United States Patent
`
`[:91
`
`Endo et al.
`
`[11]
`
`3,983,140
`
`[45] Sept. 28, 1976
`
`[54] PHYSIOLOGICALLY ACTIVE SUBSTANCES
`
`[57]
`
`ABSTRACT
`
`{'35}
`
`in ventors: Akira Endo; Masao Kurodn; Akin
`Tcrahara; Yoshio Tsojita; Cliihiro
`Tamura, all of Tokyo, Japan
`
`[73] Assignee: Sankyo Company Limited, Tokyo,
`Japan
`
`[22]
`
`Filed:
`
`May 12, 1975
`
`[21] Appl. No; 576,651
`
`[30]
`
`Foreign Application Priority Data
`'
`June ‘I_-‘, 1974
`
`49-64823
`
`[52] US.
`
`260,-343.5; l95,"8I;
`424E279
`C07!) 309330
`Int.
`Fieldof 260.*'34-3.5
`
`[51]
`{S8}
`
`[56]
`
`References Cited
`UNITED STATES PATENTS
`IUIWT4
`Thiele at 3.1 .................... .. 260;’343.5
`
`3,842,092
`
`Primary E.mmim:r—Henry J. Jiies
`Assistant E.mminer—-C. M. S. Jaisle
`Attorney, Agent, or Firm-——Flynn & Frishauf
`
`Physioiogically active substances ML-236 of the for-
`mula
`
`HC\}:::: 0
`
`OH
`
`L
`
`R
`
`@.
`
`(I3
`
`wherein R is hydrogen atom, hydroxy group or 2-
`methylbutyryloxy group having cholesterol- and lipid-
`lowering effects in blood and ‘liver and thus utility as
`hypocholesteremic
`and hypolipemic medicaments.
`They are obtained by cultivation of an ML-236-
`producing microorganism belonging to the genus Peni-
`cillium in a culture medium and subsequent recovery
`thereof from a cultured broth.
`
`4 Claims, 6 Drawing Figures
`
`1 ofll
`
`PENN EX. 2187
`
`CFAD V. UPENN
`lPR2015-01836
`
`

`
`U.S. Patent
`
`Sept. 23, 1976
`
`Sheetlof6
`
`3,983,140
`
`FIG.
`
`I
`
`2:0
`
`220
`
`240
`
`260
`
`230
`
`300
`
`WAVELENGTH -mp
`
`2 of 11
`
`PENN EX. 2187
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`U.S. Patent
`
`Sept. 23, 1976 .
`
`Sheet2of6
`
`3,983,140
`
`FIG.2
`
`600
`
`
`
`2000I800I600I400I200I000800
`
`3200
`
`
`
`
`
`WAVESNUMBERcm’
`
`I00 3 3 85
`
`NOISSIWSNVHL .I.N33«‘.-l3d
`
`3 ofll
`
`PENN EX. 2187
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`U.S. Patent
`
`Sept. zfi, 1976
`
`Sheet3of6
`
`3,983,140
`
`F I G. 3
`
`I00
`
`so
`
`5 I332.
`
`200
`
`220
`
`240'
`
`260 '
`
`290
`
`300
`
`WAVELENGTH. my
`
`4 of 11
`
`PENN EX. 2187
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`U.S. Patent
`
`Sept. 28, 1976
`
`Sheet4of6
`
`3,983,140
`
`600
`
`
`
`2000I800I600I400I200I000300
`
`40003200
`
`FIG.4
`
`I00
`
`80
`
`8
`
`o
`~:r
`
`8
`
`NOISS|WSNVE:I.L J.N33H3d
`
`
`
`
`
`WAVESNUMBERcm"
`
`5 Ofll
`
`PENN EX. 2187
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`U.S. Patent
`
`Sept. 23., 1976
`
`Sheet5 of6
`
`3,983,140
`
`FlG.5
`
`I001
`
`J
`
`1%
`Elam
`
`‘zoo
`
`'
`
`'22o '
`
`T
`240
`
`E6?)
`
`2'30
`
`300
`
`WAVE LENGTH my
`
`IPR20l 5-01836
`_
`_
` n
`
`6 of 11
`
`'
`
`PENN EX. 2187
`CFAD V. UPENN
`
`

`
`U.S. Patent
`
`Sept. 23, 1976
`
`Shect6of6
`
`3,983,140
`
`0O(
`
`O OO
`
`an
`
`O.
`
`O 2
`
`89 0
`
`3
`
`TE
`"
`n:
`33
`o
`_ 2
`
`-—
`
`no
`-
`(9
`LL
`
`O 8
`
`E<
`
`[
`§ 3
`N
`
`3
`
`8
`
`8
`
`2
`
`3
`
`Notss1wsNva.L .1r_u3:>a3d
`
`8N
`I'D
`
`§
`*
`
`7 of 11
`
`PENN EX. 2137
`CFAD V. UPENN
`IPR20l 5-01836
`
`

`
`1
`
`3,983, I 40
`
`PHYSIOLOGICALLY ACTIVE SUBSTANCES '
`
`This invention relates to new physiologically active
`substances and a ferrnentativc process for producing
`the same.
`
`5
`
`More particularly. it is concerned with a new class of
`physiologically active substances (hereinafter referred
`to as ML-236) having the formula
`
`l0
`
`H O
`I
`5‘-/
`
`CH
`
`R
`
`_
`
`(1)
`
`I5
`
`wherein R is hydrogen atom, hydroxy group or 2-
`rnethylbutyryloxy group (—OCOCH(CH3)CH,CH,,}
`and also with a process for the production of the sub-
`stances ML-236 by cultivation of an ML-236—produc—
`ing microorganism belonging to the genus Penicillium.
`The new substances MI.-236 of this invention have
`excellent physiological activities for me'di'<:in'ol use,
`more specifically, an inhibition activity of cholesterol
`biosynthesis, an antiatherosclerosis activity and an an-
`tihyperlipemia activity.
`One cause of such diseases as atherosclerosis, hyper-
`lipemia and so on is now considered to be owing to
`cholesterol deposit within a living body, especially in
`the intima of arteries.
`
`20
`
`25
`
`30
`
`2
`Under these circumstances, we have made systematic
`studies on substances obtainable from cultured broth of
`microorganisms about their inhibition activities of cho-
`lesterol biosynthesis, from a standpoint wherein inhibi-
`tion of cholesterol biosynthesis may be effective in
`preventing and treating such diseases and, as a result, it
`has been found that the substances ML-236 can be
`isolated from a cultured broth of a microorganism be-
`longing to the genus Penicillium and also that the sub-
`stances ML-236 possess potent cholesterol- and lipid-
`lowering effects in blood and liver.
`It is. accordingly, a primary object ofthis invention to
`provide a new group of the substances ML—236 of the
`above formula (1) which are highly useful as hypocho-
`lesteremic agents and hypolipemic agents. for the treat-
`ment of atherosclerosis. hyperlipemia and like diseases.
`Another object of this invention is to provide a pro-
`cess for the fermentative production of the valuable
`substance ML—236 by cultivation of an ML-236—pro-
`ducing microorganism belonging to the genus Penicil-
`Iium.
`
`Other objects and advantages of this invention will be
`apparent from the description as shown hereunder.
`In one aspect of this invention, there are provided the
`substances ML—236 of the formula (I) as briefly ex-
`plained hereinabove. It has now been clarified that the
`substances ML‘--236 of the formula (I) are separated
`into 3 kinds of substances which are designated as
`ML-236A. ML-236B and ML-236C. respectively, and
`the respective plane structures thereof have been clari-
`fied as defined below, with the NMR, mass. IR, and UV
`spectra and X-ray diffractiometries thereof.
`
`r_.i;.—256s
`
`O
`
`H
`
`“C:
`
`l"}'L—2§6B
`
`I
`
`CH5
`
`OH
`
`(I33
`
`O
`
`O
`
`HO
`
`CH
`
`O
`ojfi/\
`
`(Ib)
`
`I“'lL—2 6C
`
`_
`
`HO\
`
`/0
`
`CH3
`
`\
`
`(R3
`
`8 of 11
`
`PENN EX. 2187
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`3
`The physico-chemical properties of the substances
`ML-236A, ML-23613 and ML-236C are summarized as
`shown below.
`
`3,983,140
`
`A
`oily
`(neutral)
`
`—
`70.18
`$.75
`21.0‘.-‘
`306
`
`ML-236
`B
`white scales
`{neutral}
`
`l49—l5l"C.
`'l(l.till
`8.5!:
`2036
`390
`
`C
`oily
`(neutral)
`
`—
`'l'4_(l-1
`8.58
`17.33
`290
`
`C.,.H,..O;.
`C,;.H;..O,-,
`C,,.H,.,0..
`As shown
`All shown
`As shown
`in FIG. 5
`in FIG. 3
`in FIG.
`1
`As shown
`As shown
`As shown
`in FIG. 6
`in FIG. 4
`in FIG. 2
`Soluble in methanol. ethanol, acetone,
`ethyl acetate, chloroform and benzene
`Insoluble in n-hexane and petroleum
`ether
`
`0.21
`
`LL45
`
`(1.52
`
`(Lilli
`
`0.21
`
`0.27
`
`Nature
`
`m.p_
`Elcmcnlary
`Analysis (‘E1
`
`C
`H
`O
`Molecular Weight
`{Mass spectrum)
`Molecular Formuia
`UV spectrum
`{Methanol}
`IR spectrum
`il(Br1
`Solubility
`
`R, value
`iTLC'
`:
`Silica
`gel G}'
`
`n-hexane-
`aeerone
`I l:1l
`
`dichlorov
`methane-
`elhyl
`acetate
`[?:3}
`
`1 through 6 hereof, FIG. 1 shows the ultravi-
`In FIGS.
`olet absorption spectrum of ML-236A exhibiting max-
`ima at 229, 236 and 245 rn,u., respectively; and FIG. 2
`shows the infrared absorption spectrum of ML—236A
`showing absorption bands at 3350, 3300 and 1725
`cm", respectively.
`FIG. 3 shows the ultraviolet absorption spectrum of
`ML-23613 exhibiting maxima at 229, 236 and 245 mu.
`respectively; and FIG. 4 shows the infrared absorption
`spectrum of ML-23613 showing absorption bands at
`3500, 1740 and 1695 cm“, respectively.
`FIG. 5 shows the ultraviolet absorption spectrum of
`ML-236C exhibiting maxima at 229, 236 and 245 mu,
`respectively; and FIG. 6 shows the infra red absorption
`spectrum of ML-236C showing absorption bands at
`3350 and 1700 cm“, respectively.
`The substances ML-236 of this invention, when ap-
`plied to patients suffering from such diseases as
`atheroscrelosis, hyperlipemia and so on. may be admin-
`istered orally or parenterally in the form of a capsule, a
`tablet, an injectable preparation and the like and it is
`usually desirable to administer the substance via oral
`route. Doses may be varied depending upon the age,
`severity, body weight and other conditions of patients,
`but usual daily dosage for adult is within a range from
`about 200 mg. to 2000 mg. given in 3 or 4 divided
`doses. However, higher doses may be favorably ap-
`plied. as required.
`As fully disclosed hereinabove, the substances ML-
`236 of this invention have potent activities for the inhi-
`bition of cholesterol biosynthesis in vitro and in vivo.
`1. The activity in vitro on inhibition of cholesterol
`biosynthcsis of the substances ML.-236 may be assayed
`by the following method. Slice of rat liver and radioac-
`tive acetic acid are brought to interaction at 37°C. for
`60 minutes, the radioactive cholesterol thus biosynthe-
`sized is saponified and separated as precipitates with
`digitonin and then its radioactivity is measured to de-
`
`I0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`65
`
`4
`termine the produced cholesterol amount. Separately,
`the same procedures as above are employed except
`that ML—236 is added at the beginning of the reaction,
`thereby the biosynthesized cholesterol amount being
`determined. Thus, an effect of ML-236 can be quanti-
`tatively determined. (Sce Bricker et 31.: The Journal of
`Biological Chemistry, 24?, 4914, l9'l2]
`According to the assay method as set forth above, it
`has been shown that the substances ML-236A, ML-
`236B and ML-236C exhibit about 50% inhibition of
`cholesterol biosynthesis at
`the respective concentra-
`tions of 0.04 ug./ml.. 0.01 ,ug.l'ml. and 0.08 i.Lg.;"ml.
`Further. acute toxicity of each substance ML-236 was
`determined in mice via intraperitoneal administration
`to be 400 mg../kg. or more, which clearly shows a lower
`toxicity of the substance ML-2 36.
`ll. Effectiveness of ML—236 11:15 been confirmed by
`examining its effect on lowering the lipid content in
`blood and liver of animals with various test methods,
`representatives of which will be more fully illustrated
`below.
`I. One of two groups of rats having an average weight
`ofabout 200 g., each group consisting of 5 animals, was
`individually given by intravenous injection with 200
`mg.,l'kg. of Triton WR 1339 (trade name; available
`from Ruger Chemical Co., Ltd., U.S.A.; having an
`activity of increasing cholesterol level in blood) simul-
`taneously with intraperitoneal administration of 20
`rng.,l'lcg. of ML-236B. After 24 hours from administra-
`tion, rats were sacrificed by bleeding, blood and liver
`were collected and their cholesterol and neutral lipid
`levels were determined by a conventional method. As a
`result, it has been clear that cholesterol levels in blood
`are lowered by 14.2% and those in liver by 10.1%, as
`compared with those in the case of another group given
`with intravenous injection of Triton WR 1339 alone.
`2. Two of four groups of rats, each group consisting
`of five animals. were orally given one dose of 5 mgqlkg.
`of ML-236A suspended in gum arabic. After 3 hours
`and I8 hours from administration, animals were sacri-
`freed by bleeding and blood was collected. Cholesterol
`and neutral lipid levels in blood serum were determined
`by a conventional method. It has been clear that the
`cholesterol and neutral lipid levels after 3 hours are
`lowered by 13.5% and 49.5%, respectively, and also
`that the cholesterol level after 18 hours is lowered by
`l3.5%, while lowering of neutral lipid level is not ob-
`served, as compared with those in the case of two other
`groups administered with gum arabic alone.
`Moreover, one of two other groups of rats, each
`group consisting of five animals, were orally given 80
`mg.,l'l<g. of powdery MI.-236B in the form of a physio-
`logical saline solution. After 3 hours from administra-
`tion, cholesterol and neutral lipid levels in blood serum
`were determined in the same manner as above. It has
`been clear that the Cholesterol and neutral lipid levels
`are lowered by 20.0% and 44.2%, respectively, as com-
`pared with those in the case of another group adminis-
`tered with a physiological saline solution Containing no
`ML-23613.
`It will be apparent from the above results that the
`substances ML-236 of this invention exhibit a promi-
`nent effect in the inhibition of cholesterol biosynthesis
`in a living body and thus they are utilizable as a medica-
`ment against. for example. atherosclerosis, hyperlipe-
`min and so on.
`
`In another aspect of this invention, there is provided
`a process for producing physiologically active sub-
`9ofl1
`PENN EX. 2187
`
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`3,983,140
`
`5
`stances ML-236A, M'L—236B and"ML-236C which
`comprises cultivating an ML-236-producing microor-
`ganism belonging to the genus Penicillium and then
`recovering said substances from a cultured broth.
`The microorganisms which may be employed in this
`invention are the ML-236-producing ones belonging to
`the genus Penicillium and, as the strain proved to be
`particularly efiective for this invention,
`there is, for
`instance, mentioned Penicillium citrinum SANK l8':'6'l
`which has been deposited under an accession No. 2609
`with Technical Research Institute of Microbial Indus-
`try, Agency of Industrial Science & T_echnolo'gy'," Minis-
`try of International Trade and Industry, Japan, and also
`as NRRL-8082 in the Northern Regional Research
`Laboratory, Northern Central Region, Agricultural
`Research Service, U.S. Department of Agriculture, at
`Peoria. III., USA.
`_
`_
`Although this invention will be explained hereinbe-
`low principally with respect to the strain SANK 1876?,
`it is well-known in the art that various properties of all
`microorganisrns belonging to the genus Penicillium are
`not definite, but the microorganisms of the genus Peni-
`cillium may be easily varied naturally and artificially. It
`is, accordingly, to_be noted that all strains which are of
`the genus Pencilliurn and capable of producing ML-
`236, including varieties and mutants, are contemplated
`and usable in this invention.
`The above strain SANK I876‘.-' itself is known and its
`morphological properties are reported in the following
`literature: K. B. Raper and C. Thom; A Manual of the
`Penicillia, the Williams and Wilkins Company, i949.
`In carrying out the process of this invention, cultiva-
`tion may be satisfactorily conducted under aerobic
`condition in the same manner as commonly employed
`in the art for cultivation of a strain of the genus Penicil-
`lium. For instance, the ML-236-producing microorgan-
`ism may be grown in a culture medium, e-.g., that con-
`taining malt extract 2%, glucose 2%, peptone 1%, agar
`2% and then the culture so obtained may be inoculated
`and cultivated in a culture medium. Alternatively, the
`microorganism grown in a culture medium may be
`cultivated in another fresh culture medium to produce
`ML-23 6.
`
`10
`
`I5
`
`20
`
`25
`
`30
`
`35
`
`40
`
`6
`range and cultivation temperature may be usually of
`20°—30°C., in particular about 28°C. being preferred.
`Cultivation may be continued until ML—236 will be
`substantially accumulated in a culture medium, usually
`for 20 hours to 240 hours, preferably for 48 hours to
`[68 hours and, after cultivation, each ML-236 sub-
`stance may be isolated and recovered from a cultured
`broth by a suitable combination of various methods
`related to the properties thereof which has been eluci-
`dated by us, as illustrated in the examples given below.
`For example. there may be mentioned extraction with
`an organic solvent, e.g., ether, ethyl acetate or chloro-
`form; dissolution into a more_ polar solvent, e.g., ace-
`tone or alcohol; removal of impurities with a less polar
`solvent, e.g., petroleum ether or hexane; adsorptive
`chromatography with active Carbon or silica gel; gel
`filtration through a column of “Sephadex“ (trade
`name; available from Pharmacia Co., Ltd., U.S.A.];
`and so on. By the use of a suitable combination of these
`measures the crystalline or oily form of the present
`substance can be isolated from a cultured broth.
`The present invention will be more fully illustrated by
`way of the following examples, but they are not in-
`tended to limit the scope of this invention. Various
`modifications of the present process may be made by
`those skilled in the art, in particular, for the recovery of
`ML-236A, ML—236B and ML-236C from a cultured
`broth, in view of the above—depicted properties.
`EXAMPLE 1
`
`Into a 600 l.-volume fermenter was charged 300 l. of
`a culture medium containing glucose 2%, peptone
`(“l(yokuto," trade name; available from Kyokuto
`Seiyaku K.l(., Japan) 0.1% and malt extract 3% and
`Pem‘cr'h'ium cirrinum SANK l8'r'6'r' was inoculated
`thereon. Cultivation was conducted at a temperature of
`28°C., an aeration of 300 1.,-‘min. and an agitation of
`l90 r.p.m. for 9] hours.
`The cultured broth was filtered by a filter press to
`give 280 1. of the filtrate. The filtrate so obtained was
`adjusted to pH 4.0 with 6N hydrochloric acid and ex-
`tracted twice with 250 1. of ethyl acetate.
`The extract was concentrated to dryness under re-
`duced pressure to give 150 g. of the dried product. 120
`g. of the product was adsorbed on a column of I040 g.
`of silica gel (“Wakogel C-200": trade name; Wake
`Junyaku K.K., Japan), which was developed in turn
`with 5 l. of benzene, 7.5 1. of benzene-ethyl acetate
`(9525) and then 28 l. ofbenzene-ethyl acetate (80:20).
`Active eluates are of two fractions, namely the first
`eluted fraction named C (containing ML—236C) and
`the second eluted fraction named B (containing ML-
`236B). Further, the column was developed with 2 l. of
`acetone to give the third fraction named A (containing
`ML-236A). The former two fractions were concen-
`trated to dryness so that 2.42 g. and 2.61 g. of the dried
`products were obtained from the fractions C and B,
`respectively.
`The fraction B (2.61 g.) was dissolved in 100 ml. of
`benzene, the resulting solution was washed with 100
`ml. of water and the washings were discarded. The
`benzene layer was dried over sodium sulfate and con-
`centrated and the residue was allowed to stand over-
`
`CFAD V. UPENN
`IPR2015-01836
`
`45
`
`50
`
`SS
`
`60
`
`As medium components may be employed any of the
`well-known nutrient materials for Penicillium. For in-
`stance, as an assimilable carbon source, glucose, glyc-
`erol, Inaltose, dextrin, starch, lactose, sucrose, molas-
`ses, soybean oil, cotton seed oil, etc., preferably glu-
`cose and maltose may be employed and, as an assimila-
`ble nitrogen source, soybean meal, peanut meal, cotton
`seed meal, fish meal, corn steep liquor, peptone, rice
`bran, meat extract, yeast, yeast extract, sodium nitrate,
`ammonium nitrate, ammonium sulfate, etc. may be
`used. And, such inorganic salts as sodium -chloride,
`phosphates, calcium carbonates, etc., may be added to
`a culture medium. A minor amount of a metal salt may
`also be added, if necessary. Further, a minor amount of
`a heavy metal may be added, if necessary.
`Particularly,
`in cultivating the ML-236—producir1g
`microorganism under aerobic condition, ordinary aero-
`bic cultivation methods such as, for example, solid
`culture, culture under aeration and agitation, shaken
`culture, etc., may be favorably utilized.
`ln carrying out cultivation with aeration and agita-
`night, whereupon the so separated crystalline sub-
`stance was recovered. The substance was then recrys-
`tion, an anti-foaming agent, e.g., silicon oil, vegetable 55 '
`-oils. surfactants, etc., may be suitably employed.
`tallized once from benzene and subsequently once
`The pH of the medium may he usually within a pH
`from ethanol to give 232 mg. of ML—236B in a pure
`range of 3-9 and preferably within or around neutral
`state as white crystals. This substance was confirmed as
`1|] ofll
`PENN EX. 2187
`
`

`
`3,983,140
`
`7
`pure by a thin-layer chromatography and shows an
`inhibitory activity of about 50% on biosynthesis of
`cholesterol in a concentration of 0.01 ,ug.fml.
`EXAMPLE 2
`
`8
`- Thereafter, the column was developed with 20 l. of
`acetone, thereby eluting the third active fraction con-
`taining ML-236A.
`Each respective fraction was individually concen-
`trated to dryness.
`The dried product (I94 g.) from the ML-236A frac-
`Into a 6 ton—volume fermenter was charged 3000 l. of
`tion was dissolved in 2 l. of ethyl acetate and the result-
`the same culture medium as in Example I and Pem't-t'!-
`ing solution was extracted three times with 500 ml.
`Hum citrinum SANK 18767 was inoculated thereon.
`portion of a saturated aqueous solution of sodium car-
`Cultivation was conducted for 90 hours in the same
`as in Example 1. The cultured broth was fir ”’ 3‘.-?.§'§l‘ii Tfieéhiltiiiiiéeiéiiilsaiiitfiiéitil’§E§§i‘.‘f.§
`left‘-‘d by 3 filler PW55 ‘*0 El‘-'3 2590 1- Of “'5 fi1tT3‘5- T115
`chloride. dried over anhydrous sodium sulfate and then
`_ filtrate was concentrated under reduced pressure to
`concentrated to dryness to leave 70 g. of the dried
`450 l. and the concentrate was adjusted to pH 4.0 with
`product. The product was adsorbed on a column of l 50
`6N hydrochloric acid and then extracted with 4501. or 15 g. of silica gel
`(Wakogcl C-200). which was then
`ethyl acetate. 390 l. of the extract was concentrated to
`"”35h9d with 1
`1- Of benzene and 3 1- Of b3T1Z3"3'3th1-'1
`dryness to yield 346 g. of an oily substance, The sub-
`acetate (8:0) and eluted with 5 l. of benzene-methanol
`stance was adsorbed on a column of 5.18 kg. of silica
`gi9v':359)' Maf'“NEE"gtg%’§' “'e;‘:cC:’i$3::g;;e:c;° dryness ‘'3
`gel (“Wakogel C-200"), which was then developed in
`320
`'
`as
`'
`turn with 98 l. of n-hexane, 60 l. of n-hexane -acetone 20 licilhigrlfigdggoggcstogLiirfigzglfchgkéziifisglifilgg
`(955) and than 150 I‘ of "'hexa"e'acet°"e (ssils )‘
`were removed by filtration The filtrate was concen-
`The resulting acme fraction (42 1'3 comaining ML‘
`lrated and the concentrate was allowed to stand over-
`23 6B and other active substances] was concentrated to
`night_ whereupon ML_235B was Separated out as c,.),S_
`dryness *0 133“? 40 9 Of “'15 d1'i¢d PF0‘-l’-|°1- This PT0d'
`tals. The so separated crystals were recovered by filtra-
`UC1 W35 dl5501V¢d in 3 Sllitahli‘-' 3m01mt0f benzene and 25 tion and crystallized once from benzene and subse-
`insolubles were filtered off. The filtrate was left over-
`quently once from ethanol to give 10.5 g. of Ml..—236B
`night. whereupon ML—236B was separated out as crys-
`in a pure state.
`tals and other active substances were retained in a
`Finally, Il‘IB dried product (3.2 3.) f1’0I1'I the M1--236C
`waste solution. The crystals were collected by filtration 30 fraction was dissolved in a small amount of dichloro-
`and then recrystallized once from benzene and subse-
`'"°‘ha“5 and “"3 '°5}1!““1E 501'-“'0” W35 adsorbed 0? 3
`quently once from ethanol to obtain l2.8 g. of the pure
`column of 50 3‘ of Sihca gel (.""'a“°3°' 0200).’ which
`ML_236B
`was then developed in turn with 500 ml. of dichloro—
`'
`methane and then dichlororncthane-ethyl acetate
`35 (95:5). Fractions containing ML_-236C were collected
`:::§:rfi:;:E;t;{:1g:_dryness to give 2'1 g' of ML 236C
`what is claimed is;
`1_ A compound having the fan-nula
`
`EXAMPLE 3
`lnto a 6 ton-volume fermenter was charged 3000 I. of
`a culture medium containing glucose 2%, peptone
`(Kyokuto) 0.1% and malt extract 3% and Peniciflium
`citrinum SANK 18767 was inoculated thereon. Cultiva-
`tion was conducted at a temperature of 28°C., an aera- 40
`
`tion of 3000 Lfmin. and an agitation of 145 rpm. for
`
`96 hours.
`
`H0
`
`0
`
`g
`
`The cultured broth was filtered by a filter press to
`give 2900 l. of the filtrate. The filtrate so obtained was
`concentrated under reduced pressure to 450 I. and the 45
`concentrate was then adjusted to pH 4.0 with 6N hy-
`drochloric acid followed by extraction with 500 l. of
`wherein R is h dm en atom h dmx mu
`ethyl acetate. 500 l. of the extract was concentrated to
`p
`dryness to leave 32? g. of an oily substance. The sub- 50 methylbmyryloxi grogupl
`’
`y
`y g
`stance was adsorbed on a column of 5.5 kg. ofstlica gel
`2_ A compound according to Claim 1 wherein Said R
`(wakogel C-200}, which was then developed in turn
`is hydmxy group
`-
`with 101. of n-hexane, 60 l. of n-hexane-acetone (95:5}
`3_ A compound according to claim 1 wherein Said R
`and then "150 l. of n-hexane-acetone (85:15). Two
`is 2_mc¢hygbuty,-310,‘). group
`active fractions were -given, namely, the first eluted 55
`4, A compound according tc. c]aim 1 wherein said R
`fraction containing ML.-236C and the second eluted
`is hydrogen atom.
`fraction containing ML-23613.
`
`R
`
`CH5
`
`or 2*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`60
`
`65
`
`11 of 11
`
`PENN EX. 2187
`CFAD V. UPENN
`IPR2015-01836