. The test animals are pretreated with 0.2 
mg/kg of the muscarinic antagonist scopolamine which disrupts learning or 
are treated with scopolamine and the compound which is to be tested for 
possible reversal of the scopolamine effect. Twenty-four hours later, the 
rats are returned to the illuminated chamber. Upon return to the 
illuminated chamber, normal young rats who have been subjected to this 
training and who have been treated only with control vehicle take longer 
to re-enter the dark chamber than test animals who have been exposed to 
the apparatus but who have not received a shock. Rats treated with 
scopolamine before training do not show this hesitation when tested 24 
hours later. Efficacious test compounds can overcome the disruptive effect 
on learning which scopolamine produces. Typically, hexahydrobenzopyran 
compounds should be efficacious in this passive avoidance assay in the 
dose range of from about 0.1 mg/kg to about 100 mg/kg. 
Anxiolytic Assay 
The anxiolytic activity of the hexahydrobenzopyran compounds can be 
demonstrated in a conditioned emotional response (CER) assay. Diazepam is 
a clinically useful anxiolytic which is active in this assay. In the CER 
protocol, male Sprague-Dawley rats (250-350 g) are trained to press a 
lever on a variable interval (VI) 60 second schedule for food 
reinforcement in a standard operant chamber over weekly (five days per 
week) training sessions. All animals then receive daily 20 minute 
conditioning sessions, each session partitioned into alternating 5 minute 
light (L) and 2 minute dark (D) periods in a fixed L1D1L2D2L3 sequence. 
During both periods (L or D), pressing a lever delivers food pellets on a 
VI 60 second schedule: in the dark (D), lever presses also elicit mild 
footshock (0.8 mA, 0.5 sec) on an independent shock presentation schedule 
of VI 20 seconds. Lever pressing is suppressed during the dark periods 
reflecting the formation of a conditioned emotional response (CER). 
Drug testing in this paradigm is carried out under extinction conditions. 
During extinction, animals learn that responding for food in the dark is 
no longer punished by shock. Therefore, response rates gradually increase 
in the dark periods and animals treated with an anxiolytic drug show a 
more rapid increase in response rate than vehicle treated animals. 
Hexahydrobenzopyran compounds should be efficacious in this test procedure 
in the range of from about 0.1 mg/kg to about 100 mg/kg. 
Depression Assay 
The antidepressant activity of the hexahydrobenzopyran compounds can be 
demonstrated in a tail suspension test using mice. A clinically useful 
antidepressant which serves as a positive control in this assay is 
desipramine. The method is based on the observations that a mouse 
suspended by the tail shows alternate periods of agitation and immobility 
and that antidepressants modify the balance between these two forms of 
behavior in favor of agitation. Periods of immobility in a 5 minute test 
period are recorded using a keypad linked to a microcomputer which allows 
the experimenter to assign to each animal an identity code and to measure 
latency, duration and frequency of immobile periods. The compounds should 
be efficacious in this test procedure in the range of from about 0.1 mg/kg 
to about 100 mg/kg. 
Schizophrenia Assay 
The antidopaminergic activity of the hexahydrobenzopyran compounds can be 
demonstrated in an apomorphine-induced sterotype model. A clinically 
useful antipsychotic drug that is used as a positive control in this assay 
is haloperidol. The assay method is based upon the observation that 
stimulation of the dopaminergic system in rats produces stereotyped motor 
behavior. There is a strong correlation between the effectiveness of 
classical neuroleptic drugs to block apomorphine-induced stereotypy and to 
prevent schizophrenic symptoms. Stereotyped behavior induced by 
apomorphine, with and without pretreatment with test compounds, is 
recorded using a keypad linked to a microcomputer. Hexahydrobenzopyran 
compounds should be efficacious in this assay in the range of from about 
0.1 mg/kg to about 100 mg/kg. 
In the treatment of the clinical conditions noted above, the compounds may 
be utilized in compositions such as tablets, capsules or elixirs for oral 
administration, suppositories for rectal administration, sterile solutions 
or suspensions for parenteral or intramuscular administration, and the 
like. The compounds can be administered to patients (animals and human) in 
need of such treatment in dosages that will provide optimal pharmaceutical 
efficacy. Although the dose will vary from patient to patient depending 
upon the nature and severity of disease, the patient's weight, special 
diets then being followed by a patient, concurrent medication, and other 
factors which those skilled in the art will recognize, the dosage range 
will generally be about 5 to 6000 mg. per patient per day which can be 
administered in single or multiple doses. Preferably, the dosage range 
will be about 10 to 4000 mg. per patient per day; more preferably about 
20 to 2000 mg. per patient per day. 
In order to obtain maximal enhancement of cognitive function, the 
hexahydrobenzopyran compounds may be combined with other 
cognition-enhancing agents. These include acetylcholinesterase inhibitors 
such as heptylphysostigmine and tetrahydroacridine (THA; tacrine), 
muscarinic agonists such as oxotremorine, inhibitors of 
angiotensin-converting enzyme such as octylramipril, captopril, 
ceranapril, enalapril, lisinopril, fosinopril and zofenopril, 
centrally-acting calcium channel blockers and as nimodipine, and nootropic 
agents such as piracetam. 
In order to achieve optimal anxiolytic activity, the hexahydrobenzopyran 
compounds may be combined with other anxiolytic agents such as alprazolam, 
lorazepam, diazepam, and busipirone. 
In order to achieve optimal antidepressant activity, combinations of the 
hexahydrobenzopyran compounds with other antidepressants are of use. These 
include tricyclic antidepressants such as nortriptyline, amitryptyline and 
trazodone, and monoamine oxidase inhibitors such as tranylcypromine. 
In order to obtain maximal antipsychotic activity, the hexahydrobenzopyran 
compounds may be combined with other antipsychotic agents such as 
promethazine, fluphenazine and haloperidol.

The use of the invention is further defined by reference to the following 
examples, which are intended to be illustrative and not limiting. 
EXAMPLE 1 
Fermentation Conditions 
Vegetative mycelia of the culture were prepared by inoculating 54 mL of KF 
seed medium (Table 1-1) in a 250 mL unbaffled Erlenmeyer flask with frozen 
mycelia of Cytospora sp. MF 5658, ATCC 74091. Seed flasks were incubated 
for 3 days at 28.degree. C. and 75% relative humidity on a rotary shaker 
with a 5-cm throw at 220 rpm. Two-mL portions of the 3-day culture growth 
were used to inoculate 45 mL aliquots of a liquid production medium CYG-40 
(Table 1-2) in 250 mL unbaffled Erlenmeyer flasks. Flasks were incubated 
at 25.degree. C. and 50% relative humidity with agitation at 220 rpm on a 
rotary shaker with a 5-cm throw for up to 25 days. At harvest, 
fermentation products were extracted with 45 mL of methyl ethyl ketone per 
flask at 220 rpm for 1 hour at 25.degree. C. 
TABLE 1-1 
______________________________________ 
KF Seed Medium Trace Element Mix 
Per L Per L 
______________________________________ 
Corn Steep Liquor 
5 g FeSO.sub.4.7H.sub.2 O 
1 g 
Tomato Paste 
40 g MnSO.sub.4.4H.sub.2 O 
1 g 
Oat flour 10 g CuCl.sub.2.2H.sub.2 O 
25 mg 
Glucose 10 g CaCl.sub.2 100 mg 
Trace Element Mix 
10 mL H.sub.3 BO.sub.3 
56 mg 
pH = 6.8 (NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 
19 mg 
ZnSO.sub.4.7H.sub.2 O 
200 mg 
______________________________________ 
TABLE 1-2 
______________________________________ 
Production Medium CYG40 
Component Conc. (g/L) 
______________________________________ 
Corn Meal 50.0 
Yeast Extract 1.0 
Glucose 40.0 
______________________________________ 
EXAMPLE 2 
Isolation of Compound A 
A fifteen mL fermentation sample, from fermentation carried out as 
described in Example 1, was extracted, in succession, with equal volumes 
of methyl ethyl ketone, n-butanol, and methanol. The methyl ethyl ketone 
extract was evaporated to dryness, reconstituted in 1 mL of methylene 
chloride/methanol, 1:1, and fractionated by preparative thin layer 
chromatography (TLC) on a 20.times.20 cm silica gel 60 plate (0.25 cm 
layer thickness from E. Merck) and eluted with methylene 
chloride/methanol, 92:8. 
The single bioactive fraction thus obtained was further processed by 
preparative TLC on the same type of plate, this time using methylene 
chloride/methanol, 96:4. 
Final purification and isolation of Compound A was achieved by 
semi-preparative high performance liquid chromatography (HPLC) on a 
(Whatman) TISIL-5 ODS-3 column (4.6 mm.times.25 cm) maintained at 
40.degree. C. and eluted at 1 mL/minute with 40% aqueous acetonitrile. 
After bioassay, the active fractions were evaporated to dryness under 
vacuum to yield 2 mg of Compound A whose homogeneity was ascertained by 
proton nuclear magnetic resonance (.sup.1 H NMR), by TLC in several 
systems, and by analytical HPLC [k'=4.25, TISIL-5 ODS-3 column (4.6 
mm.times.25 cm) maintained at 40.degree. C. and eluted at 1 mL/minute with 
acetonitrile/water, 1:1] 
EXAMPLE 3 
Isolation of Compounds A, B, and C 
The presence of the co-produced minor analogs (Compounds B and C) was 
determined when purification of the major component A was carried out in a 
manner described in Example 1 on a larger scale. These analogs were not 
present in sufficient amounts to be readily identified by routine 
bioassay; rather they were recognized by their physico-chemical 
similarities with compound A. 
Accordingly, 1.4 liters of fermentation broth, prepared as detailed in 
Example 1, were extracted with an equal volume of methyl ethyl ketone. 
Compound A was present in the extract at a concentration of 175 mg/L, as 
determined by high performance liquid chromatographic (HPLC) measurements. 
The fermentation products are recovered and separated to obtain purified 
products by first extracting from the fermentation medium and thereafter 
employing HPLC and gel filtration techniques. 
Generally, the crude methyl ethyl ketone extract is evaporated under 
vacuum, reconstituted in 50 ml of methylene chloride and crudely 
fractionated on a column of silica gel (E. M. silica gel 60, 250-400 mesh) 
packed and equilibrated in methylene chloride and eluted with methylene 
chloride containing increasing amounts of methanol. Fractions containing 
Compound A are then pooled, again evaporated, and further fractionated on 
a 100 mL silica gel column packed in methylene chloride/ethyl acetate, 
90:10, and washed with the same solvent mixture, containing increasing 
concentrations of ethyl acetate. 
Examination of fractions from the second silica gel column by HPLC reveals 
the presence of the major component in early eluting volumes of methylene 
chloride/ethyl acetate, 88:12. These are evaporated down, redissolved in 
methanol and further processed by gel filtration on Pharmacia SEPHADEX 
LH-20 gel (150 mL column, eluted with methanol, volume of elution 0.7-0.8 
column volumes) and preparative HPLC [Whatman TISIL-10 ODS-3 column (20 
mm.times.25 cm) maintained at room temperature and eluted at 8 mL/minute 
with 40% aqueous acetonitrile] to produce pure compound A at k'=6.4. 
Later-eluting volumes from the second silica gel column, corresponding to 
methylene chloride/ethyl acetate, 1:2, washings, recognized as containing 
components structurally related to the main compound by the similarities 
in ultraviolet absorption spectra and color reactions on TLC plates. The 
appropriate fractions were worked up by gel filtration on SEPHADEX LH-20 
as detailed for compound A (same volume of elution). Final separation and 
purification of these minor analogs was achieved by preparative HPLC 
[TISIL-10 ODS-3 column (20 mm.times.25 cm) maintained at room 
temperature and eluted at 8 mL/minutes as follows: with 20% aqueous 
acetonitrile for 60 minutes, then with a 40 minute gradient from 20% to 
40% aqueous acetonitrile]. The minor analogs were obtained in pure form 
after 50 minutes (Compound B) and 80 minutes (Compound C) of elution. 
Homogeneity of the preparations was ascertained, after evaporation of 
solvents, by proton NMR measurements, by TLC in several systems and by 
analytical HPLC [TISIL-5 ODS-3 column (4.6 mm.times.25 cm) maintained 
at 40.degree. C. and eluted at 1 mL/minute with acetonitrile/water, k' for 
compound A: 4:6; k' for compound B: 2.4; k' for compound C: 2.91]. 
EXAMPLE 4 
Mass Spectral Characterization 
Mass spectral data were acquired for the structural analysis of Compounds 
A, B, and C on Finnigan-MAT models MAT212 and TSQ70B mass spectrometers. 
MAT212: Electron Impact (EI) mode at 90 eV. Exact mass measurements were 
performed at high resolution (HR EI) using perfluorokerosene (PFK) as an 
internal standard. 
TSQ70B: EL mode at 70 eV. Fast Atom Bombardment (FAB) mode (positive ion) 
employing as matrices 5:1 dithiothreitol/dithioerythritol (DTT/DTE), doped 
with lithium acetate and undoped. Trimethylsilyl (TMS) derivatives were 
prepared using a 1:1 mixture of bis(trimethylsilyl)trifluoracetamide 
(BSTFA)/pyridine at 50.degree. C. 
Compound A: 
The EI MS spectrum supported a MW 308 compound. The molecular formula was 
determined by exact mass measurement of the molecular ion: 
Calculated for C.sub.17 H.sub.24 O.sub.5 : 308.1624, Found: 308.1634. 
.sup.13 C NMR (CD.sub.2 Cl.sub.2): .delta. 14.0, 15.9, 22.7, 24.4, 27.3, 
30.4, 31.9, 35.0, 60.1, 61.2, 62.8, 73.5, 77.2, 139.6, 148.7, 191.6, and 
197.8. 
Compound B: 
The FAB MS spectra indicated a MW 346 compound; [M+Li].sup.+ at m/z 353 and 
[M+H].sup.+ at m/z 347 were observed. Isotope clusters for the higher mass 
ions in the EI MS spectrum pointed to the presence of one chlorine per 
molecule. The molecular formula C.sub.17 H.sub.27 ClO.sub.5, was 
determined by exact mass measurement of [M-H.sub.2 O].sup.+. 
Calculated for C.sub.17 H.sub.25 ClO.sub.4 : 328.1441, Found: 328.1435. 
The compound formed a tri-TMS derivative. 
.sup.13 C NMR (CD.sub.2 Cl.sub.2 /CD.sub.3 OD): .delta. 14.0, 22.7, 24.2, 
26.6, 27.0, 30.3, 32.2, 37.6, 68.7, 71.6, 71.8, 76.9, 79.3, 83.9, 130.9, 
163.0, and 191.8. 
Compound C: 
FAB MS results pointed to a MW 310 compound; [M+Li].sup.+ at m/z 317 was 
observed. The molecular formula was determined by exact mass measurement 
of the molecular ion: 
Calculated for C.sub.17 H.sub.26 O.sub.5 : 310.1780, Found: 310.1778. 
The compound formed a tri-TMS derivative. 
EXAMPLE 5 
Typical Pharmaceutical Compositions 
A: Dry Filled Capsules Containing 50 mg of Active Ingredient Per Capsule 
______________________________________ 
Ingredient Amount per capsule (mg) 
______________________________________ 
Compound A 50 
Lactose 149 
Magnesium stearate 
1 
Capsule (size No. 1) 
200 
______________________________________ 
Compound A can be reduced to a No. 60 powder and the lactose and magnesium 
stearate can then be passed through a No. 60 blotting cloth onto the 
powder. The combined ingredients can then be mixed for about 10 minutes 
and filled into a No. 1 dry gelatin capsule. 
B: Tablet 
A typical tablet would contain Compound A (25 mg), pregelatinized starch 
USP (82 mg), microcrystalline cellulose (82 mg), and magnesium stearate (1 
mg). 
C: Combination Tablet 
A typical combination tablet would contain a diuretic such as 
hydrochlorothiazide (25 mg) and Compound A (50 mg) pregelatinized starch 
USP (82 mg), microcrystalline cellulose (82 mg) and magnesium stearate (1 
mg). 
D: Suppository 
Typical suppository formulations for rectal administration can contain 
Compound A (0.08-1.0 mg), disodium calcium edetate (0.25-0.5 mg), and 
polyethylene glycol (775-1600 mg). Other suppository formulations can be 
made by substituting, for example, butylated hydroxytoluene (0.04-0.08 mg) 
for the disodium calcium edetate and a hydrogenated vegetable oil 
(675-1400 mg) such as Suppocire L, Wecobee FS, Wecobee M, Witepsols, and 
the like, for the polyethylene glycol. Further, these suppository 
formulations can also include another active ingredient such as another 
antihypertensive and/or a diuretic and/or an angiotensin converting enzyme 
and/or a calcium channel blocker in pharmaceutically effective amounts as 
described, for example, in C above. 
E: Injection 
A typical injectable formulation may contain Compound A (30 mg,) sodium 
phosphate dibasic anhydrous (11.4 mg), benzyl alcohol (0.01 ml), and water 
for injection (1.0 ml). Such an injectable formulation can also include a 
pharmaceutically effective amount of another active ingredient such as 
another antihypertensive and/or a diuretic and/or an angiotensin 
converting enzyme inhibitor and/or a calcium channel blocker.