United States Patent
`
`119]
`
`[11]
`
`4,231,938
`
`
`
`[45] NOV. 4, 1980
`Monaghan et al.
`
`.[54] HYPOC}lO C FERMENTATION
`PRODUCTS AND PROCESS OF
`PREPARATION
`
`[T5]
`
`inventors: Richard L. Monaglun. Somerset;
`Alfred W. Alberta, Short Hills; Carl
`H. Hoffman. Scotch Plains; George
`Albert-Schonlierg. Princeton. all of
`NJ.
`'
`
`[73] Assignee:
`
`Merck & Co., I|e., Rahway. NJ.
`
`[21] Appl. No.: 48,946
`
`[22] Filed:
`
`Jun. 15, 1979
`
`Int. Cl.’ .......................................... .. C07!) 309/30
`[51]
`[52] US. (1
`160/343.5; 560/256;
`435/125
`
`[58] Field of Search
`
`260/343.5
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`3.933.140
`4,04-9,495
`4, 1 31,322
`
`9/1916
`9/1917
`I/1979
`
`Endo et al.
`Endo el 1|.
`Eado et :1.
`
`
`
`..
`
`1&3/343.5
`195/36 R
`424/27} R
`
`'
`
`OTHER PUBLICATIONS
`
`A. Endo et 21., Eur. J. Biochem., vol. 77 (1977), pp.
`3!-J6.
`A. -Endo et al.. The Journal of Antibiotics, vol. XXIX,
`No. 12 (1916), pp. 1346-1343.
`
`A. Endo et al., FEBS Lcttera, vol. ‘#2, No. 2 (1976), pp.
`323-326.
`A. G. Brown et al., J.C.S. Perkin 1
`ll65—li69.
`'
`
`(1915). pp.
`
`Primary Exanu'ner——-Nonna S. Milestone
`Attorney, Agent. or F:'nn—'William H. Nicholson; Harry
`E Westialce, Jr.
`_
`Ansrklicr
`[57]
`Substances isolated after cultivation ofa microorganism
`belonging to the genus Aspergillus in a culture medium
`comprise a compound designated MSD803 which has
`the lactone structure:
`
`
`
`as well as its free hydroxy acid form. Together with
`salts and esters of the free acid form, these compounds
`form a class of highly active hypocholesteremic and
`hypolipemic rnedicaments.
`
`1 Claim, 2 Drawing Figures
`
`CFAD V. UPENN
`IPR20l 5-01836
`‘
`
`loflll
`
`PENN EX. 2188
`
`

`
`U.S. Patent
`
`Nov. 4, 1980
`
`Shcet'1of2
`
`4,231,938
`
`92%
`
`9.0
`
`2 "f 1"
`
`PENN EX. 2188
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`U.S. Patent
`
`Nov. 4, 1980
`
`Sheet 2 of 2
`
`4,231,938
`
`§
`
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`§
`
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`
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`
`‘
`
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`
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`
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`
`30fl0
`
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`
`_CFAD V. UPENN
`IPR2015-01836
`
`I“?
`
`?f
`
`l;
`
`

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`1
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`4,231,938
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`5
`
`I0
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`30
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`35
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`45
`
`I-IYPOCHOLESTEREMIC FERMENTATION
`PRODUCTS AND PROCESS OF PREPARATION
`
`' SUMMARY OF THE INVENTION
`
`This invention relates to hypocholesteremic products
`from the cultivation of a microfungus of the genus As-
`pergillus. More specifically. it relates to a compound of
`the formula:
`
`
`
`and to the corresponding free hydroxyaeid
`
`3
`
`CH1 -
`
`CH3
`
`0l
`
`l
`O‘-C—
`
`“'0
`
`coon
`H
`
`CH3
`
`as well as pharmaoeutieally acceptable salts of the latter
`and lower alkyl and substituted alkyl esters of the latter
`in which the possible substitcent
`is phenyl, dime-
`thylamino or acetylamino. The invention also relates to
`a process of cultivating the microfungus and isolating
`from the medium a hypocholesteremic compound of
`thevabove structures. These new compounds have ex-
`cellent properties of inhibiting cholesterol biosynthesis
`and are useful against hypercholesteremia and hyperli-
`pemta.
`
`BACKGROUND OF THE INVENTION
`
`Because of the possible connection between high
`blood cholesterol and atherosclerosis. many efforts
`have been made to find ways and substances which
`would reduce the cholesterol in the mammalian body.
`One of these ways is to inhibit in mammals the body's
`ability to synthesize cholesterol.
`Recently, Endo et al., described (U.8. Pat. Nos.
`4,049,495 and 3,933,140) a fermentation product ob-
`tained by cultivation of a microorganism of the genus
`Penicillium and isolation from the medium. They called
`it ML 236 B and determined its structure together with
`two related compounds 236 A and 236 C. Its structure,
`under the name compactin, was also determined by A.
`G. Brown, T. C. Smale. T. J. King, J. Chem. Soc. (Per-
`kin I) 1 165 (1975). This compound has been found to be
`a strong inhibitor in vivo of the biosynthesis of choles-
`terol.
`
`DESCRIPTION OF THE l-NVENTION
`
`2
`in mammals. We have further found that this substance
`comprises principally the new compound, MSDB03, of
`the above structure, accompanied by only traces of
`other compounds, none of which appears to be those
`isolated by Endo et al. This new compound of our in-
`vention does not appear to be formed in the fermenta-
`lions described by Endo. The new compound,
`MSD803, is a much more potent inhibitor of cholesterol
`synthesis in vivo than is the compound, ML236B de-
`scribed by Endo.
`'
`The compounds of this‘ invention are highly useful as
`antihypercholesteremic agents for the treatment of ath-
`erosclerosis. hyperlipemia and like diseases in humans.
`They may be administered orally or" parentally in the
`form of a capsule, in tablet. an injectable preparation and
`the like. It is usually desirable to use the oral route.
`Doses may be varied, depending on the age, severity,
`body weight and other conditions of human patientsbut
`daily dosage for adults is within a range of from about
`2 mg. to 2000 mg. (preferably 10 to 100 mg.) given in
`three or four divided doses. Higher doses may be favor-
`ably applied as required.
`_
`-
`The compounds of this invention also have useful
`antifungal activities For example, they may be used to
`control strains of Pent’:-illiurn .rp..
`'Aspergillus niger. Cla-
`dosparium sp., Cochlrbboius mi}-abeortrs and Hr‘!mi'nrhnr-
`porium cynadnoris. For those utilities they are admixed
`with suitable formulating agents. powders. emulsifying
`agents or solvents such as aqueous ethanol and sprayed
`or dusted on the plants to be protected.
`In another aspect of this invention, it relates to a
`process for producing the compounds of this invention
`which comprises cultivating a microorganism belong-
`ing to the genus Aapergillus and then recovering said
`compounds of this invention from the cultured broth.
`Based upon taxonomic studies, this Aspergillus, isolated
`and identified as a hitherto undescribed microorganism.
`has been designated MF-4833 in the culture collection
`"0 of Merck and Co., Inc., Rahway, NJ. and a. culture
`thereof has been placed on permanent deposit with the
`American Type Culture Collection, 12301 Parlrlawn
`Drive, Rocltville, Maryland 20852, and has been as-
`signed accession No. ATCC No. 2054]. Another sam-
`ple. of a similar organism, designated MF-4845 in the
`Merck culture collection. has likewise been placed on
`deposit and has been given the accession number ATCC
`20542. The latter organism is the one giving the better
`yield. Although the use of these is described in connec-
`tion with the prooess of this invention. other organisms
`of the genus Aspergillus including mutants of the above
`ones are also capable of producing MSD803 and their
`use is contemplated in carrying out the process of this
`invention.
`The morphological characteristics of the microor-
`ganisms MF4333 and MF-4845 have been found to be
`those of the genus Aspergillus. Using the criteria speci-
`tied in .the standard authority "Manual of the Aspar-
`gilli". Charles Thom and Kenneth B. Rasper, published
`by the Williams and Wilkins Company", Baltimore, Md.,
`1945, and by comparison with know species, it has been
`determined that both strains are Aspetgillns rerrens.
`The culture of these organisms to produce MSD803
`is carried out in aqueous media such as those employed
`for the production of other fermentation products. Such
`media contain sources of carbon. nitrogen and inorganic
`salts assimilable by the microorganism.
`
`We have _found that unexpectedly. the cultivation of 65
`a very different microorganism, a microfungus of the
`genus Aspergillus produces a new substance that is also
`a very potent inhibitor of the biosynthesis of cholesterol
`
`4ofl0
`
`PENN EX. 2188
`
`CFAD V. UPENN
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`
`4,231,933
`
`3
`In general, carbohydrates ‘such as sugars, for exam-
`ple, glucose, fructose, maltose, sucrose, xylose. mann.i-
`tol and the like and starches such as grains, for example,
`"oats, ryes, cornstarch; corn meal and the like can be
`used either alone or in combination as sources of assimi-
`lable carbon in the nutrient medium. The exact quantity
`of the carbohydrate source of sources utilized in the
`medium depend in part upon the other ingredients of
`the medium but, in general, the amount of carbohydrate
`usually varies between about 1% and 6% by weight of
`the medium. These carbon sources can be used individ-
`ually, or several such carbon sources may be combined
`in the medium. In general, many proteinaceous materi-
`als may be used as nitrogen sources in the fermentation
`process. Suitable nitrogen sources include for example,
`_yeast hydrolysates, primary yeast, soybean meal, cot-
`tonseed flour, hydrolysates of casein, corn steep liquor,
`distiller‘s solubles or tomato paste and the like. The
`sources of nitrogen either alone or in combination, are
`used in amounts ranging from about 0.2% to 6% 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, potamium, ammonium,
`calcium, phosphate, sulfate, chloride, carbonate. and
`like ions. Also included are trace metals such as cobalt,
`manganese, iron and magnesium.
`It should be noted that the media described in the
`Examples are merely illustrative of the wide variety of
`media which may be employed, and yet are not in-
`tended to be limitative. Specifically, the carbon sources
`used in the culture media to produce MSD803 included
`dextrose, dextrin, oat flour, oatmeal, molasses, citrate,
`soybean oil, glycerol, malt extract, cod liver oil, starch,
`ethanol, figs, sodium ascorbate and lard oil. Included as
`nitrogen sources were peptonized milk, autolyzed yeast,
`yeast RNA. tomato paste, casein, primary yeast, peanut
`meal, distillers solubles, corn steep liquor, soybean
`mean, corn meal, NZ amine, beef extract, aspargine,
`cottonseed mm! and ammonium sulfate. The major
`ionic components were CaCO3, KH;PO_.i, MgSO4.7-
`H10 and NaCl and small amounts of CoCl;.6H-30 and
`traces of Fe. Mn, Mo, B and Cu were also present.
`The fermentation is carried out at temperatures rang-
`ing from about 20' to 37' C.; however, for optimum
`results it is preferable toconduct. the ferrnentation at
`temperatures of from about 22" to 30' C. The pH of the
`nutrient media suitable for growing the Aspergillus
`culture and producing MSDSGJ can vary from about
`6.0 to 8.0.
`
`35
`
`45
`
`SO
`
`4
`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
`flasks are shaken at a constant temperature for several
`days, and at the end of the incubation period the con-
`tents of the flasks are centrifuged or filtered.
`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. Ac-
`cording to this method. the nutrient medium is made up
`in the tank and sterilized by heating at temperatures of
`up to about 120‘ C. Upon cooling, the sterilized medium
`is inoculated with a previously grown seed of the pro-
`ducing culture, and -the fermentation is permitted to
`proceed for a period of time as, for example, from 3 to
`5 days while agitating and/or aerating the nutrient me-
`dium and maintaining the temperature at about 23' C.
`This method of producing MSDB03 is particularly
`suited for the preparation of large quantities.
`The compound is conveniently isolated from the
`fermentation broth as the lactone. However, MSD803 is
`present in the fermentation broth largely as the hydrox-
`ycarboxylate (open lactone) form. It is possible to iso-
`late this form and its salts. Alternatively, the lactone
`form can be hydrolyzed with bases such as NaOH to
`yield the corresponding salts such as the sodium salts.
`The use of bases with the pharmaceutically acceptable
`cations nffords salts of these cations. Careful acidifi-
`cation of the salts affords the hydroxy acid form. Con-
`versely, the hydroxy acid can be converted to the lac-
`tone form at acidic pH. Opening the lactone, under
`catalysis. with methanol, ethanol, propanol, or butanol
`or with phenyl, dimethylamino. or acetylamino alkanols
`yields the corresponding esters of this invention.
`The physicochemical properties of MSD803 in its
`lactone form are summarized as follows:
`
`
`.
`
`3.
`
`1- 1?°‘~”1'
`2.
`hlolecular Weight
`404
`(mass spectrum)
`Cz4I{5¢,05
`Formula
`4-04.2555
`(found by mass spec-
`Irometry
`404.2563
`calculated
`1». UV 5% '
`(in aeetonitrile) :
`Maxims
`230.5 nm wilh 13% 505.‘:-'
`237.5 nm with E% 516.6
`21-6 nm with 1-3% 395.2
`
`
`Although the novel compound is produced by both
`surfce and submerged culture. it is preferred to carry
`out the fermentation in the submerged state. A small
`scale fermentation is conveniently carried out by inocu-
`lating a suitable nutrient medium with the Aspergillus
`culture and, after transfer to a production medium,
`permitting the fermentation to proceed at a constand
`temperature of about 28" C. on a shaker for several
`days.
`The fermenation is initiated in a sterilized flask of 60
`medium via one or more stages of seed development.
`The nutrient medium for tile seed stage may be any
`suitable combination of carbon and nitrogen sources.
`The seed flask is shaken in a constant temperature
`chamber at about 28' C. for 2 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
`
`65
`
`5. "C NMR chemical shifts. The spectrum has been
`recorded in CDC}: solution (20.1 mg in 0.35 ml).
`Chemical shifts are given relative to internal tetrame-
`thylsilane at zero ppm; under the experimental condi-
`tions the solvent (CDCI3) signal appears centered at
`70.0 ppm. In agreement with mass spectral data 24
`carbon atoms are observed; their chemical shifts are:
`11.5, 13.6. 16.0. 22.6, 24.], 26.6, 27.2, 30.5, 32.3, 35.9.
`36.4, 37.), 38.4, 41.3, 62.4, 67.8, 76.4, 128.4, 129.7,
`131.7, 133.2, 170.3 and 117.2 ppm.
`6. ‘H NMR Spectrum. The spectrum was recorded in
`CDC]; solution and chemical shifts are shown in
`FIG. 1 in ppm relative to internal tetrarnethylsilane at
`zero ppm.
`1. IR Spectrum. The infra red spectrum was recorded in
`a I(Br pallet preparation of a sample. It is shown in
`FIG. 2.
`'
`
`Soflll
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`6
`
`B. Isolation
`
`5
`rotation
`rotation. The specific optical
`8. Optical
`[a]p15=320.'l" has been determined on a solution of
`5.30 mg/ml Cl-l3CN. This value has been obtained by
`measuring at the sodium-D-line wave length.
`On the basis of these and other data. the structure of the
`product believed with a considerable degree of cer-
`tainty to have the chemical structure;
`
`0
`
`CH)
`
`CH3
`
`H0
`
`0
`
`CH3
`
`CH3
`
`This invention can be illustrated by the following
`examples.
`
`EXAMPLE 1
`
`A. Fermentation
`
`A tube of lyophilized culture MF-I-833 is opened
`aseptically and the contents suspended in an unbaffled
`250 ml Erlenmeyer flask (seed flask) containing approx-
`imately 20 ml of medium A. Medium A has the follow-
`ing composition:
`
`Medium A
`
`ll} g
`80 g
`10 5
`10 3
`20 5
`lmt} ml
`
`Corn steep liquor
`Tomato pate
`Oatmeal
`Glucose
`Trace Eletnent. Mix No. 2
`Distilled water
`pH 6.8 with NIOH.
`Trsee-Element Mix No. 2
`IUD Ing
`FeSO4 . 'll'l3O
`IEII} lug
`Hn5O4 . 4H;O
`25 mg
`CuCl3 . 21120
`I00 mg
`cacl, . 211,0
`55 rag
`H3303
`'9 ma
`(NH4)6M°1014- 451:0
`In mg
`ZBSCI4 . "-‘H10
`
`Distilled Deioniaed Water 1% ans
`
`5
`
`20
`
`The whole broth is centrifuged for 20-30 min. Solids
`are saved for extraction. The supernatant liquid (pH
`6-8) is charged to a950 1111 bottle and 150 ml XAD-2
`resin is added. Using an automatic Extractor, operating
`on a preset schedule. the mixture is stirred for 2 hours.
`The spent broth is then siphoned off and discarded. The _
`resin is washed twice with 200 ml of deionized water
`and the washes were discarded. 'I'here then is added a.
`charge of 3(1) 1111 of mixed solvent: isopropanol-ethyl
`acetate-dichloromethane
`25:45-30. The mixture
`is
`stirred two hours. The solvent-resin slurry is filtered on
`a Buchner or sintered glass funnel and the resin is dis-
`carded. The filtrate broth solids are stirred with I00 ml
`acetone for i hour. The mixture is then centrifuged and
`the supernatant liquor is decanted. The combined fil-
`tratm are concentrated to 15 1111.
`
`C. Testing
`The filtrates were tested as inhibitors of HMG-CoA
`reductase enzyme by the method described by Beg.
`Stonik, Brewer and Bryan (1977 FEBS Letters 30 123 to
`129) using enzymes prepared as described by Kleinsek,
`Rangatham and Porter (1977 Proc. Nat. Accd. Sci.
`‘F4,
`I431 to 1435). The positive test (over 90% inhibition at
`20 micrograms per mi1liliter—an IC5o of 2.3 micrograms
`per milliliter indicated the presence of a very potent
`inhibitor of sterol synthesis acting at the HMG-COA
`reductase level.
`
`EXAMPLE 2
`
`A. A tube of lyophilized culture of an Aspergillus sp.
`MF-4333 is opened aseptically and the contents sus-
`pended in an unbaffled 250 ml Erlenmeyer flask (seed
`flask No. 1) containing 4-0 ml of rnedium C. Medium C
`has the following compositions:
`
`
`
`Medium C
`
`Corn steep liquor
`Tomato peat-e
`Oatmeal
`Glucose
`Trace element
`Mil No. 2
`tom ml
`Distilled water
`pH as with NaOH
`
`5 5
`4-0 g
`I0 3
`ID 5
`10 g
`
`The inoculated flask is incubated for 48 hours at 28‘ C.
`on a 220 rpm shaker (2 inch throw). Two unbaffled 2
`liter Erlenmeyer flasks each containing 500 ml of me-
`dium B are then each inoculated with 10 ml per flask of
`the growth from the seed flask. Medium B has the fol-
`lowing composition:
`
` Medium B
`
`50
`
`This inoculated flask is" incubated for 24 hours at 23' C.
`on a 220 rpm shaker (2 inch throw) for 24 hours. Eight
`more unbaffled 250 ml Erlenmeyer flasks (No. 2 seed
`flask) each containing 40 ml of medium C are then each
`inoculated with 2 ml per flask of the growth from seed
`flask No. 1. These eight No. 2 seed flasks are incubated
`for 24 hours at 28' C. on a 220 rpm shaker (2 inch
`throw). Twenty, two liter unbafiled Erleruneyer flasks,
`containing 500 ml of medium B are then each inoculated
`with 14 ml per flask of the combined growth of the
`eight No. 2 seed flasks. These twenty flasks are incu-
`bated at 28', with agitation for ll days. After 11 days
`incubation,
`the contents of these twenty flasks are
`pooled.
`B. 10.2 liters of whole broth, pH 6.0, was blended in
`These two inoculated flasks are incubated for 96 hours
`a Waring blender to break up the heavy mycelial pads,
`at 28'. One flask is incubated without agitation. The 65 centrifuged and the clear supernatant decanted. After
`filtration the i0 liters of filtrate was extracted with 3
`other flask is incubated on a 150 rpm shaker (2" throw).
`liters of ethyl acetate, yielding 1820 ml of clear extract.
`After 96 hours, the contents of each flask is set aside for
`A second extraction with 3 liters of ethyl acetate
`isolation of the product.
`
`20 3
`Tomato paste
`10 3
`Primary yeast
`20 g
`CPC Starch
`5 mg
`CeCl2 . 6HzO
`ICIID ml
`Distilled water
`pH 7.2-1.4 with molt
`
`CFAD V. UPENN
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`7
`yielded 3350 ml of clear extract. The broth solids were
`extracted by stirring one hour with 2 liters of methanol
`and filtering to yield 2100 ml of filtrate.
`Aliquots of these extracts were dried and sent for
`assay by the procedure of Example l(C), with the fol-
`lowing results:
`
`8
`shaker (2 inch throw). Two, 250 ml unbaffled Erlen-
`meyer flasks each containing 40 ml of medium D are
`then each inoculated with 2 ml per flask of the growth
`from the seed flask. Medium D has the following com-
`position:
`
`S
`
`Mcdiurn D
`
`
`
`
`Volume (ml)
`ism
`3350
`2100
`
`_-—l'3’—!"":j1
`Total Solids (mg)
`I I33
`in
`13.15
`
`_
`_
`‘
`Total Units of Activity
`1,496,695
`314,900
`1,144,061
`
`'0
`
`10 3
`Lactose
`15 '
`D-mm,“ minim.
`Avgtvlyud ire-I4
`5 s
`D“N1|=d‘WI|==f
`1090 N1
`
`91: 7-0 with NIOH
`
`'5
`
`20
`
`These two inoculated flasks were incubated for 96
`hours at 28' on a 150 rpm shaker (2 inch throw). After
`96 hours incubation the contents of these two flasks is
`submitted for extraction by the procedure described in
`Example 203). Total -production in these flasks is
`1450-2000 units/ml.
`
`EXAMPLE 4
`
`25
`
`A tube of lyophilized culture of an Aspergillus, MF
`4845, is opened aseptically and the contents suspended
`in an unbaflled 250 ml Erlenmeyer flask (seed flask No.
`I} containing 4-0 ml of medium C. The inoculated flask
`is incubated for 24.43 hours at 23' c. on a 220 rpm
`shaker (2 inch throw). A portion (approx. 0.5 ml) of this
`.
`.
`.
`.
`flask. is then
`to inoculate a slant tube containing
`30 medium E. Medium E has the following composition:
`
`C. Gel Filtration
`
`All of samples from the first two extracts in Example
`2 (B) were "combined. dissolved in methanol and filtered
`to remove insoluble solids. The 30 ml of filtrate was
`loaded onto a gel filtration column (2.5 cmX200 cm,
`980 ml) packed with Sephaclex Ll-I-20 and the sample
`fractionated according to molecular size using methanol
`as solvent. With refractive index and UV. recordings as
`guides, the best fractions were identified by bioassay.
`
`
`
`T°"" 5°““‘ ‘“"‘
`F"""“°“ 1
`'
`3’
`'
`—
`_
`fig
`
`7°” "““' °""°“"“"
`‘M271
`I.
`.
`
`Medium E
`ya“ gnu“
`MallExti1ii:t
`Deltrme
`$116‘ wflfl
`PH ,9 with mo“
`
`4 5
`lo 5
`' 4 s
`‘MN;
`
`The inoculated slant tube is incubated for 11 days at
`room temperature. It is then stored at -60’ C. for 3-4
`months. A portion of the contents of this slant is then
`suspended in an unbaffled, 250 ml Erlenmeyer flask
`(No. 2 seed flask) containing 40 ml of medium C. The
`inoculated flask is incubated for 24 hours at 28' C. on a
`220 rpm shaker (2 inch throw). Six unbaffled 250 ml
`Erlenmeyer flasks (No. 3 seed flasks) containing 40 ml
`of medium C are then each inoculated with 2 ml per
`flask of the growth from the No. 2 seed flask. These six
`inoculated flasks are incubated for 48 hours at 28' C. on
`a 220 rpm shaker (2 inch throw). Six unbaffled two liter
`Erlenmeyer flasks containing 500 ml of medium F are
`each then inoculated with the contents of No. 3 seed
`flask. Medium F has the following composition:
`
`
`
`_
`”°d“""F
`Cm Ire-Bi! liquor
`$ :1“
`Sour-n MI
`Glucose
`50
`Soybean oil
`‘
`
`CICo3
`Dl“m°‘ y"““'
`P“ '5’? Wk mo“
`
`15 s
`
`* s
`5 5
`1.: 3
`gig:
`
`5;
`lmoml
`
`D. Separation and Purification
`A sample from Fraction 2 above was prefiltered
`through a 1-gram bed of Waters Bondapak C18/Porasil 35
`B and eluted with five volumes of methanol. The rneth-
`_
`anol eluate was concentrated to 0.5 ml. This sample was
`chromatographed, over several rims, on a Waters p.Cl8
`column (3.9 mm)(30 cm) with methanol: 0.05 M amnio-
`niiun phosphate, pH 2.9 (75:25), as the developing sol- ‘O
`vent. Fractions were scanned on a Beckman Spectro-
`photometer, and those showing absorption maxima at
`236 nm, with shoulders at 229 nm and 245 nm were
`combined and concentrated under reduced pressure to
`an aqueous solution. The pH of the concentrate was
`adjusted to 6.5 with 2 M potassium hydroxide and the
`active components were extracted with ethyl acetate.
`The organic layer was dried, and the residue dissolved
`in 0.3 ml methanol. The methanol
`solution was
`chromatographed as above and recycled. Cuts contain-
`ing earlier eluting component were combined, concen-
`trated to an aqueous solution and extracted with chloro-
`form. The chloroform residue was taken up in methanol
`and the solvent evaporated under nitrogen. 3.5 mg. of
`dried product was obtained and identified as the open
`lactone form. Cuts containing the second component 55
`were combined and extracted with chloroform as
`above. 0.87 mg. of dried product was obtained and
`identified as the lactone.
`Samples were sent for bioassay.
`EXAMPLE 3
`
`45
`
`50
`
`50
`
`Best Mode of Fermentation of MF-4833
`M: of lyopéiciilized culture of anh Aspergillus sp.
`
`aseptically and t e contents sus- 65
`is open
`pended in an unbaflled 250 ml Erlenmeyer flask (seed
`flask) containing 40 ml of medium C. The inoculated
`flask is incubated for 48 hours at 28' C. on a 220 rpm
`
`7 of 10
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`PENN EX. 2188
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`CFAD V. UPENN
`IPR2015-01836
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`

`
`4,231,938
`
`10
`autolyzed yeast: 0.5% wt/‘vol
`Polyglycol P2000: 0.25% wt/vol
`whose pH was adjusted to 7.0. This was sterilized 15
`minutes at 121‘ C One liter of the third stage above was
`then charged and the mixture was incubated at I30 rpm
`at 25' C. for 96 hours with an air flow of 10 cfm.
`
`B. Isolation of MSD803
`
`About 37.5 lbs. (3 bag) of a silicaceous filter aid was
`added to 110 gal. whole broth from the culture of MF-
`4833 described above and the mixture was filtered
`through an 18-inch filter press. The clarified filtrate,
`(pH 6.6) was adjusted to pH 4.0 by careful addition of
`450 ml of concentrated hydrochloric acid, and ex-
`tracted by agitation with about one-third volume (36
`gal.) of ethyl acetate. After separation, the upper sol-
`vent layer was removed, and the water phase again
`extracted with ethyl acetate (38 gal.) in a similar fash~
`ion. After separation, the two extracts were combined
`and back-washed by agitation with about twelve gal-
`lons of water. After separation, the ethyl acetate solu-
`tion was concentrated under vacuum at a temperature
`below 30' C., first in a stirred kettle. and finally in a
`rotary vacuum evaporator to a residual volume of
`slightly less than one gallon.
`_
`Approximately 1 gal. (3800 ml) of ethyl acetate con-
`centrate from the preceding extraction was further con-
`' centrated in a rotary evaporator (ca I0 mm, 4-0' C. bath)
`to a syrup and was then concentrated twice more, after
`addition of about one liter of methylene chloride in two
`portions. to free the syrup of polar solvent. The final oil
`of about 3(1) ml which contained about 250 g of solids
`by dry weight determination. was made up to about 750
`ml with ethyl acetate methylene chloride (30/70; v/v)
`and 203 g of silica gel was added and mixed in to form
`a slurry. This was layered over the top of a 14 cm by 36
`cm column bed holding 2.5 kg of the same silica gel, in
`about 7.5 1 volume, which had been packed as a slurry
`in the same solvent mixture. Development with the
`same solvent was continued until 3 liters of effluent was
`taken off as forerun.
`
`Development with ethyl acetate-methylene chloride
`(50/50; v/v) was begun. taking 300 ml effluent frac-
`tions. Twelve fractions were taken, then 100% ethyl
`acetate elution was begun, and after seven more frac-
`tions. 100% acetone elution was begun. Fractions four
`through twenty-four were assayed for bio-activity in
`the HMG-CoA Reductase inhibition assay referred to
`in Example 1. Substantial activity was found in fractions
`7 through 11. Peak activity was found in fraction 3. It
`was concentrated to an oil for further purification; dry
`wt. by solids determination was 9.0 gm.
`Fraction 3 from the silica gel column was triturated
`with 50 ml methylene chloride and filtered; the dried
`filter cake weighed 4.9 gm. The filtrate was charged to
`a 2-inch ID. by l-meter long column filled with Se-
`phadeit LH-20 dextran gel {Pharrnacia) swollen and
`equilibrated in methylene chloride, and the column was
`eluted with methylene chloride at a rate of 15 ml/min.
`MSDSD3 is eluted between 0.64 and 0.81 column
`volumes. Solvent was removed from this peak leaving a
`slightly brown residue weighing approximately 0.290
`gm. 213 mg. of this residue was taken up in L5 ml of
`CHzCl3—-CH3CN (65-35), charged to a prepacked and
`equilibrated silica gel column (EM LOBAR Size B) and
`eluted with CI-IzCl1—CH3CN (65-35) at 5 ml/min.
`Evaporation of solvent from the peak eluting between
`235 and 360 ml of eluant left 121 mg of crystalline prod-
`
`9
`The inoculated flasks are incubated for 11 days without
`agitation at 28' C. After ll days broth is delivered for
`extraction by the procedure of Example 2(3). Total
`production in these flasks is 1231 units/ml.
`EXAMPLE 5 '
`Best Mode of Fermentation with MF-4345
`
`A tube of lyophilized culture of an Aspergilltls, MF-
`4845, is opened aseptically and the contents suspended
`in an unbaffled 250 ml Erlenmeyer flask (seed flask)
`containing 4-0 ml of medium C. The inoculated flask is
`incubated for 30 hours at 28' on a 220 rpm shaker (2
`inch throw). An unbaffled. 250 ml Erlenmeyer flask "
`containing 40 ml of medium G is inoculated with 2 ml
`per flask of the growth from the seed flask. Medium G '5
`has the following composition:
`
`I0
`
`
`
`Medium (3
`
`45 g
`Dextroce
`24 3
`Peptonized milk
`2.5 g
`Autclyned yeast
`1.5 ml
`Polyglycol P2000
`1011] ml
`Distilled water
`pH ‘.-‘.0 with NIOH
`
`10
`
`This inoculated flask is incubated for 120 hours at 28' C.
`on a. 220 rpm shaker (2 inch throw). After 120 hours
`incubation, the contents of the flask is submitted for
`extraction by the procedure of Example 2 (B). Total 30
`production in the flask is 21,541! units/ml.
`EXAMPLE 6
`
`45
`
`40
`
`A. Large Scale Fermentation with MF4-8-33
`The medium used in each step of the fermentation 35
`comprised:
`Corn steep liquor: 5 g
`Tomato paste: 40 g
`Oat Flour: 10 g
`Glucose: 10 g
`Trace element solution: 10 ml
`Distilled water: 1000 ml
`adjusted to pH 6.8 with sodium hydroxide.
`The trace element solution comprised:
`FeS04.'?I-I20: l g
`MnS04.4l-I10: l g
`CuCl2.2H1O: 25 mg
`CaC12: 100 mg
`H3304: 56 mg
`(NH4)5McI702,4.4H1O: 19 mg
`Zn SO4.7H;0: 200 mg
`distilled water: 1 liter
`All mediums were checked for sterility before innocu|a-
`tion with a microorganism.
`To a 250 ml non-baffled Erlenmeyer flask was 55
`charged 40 ml of medium and the contents of one tube
`of lyophillized organism MF 4833. It was then shaken
`for 24 hours at 28' C. on a rotary shaker at 220 rpm.
`New flasks were then charged with 40 ml of medium
`and 1 ml of the first flasks’ contents and were shaken an 60
`additional 24 hours at 23' C. A 2 liter flask was then
`charged with 40.’) ml of medium and I0 ml of the second
`stage fermentation mixture and this too was shaken for
`24 hours at 28‘ C.
`A 200 gallon stainless steel fermentation vat_was then 65
`charged with 501 liters of a medium comprising:
`lactose: 2% wt/vol
`distiller solubles: 1.5% wt/vol
`
`CFAD V. UPENN
`IPR20l 5-01836
`
`
`Soflll
`
`PENN EX. 2188
`
`

`
`4,231,938
`
`-
`12
`acid is extracted with ethyl acetate. The latter solvent is
`washed once with water, dried and removed in vacuo
`with a bath temperature‘ not exceeding 30'. The hy-
`drosy acid derived slowly reverts to the lactone on
`standing.‘
`-
`-
`EXA-MP'[_.E_-9
`To a solution of 4 mg of the product of Example 6 in I
`1 Inlof absolute ethanol isadded 0.] ml 0.1 M sodium
`ethoxide in absolute ethanol. This solution is allowed to
`stand at room temperature for one hour, is then diluted
`into water and extracted twice with water, the ethyl
`acetate dried over anhydrous sodium sulfate is removed
`in vacuo to yield the ethyl ester of MSD303.
`In like manner, by the use of equivalent amounts of
`methanol. propanol. butanol,' isobutanol,
`t-butanol.
`aruylaloohol, isoamylalcohol, 2-dimethylaminoethanol.
`benzylaleoho]. phenethanol. 2—acetamidoethanol and
`the like, the corresponding esters are obtained.
`What we claim is:
`
`'
`
`1. The compound of the formula
`
`11
`uct, m.p. ]55‘—l60' C. HPLC of. this material on a EM
`RP 18 reverse-phase analytical column (B Merck
`HIBAR [1, Cat. No. 906046) using 0.05 M sodium phos- '
`phate pH 3.0-acetonitrile 45-55 as eluant at 2 ml/min._
`showed a characteristic uv absorbing peak at 1-lmin;
`- 5
`Eighty-two mg. of this material was recrystallized
`from 0.6 ml of absolute ethanol, then again l'rom_0.4 ml
`of the same solvent to afford, after drying over-night in
`a desiccator over P105,-40 mg of white feathery crys-
`tals. Analytical HPLC on the system described "above I0
`gave a single sharp peak at 1] minutes elution time.
`After further
`recrystallizations, a melting point of '
`1'.-'O‘—l'Tl' C. was obtained.
`The. product was identified by spectra. etc., as the
`lactoue form of MSD803. This material, in the in vitro 15
`HMG-CoA reductase test (of Example 1) gave an IC_-,9
`of 0.0] micrograms per milliliter.
`-
`
`'
`
`EXAMPLE 7
`Salts of MSD803
`
`_
`
`20
`
`To a solution of4-0 mg the product of Example 6 in 2
`ml of ethanol
`is added 1 ml of aqueous NaOH
`{ID-‘moles; 1 equivalent). After one hour at room tem-
`perature, the mixture is taken to drynws in vacuo to
`yield the sodium salt of the free acid tom of Msoaos. 2‘
`In like manner the potassium salt is prepared using
`one equivalent of potassium hydroxide and the calcium
`salt using one-half equivalent of C110. Other phar'tn.a-
`ceutically acceptable salts are likewise prepared using
`equivalent quantities of the appropriate base.
`EXAMPLE 8
`
`30
`
`Preparation of Free Hydroxy Acid ‘of MSD803
`The sodium salt produced in Example 7 is redissolved 35
`in 2 ml of ethanol-water (1:1) and added to 10 ml of 0.]
`N hydrochloric acid from which the liberated hydroxy
`
`45
`
`. no "H
`
`#0
`
`
`
`if!-[3
`c1t;—cH_;—t:H—'co--0
`
`-
`
`CH3
`
`I
`
`9ofl0
`
`PENN EX. 2188
`
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`
`: CERTIFICATE EXTENDING PATENT TERM
`UNDER 35 U.S.C. 156
`
`Patent No.
`
`: 4,231,933
`
`Dated
`
`: November 4, 1980
`
`-Inventor(s}
`
`: Richard I. Monaghan, Alfred W. Alberts,
`Carl H. Hoffman, George Albers-Sohonberg
`
`Patent Owner
`
`: Merck & Co., Inc.
`
`This is to certify that there has been presented to the
`
`COMMISSIONER OF PATENTS AND TRADEMARKS
`
`an application under 35 U.S.C. 156 for an exten