United States Patent [191
`Wenger
`
`[11] Patent Number:
`[45] Date of Patent:
`
`4,764,503
`Aug. 16, 1988
`
`[54] NOVEL CY CLOSPORINS
`.
`.
`_
`[75] Inventor. Roland Wenger, Riehen, Switzerland
`[73] Assrgnee: Sandoz Ltd.,"Basel, Sw1tzer1and
`[21] Appl' No’: 49’746
`[22] Filed:
`May 13, 1987
`
`[63]
`
`Related US. Application Data
`Continuation of Ser. No. 932,760, Nov. 19, 1986, aban-
`doned, which is a continuation of Ser. No. 713,259,
`Mar. 19, 1985, abandoned.
`[51] Int. 01.4 ...................... .. A61K 37/02; CO7K 5/12
`[52] US. Cl. .......................... .. 514/11; 530/317
`[58] Field of Search ......................... .. 530/321; 514/11
`56
`R f
`Ct d
`[
`1
`e e'ences ' e
`US. PATENT DOCUMENTS
`
`4,108,985 8/1978 Ruegger et a1. .................... .. 514/11
`4,210,581 7/1980 Ruegger et a1.
`530/321
`4,220,641 9/1980 Traber et a1. ....................... .. 514/11
`
`Primary Examiner-De1bert R. Phillips
`Attorney, Agent, or Firm—-—Gerald D. Sharkin; Robert S.
`Honor; Thomas O. McGovern
`[57]
`ABSTRACT
`Cyclosporins wherein the amino acid residue at the
`8-position is a (D)-acyloxy-a-amino acid residue, typi
`cally of formula
`
`X-Y-Sar4MeLeu-Z'MeLeu-Ala-Q-MeLeu-MeLeu-MeVal
`I-
`_l
`
`wherein X=—MeBmt— °r —dihYdr°-"MeBmt—,
`Y=—<1Abu—’ —A1a—1 —ThI-, —Va1—- or —-N
`‘Ia-w
`Z=—Va1—
`Or
`-—Nva—
`and
`Q=R1—CO——O—CH(R2)—CH(CO—)—NH—
`wherein R1=H, C1_4a1kyl or phenyl and R2=H or
`CH3, possess immunosuppressive, anti-in?ammatory
`and anti-parasitic activity.
`
`6 Claims, N0 Drawings
`
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`

`
`1
`
`NOVEL CYCLOSPORINS
`
`This is a continuation of application Ser. No. 932,760,
`?led Nov. 19, 1986, which in turn is a continuation of
`application Ser. No. 713,259, ?led Mar. 19, 1985, both
`now abandoned.
`The present invention relates to novel cyclosporins,
`processes for their production, their use as pharmaceuti
`cals and pharmaceutical compositions comprising them.
`The cyclosporins comprise a class of structurally
`distinctive, cyclic, poly-N-methylated undecapeptides
`commonly possessing pharmacological, in particular
`immunosuppressive, anti-in?ammatory and anti-para
`sitic activity. The ?rst of the cyclosporins to be isolated
`and the “parent” compound of the class, was the natu
`rally occurring fungal metabolite Cyclosporine, also
`known as cyclosporin A, of formula A
`
`4,764,503
`2
`ences include e.g. [Thr]2—, [Val]2—, [Nva]2— and
`[Nva]2——-[Nva]5—Cyclosporine (also known as cyclos
`porins C, D, G and M respectively), [Dihydro-MeBmt
`P-[ValP-Cyclosporine (also known as dihydrocy
`closporin D) and [(D)Ser]8— and [Dihydro-MeBmt
`]1—[(D)-Ser]8—Cyc1osporine.
`[In accordance with now conventional nomenclature
`for the cyclosporins, these are de?ned throughout the
`present speci?cation and claims by reference to the
`structure of Cyclosporine (i.e. cyclosporin A). This is
`done by ?rst indicating those residues in the molecule
`which differ from those present in Cyclosporine and
`then applying the term “Cyclosporine” to characterise
`the remaining residues which are identical to those
`present in Cyclosporine. At the same time the term
`—-dihydro——MeBmt— is employed to designate the
`residue of formula B above in which --x—y-- is —CH
`2—CH2—. Thus [Dihydro-MeBmt]1—[Val]2—Cy
`
`[- MeBmt——-aAbu—-Sar— MeLeu- Val-MeLeu-Ala-(D)Ala- MeLeu-MeLeu-MeVd
`_]
`1 1
`9
`10
`1
`2
`3
`4
`5
`6
`7
`8
`
`(A)
`
`wherein —MeBmt— represents the N-methyl-(4R)-4
`but-2E-en-1-yl-4-methyl-(L)threonyl residue of formula
`B
`
`25
`
`CH3
`
`(B)
`
`30
`
`35
`
`in which --x—y— is —CH:CH— (trans).
`Since the original discovery of Cyclosporine a wide
`variety of naturally occurring cyclosporins have been
`isolated and identi?ed and many further non-natural
`cyclosporins have been prepared by total- or semi-syn
`thetic means or by the application of modi?ed culture
`techniques. The class comprised by the cyclosporins is
`thus now substantial and includes for example the natu
`rally occurring cyclosporins A through Z [c.f. Kobel et
`al. European Journal of applied Microbiology and Bio
`technology 14, 237-240 (1982) and poster presented by
`Traber et al., 24th. Interscience Conference on Antimi~
`crobial Agents and Chemotherpy, Washington, Oct.
`8-10, (1984)]; as well as various non-natural or arti?cial
`cyclosporins, including dihydro-cyclosporins (in which
`the group —x--y—— of the —~MeBmt-— residue-see
`formula B above-is saturated, eg as disclosed in U.S.
`Pat. Nos. 4,108,985; 4,210,581 and 4,220,641), cyclospo
`rins in which the —MeBmt— residue is present in iso
`meric or N-desmethyl form [c.f. European Pat. No. 0
`034 567 and “Cyclosporin A”, Proc. Internat. Confer
`ence on Cyclosporin A, Cambridge {U.K.) September
`1981, Ed. D. J. G. White, Elsevier Press (1982)-both
`describing the total-synthetic method for the produc
`tion- of cyclosporins developed by R. Wenger] and cy
`closporins in which incorporation of variant amino
`acids at speci?c positions within the peptide sequence is
`effected (c.f. European Pat. No. 0 056 782). Examples of
`such cyclosporins as disclosed in the above art refer
`
`45
`
`60
`
`65
`
`closporine is the cyclosporin having the sequence
`shown in formula A, but in which —MeBmt— [formula
`B, —x--y—=—-CH:CI-I—- (trans)] at the 1-position is
`replaced by —dihydro-MeBmt- [formula B, —x
`—y-—=—CH2—CH2-] and -—aAbu-— at the 2-posi
`tion is replaced by —-Val—. Similarly [(D)Ser]8—Cy
`closporine is the cyclosporin having the sequence
`shown in formula A, but in which —(D)Ala— at the
`8-position is replaced by —(D)Ser—.
`In addition, amino acid residues referred to by abbre- I
`viation, e.g. —Ala—, —MeVal—- etc .
`. . are, in accor
`dance with conventional practice, to be understood as
`having the (L)-con?guration unless otherwise indi
`cated. Residue abbreviations preceded by “Me”, as in
`the case of —MeLeu- represent N-methylated resi
`dues. The individual residues of the cyclosporin mole
`cule are numbered, as in the art, clockwise and starting
`with the residue —MeBmt— or —dihydro—MeBmt——
`in position 1. The same numerical sequence is employed
`throughout the present speci?cation and claims]
`In accordance with the present invention it has now
`been found that novel cyclosporins may be obtained
`having pharmaceutical utility, in which the residue at
`the 8-position comprises an acyloxy a-amino acid resi
`due having the (D)-con?guration.
`Accordingly, in its broadest aspect, the present inven
`tion' provides: a cyclosporin wherein the amino acid
`residue at the 8-position is a (D)-acyloxy-a-amino acid
`residue, i.e. the residue of an a-amino acid of the (D)
`series wherein the side chain attaching to the a-carbon
`atom is acyloxy-substituted.
`Preferably the amino acid residue at the 8-position is
`a (D)-,B-acyloxy-a-amino acid residue, i.e. the residue of
`an a-amino acid of the (D)-series having an acyloxy
`group attached at the B-carbon atom.
`Preferred (D)-,B-acyloxy-a-amino acid residues are
`those of formula II
`
`R2
`R1-CO-O-CH(/3)
`—NH-CH-CO
`(D)
`
`(11)
`
`wherein
`
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`

`
`4,764,503
`
`4
`
`R2
`
`(Iv)
`
`3
`R1 is hydrogen, C1.4alkyl or phenyl and
`R2 is hydrogen or methyl.
`Especially preferred cyclosporins in accordance with
`the present invention are those wherein the amino acid
`residue at the 8-position is an O-acyl-(D)-seryl or O- 5
`acyl-(D)-threonyl residue, in particular an O-acyl-
`(D)seryl or O-acyl-(D)-threonyl residue of formula II
`wherein R; has the meaning given above for formula II,
`above.
`to introduce a group R1—CO—, wherein R1 has the
`In one group of cyclosporins in accordance with the
`present invention, the amino acid residue at the 8-posi- 10 meaning given above for formula II, at the B-position of
`tion is an O-acyl-(D)-seryl residue, especially an O-acyl-
`said residue IV; or
`(D)-seryl residue wherein the acyl moiety has the for-
`(b) Reducing a cyclosporin wherein the amino acid
`mula R1—CO— in which R1 has the meaning given
`residue at the l-position is —MeBmt— and the residue
`above.
`at the 8-position is a (D)-acyloxy-a-arnino acid residue,
`In a second group of cyclosporins in accordance with 15 for example a (D)-,B-acyloxy-a-amino acid residue, to
`the present invention, the amino acid residue at the
`produce the corresponding cyclosporin wherein the
`8-position is a (D)-B-acyloxy-a-amino acid residue,
`residue at the 1-position is —dihydro—-MeBmt-, e.g.
`especially an O-acyl~(D)-seryl residue, more especially
`reducing a cyclosporin of formula I as hereinbefore
`an O-acyl-(D)-seryl residue wherein the acyl moiety has
`defined, wherein X is —MeBmt-—, to produce the cor
`the formula R1--CO- in which R1 is hydrogen or 20 responding cyclosporin wherein X is —dihy
`C1.4alkyl, and the residue at the 5-position is an (L)-nor-
`dro—MeBmt-—.
`valyl residue.
`Process step (a) above may be carried out in accor
`Most preferred are cyclosporins of formulaI
`dance with standard procedures for the acylation of
`
`HO_CH
`—NI-I--CH—CO—
`(D)
`
`|-— X— Y-Sar- MeLeu-'-Z-MeLew-Ala-Q-MeLeu- MeLeu-Val-l (I)
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`
`hydroxy groups, for example by reaction with (prefera
`wherein
`30 bly 2 equivalents or, when Y=—Thr—-, 1 equivalent)
`X is —MeBmt— or —dihydro--MeBmt—,
`Y is —aAbu—, ‘—-Ala-, —Thr—, —Val— or —Nva- of an appropriate acyl—, e.g. C1-5alkanoyl— or benz
`Z is —Val-- or —Nva—, and
`oyl-halide, or corresponding —anhydride or, for formy
`Q is a residue of formula II as defined above.
`lation, by reaction with e.g. acetic-formic anhydride, at
`In formula I, Q is preferably an O-acyl-(D)-seryl or
`a temperature of e.g. from about — 10° to 50° C. The
`O=acyl-(D)-threonyl residue wherein the acyl moiety 35 reaction is carried out under anhydrous conditions,
`has the formula R1—CO— in which R1 has the meaning
`suitably in the presence of an inert solvent or diluent
`given for formula II. Y is preferably —aAbu—,
`such as methylene chloride, and in the presence of a
`—Thr-—, ——Val— or —Nva——.
`condensation agent such as 4-dimethyl-amino-pyridine.
`A group of cyclosporins in accordance with the pres-
`In this connection it is to be noted that the reaction
`ent invention are those of formula I as de?ned above, 40 proceeds with acylation occurring at the OH group of
`wherein Y is —aAbu-- or —Nva—, Z is —Val- and
`the amino acid residue at the 8-position, in preference to
`R2 is hydrogen.
`the hydroxy group of the amino acid residue at the
`A further group of cyclosporins in accordance with
`l-position.
`the present invention are those of formula I as de?ned
`Process step (b) may be carried out analogously to
`above, wherein Y is —aAbu- or —Nva~—, Z is —N- 45 known methods for reducing naturally-occurring cy
`va——, R1 is hydrogen or C1.4alkyl and R2 is hydrogen.
`closporins to the corresponding dihydrocyclosporins,
`The present invention also provides a process for the
`for example by catalytic hydrogenation, e.g. in accor
`production of a cyclosporin wherein the amino acid
`dance with the general methods disclosed in U.K. Pa
`residue at the 8-position is a (D)-acyloxy-a-amino acid
`tent Speci?cation No. 1,567,201.
`residue, for example a (D)~,B-acyloxy-a~amino acid resi- 50
`Hydrogenation is suitably effected under neutral pH
`due, e.g. for the production of a cyclosporin of formula
`conditions at temperatures of from about 20° to about
`I as de?ned above, which process comprises:
`30° C. and at atmospheric or slightly elevated pressure,
`(a) Acylating a cyclosporin wherein the amino acid
`in the presence of a catalyst such as platinum or, prefer
`residue at the 8-position is a (D)-hydroxy-a-amino acid
`ably, palladium (e.g. palladium on charcoal) in the pres
`residue, for example a (D)-B-hydroxy-a-amino acid 55 ence of an inert solvent or diluent such as ethyl acetate
`residue, e.g. acylating a cyclosporin of formula III
`or lower aliphatic alkanols such as methanol or iso
`
`X-—Y—Sar--MeLeu—Z-MeLeu—Ala—W-MeLeu—MeLeu-MeVal
`F1 2
`s
`4
`5
`6
`1
`a
`9
`1o
`11
`
`(111)
`
`wherein X, Y and Z have the meanings given above for
`formula I and W is a residue of formula IV
`
`propanol.
`Cyclosporins having a B-hydroxy-a-amino acid resi
`due at the 8-position, in particular [(D)Ser]8-Cyclospo
`65 rine and [Dihydro-MeBmt]1-[(D)Ser]8-Cyclosporine,
`suitable for use as starting materials in process step (a)
`above are known and have been described together
`with processes for their production, e.g. in the afore
`
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`

`
`4,764,503
`6
`5
`mentioned European Pat. No. 0 056 782. Other cyclos-
`prises: (c) Deprotecting a cyclosporin of formula III as
`porins having a hydroxy-a-amino acid residue at the
`de?ned above which is in O-protected form;
`8-position and required as starting materials for process
`(d) Cyclising a straight chain undecapeptide compris
`step (a), may be prepared analogously or in accordance
`ing the sequence
`
`with the general procedures of the cyclosporin total- 10 wherein Y’, Z’, W’ and X’ have the meanings given
`synthetic method described in European Pat. No. 0 034
`above for formula IIIa, said undecapeptide being in
`567 to which publication 0 056 782 cross-refers, or in
`unprotected or O-protected form and, when required,
`accordance with the procedures hereinunder described
`carrying out process step c;
`in particular in the accompanying examples.
`(e) For the production of a cyclosporin of formula
`The cyclosporins starting materials for use in process 15 IIIa wherein
`step (b) above are obtainable in accordance with the
`Y’ is —Thr—, —Val— or —Nva—,
`method of process step (a).
`Z’ is —Val—- or, when Y’ is —Nva—, —Nva—,
`Although the cyclosporin starting materials of for-
`W’ is —(D)Ser—
`mula III above speci?cally disclosed in the accompany-
`and X’ is —MeBmt—-,
`ing examples are embraced by the broad disclosure of 20 cultivating a [ThrP-Cyclosporine, [ValP-Cyclosporine,
`the aforementioned European Pat. No. 0 056 782, cer-
`[NvaP-Cyclosporine or [NvaP-[NvaP-Cyclosporine
`tain of these cyclosporins are formally novel over the
`producing fungus strain in contact with a nutrient me
`teachings of that publication, i.e. have never previously
`dium containing (D)-Serine and isolating the cyclospo
`been described as such. In accordance with the present
`rin of formula IIIa from the obtained culture medium;
`invention it has also been found that these cyclosporins 25
`(f) For the production of a cyclosporin of formula
`possess especially interesting or advantageous biologi-
`IIIa wherein X’ is —dihydro-MeBmt, reducing the corre
`cal activity or pro?le, in particular in relation to immu-
`sponding cyclosporin of formula IIIa wherein X’ is
`nosuppressive activity, and especially in relation to
`—MeBmt—.
`prevention of transplant, e.g. organ transplant, rejec-
`Undecapeptides suitable for use in process step (d)
`tion, e.g. ascompared with known cyclosporins of for- 30 above may be obtained analogously to the general
`mula III, i.e. cyclosporins of formula III speci?cally
`methods described in the above mentioned European
`disclosed in European Pat. No. 0 056 782.
`Pat. No. 0 056 782, e.g. in relation to the flow chart to
`Accordingly in a further aspect the present invention
`Example la thereof, by combination of the peptide
`also provides a cyclosporin of formula IIIa
`sequence comprising residues 8 through 11 of the cy
`
`’-—Y'—Sar—-MeLeu-Z’--MeLeu—Ala—W’—MeLeu-MeLeu-MeVal
`2
`3
`4
`s
`6
`7
`s
`9
`10
`11 _|
`
`1
`
`(Illa)
`
`50
`
`40 closporin molecule with the sequence comprising resi
`“dues 1 through 7 but with the required substitution of
`wherein
`residues at positions 2 and/or 5 and/or 8. Suitably the
`Y’ is —aAbu-—, —Thr—, —Val— or —Nva—,
`—(D)Ser— or —(D)Thr— residue at the 8-position is in
`Z’ is —Val— or, when Y’ is —aAbu- or —Nva—,
`O-protected form, e.g.in the form of the O-t-butyl de
`—Nva—
`W’ is —(D)Ser— or, when Y’ is —aAbu— and Z’ is 45 rivative. Cyclisation is carried out using the particular
`—Val—, —(D)Thr—-, and
`techniques described in the said European Patent, with
`X’ is —MeBmt- or, when Y’ is —Thr—, —Val— or
`?nal removal of O-protecting groups when present
`—Nva—, Z’ is —Val— and W’ is 13 (D)Ser—,
`[process step (c)] in accordance with techniques known
`—dihydro—MeBmt—.
`in the art of peptide chemistry.
`Specific cyclosporins of formula IIIa are:
`The preferred fungus strain for use in the method of
`(a) [(D)Thr]8-Cyclosporine
`-
`process step (e) is the strain NRRL 8044 of the species
`(b) [Thr]2-[(D)Ser]s-Cyclosporine
`Toblpocladium in?atum (Gams), a culture of which has
`(c) [Dihydro-MeBmt]1-[Thr]2-[(D)Ser]S-Cyclosporine
`been deposited with the United States Department of
`(d) [Val]2-[(D)SerF-Cyclosporine
`Agriculture (Northern Research and Development
`(e) [Dihydro-MeBmt]1-[Val]2-[(D)Ser]B-Cyclosporine 55 Division), Peoria, 111., USA and is freely available to the
`(f) [Nva]2-[(D)Ser]S-Cyclosporine
`public. A further culture of this strain has been depos
`(g) [Dihydro-MeBmt]1-[Nva]2-[(D)Ser]s-Cyclosporine
`ited with the Fermentation Research Institute, Inage,
`(h) [Nva]5-[(D)SerP-Cyclosporine; and
`Chiba City, Japan, under the code number FRI FERM
`(i) [Nva]2-[Nva]5-[(D)SerP-Cyclosporine
`p No. 2796. The morphological characteristics of said
`Of the above listed cyclosporins, (a), (b), (e), (i) and 60 strain, originally classi?ed as belonging to the species
`(i), and in particular (a), (f) and (i) are of especial inter-
`Trichoderma polysporum (Link ex Pers.), as well as
`est, having regard to their activity (e.g. immunosup-
`methods for the preparation and maintainance of pre
`pressive activity)/activity pro?le, e.g. in relation to
`and sub-cultures are fully described eg in UK patent
`cyclosporins speci?cally disclosed in European Pat.
`speci?cation No. 1,491,509.
`No. 0 056 782.
`In accordance with process step (e) the selected strain
`In addition to the foregoing the present invention also
`[e.g. Tolypocladium in?atum (Gams)] is suitably main
`provides a process for the production of a cyclosporin
`tained for a period of ca. 2 weeks at a temperature of ca.
`of formula IIIa as de?ned above, which process com-
`27° C. in a culture medium such as described in the
`
`65
`
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`

`
`8
`
`(c=1.0
`
`EXAMPLE 3
`
`54 mg of [(O-acetyl)-(D)Ser3]—Cyclosporine in 10 ml
`ethanol are hydrogenated using 10 mg palladium/char
`coal (10%) at room temperature and under normal pres
`sure. After 20 hours the obtained reaction solution is
`filtered through a thin layer of talc and the ethanol is
`evaporated off under vacuum. After further drying
`under high vacuum, the title compound is obtained:
`[a]D2°=_-205.8° (c=1.02 in CHCl3).
`EXAMPLE 4
`The following compounds may be prepared either
`analogously to example 1, starting from the correspond
`ing non-acylated cyclosporin or analogously to example
`3, by hydrogenation of the corresponding cyclosporin
`described in example 2:
`
`4,764,503
`7
`following examples, in the presence of added (D)- or
`(D,L)-serine. The amino acid precursor is suitably
`added in an amount of from about 1 to about 15 g, more
`preferably from about 4 to about 10 g/liter culture me
`dium. Suitably the culture medium also contains added
`amino acid precursor for the residue present in the de
`sired cyclosporin at position 2, e.g. in amounts of from
`about 6.0 to about 10.0, preferably about 8.0 g/liter
`culture medium. Following incubation the culture is
`harvested and the obtained cyclosporin of formula IIIa
`extracted in accordance with known techniques, e.g. by
`comminution of conidia and mycelia, followed by ex
`tractive and/or absorptive isolation. The initially ob
`tained, raw cyclosporin may thereafter be puri?ed e.g.
`chromatographically and/ or by recrystallisation, in
`particular to effect separation from other cyclosporin
`contaminants in particular “natural cyclosporin” con
`taminants.
`Process step (f) above may be carried out e.g. using
`the same methods hereinbefore described in relation to
`process step (b).
`The following examples are illustrative of the pro
`cesses of the present invention.
`
`20
`
`EXAMPLE 1
`
`25
`
`20 mg 4-dimethylaminopyridine are added to 47 mg
`30
`[(D)Ser8]-Cyclosporine (prepared in accordance with
`the method described in Example 1 or 3 of the above
`mentioned European Pat. No. 0 056 782) dissolved in 3
`ml methylene chloride. 6.1 mg of freshly distilled ace
`tylchloride in 1 ml methylene chloride are then added
`and the obtained reaction mixture is stirred for 1 hour at
`room temperature. The reaction mixture is diluted with
`50 ml methylene chloride and shaken with 30 ml H2O.
`The organic phase is separated, dried over Na2SO4,
`?ltered off and evaporated. The residue is ?ltered on 60
`g silica gel (0.062-0.20 mm) using methylene chlori
`de/ 5% methanol as eluant and collected in 25 ml frac
`tions. The title compound is recovered from fractions 4
`to 8 by thin layer chromatography using CHCl3/5 %
`methanol as carrier phase: [a]D20=—202° (0:092 in
`45
`CHC13).
`
`40
`
`35
`
`EXAMPLE 2
`The following compounds may be prepared analo
`gously to example 1 starting from the corresponding
`non-acylated cyclosporin:
`2.1 [(O-benzoyl-(D)Ser]3-Cyclosporine [Formula I:
`X=—MeBmt—,
`Y=—aAbu—,
`Z=—Val—,
`Q=—O—benzoyl—(D)Ser-1: [a]D20= —220° (0: 1.0 in
`CHC13);
`
`55
`
`Preparation of starting materials
`EXAMPLE 5
`The following compounds required as starting mate
`rials for the production of the compounds of examples
`2.2 through 2.7 may be prepared analogously to the
`known compound [(D)Ser]3-Cyclosporine, the prepara
`tion of which is described in Example 1 of European
`Pat. No. 0 056 782, with substitution of the appropriate
`residues at positions 2 and/or 5 and/ or 8 in the process
`sequence set forth in the ?ow chart to Example la of
`said patent: ‘
`
`60
`
`65
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`
`4,764,503
`10
`ised water. The spores of this culture are taken up in a
`physiological NaCl solution to give a ?nal concentra
`tion of 5X 106 spores/ml. 10 ml of this suspension are
`used for inoculation of 1 liter of a nutrient solution
`having the same composition as the culture medium of
`Example 6a, with the exception of the (D)-serine and
`(DL)-norvaline components, and incubation is effected
`at 27° C. for 3 days on a rotary shaker (200 rpm). This
`culture is used as inoculum for the producing culture,
`[Nva2]-[(D)Ser8]-Cyclosporine may be produced on
`fermenter scale as follows:
`(c) Ca. 109 spores from an agar slant of the strain
`NRRL 8044 are transferred into a stainless-steel-fer
`menter containing 20 liters of a pre-culture-medium
`comprising:
`
`10
`
`15
`
`EXAMPLE 6
`The compound of example 5.2 may alternatively be
`produced microbiologically as follows:
`20
`(a) 10 liters of a nutrient medium containing 50 g
`maltose; 5 g (DL)-norvaline; 8 g (D)-serine; 0.75 g
`KHZPO4; 0.5 g MgSO4.7HzO; 0.1 CaCl2.6H2O and 8 g
`caseinpeptone per liter are inoculated with 1 liter of a
`suspension of conidia and mycelia of the fungus strain
`NRRL 8044 taken from a 3 day old pre-culture. The
`incoculated production-medium is ?lled in 100 ml por
`tions into 100 Erlenmeyer ?asks which are then incu
`bated for 14 days at 27° on an agitator rotating at 180
`r.p.m. The mycelium is separated from the culture me
`dium and extracted in a Turrax apparatus by crushing
`30
`and stirring with 3 X3 liters of 90% methanol. The
`crushed mycelium is separated from the solvent by
`suction-?ltration and the combined ?ltrates are concen
`trated by evaporation under vacuum at a temperature of
`40° C. until the vapour consists mainly of water alone.
`35
`The obtained mixture is extracted 4>< using 0.5 liter
`1,2-dichloroethane at each extraction and the combined
`1,2-dichloroethane solutions are concentrated by evap
`oration under vacuum at a temperature of 40° C.
`The obtained residue is subjected to gel ?ltration on
`Sephadex LH-20 (1.4 kg; Pharmacia) with methanol,
`and collected in 280 ml fractions. Fractions 9-11, con
`taining a cyclosporin mixture are pooled and then sepa
`rated by silica gel column chromatography (1 kg of
`silica gel, granulate size 0.063-0.2 mm, “Merck”) using
`water saturated ethyl acetate as eluent (fractions of 500
`ml). In accordance with their polarity, [Nva2]-Cyclos
`porine elutes ?rst (fractions 7-9), followed by a mixture
`comprising [Nva2]-[(D)Ser8]—Cyclosporine and Cyclos
`porine. Separation of [Nvaz]-[(D)Ser8]-Cyclosporine
`and Cyclosporine is achieved by silica gel chromatogra
`phy (280 g, “Merck”, O.63-0.2 mm) using chloroform
`/methanol (98:2) as eluent (fractions of 100 ml). Frac
`tions 20-30, containing crude [Nva2]-[(D)Ser3]-Cyclos
`porine, are further puri?ed by medium-pressure chro
`matography on a reversed-phased silica gel column
`(“Merck” LiChropep RP 18, 260 g, granulate size
`0.04-0.063 mm) with methanol/water (85:15) as eluent,
`with collection in 25 ml fractions. The combined frac
`tions 45-55 yield pure [Nvaz]-[(D)Ser3]-Cyclosporine as
`an amorphous white powder.
`The pre-culture required for the above process may
`be obtained as follows:
`(b) The spore and mycelium suspension used for inoc
`ulation is produced from a culture of the originally
`isolated strain NRRL 8044, cultivated for 21 days at 27°
`C. on an agar medium containing 20 g of malt extract,
`20 g of agar, 4 g of yeast extract per liter of demineral
`
`65
`
`50
`
`55
`
`Fructose
`Amber EHC
`KH2OP4
`KCl
`Dist. Water to
`(PH = 5,5)
`
`75 g
`25 g
`5 g
`2.5 g
`1 liter
`
`previously sterilized for 20 minutes at 120° C. Favoura
`ble incubation conditions are a temperature of 27° C.,
`air?ow of 16 liters per minute at an overpressure of 0.5
`bar and stir rotation of 200 rpm. The developing pre
`culture is incubated for 6 days and 15 liters are then
`transferred to a stainless-steel-fermenter holding 300
`liters of production medium comprising:
`
`Maltose
`Amber EHC
`KH2PO4
`KCl
`(DL)-norvaline
`(D)-serine
`Dist. Water to
`(PH = 5.5)
`
`previously sterilized for 20 minutes at 120“ C.
`The culture is held at a temperature of 27° C., aerated
`with 120 liters air per minute at an overpressure of 0.5
`bar and stirred at 70 rpm. Foam control is performed
`by addition of a silicone emulsion.
`After incubation for 14 days the culture, which has a
`total volume of 275 liters is cooled to 10° C. and the
`mycelia removed using a Westfalia separator. The ?l
`trate is extracted by stirring 2X with ethyl acetate, the
`extracts are washed with a little water, combined and
`dried under vacuum. The mycelium is combined with
`methanol, homogenised and ?ltered. This extraction is
`repeated 2X using 90% methanol. The methanolic ex
`tracts are combined and, with addition of water, con
`centrated under vacuum. The remaining, aqueous con
`centrate is extracted 2X with ethyl acetate, the extracts
`washed with a little water, combined and concentrated
`under vacuum. The extracted aqueous phase is re
`extracted 2>< with ethyl acetate/isopropanol (8:2).
`These extracts are combined and again evaporated
`under vacuum.
`'
`The mycelial and ?ltrate extracts are ?ltered using
`50>< the amount of Sephadex LH-20 with methanol as
`eluant. The peak-fractions are then puri?ed chromato
`graphically using 100x the amount of silica gel 60 (par
`ticle size=0.04-0.063 mm) using water-saturated ethyl
`acetate as eluant. [Nva2]-Cyclosporine elutes ?rst fol
`lowed by Cyclosporine and [Nva2]-[(D)-Ser8]-Cyclos
`
`NOVARTIS EXHIBIT 2006
`Par v Novartis, IPR 2016-00084
`Page 6 of 10
`
`

`
`10
`
`20
`
`4,764,503
`12
`11
`porine. These later fractions are subjected to further
`004-0063 mm) with methanol/water (80:20) as eluant,
`chromatographic puri?cation using l40>< the amount
`to yield pure [Val2]-[(D)Ser8]-Cyclosporine.
`'
`of silica gel 60 (particle size 0.063—0.20 mm) and chloro
`form/methanol (98:2) as eluant, to yield pure [Nva2]
`[(D)Ser8]-Cyclosporine.
`EXAMPLE 7
`The compound of example 5.3 may also be produced
`microbiologically proceeding analogously to example
`6(a) but with the following modi?cations:
`(a) In the nutrient medium--replacement of (DL)
`norvaline with 10 g (L)-va1ine. Following separation of
`the mycelium from the culture medium-extraction as
`follows:
`The crushed mycelium is separated from the solvent
`by suction-?ltration and the combined ?ltrates are con
`centrated (under addition of water) by evaporation
`under vacuum at a temperature of 40° C. until the va
`pour consists mainly of water alone. The obtained mix
`ture is extracted 3 X using 5 liters ethyl acetate at each
`extraction and the combined ethyl acetate solutions are
`concentrated by evaporation under vacuum at a tem
`perature of 40° C.
`The obtained residue is subjected to gel ?ltration on
`Sephadex LH-20 (1.4 kg; Pharmacia) with methanol.
`25
`Those fractions which contain a cyclosporin mixture
`are pooled and then separated by silica gel column
`chromatography (3 kg of silicagel, granulate size
`0.020-0.045 mm, “Grace”) using water-saturated ethyl
`acetate as eluent. In accordance with their polarity,
`[Val2]-Cyclosporine elutes ?rst followed by a mixture
`comprising [Val2]-[(D)Ser8]-Cyclosporine as the major
`component. Further puri?cation of [Val2]-[(D)Ser3]
`Cyclosporine is achieved by silica gel chromatography
`(80 g, “Grace”, 0.020-0.0045 mm) using acetone/hex
`ane (1:1) as eluent. Those fractions containing crude
`[Val2]-[(D)Ser3]-Cyclosporine, are further puri?ed by
`medium-pressure chromatography on a reversed
`phased silica gel column (“Merck” LiChroprep RP 18,
`160 g, granulate size 0.04-0.063 mm) with methanol/
`water (80:20) as eluent, yielding pure [Val2]-[(D)Ser8]
`Cyclosporine as an amorphous white powder.
`(b) The pre-culture required is obtained as in example
`6(b). [Val2]-[(D)Ser8]-Cyclosporine may be produced
`on fermenter scale proceeding analogously to example
`6(c) but with the following modi?cations:
`(c) In the production medium—replacement of (DL)
`norvaline with 10 g (L)-valine. Following combination
`of the mycelium with methanol, homogenisation and
`?ltration (repeated 2X using 90% methanol)—further
`processing as follows:
`The methanolic extracts are combined and, with ad
`dition of . water, concentrated under vacuum. The re
`maining, aqueous concentrate is extracted 3X with
`ethyl acetate, the extracts washed with a little water,
`combined and evaporated under vacuum.
`The mycelial and ?ltrate extracts are ?ltered using
`50X the amount of Sephadex LH-20 with methanol as
`eluant. The peak-fractions are then puri?ed chromato
`graphically using 40X the amount of silica gel 60 (parti
`cle size=0.04-0.063 mm) using water~saturated ethyl
`acetate as eluant. [Val2]-Cyclosporine elutes ?rst fol
`lowed by Cyclosporine and [Val2]-[(D)Ser3]-Cyclospo
`rine. These later fractions are subjected to further chro
`matographic puri?cation using l00>< the amount of 65
`silica gel 60 and acetone/hexane (1:1) as eluant, and
`medium-pressure chromatography on reversed-phased
`silica gel (“Merck” LiChroprep RP 18, granulate size
`
`EXAMPLE 8
`The compound of example 5.6 may also be produced
`microbiologically proceeding analogously to example
`6(a) but with the following modi?cations:
`(a) In the nutrient medium—replacement of (DL)
`norvaline with 5 g (L)-threonine. Following incuba
`tion--extraction as follows:
`The mycelium is separated from the culture medium
`and extracted in a Turrax apparatus by crusing and
`stirring with 3X9 liters of 90% methanol. The crushed
`mycelium is separated from the solvent by suction-?l
`tration and the combined ?ltrates are concentrated
`(under addition of water) by evaporation under vacuum
`at a temperature of 40° C. until the vapour consists
`mainly of water alone. The obtained mixture is ex
`tracted 3 X using 5 liters ethyl acetate at each extraction
`and the combined ethyl acetate solutions are concen
`trated by evaporation under vacuum at a temperature of
`40° C.
`The obtained residue is subjected to gel ?ltration on
`Sephadex LH-20 (2 kg; Pharmacia) with methanol.
`Those fractions containing a cyclosporin mixture are
`pooled and then separated by silica gel column chroma
`tography (2 kg of silica gel, granulate size 0.02-0.045
`mm, “Grace”) using water-saturated ethyl acetate as
`eluent. In accordance with their polarity, Cyclosporine
`elutes ?rst, followed by [(D)Ser8]-Cyclosporine, fol
`lowed by [Thr2]-Cyclosporine and ?nally [Thr2]
`[(D)Ser8]-Cyclosporine in crude form. Further puri?ca
`tion of [Thr2]-[(D)Ser8]-Cyclosporine is achieved by
`silica gel chromatography (50 g, “Grace”, 0.02-0.45
`mm) using acetone/hexane (2:1) as eluent yielding pure
`[Thr2]-[(D)Ser8]-Cyclosporine as an amorphous white
`powder.
`(b) The pre-culture required