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`CYCLOSPORINS - NEW ANALOGUES BY PRECURSOR
`DIRECTED BIOSYNTHESISt
`
`RENE TRABER, HANS HOFMANN and HANS KOBEL
`
`SANDOZ Ltd., Preclinical Research, Biotechnology,
`CH-4002 Basel, Switzerland
`
`(Received for publication September 19, 1988)
`
`Cyclosporin A (ciclosporin), a potent and clinically important immunosuppressive drug
`(Sandimmun), represents the main component of a group of over 25 closely related, cyclic
`undecapeptides produced by the fungus Tolypocladium inflatum. By feeding experiments using
`DL-a-allylglycine as precursor, specific incorporation in position 2 was attained leading to
`[Allylgly'jcyclosporin A. Exogenously supplied L-,B-cyclohexylalanine results in the almost
`exclusive production of [MeCyclohexylala1]cyc1osporin A (replacement of methylbutenyI(cid:173)
`methylthreonine-l). D-Alanine in position 8 can be successfully substituted by D-serine.
`The new [D-SerSj analogues of thecyclosporins A, C, D and G as well as [Allylgly']cyclosporin
`A exhibit high immunosuppressive effects.
`
`Cyclosporins represent a group of biologically active secondary metabolites produced by members
`of the genus Tolypocladium in submerged culture!"). The main component in normal fermentation
`broths, cyclosporin A (INN; ciclosporin)3), has proven to be a powerful immunosuppressant (Sandi(cid:173)
`mmun) arid is used clinically in organ and bone marrow transplantations4) • Besides cyclosporin A, a
`plethora of over 25 closely related congeners (cyclosporins B to Z) have been isolated so far from large
`scale fermentations~-B). All natural cyclosporins are neutral cyclic oligopeptides composed of 11
`amino acids of which several are N-methylated and one or two belong to the D-series. A characteristic
`feature consists in the occurrence of the unique building element (2S,3R,4R,6E)-2-methylamino-3-
`hydroxy-4-methyl-6-octenoic acid (N-methyl-(4R)-4-«E)-2-butenyl)4-methyl-L-threonine, abbreviation
`MeBmt). Variations in the amino acid sequence have been observed in all positions except 3 and 8;
`some cyclosporins differ from their congeners only by N-demethylation of one amino acid residue~-B).
`The large number of minor components as well as the unusual structural units, e.g. MeBmt, L-a-
`aminobutyric acid and D-alanine, indicate a nonribosomal biosynthetic pathway for cyc1os,P()rins9• 10).
`Feeding experiments using carbon-13 labeled acetate and methionine revealed that the constituent
`amino acid MeBmt is built up by head-to-tail coupling of 4 acetate units, whereas the C-methyl in the
`carbon chain and the 7 N-methyl groups in cyclosporin A originate from the S-methyl of methioninew .
`Recently, the N-demethyJated amino acid Bmt was obtained from a cYclosporin-blocked mutant of
`Tolypocladium inflatum12).
`The course of cyclosporin biosynthesis can be strongly influenced by an exogenous supply of
`amino acid precursors to the fermentation medium. Thus, the specific amino acids L-a-aminobutyric
`acid (Abu), L-alanine, L-threonine, L-valine and L-norvaline (Nva), at position 2 in the natural cycIo(cid:173)
`sporins, are preferably well incorporated giving rise to enhanced yields of the corresponding cyclo(cid:173)
`sporins A, B, C, D and G, respectivelyIO).
`
`t Presented in part as oral communication and poster at the 16th IUPAC International Symposium on the
`Chemistry of Natural Products (Kyoto, Japan, May 29",June 3, 1988); Program and Abstract OCC 10
`and PC 79.
`
`
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`D-Ala
`
`Ala
`
`MeJ..eu
`
`Val
`
`MeJ..eu
`
`Cyclosporin A (INN; ciclosporih)
`
`In this paper we report on the successful incorporation of 'foreign' amino acids into various posi(cid:173)
`tions of the cyclosporin molecule leading to the synthesis of new analogues.
`
`Materials and Methods
`
`Strain
`The strain used in the experiments of this report is a high producing mutant ('" 500 mg cyc1osporin
`A per liter) obtained by UV treatment from the original strain of T. infiatum NRRL 8044.
`
`Cultivation
`The spore- and mycelium-suspension used for inoculation was produced from a culture of T.
`inflatum cultivated for 21 days at 27°C on an agar medium containing malt extract 20 g, yeast extract
`4 g and agar 20 g per liter of demineralized water. The spores of this precuIture were taken up in
`physiological saline to give a final concentration of 5 x 106 conidia/ml. Five m! of this suspension
`were used for inoculation of 500 ml of a nutrient solution containing maltose 50 g, the precursor (e.g.
`L-,a-cyciohexylalanine) 5 g, KH.P04 0.75 g, MgS0 4 '7H20 0.5 g, CaCI. '6H20 0.1 g and casein-peptone
`8 g per liter. The inoculated production-medium was transferred in 100 ml portions to 50 500-ml
`Erlenmeyer flasks which then were incubated for 14 days at 27°C on a rotary shaker at 180 rpm.
`For the preparation of [D-Ser] compounds, e.g. [D-Ser]cyclosporin G ([Nva', D-Ser8]cyc!osporin
`A), the fermentation medium was supplemented with DL-norvaline 5 g and D-serine 8 g per liter.
`
`Analytical Methods
`The culture medium was thoroughly mixed with an equal amount of methanol in order to extract
`the cyclosporins from the mycelial mass. After filtration an aliquot was analyzed on a HPLC
`Shandon Hypersil column (5 ,urn, 250 x 4.6 mm) at 75°C, mobile phase: acetonitrile - water - ortho-
`phosphoric acid (630: 370 : 0.1), flow: 1.5 ml/minute, detection: UV-absorptioh at 210 nm.
`
`
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`Table 1. Mobilities on TLC and HPLC.
`
`TLC (silica gel, Rf values)
`
`CycIosporin
`
`Ethyl acetate'
`
`Cyclosporin A
`[MeCyclohexylala1jcyclosporin A
`[AIlylgly"jcyclosporin A
`[D-Ser8jcyclosporin A
`[D-SerBJcyclosporin C
`[D-SerB)cyclosporin D
`[D-SerB)cyclosporin G
`• Distance: 3xlOcm. b Distance: lx10cm.
`
`0.40
`0.53
`0.48
`0.28
`0.14
`0.43
`0.37
`
`Acetone - hexaneb
`(1 : 1)
`0.37
`0.49
`0.40
`0.27
`0.14
`0.34
`0.31
`
`HPLC
`(ex values)
`
`10.00
`19.79
`10.14
`5.45
`3.64
`7.20
`6.64
`
`Chromatographic purification was monitored by the same HPLC system and by TLC (silica gel
`plates, solvent systems: ethyl acetate or acetone - hexane (1: 1), detection: iodine vapor, see also
`ref 8).
`
`Isolation and Purification Procedure
`The fermentation broth (5 liters) was separated into the mycelial cake and the culture filtrate.
`The former was homogenized three times with methanol- water (9: 1) and the combined filtrates were
`concentrated in vacuo to remove the methanol. The aqueous solution was extracted three times with
`ethyl acetate. The organic layer were combined and evaporated to dryness. The crude residue was
`subjected to gel filtration on Sephadex LH-20 with methanol and then to repeated column chromato(cid:173)
`graphy on silica gel (Merck, 0.063", 0.2 mm) using ethyl acetate saturated with water as eluent. Final
`purification, when necessary, was achieved by reversed-phase medium pressure column chromato(cid:173)
`graphy on LiChroprep RP18 (Merck) with methanol- water (4: 1) as eluent.
`
`Characterization of the New CycIosporin Analogues
`The chromatographic and physico-chemical properties are compiled in Tables 1 and 2, respec(cid:173)
`tively. The spectroscopic data (UV, IR, lH and l3C NMR) of all new cyclosporins reported are in
`agreement with the assigned structures.
`
`Biological Assays
`The immunosuppressive activity of the cyclosporins was determined in various in vitro and in vivo
`assays such as inhibition of proliferation of lymphocytes (MLR mixed lymphocyte reaction), inhibi(cid:173)
`tion of direct plaque-forming cells in mice, hemagglutination test, skin hypersensitivity reaction to
`oxazolone in mice') .
`
`Results and Discussion
`
`In a series of precursor studies published some years ago10), we observed a strong influence on
`the biosynthesis of cyclosporins exerted by exogenously supplied constituent amino acids at position
`2 (Abu, Ala, Thr, Val, Nva), resulting in enhanced yields of the desired natural cyclosporin (A, B, C,
`D or G, respectively).
`In the present study we have investigated the feasibility of incorporating foreign amino acids into
`the cyclosporin molecule. Regarding position 1, several naturally occurring variations of MeBmt
`have already been detected; deoxy analogues (cyclosporins F and K), N-methyl-L-2-aminooctanoic
`acid (cyc1osporin Z) and N-methyl-L-Ieucine (cyc1osporin 0)8).
`In feeding experiments using L-f3-
`cyclohexylalanine as a mimetic of MeBmt, this precursor was incorporated in position 1 leading to
`the almost exclusive formation of [MeCyclohexylalal]cyclosporin A. The biosynthesis of the 'normal'
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`Table 2. Physico-chemical properties of the new cyclosporin analogues.
`
`Cyclosporin
`
`MPeC)
`
`[am
`Chloroform Methanol
`
`Molecular
`formula
`
`Elemental analysis
`
`Found
`
`Calcd
`
`[MeCyclohexylala ']-
`cyclosporin A
`[Allylgly2]cyc1osporin A
`
`150~153
`
`147~151
`
`[o-Ser'lJcyclosporin A
`
`158~16O
`
`[D-Ser'l]cyclosporin C
`
`147~152
`
`[o-Ser'l]cyclosporin D
`
`152~158
`
`[o-SerBJcyclosporin G
`
`142~146
`
`-292°
`(c 0.94)
`-228°
`(c 0.75)
`-268°
`(c 0.43)
`-258°
`(c 0.39)
`-257°
`(cO.35)
`-260°
`(c 0.48)
`
`-227°
`(c 0.87)
`-175°
`(c 0.78)
`-187°
`(c 0.53)
`-178°
`(c 0.40)
`-2100
`(c 0.37)
`-19fO
`(c 0.59)
`
`C62Hl11NllOU
`
`C63Hll,NllO'2
`
`C62H111Nl1013
`
`C62Hl11N1l014
`
`C63HllSNll013
`
`C63H1l3Nll0'3
`
`C 62.5, H 9.5,
`N 13.1, 015.0
`C 62.0, H 9.4,
`N 12.4, 015.9
`C 60.6, H 9.3,
`N 12.4, 017.6
`C 60.0, H 9.2,
`N 12.3, 0 18.6
`C 61.2, H 9.4,
`N 12.4,0 16.8
`C 61.3, H 9.4,
`N 12.4,0 17.0
`
`C 62.8, H 9.4,
`N 13.0,014.8
`C 62.3, H 9.2,
`N 12.7,0 15.8
`C61.1,H 9.2,
`N 12.6,0 17.1
`C 6O.3,H 9.1,
`N 12.5, 0 18.1
`C 61.4, H 9.2,
`N 12.5, 0 16.9
`C 61.4, H 9.2,
`N 12.5,0 16.9
`
`FAB-MS
`(m/z)
`
`1,186 (M+H)+
`
`1,214 (M+H)+
`
`1,218 (M+H),'·
`
`1,234 (M+H)+
`
`1,232 (M+H)+
`
`1,232 (M+H)+
`
`i
`~
`
`~
`
`t""
`0
`I'!!j
`~
`g
`0
`tj
`I"'l rn
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`cyclosporins A, Band C was completely inhibited. Surprisingly, DL-threo-.s-cyc!ohexylserine, a
`closer analogue to MeBmt, was not employed by the organism; only the usual pattern of cydosporins
`A, Band C was found.
`In systematic trials to vary the amino acid at position 2, oL-a-allylglycine, as an example of an
`unsaturated amino acid, was provided in the fermentation medium. As expected, [AlIylgly2]cyclosporin
`A was produced in addition to a large amount of cycIosporin G containing L-norvaline in position 2.
`The formation of the latter compound can be explained by enzymatic reduction of the olefinic double
`bond.
`Additional efforts to substitute for o-alanine at place 8 by other amino acids with o-configuration,
`e.g. D-serine proved successful, yielding [D-Ser8]cyclosporin A.
`Its potent immunosuppressive activity
`(see below) prompted investigations to prepare the o-serine-8 analogues of the natural, highly active
`cyclosporins C, D and G. By combined addition of the specific amino acid of position 2 (threonine,
`valine or norvaline, respectively) and D-serine, the desired new metabolites were obtained in good yield
`(20",100 mg/liter). Separation from the natural cyclosporins, formed by competitive incorporation
`of o-alanine in position 8, was easily achieved because of the distinctive more polar character of the
`D-serine-8 congeners.
`Incorporation of foreign amino acids occurs only with a relatively limited number of specific amino
`acids; many substrates used were not incorporated into cyclosporin. For example, whereas an external
`supply of DL-2-aminobutyric acid enabled the organism to produce cyclosporin A in high quantity,
`addition of oL-3-aminobutyric acid suppressed cycIosporin biosynthesis. On the other hand, OL-4-
`aminobutyric acid exerted no effect on the course of the fermentation process.
`Regarding structure-activity relationships, biological studies with the novel analogue [MeCycIo(cid:173)
`hexylala1]cyclosporin A confirmed earlier findings (see ref 1) that modification of the essential structural
`unit MeBmt, in general, results in a more or less dramatic decrease of immunosuppressive efficacy.
`Replacement of this amino acid residue by mimetics such as N-methyl-L-2-aminooctanoic acid
`(cyc1osporin Z), N-methyl-L-.B-cycIohexylalanine or by the common amino acid N-methyl-L-leucine
`
`Table 3.
`
`Immunosuppressive activity of cycJosporins.
`
`CycJosporin
`
`Trivial name
`CyA
`CyF
`CyK
`CyZ
`CyO
`
`CyB
`CyC
`CyD
`CyG
`
`Cyclosporin A
`[(3'-Deoxy)MeBmtlJCyA
`[(3'-Deoxy)MeBmt1, VaI2]CyA
`[N-Me-2-Aminooctanoic acidljCy A
`[MeLeu1, Nva2] CyA
`[MeCyclohexylala1JCyA
`[Ala2]CyA
`+
`++
`[Thr2JCyA
`+
`[Va!2]CyA
`[Nva2]CyA
`+++
`++
`[Allylgly2]CyA
`+++
`[D-Ser8]CyA
`++
`[o-Ser8JCyC
`+++
`[D-SerB]CyD
`+++
`[D-Ser8]CyG
`.. Nat: Natural, Dbs: by directed biosynthesis.
`b + + +: Strong immunosuppressive activity, + +: moderate activity, +: weak activity, -: no significant
`activity.
`
`Activityb
`+++
`
`Source"'
`Nat
`Nat
`Nat
`Nat
`Nat
`Dbs
`Nat
`Nat
`Nat
`Nat
`Dbs
`Dbs
`Dbs
`Dbs
`Dbs
`
`
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`(as in cyclosporin 0), indeed led to only marginally potent or inactive compounds.
`On the contrary, structural variations at position 2 in the side chain of the amino acid are rather
`well tolerated, as proven by the high potency of cyclosporin A (a-aminobutyric acid), cyclosporin G
`(norvaline) and [AIlylgly']cyclosporin A. Shortened or branched alkyl residues as found in cyclosporin
`Interestingly, cyc1osporin C
`B (alanine) or cyclosporin D (valine) cause a slight decrease of activity.
`which contains a polar threonine unit in position 2 is also a strong immunosuppressant.
`Substitution of the D-alanine in position 8 by D-serine as illustrated by the series of [D-SerS](cid:173)
`cyclosporins A, C, D and G had little influence on the biological activity. The new derivatives display
`equipotent or, as in the case of [o-SerB]cyclosporin D, even higher immunosuppressive effects, com(cid:173)
`pared to the native compounds (see Table 3).
`The study presented in this communication has provided further evidence that precursor directed
`biosynthesis is a useful and efficient way to prepare cyclosporin analogues not encountered in nature.
`The successful incorporation of constituent and foreign amino acids demonstrates convincingly the
`low specificity in the biosynthesis of cyclosporins which is characteristic for a non-ribosomal biosyn(cid:173)
`thetic pathway directed by multienzyme thiotemplates. Similar results have been reported for other
`secondary metabolites from prokaryotes and eukaryotes, e.g. in the actinomycin-family 13) , the en(cid:173)
`niatins") and ergot alkaloidsl',l.).
`
`We are indebted to Mr. P. HIIlSTAND (SANDOZ Ltd., Preclinical Research) for immunopharmacological
`testing.
`
`Acknowledgment
`
`References
`
`1) VON WARTBURG, A. & R. TRABER: Cyclosporins, fungal metabolites with immunosuppressive activities.
`In Progress in Medicinal Chemistry. Vol. 25. Eds., G. P. ELLIS & G. B. WEST, pp. 1 ~33, Elsevier Science
`Publishers, Amsterdam, 1988
`2) DREYFUSS, M.; E. HARRI, H. HOFMANN, H. KOBEL, W. PACHE & H. TSCHERTER: Cyclosporin A and C,
`new metabolites from Trichoderma po/ysporum (Link ex Pers.) Rifai. Eur. J. Appl. Microbiol. 3: 125", 133,
`1976
`3) ROEGOER, A.; M. KUHN, H. LICHTI, H. R. LOOSLI, R. HUGUENIN, C. QUIQUEREZ & A. VON WARTBURG:
`Cyclosporin A, a peptide metabolite from Trichoderma po/ysporum (Link ex Pers.) Rifai, with a remark(cid:173)
`able immunosuppressive activity. Helv. Chim. Acta 59: 1075",1092, 1976
`'Ciclosporin', Progress in Allergy. Vol. 38. S. Karger, Basel, 1986
`4) BOREL, J. F. (Ed.):
`5) TRABER, R.; M. KUHN, A. RUEGGER, H. LICHTI, H. R. LooSLI & A. VON WARTBURG: The structure of
`cyclosporin C. Helv. Chim. Acta 60: 1247", 1255, 1977
`6) TRABER, R.; M. KUHN, H. R. LoOSLI, W. PACHE & A. VON WARTBURG: New cyclopeptides from Tri-
`chodermapolysporum (Link ex Pers.) Rifai: Cyclosporins B, D and E. Helv. Chim. Acta 60: 1568", 1578,
`1977
`7) TRABER, R.; H. R. LOOSLl, H. HOFMANN, M. KUHN & A. VON WARTBURG:
`Isolation and structure de(cid:173)
`termination of the new cyclosporins E, F, G, H and I. Helv. Chim. Acta 65: 1655"'1677, 1982
`8) TRABER, R.; H. HOFMANN, H. R. LOOSLI, M. PONELLE & A. VON WARTBURG: Novel cyclosporins ftom
`Tolypocladium infiatum. The cyclosporins K-Z. Helv. Chim. Acta 70: 13 '" 36, 1987
`9) ZOCHER, R.; N. MADRY, H. PEETERS & H. KLEINKAUF: Biosynthesis of cyc1osporin A. Phytochemistry
`23: 549",551, 1984
`10) KOBEL, H. & R. TRABER: Directed biosynthesis of cyclosporins. Eur. J. Appl. Microbiol. Biotechnol.
`14: 237",240, 1982
`11) KOBEL, H.; H. R. LooSLI & R. VOGES: Contribution to knowledge of the biosynthesis of cyc1osporin A.
`Experientia 39: 873-876, 1983
`12) SANGLlER, J. J. & R. TRABER:
`Isolation of N-demethyl-Cs-amino acid [(2S,3R,4R,6E)-2-amino-3-hydroxy-
`
`
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`4-methyl-6-octenoic acid], an essential building unit of cyclosporin A, from a blocked mutant of To/y-
`pocladium inflatum. 15th IUPAC Internat. Symposium on the Chemistry of Natural Products, Poster(cid:173)
`presentation, Abstract No. PC 29, Den Haag, Aug. 17", 22, 1986
`13) KATZ, E.: Controlled biosynthesis ofactinomycins. Cancer Chemother. Rep. 58: 83",91,1974
`14) ZOCHER, R.; U. KELLER & H. KLEINKAUF: Enniatin synthetase, a novel type of multifunctional enzyme
`catalizing depsipeptide synthesis in Fusarium oxysporum. Biochemistry 21: 43 ",48, 1982
`15) KOBEL, H. & J. J. SANGUER: Formation of ergotoxine alkaloids by fermentation and attempts to control
`In Antibiotics and Other Secondary Metabolites. FEMS Symposium No.5. Ed., R.
`their biosynthesis.
`HUTTER et al., pp. 233",242, Academic Press, London, 1978
`16) BEACCO, E.; M. L. BIANCHI, A. MrNGHETTI & C. SPALLA: Directed biosynthesis of analogues of ergot
`peptide alkaloids with C/aviceps purpurea. Experientia 34: 1291,.., 1293, 1978
`
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