United States Patent
`
`1191
`
`[11] Patent Number:
`
`5,025,554 .
`
`Bartizal et al.
`
`[45] Date of Patent:
`
`Jun. 25, 1991
`
`C. D. Poulter et al., J. Am. Chem. Soc. 111. 3734.
`(I989).
`E. J. Corey ct al., J. Am. Chem. Soc, 98. 1291, (1976).
`
`Primary Examiner—Thurn1an Page
`Assistant E_xam:'ner—D0nald R. McPhail
`Attorney, Agent, or Ffrm—Me1vin Winokur; Joseph F.
`DiPrima
`
`[ST]
`
`ABSTRACT
`
`This invention relates to a method of inhibiting fungal
`growth by employing an antifungal amount of a com-
`pound of formula (I):
`
`DA‘:
`
`no 1
`
`5 0
`
`(11
`
`Me
`
`[54] METHOD OF INHIBITING FUNGAI.
`GROWTH USING SQUALENE SYNTHETASE
`INHIBITORS
`
`[75]
`
`Inventors: Kenneth F. Bartlzal, Somerset; Jane!
`C. Ollishi, Mountainsidn, both of NJ.
`
`[73] Assignec: Merck & C0,, Inc., Rahway, NJ.
`
`[21] Appl.N0.: 582,303
`
`[22] Filed:
`
`Sep. 13, 1990
`
`A61K 31/335
`Int.Cl.5
`[51]
`
`[521 U.S.Cl.
`424/404; 424/79;
`424/405; 424/409; 514/450; 514/464; 514/824;
`549/363; 549/397
`[58] Field of Search ................ 549/363, 397; 514/450,
`514/824, 464; 424/79, 404, 405, 409
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`................... .. 424/404 X
`. 3,734,757 5/l9T3 Wiles at al.
`4,790,989 12/1988 Hunter at £11.
`.
`424/404
`
`4,865,344 9/I989 Blank ct 8.1.
`424/'4-O-I X
`4.3Tl.'.’2l 10/1939 Hillel’ ........... ..
`514/I02
`.................. .. 424/404 X
`4,915,940 4/I990 Saitnh at al.
`
`OTHER PUBLICATIONS
`
`S. A. Billcr ct al., 4'. Med. Chem., 31, 1869, (I988).
`
`6 Claims, No Dnwings
`
`1uf9
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`PENN EX. 2199
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`5,026,554
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`METHOD OF INHIBITING FUNGAL GROWTH
`USING SQUALENE SYNTHETASE INHIBITORS
`
`BACKGROUND OF THE INVENTION
`Hypercholesterolemia is known to be one of the
`prime risk factors for ischemic cardiovascular disease.
`such as arteriosclerosis. Bile acid sequestrants have been
`used to treat this condition; they seem to be moderately
`effective but they must be consumed in large quantities, 10
`i.e. several grams at a time and they are not very palat-
`able.
`.
`
`5
`
`2
`
`H0
`
`0“:
`
`C
`
`M
`
`'
`0
`||
`
`/
`
`M:
`
`?
`
`0
`
`0
`
`(I)
`
`_
`
`6
`
`C022‘
`C0323
`
`0
`3
`
`0“
`C0323
`
`l5
`
`wherein Z1, Z2 and Z; are each independently selected
`from
`(a)
`
`. h
`d
`b .
`Emma Wu
`
`f h
`b
`3 mcm Cr 0 t C group
`
`now commercially
`MEVACQR ® (lovastatin),
`available, is one of a group of very active antihypercho-
`lesterolemic agents that function by limiting cholesterol
`biosynthesis by inhibiting the enzyme, HMG-COA re-
`ductase.
`.
`1k 1_
`C’
`Squalene synthetase is the enzyme involved in the
`Cwalkyl’
`first committed step of the de novo cholesterol biosyn-
`lisa. Y
`thetic pathway. This enzyme catalyzes the reductive 20 (C)
`O ’
`dimerization of two molecules of famcsyl pyrophos-
`flit s=h=nv1subsW=d with m-=*h¥'- m=th°*v- ha1°s="
`fchdaii.3?ti,°ii'?oi§§ZiZi°;$i°d‘?e’I3l"§$h?I$§‘§§d°§$§§1§-
`athways to ubiquinone’ dolichol and isopcmcnyl 25 a phiirciiiaggbtlicalliyogel-izigiigbig git of a compound of of
`
`inhibiting squalene synthetase
`Previous efforts at
`have employed pymphosphate or pyrophosph-ate an}
`.
`.
`7
`7
`gftiioggafi:)nrfi§?ar:§O;I;cEflfljegsgfilgfildfgfgffgllgjgj
`and E. J. Corey and R. Volante. J. Am. Chem. Soc.; 93,
`
`formula (I).
`.
`.
`.
`In one embodiment of the present invention are those
`compounds of formula (1) wherein the relative stereo-
`chemical configuration of the 2,8-dioxabicyclo{3.2.1]oc-
`‘W ""3 *5 “‘ ‘h°‘’"’' b°1°“f‘
`
`C0223
`
`CO 221
`
`C0223
`
`Me
`
`1291 (1976) S‘ Baler (U_S_ Pat No_ 4’8.”’721)dcsCn.bcs 45 Throughout this Specification and claims where stereo-
`.
`.
`.
`.
`.
`.
`hemistry is described for the dioxabicyclo[3.2.1]octane
`isoprenoid {phosphinylmethyhphosphonates as 1nh1bi-
`C.
`.
`.
`.
`.
`.
`tors of squalene symhcmst
`ring the configuration implied is relative. The actual
`The present invention provides nonphosphorus con-
`COEEES:-E:i3?u:::tElegafiigoelfiigggglitngffiiTgfigglisl-3:;
`taming Inhibitors of Squalene symhctasa
`pounds of structural formula (I) wherein the relative
`DETAILED DESCRIPTION OF THE
`50- configuration at positions 3,6 and 7 is as shown below:
`INVENTION
`
`‘ —
`
`as
`
`invention is directed to novel com»
`The present
`1?>0111'ldS Of Structural formula (1) which are squalene
`synthetase inhibitors:
`
`In one class of this embodiment are those compounds
`of structure (I) wherein the relative configuration at the
`4-position is as shown below:
`
`2 Hf 9
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`Exemplifying this class is the compound wherein Z1,
`Z2 and 23 are each hydrogen or a pharmaceutically
`acceptable salt thereof. The compound wherein Z1, Z3
`and 23 are each hydrogen is hereafter referred to as
`Compound A.
`Further illustrating this class are those compounds in
`which one or more of Z1, Z; or Z; is C1_5aIkyl or C1_
`salkyl substituted with phenyl or substituted phenyl
`wherein the substituent is methyl, methoxy, halogen or
`hydroxy. In a specific illustration, Z1, Z2 and Z3 are
`each methyl. This compound is hereafter referred to as
`Compound B.
`The compounds of formula (1) are prepared in an
`aerobic fermentation procedure employing a novel fun-
`gal culture, MF5465, identified as Leptodontium elarfus.
`Although the use of this organism is specifically de-
`scribed herein, other species of the genus Leptodontium
`including mutants of the above described organism are
`also capable of producing compounds of this invention.
`The culture MF54:65 is that of a fungus, a lignicolous
`Hyphomycete, Lepradontium efatius,
`isolated from
`wood in the Joyce Kilmer Memorial Forest in North
`Carolina. This culture has been deposited with the
`American Type Culture Collection at 12301 Parklawn
`Drive, Rockville, Md. 20852 as ATCC 74011.
`The culture MF5465,
`identified as Leptodontium
`elarius exhibits the following morphological features.
`Colonies attaining 12-15 mm in 7 days on oatmeal
`agar {Difco), with both aerial and submerged n1yce-
`litlm. Colony surface flat to appressed in side view,
`minutely velvety with a metallic sheen towards the
`margins, dull towards the center, hyaline at the margin,
`but soon becoming pale to dark gray, finally black,
`often developing olivaceous colors in age, Pallid Neu-
`tral Gray, Light Gull Gray, Deep Gull Gray, Dark
`Gull Gray, Slate-Gray, Deep Olive-Gray, Olive-Gray,
`(capitalized color names from Rid gway, R. 1912. Color
`Standards and Nomenclature, Washington, D.C.), with
`similar reverse pigmentation, without exudates diffus-
`ible pigments or odors.
`Conidiogenous cells holoblastic, arising as the termi-
`nal cells of relatively undifferentiated conidiophores,
`with tapered, subulate apices, with the conidiogenous
`loci confined to the extreme apex. Occasionally with
`undifferentiated conidiogenous loci directly on vegeta-
`tive hyphae. Developing conidia adhere to conidio-
`phore terminus in a thin, irregular to ladder—like rachis
`in groups of up to 4-15 conidia. Conidiophores originat-
`ing as undifferentiated branches at right or subacute
`angles from vegetative hyphae, gradually elongating,
`remaining simple or forming l—3—branch points, usually
`at right to subacute angles, usually clustered in small
`groups when viewed from above, 1-3 septate, cylindri-
`cal to conical with tapered apices hyaline when young
`but developing olivaceous to olivaceous gray pigments
`from the base upward in age, with walls slightly thicker
`than those of vegetative hyphae, 20—65>< 3-5 ,u.m. Co-
`nidia formed abundantly on common media such as
`
`oatmeal, malt extract, or corn meal agar, 3.5-5 pm X 1-2
`pm, aseptate, smooth, thin-walled, allantoid, suballan-
`mid, to short cylindrical, or narrowly elliptical, often
`with a small proximal scar or apiculus, without visible
`slime
`or
`gelatinous materials.
`I-Iyphae
`septate,
`branched, cylindrical or occasionally inflated, up to 5
`_u,I1'l in diameter.
`Compounds of this invention can be obtained by
`culturing the above noted microorganism in an aqueous
`nutrient medium containing sources of assimilable car-
`bon and nitrogen, preferably under aerobic conditions.
`Nutrient media may also contain mineral salts and de-
`foaming agents.
`The preferred sources of carbon in the nutrient me-
`dium are carbohydrates such as glucose, glycerin,
`starch, dextrin, and the like. Other sources which may
`be included are maltose, mannose, sucrose, and the like.
`In addition, complex nutrient sources such as oat flour,
`corn meal, millet, corn and the like may supply utiliz-
`able carbon. The exact quantity of the carbon source
`which is used in the medium will depend, in part, upon
`the other ingredients in the medium, but is usually found
`in an amount ranging between 0.5 and 5 percent by
`weight. These carbon sources can be used individually
`in a given medium or several sources in combination in
`the same medium.
`The preferred sources of nitrogen are amino acids
`such as glycine, methionine, proline, threonine and the
`like, as well as complex sources such as yeast extracts
`(hydrolysates, autolysates), dried yeast, tomato paste.
`soybean meal, peptone, corn steep liquor, distillers solu-
`bles, malt extracts and the like.
`Inorganic nitrogen
`sources such as ammonium salts (eg. ammonium nitrate,
`ammonium sulfate, ammonium phosphate, etc.) can also
`be used. The various sources of nitrogen can be used
`alone or in combination in amounts ranging between 0.2
`to 90 percent by weight of the medium.
`The carbon and nitrogen sources are generally em-
`ployed in combination, but need not be in pure form.
`Less pure materials which contain traces of growth
`factors, vitamins, and mineral nutrients may also be
`used. Mineral salts may also be added to the medium
`such as (but not limited to) calcium carbonate, sodium
`or potassium phosphate, sodium or potassium chloride,
`magnesium salts, copper salts, cobalt salts and the like.
`Also included are trace metals such as manganese, iron,
`molybdenum, zinc, and the like. In addition, if neces-
`sary, a defoaming agent such as polyethylene glycol or
`silicone may be added, especially if the culture medium
`foams seriously.
`The preferred process for production of compounds
`of this invention consists of inoculating spores or myce-
`lia of the producing organism into a suitable medium
`and then cultivating under aerobic condition.
`The fermentation procedure generally is to first inoc-
`- ulate a preserved Source of culture into a nutrient seed
`3of9
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`medium and to obtain,' sometims through a two step
`process, growth of the organisms which serve as seeds
`in the production of the active compounds. After inocu-
`lation, the flasks are incubated with agitation at temper-
`atures ranging from 20° to 30° C., preferably 25‘ to 28°
`C. Agitation rates may range up to 400 rpm, preferably
`200 to 220 rpm. Seed flasks are incubated over a period
`of 2 to 10 days, preferably 2 to 4 days. When growth is
`plentiful, usually 2 to 4 days, the culture may be used to
`inoculate production medium flasks. A second stage
`seed growth may be employed, particularly when going
`into larger vessels. When this is done, a portion of the
`culture growth is used to inoculate a second seed flask
`incubated under
`similar conditions but employing
`shorter time.
`After inoculation, the fermentation production me-
`dium is incubated for 3 to 30 days, preferably 4 to 14
`days, with or without agitation (depending on whether
`liquid or solid fermentation media are employed). The
`fermentation is conducted aerobically at temperatures
`ranging from 20° to 40° C. If used, agitation may be at
`a rate of 200 to 400 rpm. To obtain optimum results, the
`temperatures are in the range of 22' to 28° C., most
`preferably 24” to 26' C. The pH of the nutrient medium
`suitable for producing the active compounds is in the
`range of 3.5 to 8.5, most preferably 5.0 to 7.5. After the
`appropriate period for production of the desired com-
`pound, fermentation flasks are harvested and the active
`compound isolated.
`An alcoholic solvent is employed to extract a com-
`pound of this invention from the solid fermentation
`medium. The preferred solvent for extraction of the
`solid fermentation is methanol. The mixture of alcoholic
`solvent and fermentation broth is vigorously stirred and
`filtered, and water added to the filtrate. The aqueous
`methanol extract is then adsorbed on an anion exchange
`resin. The preferred resin is Dowex-l (Cl"). The active
`compound can be eluted from Dowex-l using a high
`salt eluant; the preferred eluant is 3% ammonium chlo-
`ride in 90% methanol/’water. After elution from the ion
`exchange resin, the active compound may be recovered
`from the eluate by diluting the eluate with water, lower-
`ing the pH to 2.5, and extracting into an organic solvent;
`the preferred sol vent for extraction is dichloromethane.
`The organic extract is then evaporated to afford par-
`tially purified active compound.
`The active compound is further purified by chro-
`matographic separation which may be carried out by
`employing reverse phase chromatagraphy. The pre-
`ferred adsorbent
`for
`this chromatography is a C8
`bonded phase silica gel. The preferred eluant for re-
`verse phase chromatography is a mixture of acetonitrile
`and water buffered at a low pH. such as 0.1% phos-
`phoric acid, or trifluoroacetic acid.
`The present invention is also directed to a method of SS
`inhibiting cholesterol biosynthesis which comprises the
`administration to a subject in need of such treatment a
`nontoxic therapeutically effective amount of a com-
`pound represented by structural formula (I) and phar-
`rnaceutically acceptable salts thereof. Specifically, the
`compounds of this invention are useful as antihypercho-
`lesterolemic agents for the treatment of arteriosclerosis, _
`hyperlipidemia, familial hypercholesterolemia and the
`like diseases in humans. They may be administered
`orally or parenterally in the form of a capsule, a tablet,
`an injectable preparation or the like. It is usually desir-
`able to use the oral route. Doses may be varied, depend-
`ing on the age, severity, body weight and other condi-
`
`6
`tions of human patients, but daily dosage for adults is
`within a range of from about 20 mg to 2000 mg (prefera-
`bly 20 to 100 mg) which may be given in two to four
`divided doses. Higher doses may be favorably em-
`ployed as required.
`The pharmaceutically acceptable salts of the com-
`pounds of this invention include those formed from
`cations such as sodium, potassium, aluminum, calcium,
`lithium. magnesium, zinc, and from bases such as ammo-
`nia, ethylenediamine, N-methyl-glucamine, lysine, argi-
`nine, ornithine, choline, N,N'-dibenzylethylenediauiine,
`chloroprocaine, diethanolamine, procaine, N-benzy]-
`phenethylamine, diethylamine, piperazine, tris(hydrox-
`ymethyl)aminomethane,
`and
`tetramethylammonium
`hydroxide. The salts included herein encompass those
`wherein one, two or all three of the carbonyl groups are
`in the salt form.
`The compounds of this invention may also be coad-
`ministered with pharmaceutically acceptable nontoxic
`cationic polymers capable of binding bile acids in a
`non-reabsorbable form in the gastrointestinal tract. Ex-
`amples of such polymers include cholestyramine, coles-
`tipol and poly[methyl-(3-trimethylaminopropylfimino-
`trimethylene dihalide]. The relative amounts of the
`compounds of this invention and these polymers is be-
`tween l:l00 and l:l5,000.
`The intrinsic squalene synthetase inhibitory activity
`of representative compounds of this invention was mea-
`sured by the standard in vitro protocol described below:
`
`Preparation of Microsomes
`
`Male, Charles River CD rats (120 to 150 g) were fed
`a diet containing 0.1% lovastatin for 4 days. The livers
`from these rats were homogenized in 5 volumes (ml/g)
`of ice cold 50 mM HFPES (4-(2-hydroxyethy1)- l-piper-
`azine-ethanesulfonic
`acid],
`5
`mM EDTA(e-
`thylenediaminetetraacetic acid) pH 7.5 with a Potter-
`Elvehjem type tissue grinder. The homogenate was
`centrifuged twice at 20,000>< g for 15 minutes at 4' C..
`discarding the pellet each time. The supernatant was
`then centrifuged at l00,000)( g for 1 hour at 4" C. The
`resulting microsomal pellet was resuspended in a vol-
`ume of the above homogenizing buffer equal to one-
`fifth the volume of the original homogenate. This mi-
`crosornal preparation has a protein concentration of
`about 7 mg/ml. The microsomal suspensions were
`stored in aliquots at —-70° C. Squalene synthetase activ-
`ity in these aliquots is stable for at least several months.
`
`Partial Purification of Prenyl Transferase
`
`Prenyl transferase was purified to use in the enzy-
`matic synthesis of radiolabelled farnesyl pyrophos-
`phate. Prenyl transferase was assayed by the method of
`Rilling (Methods in Enzymology 110, 125-129 (1985))
`and a unit of activity is defined as the amount of enzyme
`that will produce 1
`,u. mole of farnesyl pyrophosphate
`per minute at 30' C. in the standard assay.
`The livers of 23 forty-day old male rats that had been
`fed 5% cholestyrarnine plus 0.1% lovastatin were ho-
`mogenized in a Waring blender in 1
`liter of 10 mM
`mercaptoethanol, 2 m.M EDTA, 25 ,u.M leupeptin,
`0.005% phenylmethyl sulfonyl fluoride pH 7.0 contain-
`ing 0.l trypsin inhibitor units of aprotinin/ml. The ho-
`mogenate was centrifuged at 20.000)( g for 20 minutes.
`The supernatant was adjusted to pH 5.5. with 6N
`HO_Ac and centrifuged at l00,000)<g for 1 hour. This
`supernatant was adjusted to pH 7.0 with 3N KOH and
`a 35-60% ammonium sulfate fraction taken. The 60%
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`pellet was redissolvedin 60 ml of 10 mM potassium
`phosphate, 10 mM mercaptoethanol.
`1 mM EDTA pH
`7.0 (Buffer A) and dialyzed against two 1 liter changes
`of Buffer A. This dialyzed fraction was applied to a
`l2.5)(5 crn column of DEAE-sepharose 4B equili-
`brated with Buffer A. The column was washed with 700
`ml of Buffer A and a 1 liter gradient from Buffer A to
`100 mM potassium phosphate, 10 mM mercaptoethanol.
`1 mM EDTA pH 7.0. Fractions having a specific activ-
`ity greater than 0.20 units/mg were combined, solid
`ammonium sulfate was added to bring to 60% satura-
`tion and pelleted. The pellet was dissolved in 8 ml of 10
`mM Tris, 10 mM B-mercaptoethanol pH 7.0 {Buffer B).
`The redissolved pellet was taken to 60% saturation with
`ammonium sulfate by adding 1.5 volumes of saturated
`ammonium sulfate in Buffer B. This ammonium sulfate
`
`suspension contained 3.5 units/ml with a specific activ-
`ity of 0.23 units/rng and was free of isopentenyl pyro-
`phosphate isomerase activity. This ammonium sulfate
`suspension was used for the synthesis of {4-’4C]farnesyl-
`pyrophcsphate and its activity was stable stored at 4° C.
`for at least 6 months.
`
`10
`
`15
`
`20
`
`25
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`30
`
`Enzymatic Synthesis of [4-1“C]farnesyl-pyrophosphate
`The solvent (ethanol: 0.l5N NH4OH, 1:1) was re-
`moved from 55 p.Ci of [4-“*C]isopentenyl pyrophos-
`phate(47.9 p.Ci/prnole) by rotary evaporation. Six hun-
`dred microliters of 100 mM Tris, 10 mM MgCl1, 4 mM
`dithiothreitol pH 15 was added and the solution was
`transferred to a 1.5 ml Eppendorf centrifuge tube. Gera-
`nyl-pyrophosphate, 250 pl of a 20 mM solution, and 50
`pl of the ammonium sulfate suspension of prenyl trans-
`ferase were added to initiate the reaction. This incuba-
`tion contained 5 pmoles of geranyl pyrophosphate, 1.15
`pmoles of isopentenyl pyrophosphate, 6 umoles of 35
`Mgclz of 0. 13 units of prenyl transferase in a volume of
`900 pl. The incubation was conducted at 3?’ C. During
`the incubation,
`the mix turned cloudy white as the
`newly formed magnesium complex of farnesyl pyro-
`phosphate precipitated out of
`solution. The
`[-1- do
`1_‘*C]farnesyl pyrophosphate was collected by centrifu-
`gation for 3 minutes at
`l4,{X)0 rpm in an Eppendorf
`centrifuge tube, the supernatant removed, and the pellet
`was dissolved in 1.0 ml of 50 mM HEPES, 5 mM
`EDTA, pH 7.5 . The yield was 50.7 itCi (92%) of [4- 45
`"‘C]f'arnesyl pyrophosphate. The [4-”C]farr1esyl pyro-
`phosphate was stored in aliquots at -70“ C.
`
`Squalene Synthetase Assay
`
`Reactions were performed in 16 X 125 mm screw cap 50
`test tubes. A batch assaymix was prepared from the
`following solution:
`
`
`
`ul per assay
`20
`10
`10
`10
`10
`3.0
`
`volume for
`St] assays
`1000
`500
`500
`500
`500
`150
`
`55
`
`60
`
`8
`with the original homogenizing buffer. For each reac-
`tion, 87 pl of the assay mix was taken with 3 pl of an
`inhibitor solution (DMSO or MeOH in the controls),
`warmed to 30“ C. in a water bath and then the reaction
`
`was initiated by the addition of 10 pl of the l:l20 dilu-
`tion of microsomal protein {[16 pg protein total in the
`assay). The reactions were stopped after 20 minutes by
`the addition of 100 pl of a 1:1 mix of 4-0% KOH with
`95% EtOH. The stopped mix was heated at 65° C. for
`30 minutes, cooled, 10 ml of heptane was added and the
`mix was vortexed. Two g of activated alumina was then
`added. the mix vortexed again, the alumina allowed to
`settle and 5 ml of the heptane layer was removed. Ten
`ml of scintillation fluid was added to the heptane solu-
`tion and radioactivity was determined by liquid scintil-
`lation counting.
`Percent inhibition is calculated by the formula:
`
`[Sample — Blank]
`‘‘ [Control — Blank]
`
`[1
`
`100
`
`“:50 values were determined by plotting the log of
`the concentration of the test compound versus the per-
`centage inhibition. The IC5o is the concentration of
`inhibitor that gives 50% inhibition as determined from
`these plots.
`Representative of the intrinsic squalene synthetase
`inhibitory activities of the compounds of this invention
`is the 1C5a data tabulated below:
`
`Squalene Synlhetase
`1C5oCompound
`
`9nM
`Compound A
`
`The present compounds also demonstrate broad spec-
`trum antifungal activity as determined by broth and
`agar dilution methods. The compounds are particularly
`active towards filamentous fungi and yeasts including
`Candida afbicans and Crjzprococcus neoformans. The
`sensitivity of filamentous fungi and yeast was deter-
`mined using inhibitor dilution assays in microtiter for-
`mat. The compounds were dissolved in DMSO at 2
`mg/ml and serially diluted in 0.1M phosphate buffer,
`pH 7.0 in the microtiter dish from 100 to 0.006 pg/ml.
`A standardized spore suspension for testing the filamen-
`tous fungi was prepared by inoculating Antibiotic Me-
`_dit1m #3 Containing 1.5% agar with spores such that
`l.5>< l03colony forming units were added per well. The
`microtiter wells were filled with 50 pl of buffer contain-
`ing compound and 50 pl of inoculated medium.
`The sensitivity of yeasts was determined by inoculat-
`ing yeast nitrogen base containing l% dextrose
`(YNB/G) with aliquots of an overnight yeast culture
`grown in Yeast Morphology (Y M) media at 35° C. and
`diluting in YNB/G to yield a final concentration of
`l.5~7.5 X 103 colony forming units/well. To test
`the
`sensitivity of yeast, compound was solubilized in 10
`percent aqueous DMSO at 2.56 mg/ml. The compound
`was diluted serially in YNB/G from 128 to 0.06 pg/ml
`and further diluted 1:10 in YNB/G. The wells were
`filled with 150 pl of media containing drug. The mini-
`mum inhibitory concentration (MIC) is defined as the
`lowest concentration to prevent growth after an incuba-
`tion for 42 hours, at 28° C. for the filamentous fungi and
`24 to 48 hours. at 35° C. for the yeasts. Representative
`
`250 mm Hepes pH 7.5
`1.
`2. Na!’ 1I0 mM
`3. MgCl1 55 mM
`4. Dithiothreitol 30 mid
`5. NADPH 10 mM (made fresh)
`6.
`[4-1‘C]l'arnesy|~pyrophosphate
`47.9 pct/umole. and
`0.025 uCiJ3.0 in
`‘I. H30 1200 24
`
`
`
`
`This assay mix was degassed under a vacuum and 65
`flushed with N1. Solutions of the squalene synthetase
`inhibitors were prepared either in DMSO or MeOH and
`in M20 dilution of the rnicrosomal protein was made
`
`5uf9
`
`PENN EX. 2199
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`9
`of the antifungal activity are the minimum inhibitory
`concentration data shown below.
`
`5,026,554
`
`10
`,
`tion, the composition may be applied by injection or
`may be administered orally.
`For non-medical application, the product of the pres-
`ent
`invention, either singly or as a mixture, may be
`employed in compositions in an inert-carrier which
`includes finely divided dry or liquid diluents, extenders,
`tillers, conditioners and excipients,
`including various
`clays, diatomaceous earth, talc, and the like, or water
`and various organic liquids such a lower alkanols, for
`example ethanol and isopropanol, or kerosene, benzene,
`toluene and other petroleum distillate fractions or mix-
`tures thereof.
`These compositions may be employed by applying to
`the surface of or incorporating in the medium to be
`protected. For the control of rice blast,
`tomato late
`blight, tomato early blight, wheat leaf rust, bean pow-
`dery mildew and tomato Fusarium wilt, the composi-
`tions may be applied directly to the plant
`in topical
`application or administered to the soil for systemic ap-
`plication. The method comprises administering to the
`affected plant, soil or medium to be protected an an-
`tifungally effective amount of the compound of For-
`mula I.
`
`The following examples illustrate the preparation of
`the compounds of formula (I) and their incorporation
`into pharmaceutical compositions and, as such, are not
`to be considered as limiting the invention set forth in the
`claims appended hereto.
`The composition of media employed in the following
`Examples are listed below:
`
`KF SEED MEDIUM
`
`Trace Element Mix #2
`
`1 JL
`5 g
`FeSO4.‘Il-I20
`l.0
`40 g
`MrrS04.-H-I20
`1.0
`10 g
`CuC12.2H;O
`0.025
`10 g
`Cat;lg.2H30
`0.]
`H3303
`0.056
`(NH4)5MDT014.4H20
`0.019
`Zni-04.'H-I30
`0.2
`
`dissolved in [L 0.6 N HC1
`
`Corn Steep Liquor
`Tomato Paste
`Oat Flour
`Ccrelosc
`Trace Element
`10 ml
`Mix #2
`1000 ml
`Distilled Water
`pH adjusted to 6.8 tpresterile)
`S0 mls./nonbaffied 250 mls
`Erlenmeyer flask
`autoclave 20 minutes U21’ (2..
`15 psi)
`
`10
`
`I5
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`Minimum Inhibitory Concentration lmcgfntli
`
`Organism
`Compound A
`Filamentous Fungi
`Aspergiflusflavus MF3-83
`/l. nidufanx R2]
`Yeast
`Candida afbiearu MY1055
`C.
`trcpr'cufi'.t MYl0l2
`C. pampsifasir MYIOIO
`Crypt. mofor.-Harts M‘t’l05l
`
`88I
`
`16
`
`invention is also directed to a
`Thus the present
`method of treating fungus infections which comprises
`the administration to an organism in need of such treat-
`ment a nontoxic therapeutically effective amount of a
`compound represented by the structural formula (I) and
`pharmaceutically acceptable salts thereof. Based on the
`above MIC data it is determined that generally from 2
`to about 20 mg/kg should be employed as a unit dosage
`in an antifungal treatment.
`The compounds of this invention are adaptable to
`being utilized in various applications of antifungal com-
`positions. In such use, compounds may be admixed with
`a biologically inert carrier, generally with the aid of a
`surface active dispersing agent, the nature of which
`would vary depending on whether the use is for the
`control of pathogens infecting mammals such as man, or
`birds or reptiles, or for control of fungi in agriculture
`such as in soil or plant parts, or for the control of fungi
`in inanimate objects.
`In compositions for medical applications, the com-
`pounds may be admixed with a pharmaceutically ac-
`ceptable carrier, the nature of which will vary depend-
`ing on whether the composition is to be topical, paren-
`teral or oral.
`If said application is to be topical, the drug may be
`formulated in conventional creams and ointments such
`as white petroleum, anhydrous lanolin, cetyl alcohol,
`cold cream, glyceryl monostearate, rose water and the
`like.
`
`For parenteral applications, the compounds may be
`formulated in conventional parenteral solutions such as
`0.85 percent sodium chloride or 5 percent dextrose in
`water; or other pharmaceutically acceptable composi-
`tions.
`
`Compositions for oral administration may be pre-
`pared by intimately mixing the component drugs with
`any of the usual pharmaceutical media, including, for
`liquid preparations, liquid carriers such as water, gly-
`cols, oils, alcohols, and the like; and for solid prepara-
`tions such as capsules and tablets, solid carriers such as
`starches, sugars, kaolin, ethyl cellulose, surface active
`dispersing agents, generally with lubricant such as cal-
`cium stearate,
`together with binders, disintegrating
`agents and the like.
`These compositions are then administered in amounts
`suflicient
`to obtain the desired antifungal effect. For
`medical application, the method comprises administer-
`ing to a subject in need of treatment a therapeutically
`effective antifungal amount of a compound of Formula
`I. The appropriate closes will vary depending on age.
`severity, body weight and other conditions. For topical
`application the compositions are applied directly to the
`area where control is desired. For internal administra-
`
`
`
`Fl
`
`Production Media
`BRF
`
`Brown rice
`Base liquid #2
`Base liquid #2
`Yeast extract
`Sodium tartrate
`KH 3PO4
`distilled water
`
`5.0 g/llask
`20.0 mlsfflask
`g/L
`1.0
`0.5
`0.5
`l0C(].0 mls
`
`Cracked corn
`Base liquid #3
`Base liquid #3
`Ardamine PH
`K.H2P0¢
`MgSO4.?H2O
`Sodium tartnte
`FeSO4.'iH3O
`ZnSO4.'l‘H-20
`distilled H;O
`(no pH adjustment)
`
`10.0 gfflask
`10.0 mls/flask
`g,/L
`0.2
`0.1
`0.1
`0.]
`0.0]
`0.0]
`1030.0 mls
`
`(no pH adjustment)
`
`autoclave 15 minutes
`021' C.. 15 psi]
`add l5.0 ml: distilled
`H30/llask
`autoclave 20 minutes
`(l2l' C... 15 psi}
`.
`
`autoclave 15 minutes
`(l2l'C.. 15 psi}
`add l5.0 rnls distilled
`H10,/l'lask
`autoclave 10 minutes
`H21’ (2.. _l5 psi)
`
`6of9
`
`PENN EX. 2199
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`5,026,554
`
`12
`
`EXAMPLE 3
`
`11
`
`EXAMPLE 1
`
`Preparation of Compound A
`
`A. Culturing MF5465
`
`Culture MF5465, inoculated from a soil tube using
`one glass scoop of soil, was grown in 3 [CF seed medium
`flasks for 74 hours at 25' C.. 220 rpm, 85% humidity.
`The flasks were then pooled, and sterile glycerol added
`to obtain a final concentration of 10%. The contents
`were mixed and 2.0 ml aliquots were dispensed asepti-
`cally into sterile cryotubes. The vials were frozen and
`maintained at -80‘ C.
`
`Three vails containing frozen vegetative mycelia
`were defrosted and transfered, one to each of three KF
`seed medium flasks. These seed flasks were incubated
`for 71 hours at 25° C., 220 rpm, 85% humidity. At com-
`pletion of the incubation,
`the three KF flasks were
`pooled and the seed was used to inoculate 56 F1 pro-
`duction medium flasks. Care was taken to manually
`distribute seed growth throughout the solid production
`medium. Production flasks were incubated statically at
`25" C. for 21 days. Flasks were harvested as follows: 45
`rnls 75% methanol was added to each production flask;
`growth was manually broken apart into small fragments
`by use of a glass pipette; flasks were re-stoppered and
`placed onto a gyrotory shaker and agitated for 30 min-
`utes at 220 rpm while the extraction proceeded. After
`shaking,
`the contents of the individual
`flasks were
`pooled by pouring the solvent-extract off the mycelial
`covered corn and into a 2 liter Erlenmeyer flask. Con-
`tents of each flask were then subjected to a second
`extraction with another 45 rnls 75% methanol. Extrac-
`tion proceeded as above with the resultant extracts
`being pooled into a second 2 liter Erlenmeyer flask.
`
`B. Isolation of Compound A
`
`The extracts from above (4800 mL) were loaded onto
`a DOWEX-1 column (500 mL resin) at a rate of 20
`mL/min. The column was then washed with 50% me-
`thanol/'water (300 mL), and 90% methanol/water (500
`mL), and then eluted with 3% ammonium chloride in
`90% methanol/water. Six fractions (500 mL) were col-
`lected. The first 3 fractions were combined, diluted
`with water (1 L), and adjusted to pH 2.5 with cone.
`hydrochloric acid. The acidified eluate was extracted
`with dichloromethane (2 X 500 mL). Evaporation of the
`dichloromethane extract afforded an oily residue (402
`mg). The residue was dissolved in methanol (1.2 ml) and
`loaded on a prep HPLC column (Dynamax 60A, 8 um
`C3, 24.6>(250 mm with guard column). The column
`was eluted with 72% acetonitrile/18% (0.1% phos-
`phoric acid in water) with a 10 mL/min flow rate. Col-
`lecting 5 ml. fractions, the desired compound eluted in
`fractions 29-34. Fractions 29-34 were combined and
`ethyl acetate (30 mL) was added. After washing with
`water (10 mL), the organic layer was evaporated to
`give Compound A as an oil.
`EXAMPLE 2
`
`Preparation of an Ammonium Salt
`
`I0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`A 0.1 mmol sample of the free acid of a compound of
`- formula (I) is dissolved in 10 ml of ethyl acetate. The
`resulting solution is saturated with gaseous ammonia
`upon which the ammonium salt precipitates from solu-
`tion.
`
`65
`
`Preparation of a Potassium Salt
`
`A solution of0.l mmol ofthe free acid ofa compound
`of formula (I) in 10 ml of methanol is treated with an
`aqueous or methanolic solution containing 0.3 mmol of
`potassium hydroxide. Evaporation of the solvent af-
`fords the tri-potassium salt. Addition of between 0.] and
`0.3 mmol of potassium hydroxide yields analogously
`mixtures of the mono-potassium, di—potassium and tri-
`potassium salts whose composition depends upon the
`exact amount of potassium hydroxide added.
`In a similar fashion the sodium and lithium salts can
`be formed.
`
`EXAMPLE 4
`
`Preparat