United States Patent [191
`Okuhara et al.
`
`[11] Patent Number:
`[45] Date of Patent:
`
`4,929,611
`May 29, 1990
`
`[54] METHOD FOR INIMUNOSUPPRESSION
`
`[75] Inventors: Masakuni Okuhara; Hirokazu
`Tanaka; Toshio Goto, all of Ibaraki;
`Tohru Kino, Tsuchiura; Hiroshi
`Hatanaka, Ibaraki, all of Japan
`[73] Assignee: Fujisawa Pharmaceutical Company,
`Ltd., Osaka, Japan
`[21] Appl. No.: 868,749
`[22] Filed:
`May 30, 1986
`
`[63]
`
`Related US. Application Data
`Continuation-impart of Ser. No. 799,855, Nov. 24,
`1985, Pat. NO. 4,894,366.
`Foreign Application Priority Data
`[30]
`Dec. 3, 1984 [GB] United Kingdom ............... .. 8430455
`Feb. 5, 1985 [GB] United Kingdom ............... .. 8502869
`Apr. 1, 1985 [GB] United Kingdom ............... .. 8508420
`
`[51] Int. 01.5 . . . . . .
`
`. . . . . . . . .. A61K 31/395
`
`[52] US. Cl. .................................... .. 514/183; 514/63;
`514/291; 514/411; 540/452; 540/456
`[58] Field of Search ................ .. 540/452, 456; 514/63,
`514/291, 411, 183
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`3,244,592 4/1966 Arai ................................... .. 514/183
`
`OTHER PUBLICATIONS
`J. A. Findlay: Canadian Journal of Chemistry, vol. 58
`(1980),~pp. 579-590.
`Arai et al., Journal of Antibiotics, vol. 15, (1962), pp.
`231-232.
`
`Korzybski, “Antibiotics-Origin Nature and Proper—
`ties”, vol. II, 1978.
`Primary Examiner-Robert T. Bond
`Attorney, Agent, or Firm-Oblon, Spivak, McClelland,
`Maier & Neustadt
`ABSTRACT
`[57]
`This invention relates to tricyclo compounds useful for
`treatment and prevention of resistance by transplanta
`tion, graft-versus-host diseases by medulla ossium trans
`plantation, autoimmune diseases, infectious diseases,
`and the like, which can be represented by the following
`formula:
`
`R1
`
`(1)
`
`OCH; OCH3
`
`to a process for their production, to a pharmaceutical
`composition containing the same and to a use thereof.
`
`5 Claims, No Drawings
`
`NOVARTIS EXHIBIT 2016
`Par v Novartis, IPR 2016-00084
`Page 1 of 40
`
`

`
`1
`
`4,929,611
`
`METHOD FOR IMIVIUNOSUPPRESSION
`
`RELATED APPLICATION
`
`This application is a continuation-in-part of Ser. No.
`06/799,855, ?led Nov. 24, 1985, now US. Pat. No.
`
`(I)
`
`15
`
`25
`
`35
`
`4,894,366.
`'
`This invention relates to novel tricyclo compounds
`having pharmacological activities, to a process for their
`production and to a pharmaceutical composition con~
`taining the same.
`More particularly, it relates to novel tricyclo com
`pounds, which have pharmacological activities such as
`immunosuppressive activity, antimicrobial activity, and
`the like, to a process for their production, to a pharma
`ceutical composition containing the same and to a use
`thereof.
`Accordingly, one object of this invention is to pro
`vide a novel tricyclo compounds, which are useful for
`treatment and prevention of resistance by transplanta
`tion, graft-versus~host diseases by medulla ossium trans
`plantation, autoimmune diseases, infectious diseases,
`and the like.
`'
`Another object of this invention is to provide a pro
`cess for production of the tricyclo compounds by fer
`mentation processes and synthetic processes.
`A further object of this invention is to provide a
`pharmaceutical composition containing, as active ingre
`dients, the tricyclo compounds.
`Still further object of this invention is to provide a use
`of the tricyclo compounds for manufacturing a medica
`ment for treating and preventing resistance by trans
`plantation, graft-versus-host diseases by medulla ossium
`transplantation, autoimmune diseases, infectious dis
`eases, and the like.
`With respect to the present invention, it is to be noted
`that this invention is originated from and based on the
`?rst and new discovery of new certain speci?c com
`pounds, FR-900506, FR-900520, FR-900523 and FR
`900525 substances. In more detail, the FR-900506, FR
`900520, FR~900523 and FR-900525 substances were
`?rstly and newly isolated in pure form from culture
`broths obtained by fermentation of new species belong
`ing to genus Streptomyces.
`And, as a result of an extensive study for elucidation
`of chemical structures of the FR-900506, FR-900520,
`FR-9QO523 and FR-900525 substances, the inventors of
`this invention have succeeded in determining the chem
`ical structures thereof and in producing the tricyclo
`compounds of this invention.
`The new tricyclo compounds of this invention can be
`represented by the following general formula:
`
`45
`
`65
`
`OCH3 OCH3
`
`wherein
`R1 is hydroxy or protected hydroxy,
`R2 is hydrogen, hydroxy or protected hydroxy,
`R3 is methyl, ethyl, propyl or allyl,
`n is an integer of l or 2, and
`the symbol of a line and dotted line is a single bond or
`a double bond,
`.
`and salts thereof.
`Among the object compound (I), the following four
`speci?c compounds were found to be produced by
`fermentation.
`(l) The compound (I) wherein R1 and R2 are each
`hydroxy, R3 is allyl, n is an integer of 2, and the symbol
`of a line and dotted line is a single bond, which is enti
`tled to the FR-900506 substance;
`(2) The compound (I) wherein R1 and R2 are each
`hydroxy, R3 is ethyl, n is an integer of 2, and the symbol
`of a line and dotted line is a single bond, which is enti
`tled to the FR-900520 substance (another name: the WS
`7238A substance);
`(3) The compound (I) wherein R1 and R2 are each
`hydroxy, R3 is methyl, n is an integer of 2, and the
`symbol of a line and dotted line is a single bond, which
`is entitled to the FR-900523 substance (another name:
`the WS 7238B substance); and
`(4) the compound (I) wherein R1 and R2 are each
`hydroxy, R3 is allyl, n is an integer of l, and the symbol
`of a line, and dotted line is a single bond, which is enti
`tled to the Ell-900525 substance.
`With respect to the tricyclo compounds (I) of this
`invention, is to be understood that there may be one or
`more conformer(s) or stereoisomeric pairs such as opti
`cal and geometrical isomers due to asymmetric carbon
`atom(s) and double bond(s), and such isomers are also
`included within a scope of this invention.
`According to this invention, the object tricyclo com
`pounds (I) can be prepared by the following processes.
`[I] FERMENTATION PROCESSES
`
`7 Species belonging
`to the genus
`Streptomyces
`
`Fermentation ;
`
`FR-900506 substance
`FR-900520 substance
`FR-900523 substance and
`FR-900525 substance
`
`NOVARTIS EXHIBIT 2016
`Par v Novartis, IPR 2016-00084
`Page 2 of 40
`
`

`
`4,929,611
`
`[II] SYNTHETIC PROCESSES
`
`5
`
`(2) Process 2 (Introduction of Hydroxy-Protective
`
`(1) Process 1 (Introduction of Hydroxy-Protective
`
`Group)
`
`Group)
`
`CH3
`
`OH
`
`0
`
`CH3
`
`CH3
`
`OCH} OCH3
`
`(Ia)
`or a salt
`thereof
`
`Introduction of Hydroxy-
`Protective Group
`
`15
`
`20
`
`R!
`
`25
`
`CH3O
`
`3O
`
`35
`
`40
`
`45
`
`i
`OCH} OCH‘
`'
`“
`
`(1C)
`or a salt
`thereof
`
`Introduction of Hydroxy~
`PFOIECYWE Group
`
`CH3
`
`OH
`0
`
`CH3
`CH3
`
`CH3
`
`OH
`0
`
`CH3
`CH3
`
`OCH; OCH3
`
`(Ib)
`or a salt
`thereof
`
`65
`
`OCH; OCH3
`
`(Id)
`or a salt
`thereof
`
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`Page 3 of 40
`
`

`
`5
`
`4,929,611
`
`(3) Process 3 (Formation of Double Bond)
`
`6
`
`-continued
`
`CH3
`
`OH
`0
`
`CH3
`CH3
`
`(1B)
`or a salt
`thereof
`
`OCH3 OCH 3
`
`Base
`
`1O
`
`15
`
`20
`
`25
`
`CH3
`
`OH
`
`0
`
`CH3
`
`CH3
`
`OCH; OCH3
`
`(1h)
`or a salt
`thereof
`
`(5) Process 5 (Reduction of Allyl Group)
`
`CH3
`
`01-1
`0
`
`CH3
`CH}
`
`OCH3 OCH;
`
`(10
`or a salt
`thereof
`
`40
`
`CH
`3
`
`O“
`0
`
`(4) Process 4 (Oxidation of Hydroxyethylene Group) 45
`
`OCH; OCH3
`
`CH1CH=CH]
`
`CH3
`
`CH3
`
`(Ii)
`or a salt
`thereof
`
`Reduction
`
`CH3
`
`0H
`0
`
`CH3
`CH3
`
`OCH; OCH;
`
`0
`
`CHQCHzCH;
`
`(is)
`or a salt
`thereof
`
`H
`C 3
`
`OH
`
`0
`
`CH},
`
`CH3
`
`Oxidation of
`Hydroxyethylene Group
`
`65
`
`OCH; OCH;
`
`(Ii)
`or a salt
`thereof
`
`NOVARTIS EXHIBIT 2016
`Par v Novartis, IPR 2016-00084
`Page 4 of 40
`
`

`
`7
`(6) Process 6 (Removal of the carboxy-protective
`group)
`
`10
`
`20
`
`25
`
`30
`
`35
`
`CH3
`
`on
`0
`
`CH3
`CH3
`
`OCH; OCH3
`
`(1k)
`or a salt
`thereof
`
`Removal of the
`carboxy-protective group
`
`C
`H3
`
`on
`
`0
`
`CH3
`
`CH3
`
`OCH; OCH3
`
`(11)
`or a salt
`thereof
`
`4,929,611
`8
`silyl, propyl-diphenylsilyl, tert-butyl-diphenylsilyl,
`etc.), and the like, in which the preferred one may be
`tri(C1-C4)alkylsilyl and C1-C4alkyl-diphenylsilyl,
`and the most preferred one may be tert-butyl-dime
`thylsilyl and tert-butyl-diphenylsilyl;
`acyl such as aliphatic acyl, aromatic acyl and aliphatic
`acyl substituted with aromtic group, which are de
`rived from carboxylic, sulfonic and carbamic acids;
`and the like.
`The aliphatic acyl may include lower alkanoyl which
`may have one or more suitable substituent(s) such as
`carboxy (e.g. formyl, acetyl, propionyl, buty'ryl, isobu
`tyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, carboxya
`cetyl, carboxypropionyl, carboxybutyryl, carboxyhex
`anoyl,
`etc.),
`cyclo(lower)alkyloxy(lower)alkanoyl
`which may have one or more suitable substituent(s)
`such as lower alkyl (e.g. cyclopropyloxyacetyl, cy
`clobutyloxypropionyl, cycloheptyloxybutyryl, men
`thyloxyacetyl, menthyloxypropionyl, menthyloxybuty
`ryl, menthyloxyheptanoyl, menthyloxyhexanoyl, etc.),
`camphorsulfonyl, lower alkylcarbamoyl having one or
`more suitable substituent(s) such as carboxy and a pro
`tected carboxy, for example, carboxy(lower)alkylcar
`bamoyl (e.g. carboxymethylcarbamoyl, carboxyethyl
`carbamoyl, carboxypropylcarbamoyl, carboxybutylcar
`bamoyl, carboxypentylcarbamoyl, carboxyhexylcar
`bamoyl, etc.) protected carboxy(lower)alkylcarbamoyl
`such as tri(lower)alkylsilyl(lower)alkoxycarbonyl(
`lower)alkylcarbamoyl (e.g. trimethylsilylmethoxycar
`bonylethylcarbamoyl,
`trimethylsilylethoxycarbonyl
`propylcarbamoyl, triethylsilylethoxycarbonylpropyl
`carbamoyl,
`tert-butyldimethylsilylethoxycarbonyl
`propylcarbamoyl, trimethylsilylpropoxycarbonylbutyl
`carbamoyl, etc.), and the like.
`The aromatic acyl may include aroyl which may
`have one or more suitable substituent(s) such as nitro
`(e.g. benzoyl, toluoyl, xyloyl, naphthoyl, nitrobenzoyl,
`dinitrobenzoyl, nitronaphthoyl, etc.), arenesulfonyl
`which may have one or more suitable substituent(s)
`such as halogen (e.g. benzenesulfonyl, toluenesulfonyl,
`xylenesulfonyl, naphthalenesulfonyl, fluorobenzenesul
`fonyl, chlorobenzenesulfonyl, bromobenzenesulfonyl,
`iodobenzenesulfonyl, etc.), and the like.
`The aliphatic acyl substituted with aromatic group
`may include ar(lower)alkanoyl which may have one or
`more suitable substituent(s) such as lower alkoxy and
`trihalo(lower)alkyl (e.g. phenylacetyl, phenylpropio
`nyl, phenylbutyryl, 2-trifluoromethyl-2-methoxy-2
`phenylacetyl, 2-ethyl-2-tri?uoromethyl-Z-phenylacetyl,
`2-tri?uoromethyl-2-propoxy-Z-phenylacetyl, etc.), and
`the like.
`The more preferred acyl group thus de?ned may be
`C1-C4alkanoyl which may have carboxy, cyclo(C5—C6
`alkyloxy(C1-C4)alkanoyl having two (C1—C4)alkyl
`groups on the cycloalkyl moiety, camphorsulfonyl,
`carboxy(C1—C4)-alkylcarbamoyl, tri(C1—C4)alkylsi1yl(
`C1—C4)alkoxycarbonyl(C1—C4)alkylcarbamoyl, benzoyl
`which may have one or two nitro, benzenesulfonyl
`having halogen, phenyl(C1-C4)alkanoyl having C1-C
`4alkoxy and trihalo(C1—C4)alkyl, and the most preferred
`one may be acetyl, carboxypropionyl, menthyloxya
`cetyl, camphorsulfonyl, benzoyl, nitrobenzoyl, dinitro
`benzoyl, iodobenzenesulfonyl and 2-tri?uoromethyl-2
`methoxy-Z-phenylacetyl.
`Suitable “protected carboxy(lower)alkylcarbamoyl”
`and "carboxy(lower)alkylcarbamoyl" moieties of the
`“protected carboxy(lower)alkylcarbamoyloxy" and
`
`in which
`R‘, R2, R3, 11 and the symbol of a line and dotted line are
`each as de?ned above,
`RG1 and R,,2 are each protected hydroxy,
`R1,1 is protected carboxy(1ower)alkylcarbamoyloxy,
`RC1 is carboxy(lower)alkylcarbamoyloxy, and
`Rbz is a leaving group.
`Particulars of the above de?nitions and the preferred
`embodiments thereof are explained in detail as follows.
`The term “lower” used in the speci?cation is in
`tended to mean 1 to 6 carbon atoms, unless otherwise
`indicated.
`Suitable hydroxy-protective group in the “protected
`hydroxy” may include:
`l-(lower alkylthio)(lower)alkyl such as lower alkylthi
`omethyl (e. g. methylthiomethyl, ethylthiomethyl,
`propylthiomethyl, isopropylthiomethyl, butylthi
`omethyl, isobutylthiomethyl, hexylthiomethyl, etc.),
`and the like, in which the preferred one may be C1-C
`4alkylthiomethyl and the most preferred one may be
`methylthiomethyl;
`trisubstituted silyl such as tri(lower)alkylsilyl (e.g. tri
`methylsilyl, triethylsilyl, tributylsilyl, tert-butyl
`dimethylsilyl, tri-tert-butylsilyl, etc.), lower alkyl
`diarylsilyl (e.g. methyl-diphenylsilyl, ethyl-diphenyl
`
`45
`
`55
`
`65
`
`NOVARTIS EXHIBIT 2016
`Par v Novartis, IPR 2016-00084
`Page 5 of 40
`
`

`
`4,929,611
`9
`"carboxy(lower)alkylcarbamyloxy” groups may in
`clude the same as those exempli?ed in the explanation
`of the hydroxy-protective group mentioned above.
`Suitable “leaving group” may include hydroxy,
`acyloxy in which the acyl moiety may be those as exem
`plified above, and the like.
`The processes for production of the tricyclo com
`pounds (I) of this invention are explained in detail in the
`following.
`[I] FERMENTATION PROCESSES
`The FR-900506, FR-900520, FR-900523 and FR~
`900525 substances of this invention can be produced by
`fermentation of FR-900506, FR-900520, FR-900523 and
`/or F R-900525 substance(s)-producing strains belonging
`to the genus Streptomyces such as Streptomyces tsuku
`baensis No. 9993 and Streptomyces hygroscopt'cus sybsp.
`yakushimanensis No. 7238 in a nutrient medium.
`Particulars of microorganisms used for the produc
`tion of the FR-900506, FR~900520, FR-900523 and FR
`900525 substances are, explained in the following.
`[A] The FR-900506, FR-900520 and FR-900525 sub
`stances of this invention can be produced by fermenta
`tion of a FR-900506, FR-900520 and/or FR-900525
`substance(s)-producing strain belonging to the genus
`Streptomyces such as Streptomyces tsukubaensis No.
`9993 in a nutrient medium.
`
`20
`
`10
`Morphological observations were made with light
`and electron microscopes on cultures grown at 30° C, for
`14 days on oatmeal agar, yeast-malt extract agar and
`inorganic salts-starch agar. The mature sporophores
`formed Recti?exibiles with 10 to 50 or more than 50
`spores in each chain. The spores were oblong or cylin
`drical, 0.5—0.7><O.‘7-0.8 pm in size by electron micro
`scopic observation. Sport surfaces were smooth.
`[2] Cultural Characteristics:
`Cultural characteristics were observed on ten kinds
`of media described by Shirling and Gottlieb as men
`tioned above, and by Waksman (Wakstnan, S. A.: The
`actinomycetes, vol. 2: Classi?cation, identi?cation and
`description of genera and species. The Williams and
`Wilkins Co., Baltimore, 1961).
`The incubation was made at 30° C. for 14 days. The
`color names used in this study were based on Guide to
`Color Standard (manual published by Nippon Shikisai
`Kenkyusho, Tokyo). Colonies belonged to the gray
`color series when grown on oatmeal agar, yeast-malt
`extract agar and inorganic salts-starch agar. Soluble
`pigment was produced in yeast-malt extract agar but
`not in other media. The results are shown in Table 1.
`
`THE MICROORGANISM
`The microorganism which can be used for the pro
`duction of the FR-900506, FR-900520 and/or FR
`900525 substances is FR-900506, FR-90052O and/or
`FR‘900525 substance(s)-producing strain belonging to
`the genus Streptomyces, among which Slreptomyces
`tsukubaensis No. 9993 has been newly isolated from a
`soil sample collected at Toyosato-cho, Tsukuba-gun,
`Ibaraki Prefecture, Japan.
`A lyophilized sample of the newly isolated Streptomy
`ces tsukubaensis No. 9993 has been deposited with the
`Fermentation Research Institute, Agency of Industrial
`Science and Technology (No. 1-3, Higashi l-chome,
`Yatabemachi Tsukuba-gun, Ibaraki Prefecture, Japan)
`under the deposit number of FERM P-7886 (deposited
`date: Oct. 5th, 1984), and then converted to Budapest
`Treaty route of the same depository on Oct. 19, 1985
`under the new deposit number of FERM BP-927.
`It is to be understood that the production of the novel
`FR-900506, FR-90052O and/or FR-900525 substance(s)
`is not limited to the use of the particular organism de
`scribedherein, which is given for the illustrative pur
`pose only. This invention also includes the use of any
`mutants which are capable of producing the FR-900506,
`FR-900520 and/or FR-900525 substances including
`natural mutants as well as arti?cial mutants which can
`be produced from the described organism by conven
`tional means such as irradiation of X-rays, ultra-violet
`radiation,
`treatment
`with
`N-methyl-N’-nitro-N
`nitrosoguanidine, Z-aminopurine, and the like.
`The Streptomyces tsukubaensis No. 9993 has the fol
`lowing morphological, cultural, biological and physio
`logical characteristics.
`[1] Morphological Characteristics:
`The methods described by Shirling and Gottlieb
`(Shirling, E. B. and D. Gottlieb: Methods for character
`ization of Streptomyces species. International Journal
`of Systematic Bacteriology, 16, 313-340, 1966) were
`employed principally for this taxonomic study.
`
`35
`
`40
`
`45
`
`55
`
`65
`
`TABLE 1
`Cultural Characteristics of Strain No. 9993
`and Streptomvcer misakt'enris lFO 12891
`Cultural characteristics
`IFO 12891
`
`No. 9993
`
`Medium
`
`Oatmeal Agar
`
`Yeast-Malt
`Extract Agar
`
`G Moderate
`A Gray
`R Pale Pink
`5 None
`G Moderate
`A Light Gray
`R Dull Reddish Orange
`S Dull Reddish Orange
`Inorganic Salts- G Moderate
`Starch Agar
`A Pale Yellow Orange to
`Light Gray
`R Dark Orange
`
`Glucose-
`Asparagine
`Agar
`
`Glycerin‘
`Asparagine
`Agar
`
`Czapek Agar
`
`S None
`G Poor
`A White
`R Pale Brown
`
`S None
`G Moderate
`A Pale Pink to White
`R Pale Pink
`
`5 None
`G Poor
`A None
`R Pale Pink
`
`S None
`Nutrient Agar G Poor
`A White, Poor
`R Colorless
`S None
`Potato-Dextrose G Poor
`Agar
`A None
`R Pale Pink
`S None
`Tyrosine Agar G Moderate
`A White
`
`R Dull Reddish Orange
`
`Peptone-Yeast
`Extract-Iron
`Agar
`
`5 None
`G Poor
`A None
`R Colorless
`
`Moderate
`Grayish White
`Colorless
`None
`Moderate
`Grayish White
`Light Brown
`None
`Moderate
`Grayish White
`
`Pale Yellowish
`Brown
`None
`Moderate
`Grayish White
`Pale Yellowish
`Brown
`Pale Brown
`Moderate
`Grayish White
`Pale Yellowish
`Brown
`Pale Brown
`Abundant
`Grayish White
`Dark Orange to
`Dark Brown
`None
`Poor
`White
`Colorless
`None
`Moderate
`Yellowish Gray
`Brown
`None
`Moderate
`Grayish White to
`Light Gray
`Dark Orange to
`Black
`None
`Poor
`None
`Colorless
`
`NOVARTIS EXHIBIT 2016
`Par v Novartis, IPR 2016-00084
`Page 6 of 40
`
`

`
`11
`TABLE l-continued
`Cultural Characteristics of Strain No. 9993
`and Streptomyces misakienris IFO 12891
`Cultural characteristics
`IFO 12891
`
`No. 9993
`
`S None
`
`None
`
`Medium
`
`Abbreviation:
`G = Growth,
`R = Reverse Side Color.
`= Aerial Mass Color.
`5 = Soluble Pigment,
`
`4,929,611
`
`12
`TABLE 3
`Carbon Sources Utilization of Strain No. 9993 and
`Streptomyces misakiensi's lFO 12891
`
`Carbon
`Sources
`D-Glucose
`Sucrose
`Glycerin
`D-Xylose
`D-Fructose
`Lactose
`Maltose
`
`Rhamnose
`
`10
`
`Raffinose
`D-Galactose
`L-Arabinose
`D-Mannose
`D-Trehalose
`lnositol
`D-Mannitol
`lnulin
`Cellulose
`Salicin
`Chitin
`Sodium Citrate
`Sodium Succinate
`Sodium Acetate
`Symbols:
`+2 utilization
`:: doubtful utilization
`—: no utilization
`
`No. 9993
`
`IFO 12891
`
`+
`
`—
`
`l
`
`lll+l
`
`l
`
`—
`
`l
`
`II
`
`I
`
`l
`
`+
`
`l
`
`l
`
`1
`
`The cell wall analysis was performed by the methods
`of Becker et al. (Becker, 8., M. P. Lechevalier, R. E.
`Gordon and H. A. Lechevalier: Rapid differentiation
`between Nocardia and Streptomyces by paper chroma
`tography of whole cell hydrolysates: Appl. Microboil,
`l2, 42l—423, 1964) and Yamaguchi (Yamaguchi, T.:
`Comparison of cell‘ wall composition of morphologi
`cally distinct actinomycetes: J. Bacteriol., 89, 444-453,
`1965). Analysis of whole cell hydrolysates of the strain
`No. 9993 showed the presence of LL-diaminopimelic
`acid. Accordingly, the cell wall of this strain is believed
`to be of type I.
`[3] Biological and Physiological Properties:
`Physiological properties of the strain No. 9993 were
`determined according to the methods described by
`Shirling and Gottlieb as mentioned above. The results
`are shown in Table 2. Temperature range and optimum
`temperature for growth were determined on yeast-malt
`extract agar using a temperature gradient incubator
`(made by Toyo Kagaku Sangyo Co., Ltd.). Tempera
`ture range for growth was from 18° to 35° C. with
`optimum temperature at 28° C. Milk peptonization and
`gelatin liquefaction were positive. Melanoid pigment
`production was negative.
`
`25
`
`35
`
`TABLE 2
`Physiological Properties of Strain No. 9993 and
`Streptomyces misakiensis IFO 12891
`
`Physiological
`properties
`
`, No. 9993
`
`[PO 12891
`
`Temperature Range for Growth
`Optimum Temperature
`Nitrate Reduction
`Starch Hydrolysis
`Milk Coagulation
`Milk Peptonization
`
`18° C.-35° C.
`28° C.
`Negative
`Negative
`Negative
`Positive
`
`Melanin Production
`Gelatin Liquefaction
`H38 Production
`NaCl Tolerance (%)
`
`Negative
`Positive
`Negative
`53%
`
`12° C.-35° C.
`28° C.
`Negative
`Positive
`Negative
`Weakly
`Positive
`Negative
`Negative
`Negative
`3% <, <5%
`
`45
`
`Utilization of carbon sources was examined accord
`ing to the methods of Pridham and Gottlieb (Pridham,
`T. G. and D. Gottlieb: The utilization of carbon com
`60
`pounds by some Actinomycetales as an aid for species
`determination: J. BacterioL, 56, 107-114, 1948). The
`growth was observed after 14 days incubation at 30° C.
`Summarized carbon sources utilization of this strain is
`shown in Table 3. Glycerin, maltose and sodium succi
`nate could be utilized by the strain No. 9993. Further,
`doubtful utilization of D-glucose, sucrose, D-mannose
`and salicin was also observed.
`
`65
`
`Microscopic studies and cell wall composition analy
`sis of the strain No. 9993 indicate tha this strain belongs
`to the genus Streptomyces Waksman and Henrici 1943.
`Accordingly, a comparison of this strain was made
`with various Streptomyces species in the light of the
`published descriptions [International Journal of System
`atic Bacteriology, 18, 69 to 189, 279 to 392 (1968) and
`19, 391 to 512 (1969), and Bergy’s manual of Determina
`tive Bacteriology 8th Edition (1974)].
`As a result of the comparison, the strain No. 9993 is
`considered to resemble Streptomyces aburavl'ensz's Ni
`shimura et. al., Streptomyces avellaneus Baldacci and
`Grein and Streptomyces misakz‘ensis Nakamura. There
`fore, the cultural characteristics of the strain No. 9993
`were compared with the corresponding Streptomyces
`aburaviensz's IFO 12830, Streptomyces avellaneus IFO
`13451 and Streptomyces misakiensis IFO 12891. As a
`result, the strain No. 9993 was the most similar to Strep
`romyces misakiensis IFO 12891. Therefore, the strain
`No. 9993 was further compared with Streptomyces
`misakiensis IFO 12891 as shown in the above Tables 1,
`2 and 3. From further comparison, the strain No. 9993
`could be differentiated from Streptomyces misakl'ensis
`IFO 12891 in the following points, and therefore the
`strain No. 9993 is considered to be a new species of
`Streptomyces and has been designated as Streptomyces
`tsukubaensis sp. nov., referring to the soil collected at
`Tsukuba-gun, from which the organism was isolated.
`
`Difference from Streptomyces misakiensis IFO 12891
`Cultural characteristics of the strain No. 9993 are
`different from the Streptomyces misakiensis IFO 12891
`on oatmeal agar, yeast-malt extract agar, glucose
`asparagine agar, Czapek agar and potato-dextrose agar.
`Starch hydrolysis of the strain No. 9993 is negative,
`but that of the Streptomyces misakiensis IFO 12891 is
`positive.
`Gelatin liquefaction of the strain No. 9993 is positive,
`but that of the Streptomyces misakiensis IFO 12891 is
`negative.
`
`NOVARTIS EXHIBIT 2016
`Par v Novartis, IPR 2016-00084
`Page 7 of 40
`
`

`
`4,929,611
`13
`In carbon sources utilization, the strain No. 9993 can
`utilize glycerin, maltose and sodium succinate, but the
`Streptomyces misakiensis IFO 12891 can not utilize
`them. And, the strain No. 9993 can not utilize D-galac
`tose and inulin, but the Streptomyces misakiensis IFO
`12891 can utilize them.
`
`14
`Agitation and aeration of the culture mixture may be
`accomplished in a variety of ways. Agitation may be
`provided by a propeller or similar mechanical agitation
`equipment, by revolving or shaking the fermentor, by
`various pumping equipment or by the passage of sterile
`air through the medium. Aeration may be effected by
`passing sterile air through the fermentation mixture.
`The fermentation is usually conducted at a tempera
`ture between about 20° C. and 40° C., preferably
`25°—35° C., for a period of about 50 hours to 150 hours,
`which may be varied according to fermentation condi
`tions and scales.
`Thus produced FR-900506, FR-900520 and/or FR
`900525 substance(s) can be recovered from the culture
`medium by conventional means which are commonly
`used for the recovery of other known biologically ac
`tive substances. The FR-900506, FR-900520 and FR
`900525 substances produced are found in the cultures
`mycelium and ?ltrate, and accordingly the Ell-900506,
`FR-900520 and FR-900525 substances can be isolated
`and puri?ed from the mycelium and the ?ltrate, which
`are obtained by ?ltering or centrifuging the cultured
`broth, by a conventional method such as concentration
`under reduced pressure, lyophilization, extraction with
`a conventional solvent, pH adjustment, treatment with
`a conventional resin (e.g. anion or cation exchange
`resin, non-ionic adsorption resin. etc), treatment with a
`conventional adsorbent (e.g. activated charcoal, silicic
`acid, silica gel, cellulose, alumina, etc). crystallization,
`recrystallization, and the like.
`
`PHYSICAL AND CHEMICAL PROPERTIES OF
`FR-900506, FR-90052O AND FR-900525
`SUBSTANCES
`The FR-900506, FR-900520 and FR-900525 sub
`stances produced according to the aforementioned pro
`cess possess the following physical and chemical prop
`erties.
`
`FR-900506 Substance
`(1) Form and Color: white powder.
`(2) Elemental Analysis:
`
`C:
`
`64.72%,
`64.59%
`
`H:
`
`8.78%.
`8.74%
`
`N:
`
`1.59%
`1.62%
`
`(3) Color Reaction:
`Positive: cerium sulfate reaction, sulfuric acid reac
`tion, Ehrlich reaction, Dragendorff reaction and iodine
`vapor reaction.
`Negative: ferric chloride reaction, ninhydrin reaction
`and Molish reaction.
`(4) Solubility:
`Soluble: methanol, ethanol, acetone, ethyl acetate,
`chloroform, diethyl ether and benzene.
`Sparingly Soluble: hexane, petroleum ether.
`Insoluble: water.
`(5) Melting Point: 85°-90° C.
`(6) Speci?c Rotation: [a]D23: —73° (c=0.8, CI-ICl3).
`(7) Ultraviolet Absorption Spectrum: end absorption.
`(8) Infrared Absorption Spectrum: vmaxCHC13: 3680,
`3580, 3520, 2930, 2870, 2830, 1745, 1720, 1700, 1645,
`1450, 1380, 1350, 1330, 1310, 1285, 1170, 1135, 1090,
`1050, 1030, 1000, 990, 960(sh), 918 cm-1.
`(9) 13C Nuclear Magnetic Resonance Spectrum:
`
`15
`
`20
`
`25
`
`30
`
`35
`
`PRODUCTION OF FR-900506, FR-900520 AND
`FR-900525 SUBSTANCES
`The novel FR-900506, FR-900520 and FR-900525
`substances of this invention can be produced by cultur
`ing a FR-900506, FR-900520 and/or FR-900525 sub
`stance(s)-producing strain belonging to the genus Strep~
`tomyces (e.g., Streptomyces tsukubaensis No. 9993,
`FERM BP-927) in a nutrient medium.
`In general, the FR-900506, FR-900520 and/or FR
`900525 substance(s) can be produced by culturing the
`Ell-900506, FR-900520 and/or FR-900525 substance(s)~
`producing strain in an aqueous nutrient medium con-
`taining sources of assimilable carbon and nitrogen, pref
`erably under aerobic conditions (e.g. shaking culture,
`submerged culture, etc.).
`The preferred sources of carbon in the nutrient me
`dium are carbohydrates such as glucose, xylose, galac
`tose, glycerin, starch, dextrin, and the like. Other
`sources which may be included are maltose, rhamnose,
`raf?nose, arabinose, mannose, salicin, sodium succinate,
`and the like.
`'
`The preferred sources of nitrogen are yeast extract,
`peptone, gluten meal, cottonseed meal, soybean meal,
`corn steep liquor, dried yeast, wheat' germ, feather
`meal, peanut powder etc., as well as inorganic and or
`ganic nitrogen compounds such as ammonium salts (e. g.
`ammonium nitrate, ammonium sulfate, ammonium
`phosphate, etc), urea, amino acid, and the like.
`The carbon and nitrogen sources, though advanta
`geously employed in combination, need not be used in
`their pure form, because less pure materials which con
`tain traces of growth factors and considerable quantities
`of mineral nutrients, are also suitable for use. When
`40
`desired, there may be added to the medium mineral salts
`such as sodium or calcuim carbonate, sodium or potas
`sium phosphate, sodium or potassium chloride, sodium
`or potassium iodide, magnesium salts, copper salts, co
`balt salt and the like. If necessary, especially when the
`culture medium foams seriously, a defoaming agent,
`such as liquid paraffin, fatty oil, plant oil, mineral oil or
`silicone may be added.
`As the conditions for the production of the FR
`900506, FR-900520 and FR-900525 substances in mas
`sive amounts, submerged aerobic cultural conditions are
`preferred therefor. For the production in small
`amounts, a shaking or surface culture in a ?ask or bottle
`is employed. Furthermore, when the growth is carried
`out in large tanks, it is preferable to use the vegetative
`form of the organism for inoculation in the production
`tanks in order to avoid growth lag in the process of
`production of the FR-900506, FR-900520 and FR
`900525 substances. Accordingly, it is desirable ?rst to
`produce a vegatative inoculum of the organism by inoc
`60
`ulating a relatively small quantity of culture medium
`with spores or mycelia of the organism and culturing
`said inoculated medium, and then to transfer the cul
`tured vegatative inoculum aseptically to large tanks.
`The medium, in which the vegetative inocululm is pro
`duced, is substantially the same as or different from the
`medium utilized for the production of the FR-900506,
`FR-900520 and FR-900525 substances.
`
`45
`
`55
`
`65
`
`NOVARTIS EXHIBIT 2016
`Par v Novartis, IPR 2016-00084
`Page 8 of 40
`
`

`
`4,929,611
`
`16
`This white powder of the FR-900506 substance could
`be transformed into a form of crystals by recrystalliza
`tion thereof from acetonitrile, which possess the follow
`ing physical and chemical properties.
`(1) Form and Color: colorless prisms.
`(2) Elemental Analysis:
`
`5
`
`10
`
`C:
`
`64.30%,
`64.20%,
`
`H:
`
`8.92%,
`8.86%,
`
`N:
`
`1.77%
`1.72%.
`
`(3) Melting Point: 127°-129° c.
`(4) Speci?c Rotation: [a]D23: -—84.4° (c: 1.02,
`CHCl3).
`(5) 13C Nuclear Magnetic Resonance Spectrum:
`
`15
`
`6(ppm,
`212.59 (5)
`196.18 (s)
`coon); { 212.45 (s), { 192.87 (s),
`164.90 (5)
`138.89 (5)
`( 166.01 (5), { 139.67 (s),
`132.52 (5)
`130.27 (d)
`{ 131.99 (5), { 130.21 (d),
`116.57 (1)
`97.35 (5)
`
`{ 116.56(t), { 9876(5).
`
`77.79 (6)
`75.54 (d)
`{ 7S.22(d), { 76.97 (d),
`73.72 (d)
`70.05 (<1)
`
`( 72.57 (d), { 6915(6).
`53.03 (d)
`48.85 (t)
`( 53.13 (a), ( 48.62 (1),
`3940(1),
`31.58 (l),
`
`30.79 (1),
`
`26.46 (a),
`
`24.65 (1),
`
`14.06 (q)
`9.69 (q)
`{ 14.23 (q). < 9.98 (q).
`
`169.07 (s)
`{ 168.9O(s),
`135.73 (11)
`{ 135.60 (d),
`122.87 (d)
`123.01 (d),
`84.41 (d),
`
`73.93 (a)
`{ 73.09 (d),
`56.75 (0),
`
`40.33 (d)
`{ 40.85 (d),
`
`27.72 (t)
`
`2634(t),
`
`20.45 (q)
`
`19.73 (q).
`
`5(ppm,
`211.98 (s)
`196.28 (5)
`CDC13): { 211.74 (5), { 193.56 (s),
`164.85 (5)
`138.76 (5),
`165.97 (5), < 139.51 (5),
`
`168.97 (5)
`{ 168.81 (5),
`13573 (d)
`135.63 (d),
`
`132.38 (5)
`13039 ((11)
`131.90 (5). ( 130.17 (d).
`
`12282 (d)
`122.96 (d).
`
`84.29 (11).
`
`00.89111)
`09.00 (d).
`
`48.76 (I)
`( 48.31 (t).
`30.72 (t).
`
`97.19 (5)
`( 03.63 (5).
`77.5201)
`{ 76.97 (d).
`
`529701)
`{ 52.82 (d).
`31.62 (t).
`
`21.12 (t)
`
`20.86 (I).
`
`15.88 (q)
`15.75 (q),
`
`20.33 (q)
`
`19.74 (q).
`
`13.89 (q)
`14.05 (q),
`
`116.43 (I).
`
`77.8401)
`78.21 (d).
`
`56.63 (d)
`54.87 (cl).
`40.21 (d)
`
`40.5401),
`
`24.56 (t).
`
`16.17 (q)
`{ 16.10 (q),
`
`9.64 (q)
`
`{ 9-9661).
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`the chart of which being shown in FIG. 1,
`(10) 1H Nuclear Magnetic Resonance Spectrum: the
`chart of which being shown in FIG. 2.
`(11) Thin Layer Chromatography:
`
`Stationary Phase
`
`silica gel plate
`
`Developing
`Solvent
`
`chloroformzmethanol
`(10:1, v/v)
`ethyl acetate
`
`Rf Values
`
`0.58
`
`0.52
`
`(12) Property of the Substance: neutral substance.
`With regard to the FR-900506 substance, it is to be
`noted that in case of measurements of 13C and 1H nu
`clear magnetic resonance spectra, this substance
`showed pairs of the signals in various chemical shifts.
`The FR-900506 substance thus characterized further
`possesses the following properties.
`(i) The measurements of 13C Nuclea