Sulfircin and derivatives thereof as therapeutic agents

Novel sesterterpene compounds are derived from marine sponges of the genus Ircinia. These compounds and pharmaceutical compositions containing them as active ingredients are useful in the treatment of fungal infections.

FIELD OF THE INVENTION 
This invention relates to new organic compounds and compositions that have 
useful therapeutic properties. More particularly, the invention relates to 
novel sesterterpene compounds derived from marine organisms, i.e., sponges 
of the genus Ircinia, compositions comprising such compounds and their 
methods of use for therapeutic and other purposes. 
BACKGROUND OF THE INVENTION 
Prevention of the growth of fungi and prevention of infections and maladies 
caused by fungi to mammals and plants has received much attention by 
scientists and industry since fungi may cause various diseases and 
infections in man including mycotic disease, e.g., pulmonary candidiasis 
and pulmonary aspergillosis. Certain yeastlike organisms e.g., 
Cryptotococcus neoformans, may cause serious infections of the central 
nervous system. More commonly known fungal infections in humans and 
mammals include ringworm, which are fungal infections of hair and nail 
areas, as well as resistant infections of the skin. Many other fungal 
infections inflict humans and other mammals in the areas of skin, mucous 
membranes, intestinal tract, vaginal area and lungs. 
Plants are also attacked by various fungi. Damage caused by fungal 
infection to agriculture amounts to billions of dollars annually. Various 
inorganic and organic fungistats and fungicides have been tried, but with 
limited success. It is, of course, important for the fungicide to kill the 
fungi, but not the plant and to leave no toxic residue on the food product 
of the plant. 
Various methods have been utilized to combat fungal infection in 
agriculture, including foliage fungicides wherein plants are coated with a 
weather-resistant fungicide. Seed treatment and soil treatment methods are 
also know using fungicides safe for seeds and resistant to degradation by 
soil or soil borne microorganisms. Chemotherapeutants are fungicides which 
permeate the plant to protect new growth or eliminate infections which 
have already occurred within the plant. Agricultural fungistats and 
fungicides and their application must meet very stringent requirements and 
regulations that have been promulgated by governmental authorities. 
Considerable research and resources have been devoted to combating fungal 
infections in both mammals and plants. While some antifungal agents and 
methods have been developed that aid in inhibiting the spread of fungi and 
fungi caused diseases in both mammals and plants and in treating fungi 
infected hosts, new methods and antifungal compositions are needed. The 
present invention concerns the discovery of novel compounds that help to 
fill such need. 
Hence, an object of this invention is the provision of new organic 
compounds and compositions which have useful therapeutic properties. 
Another object is the provision of novel sesterterpene compounds derived 
from a marine organism, i.e., sponges of the genus Ircinia aff. I. 
paucifilamentosa, new compositions comprising such compounds and their 
methods of use for antifungal and other purposes. 
The full scope of applicability of the present invention will become 
apparent from the detailed description given hereinafter; it should be 
understood, however, that the detailed description, while indicating 
preferred embodiments of the invention, is given by way of illustration 
only, since various changes and modifications within the spirit and scope 
of the invention will become apparent to those skilled in the art from 
this detailed description. 
SUMMARY OF THE INVENTION 
The objects of the invention are accomplished, in part, by the provision of 
novel compounds of the formulae I & II: 
##STR1## 
wherein: A is SO.sub.3 X, R or OCOR.sup.1 ; 
R is hydrogen or R.sup.1, 
R.sup.1 is alkyl, preferably lower alkyl, 
X is H, Z or M, 
M is alkali metal ion, 
Z is NH.sub.2 .dbd.C(NR.sub.1).sub.2.sup.+ or NHR.sub.3.sup.+, 
Compounds of formula II are dihydro derivatives of the compounds of formula 
I. 
Specific examples of: 
R.sup.1 are C1-C5 alkyl, including methyl, ethyl, propyl, isopropyl, butyl, 
isobutyl, & amyl; isooctyl; hexyl; dodecyl; etc. 
M are Na.sup.+, K.sup.+, Li.sup.+. 
Z are protonated counterions of ammonia, guanidine, N-methyl guanidine, 
N,N-dimethyl guanidine, tetramethyl guanidine, N-methyl,N'-ethyl 
guanidine, methyl amine, dimethyl amine, amylamine, ethyl amine, triethyl 
amine, methyl dipropyl amine, isooctyl amine, etc. 
Preferred compounds of the invention are those of the formula III in which 
the substituents are as noted above: 
##STR2## 
and the dihydro derivatives thereof. 
In preferred embodiments of the invention, the new compounds are 
substantially pure. 
Also provided by discoveries of the invention are pharmaceutical 
compositions containing between about 0.01 to 50%/w of one of the new 
compounds of the invention or a mixture of two or more of such compounds 
and one or more non-toxic, compatible ingredient, e.g., carrier, diluent 
and/or adjuvant. 
Additionally provided by discoveries of the invention are agricultural 
fungicidal compositions containing between about 1 to 70%/w of one of the 
new compounds of the invention or a mixture of two or more of such 
compounds and one or more compatible ingredients, e.g., diluent, carrier, 
wetting agent, herbicide, adhesive agent, etc. 
The invention provides a variety of processes for the production of 
compounds of the invention. A preferred method of producing compounds of 
the formula III comprises the steps of collecting marine sponge of the 
genus Ircinia, contacting such sponge with a selected organic solvent 
system to obtain an extract, fractioning the extract and isolated 
sesterterpene compounds of formula III from the fractionated extract. 
In further preferred methods of the invention, compounds of the formula II 
are made by hydrogenation of the compounds I in the presence of a 
hydrogenation catalyst. Also, ion-exchange, hydrolysis, alkylation, 
acetylation and other known synthesis type reactions may be used pursuant 
to known procedures to add or modify various groups in the preferred 
compounds III to produce other compounds according to the formulae I & II. 
As a result of the discoveries by the invention of the new compounds, 
skilled chemists will be able to use known procedures to synthesize these 
compounds from available stock substances. 
The objects are further accomplished according to the invention by the 
discovery that growth of fungi can be inhibited by contact with an 
effective amount of the new compounds. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
A more complete understanding of the invention can be obtained by reference 
to preferred embodiments of the invention which are illustrated by the 
following specific examples of compounds, compositions and methods of the 
invention. It will be apparent to those skilled in the art that the 
examples involve use of materials and reagents that are commercially 
available from known sources, e.g., chemical supply houses, so no details 
are given respecting them.

EXAMPLE 1 
This example concerns the preparation of sulfircin, i.e., the compound of 
the formula IV: 
##STR3## 
Marine sponge of the genus Ircinia aff. I. paucifilamentosa Vacelet, 1961 
was collected at a depth of 120 meters off Fresh Creek, Andros, Bahamas. 
Ircinia aff. I. paucifilamentosa Vacelet, 1961 is an amorphous to 
spherical sponge, grey to white alive, white in ethanol; surface conulose, 
with conules 2-3 mm in height and 3-4 mm apart. Choanosome is typical for 
the genus Ircinia. Spongin filaments are unusually thick (6 um) and 
terminal knobs are large and ovoid (14-16 um long). The specimens, all 
collected from deep water environments in the Bahamas, may be conspecific 
with the Mediterranean species Ircinia paucifilamentosa Vacelet, 1961 
(Rev. Trav. Inst. Peches marit., 25(3); 351-354). Taxonomic reference 
samples are deposited at the Harbor Branch Oceanographic 
Institution/Indian River Coastal Zone Museum. 
An extract of the sponge is prepared by homogenizing in a blender. The 
crude extract is filtered and then concentrated by distillation under 
reduced pressure yielding a pale yellow oil. This oil is chromatographed 
under reverse phase vacuum liquid chromatographic conditions using a C-18 
stationary phase and a step gradient of acetonitrile/water as eluent. The 
fraction eluting with acetonitrile/water (4:1) contains a microcrystalline 
solid which is recrystallized from a dichloromethane/methanol/water 
mixture to give sulfircin (IV). 
Using typical spectral measurement techniques and apparatus, the following 
spectral data are determined for sulfircin: 
.sup.13 C NMR (90 Mhz, CDCl.sub.3 -d.sub.4 -methanol): 
158.3 s, 142.7 d, 138.9 d, 135.8 s,125.2 s, 122.2 d, 111.1 d, 83.9 d, 50.1 
d, 48.8 d, 42.3 t, 38.7 t, 37.8q (2C), 37.0 d, 36.5 s, 33.2 q, 33.1 s, 
33.1 t, 28.3 t, 26.6 t, 24.9 t, 23.9 t, 22.4 q, 21.9 q, 18.9 t, 13.6 q, 
13.1 q. 
.sup.1 H NMR (360 MHz, CDCl.sub.3 -d.sub.4 -methanol): 
7.25 (t J=1.2 Hz), 7.01 (bs), 6.07 (d J=1.2 Hz), 5.18 (bs), 4.33 (dd 
J=11.3, 3.1 Hz), 2.844 (6H, s), 2.24 (2H, t J=7.5 Hz), 2.10 (2H, m), 2.04 
(2H, m), 1.73 (2H, m), 1.60 (m), 1.51 (3H, bs), 1.50 (m), 1.42 (m), 1.33 
(m), 1.22 (2H, m), 1.21 (m), 1.18 (m), 1.05 (m), 1.04 (m), 0.99 (m), 0.97 
(m), 0.97 (m), 0.76 (3H, d J=7.0 Hz), 0.69 (3H, s), 0.65 (3H, s), 0.55 
(3H, s). 
IR: (KBr) .nu.cm-1: 
3340, 3200, 2920, 1620, 1445, 1380, 1360, 1210, 1050, 1020, 960, 900, 870, 
820, 746, 775. 
UV: MeOH .lambda.max=208; (.epsilon.=1024). 
MS: (FAB) M/Z=627.49 (IV +N,N-dimethylguanidine). 
EXAMPLE 2 
This example concerns sulfircin A, a dihydro derivative of sulfircin which 
is represented by the following formula: 
##STR4## 
A portion of sulfircin and a small amount of hydrogenation catalyst, e.g., 
Pd/C, Pt/C or Raney Ni are mixed in a suitable solvent, e.g., ethanol or 
methanol. The mixture is stirred in the presence of hydrogen in a 
hydrogenation apparatus capable of operation at elevated pressure, e.g., 
Parr apparatus, to produce dihydrosulfircin. If the reaction is too slow, 
it is facilitated by making the media slightly acidic by addition of a 
trace amount of HCl or like acid. 
EXAMPLE 3 
This example concerns in vitro fungicidal activity of compounds of the 
invention against the fungal pathogen Candida albicans. 
ASSAY METHOD 
Preparation of Inocula 
C. albicans (CA) is grown on Sabouraud dextrose agar to produce single 
colonies, one of which is used to inoculate Sabouraud dextrose broth. This 
broth is incubated at 37.degree. C. with shaking at 200 rpm for 18 hrs., 
the resultant culture is frozen with 10% (v/v) glycerol at -80.degree. C. 
and used as the innoculum for the anti-Candida assay. 
Aspergillus nidulans (AN): AN (ATCC strain 36321) is grown from a spore 
stock on the surface of a YAG plate (YAG: Yeast extract 0.5%; glucose 2%; 
agar 2%) at 30.degree. C. until the colony sporulates, at this point the 
colony is green (usually within one week). The spores are harvested from 
this plate by washing with 0.1% (v/v) Triton X-100 (filter sterilized) and 
spores are then washed with distilled water before freezing at -80.degree. 
C. in the presence of 10% (v/v) glycerol. 1. AN Disc diffusion assay 
AN is inoculated into melted yeast extract glucose agar at 45.degree. C. to 
give a cell density of approximately 1000 spores/ml. Plates are prepared 
with 10 ml of the seeded agar in a 10 cm.times.10 cm petri dish. These 
plates are stored at 4.degree. C. until needed for the assay. 
Paper discs (6.35 mm) are impregnated with the test substance and allowed 
to dry. They are then placed onto the surface of the test plate prepared 
as detailed above. Plates are incubated overnight at 30.degree. C. after 
which the zones of growth inhibition are read, being expressed as the 
diameter of the zone in millimeters. 
2. Candida albicans (CA): Minimum Inhibitory Concentration (MIC) 
Two-fold dilution for the test compound are prepared in 50 ul volumes of 
Sabouraud dextrose broth using 96-well microtiter plates. An inoculum of 
CA is added in a small volume to give a cell density of about 1000 
cells/ml. Plates are incubated at 37.degree. C. overnight. 10 ul of 
triphenyl tetrazolium chloride (1%w/v) is then added to each well and a 
further 2 hour incubation results in a deep coloration of the 
microorganism. The MIC is the lowest concentration of the test compound 
which completely inhibits growth of the CA. 
Assay Data for Sulfircin 
CA MIC 25 ug/ml. 
AN Disc.degree. diffussion assay, 12 mm zone at 25 ug/disc 
EXAMPLE 4 
Preparation of sulfircin B, i.e., the free acid compound: 
##STR5## 
A portion of sulfircin dissolved in methanol is passed into an anion 
exchange column. Thereafter, the column is eluted with dilute HCl to yield 
sulfircin B. In related manner, sulfircin derivatives containing other 
counterions then N,N-dimethyl guanidine, e.g., alkali metal, ammonia, 
guanidines, amines, etc., can be prepared by the use of other eluents than 
HCl, e.g., acetic acid, formic acid, ammonium hydroxide, ammonium 
floroborate, dilute NaOH solution, etc. 
EXAMPLE 5 
Preparation of sulfircin C, the compound: 
##STR6## 
A portion of sulfircin is reacted with 25% w/w caustic soda solution and 
sulfircin C is recovered by extraction from the reaction mixture with 
chloroform. The methyl ether of sulfircin C, i.e. sulfircin D, is prepared 
by reaction of sulfircin C with methyl iodide in the presence of base, 
e.g., potassium t-butoxide. 
EXAMPLE 6 
Preparation of sulfircin E, the compound: 
##STR7## 
A portion of sulfircin C is reacted with a mixture of acetic anhydride and 
pyridine. Sulfircin E is recovered by extraction with chloroform from the 
reaction mixture. 
Discussion of Variables 
The scope of the invention is not limited by the specific examples and 
suggested procedures and uses related herein since modifications can be 
made within such scope from the general information provided by this 
specification to those skilled in the art. In addition to hydrogenated 
derivatives as exemplified above, fluorinated and salt derivatives may be 
prepared and have pharmaceutical activity. 
Therapeutic and prophylactic application of the new compounds and 
compositions containing them can be contemplated to be accomplished by any 
suitable method and technique presently or prospectively known to those 
skilled in the art. Further, the compounds of the invention have use as 
starting materials or intermediates for the preparation of other useful 
compounds and compositions. 
The administration of sulfircin and other compounds of the invention is 
useful for treating fungal infections. Thus, pharmaceutical compositions 
containing compounds of the invention as active ingredients are useful in 
prophylactic or therapeutic treatment of humans or other mammals infected 
with or likely to be infected with fungii. 
The dosage administered will be dependent upon the identity of the fungal 
infection, the type of host involved, its age, health, weight, kind of 
concurrent treatment, if any, frequency of treatment, therapeutic ratio 
and like considerations. Advantageously, dosage levels of the administered 
active ingredients can be: dermal, 1 to about 500 mg/kg; orally, 0.01 to 
200 mg/kg; intranasal 0.01 to about 100 mg/kg and aerosol 0.01 to about 50 
mg/kg of animal body weight. 
Expressed in terms of concentration, the active ingredient of the invention 
can be present in the new compositions for localized use dermally, 
intranasally, bronchially, intravenously, intravaginally, intramuscularly 
or orally in a concentration of from about 0.01 to about 50% w/w of the 
composition, and especially from about 0.1 to about 30% w/w of the 
composition. 
The compositions of the invention are advantageously used in a variety of 
forms, e.g., tablets, ointments, capsules, pills, powders, aerosols, 
granules and oral solutions or suspensions and the like containing the 
indicated suitable quantities of the active ingredient. Such compositions 
are referred to herein and in the accompanying claims generically as 
"pharmaceutical compositions". Typically, they can be in unit dosage form, 
namely, in physically discrete units suitable as unitary dosages for human 
or animal subjects, each unit containing a predetermined quantity of 
active ingredient calculated to produce the desired therapeutic or 
prophylactic effect in association with one or more pharmaceutically 
acceptable other ingredients, e.g., diluent or carrier. 
Where the pharmaceutical compositions are aerosols, the active ingredients 
can be packaged in pressurized aerosol containers with a propellant, e.g., 
carbon dioxide, nitrogen, propane, etc. with the usual adjuvants such a 
cosolvents, wetting agents, etc. 
Where the pharmaceutical compositions are ointments, the active ingredient 
can be mixed with a diluent vehicle such as cocoabutter, viscous 
polyethylene glycols, hydrogenated oils and such mixtures can be 
emulsified if desired. 
In accordance with the invention, pharmaceutical compositions comprise, as 
an active ingredient, an effective amount of one or more of the new 
compounds and one or more non-toxic, pharmaceutically acceptable 
ingredient. Examples of such ingredients for use in the compositions 
include, ethanol, dimethyl sulfoxide, glycerol, silica, alumina, starch, 
calcium carbonate, talc, flour, and equivalent non-toxic carriers and 
diluents. 
In preferred embodiments for production of the new compounds by extraction 
from marine sponges, etc., suitable organic solvent systems for extraction 
can be selected from methanol, ethyl acetate, acetone, diethyl ether, 
t-butyl methyl ether, ethanol, and isopropanol. Mixtures of two or more of 
such solvents in various ratios and combinations are advantageous. 
Compounds of the invention are synthesized and/or isolated by various 
fractionation and chromatographic techniques from the extracts obtained as 
disclosed. Preferred isolation procedures include various chromatography 
techniques, e.g., countercurrent chromatography with suitable columns, 
including multi-layer planetary coil columns. A variety of solvents are 
available for use as single or mixed eluents, such as tetrahydrofuran, 
methanol, ethyl acetate, acetonitrile, n-propanol, n-butanol, water, and 
equivalent solvents. Further purifications using such procedures may also 
be carried out on the recovered extractions. Preferred isolation 
techniques for further purification include chromatographic operations 
such as high-pressure, liquid chromatography with suitable columns and 
suitable solvents.