United States Patent
`Terahara et al.
`
`119]
`
`[11] Patent Number:
`[451 Date of Patent:
`
`4,537,859
`* Aug. 27, 1985
`
`[54] PROCESS FOR PREPARING
`3-HYDROXY-ML-236B DERIVATIVES
`KNOWN AS M-4 AND M-4’
`
`[57]
`
`ABSTRACI‘
`
`Compounds of formula (I):
`
`[75]
`
`Inventors: Akira Terahara; Minoru Tanaka,
`both of Hiromachi, Japan
`
`[73] Assignee:
`
`Sankyo Company, Limited, Tokyo,
`Japan
`
`[ * ] Notice:
`
`The portion of the term of this patent
`subsequent to Oct. 18, 2000 has been
`disclaimed.
`
`[21] Appl. No.: 442,840
`
`[22] Filed:
`
`Nov. 18, 1982
`
`Foreign Application Priority Data
`[30]
`Nov. 20, 1981 [JP]
`Japan ................................ 56-186641
`
`[51]
`
`Int. Cl.3 .......................... C12P 7/42; C12P 7/62;
`C12N 1/20
`-
`[52] U.S. c1. .................................. .. 435/146; 435/135;
`435/253; 435/872
`[58] Field of Search .............. .. 435/135, 136, 146, 253
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`.................... .. 435/136
`3,281,330 10/1966 Fonken et al.
`
`.. ...
`.. .. .. 435/121
`3,392,171
`7/1968 Fonken et al.
`4,410,629 10/1983 Terahara et al.
`................... 435/135
`
`Primary Examiner--Alvin E. Tanenholtz
`Attorney, Agent, or Fz'rm—Frishauf, Holtz, Goodman &
`Woodward
`
`\\\\‘OH
`
`
`
`111:
`
`HO
`
`(wherein -- OH represents< OH or 11111111 -OH), that
`is to say M-4 carboxylic acid and M-4’ carboxylic acid, as
`well as pharmaceutically acceptable salts and esters
`thereof and the corresponding ring-closed lactones may
`be prepared by contacting an ML-236B compound with a
`microorganism of the genus Nocardia or a cell-free,
`enzyme-containing extract thereof and then, if neces-
`sary, subjecting the resulting product to one or more of
`the following reactions: hydrolysis, salification, esterifi-
`cation and lactonisation. The resulting M-4 and M-4’
`derivatives have the ability to inhibit the biosynthesis of
`cholesterol and are therefore of value in the therapy
`and/or prophylaxis of hyperlipaemia and arteriosclero-
`SIS.
`
`22 Claims, No Drawings
`
`Mylan Exhibit 1026, Page 1
`
`Mylan Exhibit 1026, Page 1
`
`

`
`1
`
`4,537,859
`
`PROCESS FOR PREPARING
`3-HYDROXY-ML-236B DERIVATIVES KNOWN AS
`M-4 AND M-4’
`
`BACKGROUND TO THE INVENTION
`
`The present invention relates to a process for prepar-
`ing certain 3-hydroxy-ML-236B derivatives known as
`M-4 and M-4’, as well as salts and esters of these com-
`pounds.
`ML-236B, which can exist in the form of an acid
`(known as “ML-236B carboxylic acid”) or a lactone
`(known as “ML-236B lactone”), is disclosed in United
`Kingdom Patent Specification No. 1,453,425 and, in its
`lactone form, has the formula:
`
`
`
`Subsequently, United Kingdom Patent Specification
`No. 1,555,831 disclosed a variety of salts and esters of
`‘ ML-236B. ML-236B and its salts and esters were found
`to inhibit the biosynthesis of cholesterol by competing
`with 3-hydroxy-3-methylglutaryl coenzyme A reduc-
`tase, which is the rate-determining enzyme for chloes-
`terol biosynthesis; these compounds were thus found to
`exhibit a very marked ability to reduce serum choles-
`terol levels.
`
`Subsequently, certain 3-hydroxy-ML-236B deriva-
`tives were isolated as products of the animal metabolism
`of ML-236B lactone and similar derivatives were found
`to be produced by the enzymatic hydroxylation of ML-
`236B lactone or carboxylic acid or salts or esters
`thereof, effected by means of various microorganisms of
`the genera Absidia, Cunninghamella, Syncephalastrum,
`Streptomyces, Mucor, Rhizopus, Zygorinchus, Cir-
`cinella, Actinomucor, Gongronella, Phycomyces, Mor-
`tierella, Pycnoporus and Rhizoctonia. These processes
`are disclosed in U.S. Pat. No. 4,346,227, filed 5th June
`1981, by A. Terahara and M. Tanaka and the com-
`pounds thus produced are described in that patent appli-
`cation as M-4, M-4’, IsoM-4 and IsoM-4’. These com-
`pounds were found to have an ability to inhibit the
`biosynthesis of cholesterol which is at least comparable
`with and, in some instances, substantially exceeds that
`of ML-236B itself.
`ML-236B and its derivatives, including the M-4 and
`M-4’ compounds, are thus of therapeutic value for the
`treatment of hyperlipaemia and the prophylaxis of arte-
`riosclerosis.
`
`BRIEF SUMMARY OF INVENTION
`
`We have now discovered that M-4 and M-4’ can also
`be produced from ML-236B and various derivatives
`thereof by treatment with a microorganism of the genus
`Nocardia or a cell-free, enzyme-containing extract
`thereof. The use of microorganisms of the genus Nocar-
`dia has the advantage over the use of those microorgan-
`
`2
`isms disclosed in U.S. Pat. No. 4,346,227 that the M1-
`
`236B compound employed as substrate can be present in
`the reaction medium to a much higher concentration
`than when the prior art microorganisms were used. This
`is most surprising as the ML-236B compounds have
`been found to possess antifungal and antibiotic proper-
`ties.
`
`Accordingly, the present invention provides a pro-
`cess for preparing a compound of formula (I):
`
`(1)
`
`
`
`OH),
`(wherein --v~ OH represents '—--I OH or
`pharrnaceutically acceptable salts and esters thereof and
`the corresponding ring-closed lactones, which process
`comprises contacting an ML-236B compound selected
`from the group consisting of ML-236B carboxylic acid,
`having the formula (II):
`
`(11)
`
`
`
`salts and esters thereof and the corresponding ML-236B
`lactone with a hydroxylation enzyme produced by a
`microorganism of the genus Nocardia;
`if necessary,
`subjecting the resulting product to one or more reac-
`tions selected from the group consisting of hydrolysis,
`salification, esterification and lactonisation; and isolat-
`ing the product from the reaction mixture.
`
`DETAILED DESCRIPTION OF INVENTION
`
`The compound of formula (I) in which--wOH
`represents —— OH is called M-4 carboxylic acid and
`the corresponding salts and esters are known as M-4
`carboxylates. The compound of formula (I) in which
`«~v~vOH represents OH is referred to as M-4’
`carboxylic acid and the corresponding salts and esters
`are referred to as M-4’ carboxylates.
`The ring-closed lactones corresponding to the com-
`pounds of formula (I) may be represented by the for-
`mula (Ia):
`
`l0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Mylan Exhibit 1026, Page 2
`
`Mylan Exhibit 1026, Page 2
`
`

`
`4,537,859
`
`
`
`(la)
`
`5
`
`10
`
`OH and 15
`wherein --zw OH represents '-—-I OH or
`are known as M-4 lactone and M-4’ lactone respec-
`tively.
`The ring-closed lactone corresponding to ML-236B
`carboxylic acid of formula (II) may be represented by
`the formula (Ila):
`
`20
`
`(Ila)
`
`‘El
`H3C/\4:/\
`CH3
`
`25
`
`30
`
`and is known as ML-236B lactone.
`
`40
`
`Preferred species of the genus Nocardia for use in the 35
`process of the present invention are Nocardia autotro-
`phica, Nocardia asteroides, Nocardia farcinica and Nor-
`cardia coeliaca, particularly Nocardia autotrophica. Of
`these species, the following strains are preferred:
`Nacardia autotrophica FERM P-6181 (SANK 62781);
`Nocardia autotrophica subsp. canberrica subsp. nov.
`FERM P-6182 (SANK 62881);
`Nocardia autotrophica subsp. amethystina subsp. nov.
`FERM P-6183 (SANK 62981);
`Nocardia autotrophica IFO 12743 (SANK 91279);
`Nocardia asteroids IFO 3424 (SANK 62065);
`Nocardia farcinica ATCC 3318 (SANK 64265) and
`Nocardia coeliaca ATCC 17040 (SANK 63665).
`Of the above preferred strains, Nocardia autotrophica
`IFO 12743, Nocardia asteroides IFO 3424, Nocardia 50
`farcinica ATCC 3318 and Nocardia coeliaca ATCC
`17040 are all known strains which are freely and pub-
`licly available from the appropriate culture collection,
`i.e. the Institute for Fermentation, Osaka, Japan (IFO)
`or the American Type Culture Collection, U.S.A. 55
`(ATCC), under the accession numbers given.
`The strains of Nocardia autotrophica identified by the
`accession numbers FERM P-6181, FERM P-6182 and
`
`45
`
`4
`FERM P-6183 are all new strains of the microorganism,
`newly isolated from soil and deposited on the sixteenth
`day of October 1981 at
`the Fermentation Research
`Institute, Ibaraki-Ken, Japan (FERM).
`The morphological and physiological properties of
`the newly isolated microorganisms were determined
`using conventional media and the methods described by
`Shirling and Gottlieb [International Journal of System-
`atic Bacteriology 16, 313-340 (1966)li
`together with
`several supplementary tests. Observations of the culture
`were made after incubation at 28° C. for 2 weeks. The
`colour names used were assigned according to the
`“Guide to Colour Standard” (a manual published by
`Nippon Shikisai Kenkyusho, Tokyo, Japan). The char-
`acteristics of the cultures were compared with those of
`various known species of actinomycetes described in
`“The Actinomycetes, Vol. 2” by Waksman, “The ISP
`Report” by Shirling and Gottlieb, “Bergey’s Manual of
`Determinative Bacteriology”, 8th edition and other re-
`cent literature concerning the taxonomy of the family
`Nocardiaceae. The new microorganisms are identified
`by their FERM accession numbers.
`
`Morphological characteristics
`TABLE 1
`FERM
`P-6181
`
`FERM FERM
`P-6182
`P-6183
`
`Spore chain morphology
`Branching
`Fragmentation
`Surface structure of segmented
`hyphae (spores)
`Other organs
`
`RF = rectus-tlexibilus
`
`RF
`simple
`yes
`smooth
`
`RF
`simple
`yes
`smooth
`
`‘
`
`RF
`simple
`yes
`smooth
`
`knots, nest-
`like tangles
`
`knots
`
`none
`
`Growth on taxonomic media
`All of the new strains showed good growth on a
`variety of media.
`Strain FERM P-6181 had white aerial mycelia on a
`yellowish-grey to pale yellow-orange growth. In cer-
`tain media a pale yellow-brown soluble pigment was
`observed, but only to a small extent.
`Strain FERM P-6182 had brown-white to pale yel-
`low-orange aerial mycelia on a greyish-yellow-brown
`growth. No soluble pigment was observed.
`Strain FERM P-6183 had a brown-white to pale
`yellow-orange growth and ‘brown-violet spots were
`observed as the cultivation proceeded. Brownish-grey
`aerial mycelia were present on all media, except for the
`yeast-malt medium.
`The culture properties on the 14th day of cultivation
`at 28° C. in a variety of media are shown in Table 2. The
`abbreviations used in the Table are as follows:
`
`_
`G=g1'0Wfh;
`AM==aeT1a1 I113/081111111;
`R=1'€V€fSC;
`.
`SP=-Soluble P183133‘-
`
`Media
`
`TABLE 2
`FERM P-6181
`FERM P-6182
`
`Yeast-
`malt
`agar
`(ISP 2)
`
`G
`
`Very good,
`pale yellow-
`brown (6-7-9)
`
`AM Abundant,
`white
`
`R
`
`Dull, yellow-
`orange (8-8-8)
`
`Very good,
`brown
`(6-4-l)
`
`Abundant
`brownish-
`white (29.7)
`Brown (4-4-7)
`
`FERM P-6183
`
`Very good, brown-
`white (2-9-8) to
`greyish-red-brown
`(4-3-5)
`Trace, white
`
`Brown-white (29-8)
`to greyish-red-
`
`Mylan Exhibit 1026, Page 3
`
`Mylan Exhibit 1026, Page 3
`
`

`
`5
`TABLE 2-continued
`
`
` Media FERM P-6181 FERM P-6182 FERM P-6183
`
`brown (4-3-5)
`None
`
`4,537,859
`
`None
`
`Oatmeal
`agar
`(ISP 3)
`
`Starch/
`inorganic
`salt
`agar
`(ISP 4)
`
`G1ycerin/
`asparagine
`agar
`(ISP 5)
`
`Tyrosine
`agar
`(ISP 7)
`
`Sucrose
`nitrate
`agar
`
`Glucose/
`asparagine
`agar
`
`Nutrient
`agar
`
`Water
`agar
`
`SP
`
`G
`
`Yellow-brown
`(S-7-9)
`Good, pale
`brown (2-8-9)
`
`AM Fair, white
`
`R
`
`SP
`
`G
`
`Yellowish-
`brown (4-7-9)
`Pale yellow-
`brown (4-8-9)
`Good,
`yellowish-grey
`(2-9-10)
`
`AM Fair, white
`
`R
`
`SP
`G
`
`Pale yellow
`(3-9-10)
`None
`Good, pale
`brown (2-8-9)
`
`AM Abundant,
`white
`
`R
`
`Pale yellow-
`brown (6-8-9)
`
`G
`
`SP
`
`Pale yellow-
`brown (6-9-l 1)
`Very good,
`pa1e-yellow-
`orange (3-8-8)
`AM Abundant,
`white
`
`R
`
`Yellowish-
`grey (1-9-10)
`
`G
`
`SP
`
`Pale yellow-
`brown (6-7-9)
`Good, pale
`yellow-brown
`(2-9-9)
`AM Fair, white
`
`R
`SP
`G
`
`Yellowish-
`grey (1-9-10)
`None
`Very good,
`pale yellow-
`orange (2-9-9)
`
`AM Fair, white
`
`.
`
`R
`
`Pale yellow-
`brown (4-8-9)
`
`G
`
`SP
`
`Pale yellow-
`brown (4-8-9)
`Good,
`yel1owish-
`grey (2-9-10)
`AM Fair, white
`
`R
`SP
`G
`
`Yellowish-
`grey (4-9-10)
`None
`Poor,
`yellowish-
`grey (1-9-10)
`AM Fair, white
`
`Very good,
`pale yellow-
`orange (2-9-9)
`Abundant, pale
`yellow-brown
`(2-9-9)
`Pale yellow-
`brown (4-8-9)
`None
`
`Very good, dark
`red-brown (4-3-4)
`
`Fair, pale pink
`(2-8-4)
`
`Brown-violet
`(3-3-2)
`None
`
`Poor,
`yellowish-grey
`(1-9-10)
`
`Very good,
`brown-violet
`(3-3-2)
`
`Abundant, pale Good, bright
`yellow-orange
`brown-grey
`(2-9-9)
`(2-8-2)
`Pale yellow-
`Dark red-brown
`orange (2-9-9)
`(4-3-4)
`None
`None
`Good, greyish- Very good, pale
`yellow-brown
`brown (2-9-9) to
`(4-5-7)
`brown-violet ,
`(3-3-2)
`Abundant, white
`
`Abundant,
`brown-white
`(1-8-6)
`Brown (4-4-6)
`
`None
`
`Very good,
`greyish-yellow-
`brown (4-5-7)
`Abundant,
`brown-white
`(2-9-7)
`Bright brown
`(6-5-7)
`
`None
`
`Poor, pale
`yellow-brown
`(2-9-9)
`Abundant,
`brown-white
`(2-9-7)
`Brown-white
`(1-9-6)
`None
`Good, greyish
`yellow-brown
`(4-5-7)
`
`Abundant,
`bright brown-
`white (1-7-6)
`Greyish-red—
`brown (4-3-6)
`
`None
`
`Very good,
`pale yellow-
`brown (6-8-9)
`Pale yellow-
`orange (2-9-9)
`Pale yellow-
`brown (6-8-9)
`None
`Poor,
`colourless
`
`Abundant,
`white
`
`Pale yellow-
`orange (2-9-9)
`to greyish—red-
`brown (4-3-6)
`None
`
`Good, greyish-
`brown (4-6-6)
`
`Trace, white
`
`Pale yellow-orange
`(2-9-9) to brown-
`violet (3-3-2)
`None
`
`Poor, pale yellow-
`orange (2-9-9)
`
`Fair, white
`
`,
`
`Pale yellow-orange
`(2-9-9)
`None
`Very good, pale
`yellow-orange
`(2-9-9) to brown-
`violet (3-3-2)
`Fair, white
`
`Pale yellow-orange
`(2-9-9) to greyish-
`red-brown (4-3-6)
`None
`
`Good, pale yellow-
`orange (2-9-9)
`
`Trace, white
`
`Pale yellow-orange
`(2-9-9)
`None
`Poor, pale yellow-
`orange (2-9-9)
`
`Fair, white
`
`Mylah Exhibit 1026, Page 4
`
`Mylan Exhibit 1026, Page 4
`
`

`
`7
`TABLE 2-continued
`Media
`FERM P-6181
`FERM P-6182
`FERM P-6183
`
`
`4,537,859
`
`Potato/
`carrot
`extract
`agar
`
`R
`SP
`G
`
`Yellowish—
`grey (l-9-10)
`None
`Poor,
`yellowish-
`grey (l-9-10)
`
`AM Fair, white
`R
`Yellowish-
`grey (1-9-l0)
`SP
`None
`
`Pale yellow-
`orange (2-9-9)
`None
`Poor,
`colourless
`
`Pale yellow-orange
`(2-9-9)
`None
`Poor, pale yellow-
`orange (2-9-9)
`
`Fair, white
`Pale yellow-
`orange (2-9-9)
`None
`
`Fair, white
`Pale yellow-orange
`(2-9-9)
`None
`
`8
`
`I
`_
`Physiological properties
`The~physiological properties of the new strains are
`shown in Table 3. The test for melanoid pigment forma-
`tion was carried out in three media, as follows:
`Medium 1: Tryptone-yeast extract broth (ISP 1);
`Medium 2: Peptone-yeast extract-iron agar (ISP 6);
`Medium 3: Tyrosine gear (ISP 7).
`TABLE 3
`FERM FERM
`FERM
`
`P-6181
`P-6182
`P-6183
`—
`—
`—
`—
`—
`--
`+
`—
`+
`-
`+
`—-
`
`Nitrate reduction
`Starch hydrolysis
`Urea decomposition
`Lysozyme resistance
`Melanoid pigment formation
`
`15
`
`20
`
`25
`
`g:il‘;;°:ESe
`D_xy1Ose
`D-Fructose
`L-Rh
`im.;;’i"“°‘”
`Sucrose
`-
`to
`$831108;
`Contign
`_
`_
`I f ;'lti'1':1d‘umiSed.
`: ; mi mi}l,iSed_
`’
`
`1
`
`TABLE 4
`
`gglfgf
`
`I:f6}§"2I
`
`girfgg
`
`:
`+
`+
`1:
`+
`+
`+
`_
`
`:
`_
`+
`—
`+
`-
`—
`t
`
`:
`+
`+
`i
`+
`-
`—
`E
`
`—
`__
`_
`
`—
`_
`_
`
`Mediuml
`Medium 2
`Medium 3
`Acid 2r0dI1<-‘lion from
`arabinose
`+
`—
`+
`
`1’_‘;'tli‘.;:se
`:
`:
`NE
`_
`1 ;
`NG = no growth.
`
`—
`_
`_
`
`30
`
`Utilisation of carbohydrates
`The utilisation of carbohydrates by the new strains is 40
`shown in Table 4. The medium used was Pridham-Gott-
`lieb agar (ISP 9) and determination was made after
`cultivation at 28° C. for 14 days.
`
`45
`
`
`
`TEST
`
`Growth
`colours
`
`Decomposition
`of urea
`Resistance to
`lysozyme
`Acid production
`from:
`arabinose
`xylose
`raffinose
`
`arabinose
`xylose
`rhamnose
`
`FERM
`P-6181
`AM white
`
`G
`
`yellowish-
`grey to
`pale yellow-
`orange
`+
`
`—
`
`+
`+
`+
`
`+
`+
`+
`
`.
`1
`11
`C H
`.
`e wa ana ysis
`Paper chromatographic analyses were performed on
`acid hydrolyzates of each of the three new strains, fol-
`lowing the method of B. Becker et al. [Applied Microbi-
`ology, 13, 236 (l965)] and that of M. P. Lechevalier et
`35 al. [“The Actinomycetales” by H. Prauser, 311 ( 1970)].
`Meso-2,6-diaminopimelic acid was found in the cell
`walls and arabinose and galactose were found as saccha-
`ride components of the whole cell, thus confirming that
`each of the strains had cellular components of the type
`IV-A.
`The results of these taxonomic studies demonstrate
`that all strains belong to the genus Nocardia. Of the
`known species of Nocardia, the characteristics of the
`new strains are most closely related to those of Nocardia
`autotrophica [International Journal of Systematic Bacte-
`riology, 30, 337 (l980)[, except only for the differences
`shown in Table 5. In the Table, symbols and abbrevia-
`tions are as given in the corresponding Tables 1-4.
`TABLE 5
`FERM
`P-6182
`white to
`pale yellow-
`orange
`greyish-
`yellow-
`brown
`
`FERM
`P-6183
`white to
`brownish-
`grey
`brown-
`violet
`
`Nocardia
`autotrapnica
`white to pale
`yellow
`
`pale yellow to
`yellowish-grey
`
`—
`
`+
`
`—
`—
`—
`
`—
`—
`—
`
`'
`
`+
`
`—
`
`+
`+
`NG
`
`+
`+
`i
`
`+
`
`—
`
`+
`+
`—
`
`+
`+
`+
`
`Mylan Exhibit 1026, Page 5
`
`Mylan Exhibit 1026, Page 5
`
`

`
`4,537,859
`
`10
`
`9
`TABLE 5-continued
`Nocardia
`FERM
`FERM
`FERM
`autatropnica
`P-6181
`P-6182
`P-6183
`+
`+
`—
`-
`SUCIOSE
`
`raffinose + + — —
`
`
`
`
`
`
`TEST
`
`,
`
`Strain FERM P-6181 and Nocardia autotrophica re-
`semble each other in their morphological, cultural and
`physiological characteristics and it was thus concluded
`that this strain belonged to the species Nocardia autotro-
`phica.
`Strain FERM P-6182 differed'from Nocardia autotro-
`phica in its decomposition of urea, its resistance to lyso-
`zyme, its acid production from carbohydrates and its
`utilisation of carbohydrates. However, these differences
`are not sufficient that strain FERM P-6182 should be
`considered a new species; it is therefore regarded as a
`new subspecies of Nocardia autotrophica. This strain
`was accordingly named as Nocardia autotrophica subsp.
`canberrica subsp. nov..
`Strain FERM P-6183 differed from Nocardia autotro-
`phica in its growth colour and utilisation of rhamnose,
`sucrose and raffinose. These differences likewise were
`not sufficient that this strain should be considered to be
`a new species and it is therefore regarded as a subspe-
`cies of Nocardia autotrophica. Ithas been named Nocar-
`dia auzotraphic subsp. amethystina subsp. nov.
`As with all microbial strains, the new strains FERM
`P-6181, FERM P-6182 and FERM P-6183 are unstable
`in their properties and are readily mutated by such
`artificial mutating agents as ultraviolet radiation, high
`frequency electromagnetic waves, nuclear radiation
`and chemical mutating agents. Any mutants obtained
`from these strains and possessing the desired activities
`can be employed in the process of the present invention.
`Of the various microorganisms of the genus Nocardia
`which can be employed in the process of the invention,
`we particularly prefer to employ the three new strains,
`that is to say Nocardia autatrophica FERM P-6181,
`Nocardia autotrophica subsp. canberrica FERM P-6182
`or Nocardia autotrophica subsp. amethystina FERM
`P-6183.
`
`The enzymatic hydroxylation process of the present
`invention can be effected by contacting the ML-236B
`compound with the chosen microorganism of the genus
`Nocardia or with a cell-free, enzyme-containing extract
`thereof.
`
`This process of the invention is preferably carried out
`in one of three ways:
`(a) adding the starting ML-236B compound to the
`culture medium during the cultivation of the con-
`verting microorganism and then continuing with
`the cultivation;
`'
`(b) collecting a culture of the converting microorgan-
`ism and contacting the collected cells with the
`starting ML-236B compound; or
`(c) preparing a cell-free, enzyme-containing extract
`from the cells of the converting microorganism and
`contacting this‘ extract with the starting ML-236B
`compound.
`.
`Cultivation of the converting microorganism of the
`genus Nocardia can be carried out by conventional
`means in a conventional culture medium containing
`nutrients well known for use with such microorganisms.
`Thus, as is well known, such culture media contain
`sources of assimilable carbon and of assimilable nitro-
`gen and often inorganic salts. Examples of sources of
`assimilable carbon include glucose, sucrose, starch,
`
`10
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`glycerin, millet jelly, molasses and soybean oil. Exam-
`ples of sources of assimilable nitrogen include soybean
`solids (including soybean meal and soybean flour),
`wheat germ, meat extracts, peptone, corn steep liquor,
`dried yeast and ammonium salts, such as ammonium
`sulphate. If required, inorganic salts, such as sodium
`chloride, potassium chloride, calcium carbonate or
`phosphates, may also be included. Also, if desired, other
`additives capable of promoting the production of hy-
`droxylation enzymes may be employed in appropriate
`combinations. The particular cultivation technique is
`not critical to the process of the invention and any
`techniques conventionally used for the cultivation of
`microorganisms may equally be employed with the
`present invention. In general, of course, the techniques
`employed will be chosen having regard to industrial
`efficiency. Thus, liquid culture is generally preferred
`and the deep culture method is most convenient from
`the industrial point of view.
`Cultivation will normally be carried out under aero-
`bic conditions and at a temperature within the range
`from 20“ to 37° C., more preferably from 26° to 28° C.
`Method (a) is carried out by adding the starting ML-
`236B compound to the culture medium in the course of
`cultivation. The precise point during the cultivation at
`which the starting compound is added will vary de-
`pending upon the cultivation equipment, composition of
`the medium, temperature of the culture medium and
`other factors, but it is preferably at the time when the
`hydroxylation capacity of the microorganism begins to
`increase and this is usually 2 or 3 days after beginning
`cultivation of the microorganism. The amount of the
`ML-236B compound added is preferably from 0.01 to
`5.0% by weight of the medium, more preferably from
`0.05 to 0.5%, e.g. from 0.05 to 0.1% by weight. After
`addition of the ML-236B compound, cultivation is con-
`tinued aerobically, normally at a temperature within the
`ranges proposed above. Cultivation is normally contin-
`ued for a period of from 3 to 5 days after addition of the
`ML-236B compound.
`In method (b), cultivation of the microorganism is
`first carried out under conditions such as to achieve its
`maximum hydroxylation capacity; this capacity usually
`reaches a maximum between 4 and 5 days after begin-
`ning the cultivation, although this period is variable,
`depending upon the nature and temperature of the me-
`dium, the species of microorganism and other factors.
`The hydroxylation capacity of the culture can be moni-
`tored by taking samples of the culture at suitable inter-
`vals, determining the hydroxylation capacity of the
`samples by contacting them with an ML-236B com-
`pound under standard conditions and determining the
`quantity of M-4 and M-4’ compound obtained and plot-
`ting this capacity against time as a graph. When the
`hydroxylation capacity has reached its maximum point,
`cultivation is stopped and the microbial cells are col-
`lected. This may be achieved by subjecting the culture
`to centrifugal separation, filtration or similar known
`separation methods. The whole cells of the cultivating
`microorganism thus collected are preferably then
`
`Mylan Exhibit 1026, Page 6
`
`Mylan Exhibit 1026, Page 6
`
`

`
`4,537,859
`
`11
`-
`washed with a suitable washing liquid, such as physio-
`logical saline or an appropriate buffer solution.
`Contact of the collected cells of the microorganism of
`the genus Nocardia with the ML-236B compound is
`generally effected in an aqueous medium, for example in
`a phosphate buffer solution at a pH value of from 5 to 9.
`The reaction temperature is preferably within the range
`from 20“ to 45° C., more preferably from 25° to 30° C.
`The concentration of the ML-236B compound in the
`reaction medium is preferably within the range from
`0.01 to 5.0% by weight. The time allowed for the reac-
`tion is preferably from 1 to 5 days, although this may
`vary depending upon the concentration of the ML-
`236B compound in the reaction mixture, the reaction
`temperature, the hydroxylation capacity of the micro-
`organism (which may, of course, vary from species to
`species and will also, as explained above, depend upon
`the cultivation time) and other factors.
`The cell-free, enzyme-containing extract employed in
`method (c) may be obtained by breaking down the
`whole cells of the microorganism obtained as described
`in relation to method (b) by physical or chemical means,
`for example by grinding or ultrasonic treatment to pro-
`vide a disintegrated cellular mass or by treatment with
`a surface active agent or an enzyme to produce a cellu-
`lar solution. The resulting cell-free extract is then con-
`tacted with the starting ML-236B compound under the
`-same conditions as are described above in relation to
`’ method (b).
`After completion of the conversion reaction by any
`of the above methods, the desired compound can be
`directly isolated, separated or purified by conventional
`means. For example, separation and purification can be
`effected by filtering the reaction mixture, extracting the
`resulting filtrate with a water-immiscible organic sol-
`vent (such as ethyl sulphate), distilling the solvent from
`the extract, subjecting the resulting crude compound to
`column chromatography, (for example on silica gel or
`alumina) and eluting the column with an appropriate
`eluent.
`
`Where the M-4 or M-4’ compound converted by the
`—microorganism is not the desired form of that com-
`pound, then the product of the conversion reaction may
`be subjected to one or more further reactions such as
`hydrolysis, salification, esterification or lactonisation by
`conventional methods, as described in more detail here-
`after. Such additional reactions may be carried out prior
`to, after or in the course of the separation and purifica-
`tion stages described above, preferably in the course of
`these stages.
`The hydroxylation enzyme active in the process of
`the invention has no effect in itself on the carboxy group
`of the ML-236B compound and hence, other factors
`being equal, ML-263B lactone would give M-4 and/or
`M-4’ lactone, ML-236B carboxylic acid would give M-4
`and/or M-4’ carboxylic acid, and a salt or ester of ML-
`236B carboxylic acid would give the same salt or ester
`of M-4 and/or M-4’ carboxylic acid. However, this may
`be affected by other factors, especially the pH value of
`the reaction mixture, in a way which is predictable by
`ordinary laws of chemistry.
`The ML-236B starting compound may be the free
`ML-236B carboxylic acid of formula (11),
`its corre-
`sponding lactone of formula (Ila) or a salt (e.g. metal,
`amino acid or amine salt) or ester (particularly alkyl
`ester) thereof.
`Preferred metal salts are salts with alkali metals, such
`as sodium or potassium, salts with alkaline earth metals,
`
`12
`such as calcium, or salts with other metals such as mag-
`nesium, aluminium, iron, zinc, copper, nickel or cobalt,
`of which the alkali metal, alkaline earth metal, magne-
`sium and aluminium salts are preferred, the sodium,
`calcium and aluminium salts being most preferred.
`Preferred amino acids to form amino acid salts are
`basic amino acids, such as arginine,
`lysine, histidine,
`a,,8-diaminobutyric acid or omithine.
`Preferred amines to form amine salts include t-octyla-
`mine, dibenzylamine, dichlorohexylamine, morpholine,
`alkyl esters of D-phenylglycine and D-glucosamine.
`ML-236B esters are preferably the alkyl esters, such
`as the methyl, ethyl, propyl, isopropyl, butyl, isobutyl
`or pentyl esters, of which the methyl ester is preferred.
`However, other esters may be employed if desired.
`Of the ML-236B starting materials, the alkali metal
`salts, e. g. the sodium or potassium salts, are particularly
`preferred, the sodium salt being most preferred as we
`have found that this gives the best conversion of the
`ML-236B compound into the desired M-4 or M-4’ com-
`pound.
`Where the product obtained by the enzymatic hy-
`droxylation process of the present invention is a salt of
`the carboxylic acid of formula (I), the free carboxylic
`acid itself can be obtained by adjusting the pH of the
`filtrate to a value of 4 or less, preferably to a value of
`from 3 to 4. Any organic acid or mineral acid may be
`employed, provided that it has no adverse effect upon
`the desired compound. Examples of the many acids
`which are suitable include trifluoroacetic acid, acetic
`acid, hydrochloric acid and sulphuric acid. This carbox-
`ylic acid may itself be the desired product or it may be,
`and preferably is, subjected to subsequent reactions, as
`described below, optionally after such treatments as
`extraction, washing and dehydration.
`Metal salts of the carboxylic acids of formula (I) may
`be obtained by contacting a hydroxide, carbonate or
`similar reactive compound of the chosen metal in an
`aqueous solvent with the carboxylic acid of formula (I).
`The aqueous solvent employed is preferably water, or it
`may be a mixture of water with an organic solvent,
`preferably an alcohol (such as methanol or ethanol), a
`ketone (such as acetone), an aliphatic hydrocarbon
`(such as hexane) or an ester (such as ethyl acetate). We
`particularly prefer to use a mixture of a hydrophilic
`organic solvent with water. Such reactions are nor-
`mally conducted at ambient temperature but they may,
`if desired, be conducted with heating.
`Amine salts of the carboxylic acids of formula (I) may
`be obtained by contacting an amine in an aqueous sol-
`vent with the carboxylic acid of formula (1). Suitable
`aqueous solvents include water and mixtures of water
`with alcohols (such as methanol or ethanol), ethers
`(such as tetrahydrofuran), nitriles (such as acetonitrile)
`or ketones (such as acetone); we particularly prefer to
`use aqueous acetone as the solvent for this reaction. The
`reaction is preferably carried out at a pH of from 7 to
`8.5 and preferably at a temperature of ambient or below,
`more preferably a temperature of from 5° to 10° C. The
`reaction goes immediately to completion. Alternatively,
`a metal salt of the carboxylic acid of formula (I) (which
`may have been obtained as described above) can be
`dissolved in an aqueous solvent, after which a mineral
`acid salt (for example the hydrochloride) of the desired
`amine is added, employing the same reaction conditions
`as when the amine itself is reacted with the carboxylic
`acid of formula (I), and the desired product is then
`obtained by a salt exchange reaction.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Mylan Exhibit 1026, Page 7
`
`Mylan Exhibit 1026, Page 7
`
`

`
`13
`Amino acid salts of the carboxylic acids of formula (I)
`may be obtained by contacting an amino acid in aqueous
`solution with the carboxylic acid of formula (1). Suit-
`able aqueous solvents include water and mixtures of
`water with alcohols (such as methanol or ethanol) or
`ethers (such as tetrahydrofuran). The reaction is prefer-
`ably carried out with heating, for example at a tempera-
`ture of from 50° to 60° C.
`Esters, preferably alkyl esters, of the carboxylic acids
`of formula (I) may be obtained by contacting the car-
`boxylic acid of formula (I) with an appropriate alcohol.
`We prefer to carry out this reaction in the presence of
`an acid catalyst, for example a mineral acid (such as
`hydrochloric acid or sulphuric acid), a Lewis acid (for
`example boron trifluoride) or an ion exchange resin.
`The solvent employed for this reaction is not critical,
`provided that it does not adversely affect the reaction;
`suitable solvents include benzene, chloroform and
`ethers. Alternatively, the desired product may be ob-
`tained by contacting the carboxylic acid of formula (I)
`with a diazoalkane, in which the alkane moiety may be
`substitued or unsubstituted. This reaction is usually
`effected by contacting the acid with an ethereal solution
`of the diazoalkane. As a further alternative, the ester
`may be obtained by contacting a metal salt of the car-
`boxylic acid of formula (I) with a halide, preferably an
`alkyl halide,
`in a suitable solvent; preferred solvents
`include dimethylformamide, tetahydrofuran, dimethyl
`sulphoxide and acetone. All of the reactions for produc-
`ing esters are preferably effected at about ambient tem-
`perature, if required by the nature of the reaction sys-
`tem, the reactions may be conducted with heating.
`Lactones of the carboxylic acids of formula (I) may
`be obtained by contacting the carboxylic acid of for-
`mula (I) with a catalytic amount of an acid, which may
`be organic or inorganic. We prefer to use such organic
`and mineral acids as trifluoroacetic acid, hydrochloric
`acid and sulphuric acid. This reaction is preferably
`effected at about ambient temperature.
`Examples of the salts and esters of the M-4 and M-4’
`compounds produced by the processes of the invention
`include those given above as salts and esters of ML-
`236B, for use as the starting material.
`The M-4 and M-4’ derivatives thus obtained can be
`isolated, separated or purified by convention means, for
`example by adsorption on a carrier (such as active car-
`bon or silica gel), or ion exchange chromatography, or
`gel filtration by a Sephadex (trade mark) column, or by
`extraction with an organic solvent, such as an ether,
`ethyl acetate or chloroform; if desired, and it is nor-
`mally preferred, a combination of these techniques may
`be employed.
`The M-4 and M-4’ isomers can be separated from
`each other after completion of the conversion reaction
`or at any appropriate point during the reactions or dur-
`ing the above—described separation or purification pro-
`cesses.
`
`The invention is further illustrated by the f