CA2224715A1 - Macrolides - Google Patents

Abstract

A novel class of macrolides in which i) positions 2 to 6 inclusive of the macrocyclic ring are provided by a piperidazinyl carboxylic acid residue; and/or ii) positions 7 to 9 inclusive of the macrocyclic ring are provided by an aromatic .alpha.-amino acid residue; and/or iii) positions 10 to 12 inclusive of the macrocyclic ring are provided by an aliphatic .alpha.-amino acid residue, preferably comprising two, or especially all three of the characteristic structural features i), ii) and iii), more especially a compound of formula (IX) is provided having immunosuppressant and antiinflammatory properties and protected and ring-open forms thereof.

Description

MACROLIDES

The present invention relates to a novel class of macrolides having valuable ph7...... ~rA~ and r_lated activity. For con-_nience colllpou"ds of this novel macrolide class are refi,.l~d to herein collectively as "S~nelifrPhrin~".

The first of the Sangli~s were icol~cl from actin--mycete fe ".~ .l t;or broths. These are the Sanglifehrins A through D of forrn~ P-~,~ . r".A~ 0 Me ~ 3~,0 OH ~O ~ ,o, ~ ~12 ~

4 ~~ 6,l1 ~ N ~ s5Me56 Me3 5 58 ~ 3Me57 44Sanglifehrin A 61 62 OH

OH OH Me ~0 Me ~ O OH I ~ R ~ I
O~ ~ Me O ~ N ~ Me Me Sanglifehrin B
OH

wo s7/02285 r~lih~s/02ss2 Me-o~Me OH o HO ~
Me ~ ~O OH 1O Y 1~l ~ N O
Me O ~ N ~ Me Sanglifehrin C
OH

Me-o ~Me OH O
Me Me Me ~

Me ~ ~ o MeN ~ O
~N ~ O ~ N

Me Sangllfehr1n D
OH

As can be seen the macrocyclic ring of S~nglirf k. ;,~c A to D is of entirely novel structure cha~ tc ;~d in that i) positions 1 to 6 co,ll~,ise a 3~1~Ayl,i~,;cl~7inyl carboxylic acid residue, ii) positions 7 to 9 an aromatic a-arnino acid residue, and iii) positions 10 to 12 an ~lirh~tic a-arnino acid residue. The rPn-~in~Pr of the macrocyclic ring is comrricPd by an hydroxy carboxylic acid residue, providing, in the case of ~S~r~gl;f~h-;nc A to D a further 11 carbon atoms in the pl Ullaly Illa~ ;y~;liC ring.

In âccoldance with conventional ylacL;ce in macrolide cl. ..;cl,y the atorns of the Sanglifehrin primany mac .c~yclic ring are llum~,~d as in~lir~- ~ above for Sanglifehrin A, st~ting with the carbon atom of the c~l~nyl group of the macrocylic lactone linkage as position 1.

WO g7/02285~ ~,1i~35/029S2 Sanglifi k~ C A to D are also cha~1 by the prescnce of a novel bicyclic spiro system attached at the 23 poCitinn of the ~ ic ring via a hydocarbyl linker group.

s ~ ;r~ h~ c A to D may be ~ Ld to e.~t~ _ ch- .,,;~1 marir~ inn to obtain yet further macrolides of the Sangl;r~k~ dass. Such manirll~tionc includeclea~..g_ of the --r ~clic ring, in particular at the lactone oxy-group, cleavage of the linker group between the macrolide and spiro ring ~ ms, and manipulation, e.g.
protection~ derivatisation or other çl f ~ ;rAl ~l.odirlcation of ;"~ ih. -t groupings; for exarnple, as h~ ,hl~fte, ~çs~ ;kA Furthcr means of .~,~;r,r ~ion will be apparent to those skilled in the art.

In acco..l~ce with the i~ lion it has been found that San~;fih.;..c, in particular those in which the spiro ring system is present, as in the case of S~n~);r k. ;.~c A to D, have a chl~ .;cl;r and entirely novel profile in terrns of their biological activity. In particular they have been found to exhibit the following c~....hi~ ~ion of activihes:
- cyclophilin binding activity;
- immllno~up~l~,ssi./e activity;
- inhibition of both B-cell and T-cell proliferation - they do not, ho~ _l, have FK binding protein binding activity or c~k ;.. ;i~
inhibiting activity.

The Sanglifehrins can accol lh~gly be seen as providing an çYriti~ and novel class of ;~ -osu~.lJ~ssa~ll and ~ntiinn~.. -~ol~ cc-.~ lc In particular the I;r k.;i-c have an achivity profile that differs from that of pl~io~sly known osu~ ssant and A.~ti;l.fl;..lllll /t~y co.-.l o~ 1s such as ~ lo~..;,-c and macrolides, e.g. of the raparnycin and FK 506 class, indicating that the S~gl;r~h.;~c have a dirr~ ;"t mode of achon than such previous colllpoullds. Thus the Sangl;f~k- ;I~C
provide a novel category of drug ~ s~ r~ both in terms of s~ Ule, and activity which may be ~ntiripA-~d to m~teriAlly extend the bounds of ;-.. ~ ~o~ lessi-_ and/or ~ntiinfl~ ol~ therapy; for example, to avoid or reduce undesirable side effects of W O g7/02285 r~l/hl~5102gS2 pl.,~iOU~ no~ ,l~c~ivc and nn~ .na-.. ~O~ therapies and/or to illl~)lU~_ or extend such therapy to new disease areas or new patient c 'Pg~ 5 s~ngl;r h. ;i~c, e.g. in which the .llacl~lide ring is in ring-opened form, in which the 26 and 27 ~ nc in the hydrocarbyl linkcr l~t~.~n the lll&lvLdlc and spiro ring systems are both hyd~v"y ~ub~ cd, or in which the spiro residue ~tt~~hP~ to the lll&lu~;yclic ring has been cleaved or tmncated, gPnPrally lack some or all of the combination of San~;r~-h~ ;n cha~ I ;r ;~LviL~s. For example, San~,1ir h. ;nc inwhich the spiro residue is cleaved typically posscss cy~lophilin binding activity but do not possess significant ;.. - ~ .o~ activity. As will be apparent to those skilled in the art, ho~ r, such co...~ ...Ac provide valuable co...l~lu--.t~ e-...~Ai~tes or key building blocks for the ple~&alion of further novel S~ngl;r~ k~ c~ and hence further extend the the.~ ic potential of the Sangl;r h- ;-- class.

In that its pl.,sel.ce appears material to the biological activity, e.g. of the ~nglifehrinc A to D, the bicyclic spiro system too may be viewed as providing a Sll~ lUlalC(:IlllpOne-~l with key biological ci~ifit~nce, useful as a sl,uclul~l co...pQn~n~
for further derivatisation or moAifi~ ion both in relation to the prod~cti~ n of further Sanglifehrins or for applicdlion in the de.i~ n or ~ ;f.~AI;on of other drugsub~lances; for eY~mp'e to modify the activity of other ;.. ~.o~uppl~ssi~e drug s~ s of the macrolide class.

As intlic~tp~l~ the Sanglifehrins IG~l~sent a novel class of macrolide colll~unds of entirely novel and wholly cl~ t~, ;cl ;r S~ lule.

Accordingly in a first aspect the in~e.ltio.l provides:
a macrolide in which i) pocitionc 2 to 6 inclusive of the Illacl~n;y~lic ring are provided by a pj~rid~7inyl call,o~ylic acid residue; and/or ii) positions 7 to 9 inclusive of the Illa.,lu~;yclic ring are provided by an aromatic a-amino acid residue; andlor W 0 97/02285 rCTn~6/029S2 iii) positiQnc 10 to 12 inclusive of the ~lo-,y..lic ring are prwided by an aliphatic a-amino acid residue, in free or protected form, or a salt thereof.

Suitably the macrolides of the invention co~n~ two, çsreri~lly all three of the characteristic structural features i), ii) and iii).

The pire~id~7inyl ca lwAylic acid residue is suitably a 1,2 pire~ 7in-3-carboxy-l-yl residue of which the carboxy moiety oc r-'S the l-posi~ion~ and the 1-nitrogen atom the 6-position of the ll~&lu~;yclic ring, e.g. a residue of formula I

O H
--C j~N--3 s wherein the assigned nu,..~ replesent the position of the atoms of the residue in the macrocyclic ring. This residue may be ring ~ub~liluled or u~ Jbslil~lçd Suitably it is unsubstituted.

The a-amino moiety of the aromatic a-amino cid residue suitably oc~up:es the 9-position of the .,lacl~yclic ring. Suitably the aromatic a-amino ~id is a phenyl~l~nine, especially 3-OH-phenyl~l~nine. residue in free or ~1~ ctr,d form.

The a-amino moiety of the ~lirh~ir a-amino cid residue suitably occ.r:es the 12-position of the ...ac.~yclic ring. Suitably the aliphatic a-amino cid residue is a valine residue in free or protected form.

The rem~inder of the .llaclu~;yclic ring suitably CG~ ;ces a hydroxy carboxylic acid residue, the oxy-moiety of which comrlçtçs the l..a~f~;yclic lactone linkage and the carbonyl moiety of which forms an amide linkage with the a-aminogroup at position 12 of of the l~l&l~yclic ring. The said hydroxy carboxylic acid residue suitably has a chain length of from 6 to 20, more suitably 11 carbon atoms. It WO 97/0228s PcTlEr96lo29S2 rnay be ~lb~ ed or ~ ub~ t.,d and/or contain one or more ulls~tulated link~s in particular c~ml~ ive double bonds along its length. More suitably the lc ~.~inA~r of the ll~,~yclic ring co...r.;ces a 1l-oxyç~d~."rl-ll-yl, espe~ lly 11-oxy-6,8-entlecn~lienoyl-ll-yl, residue optionally ~ ed e.g. in the 2, 3, 4 and/or 5 position. More suitably the said hydoxy carboxylic acid residue is a residue of formula II.

I 1 R2 O ~ CH2 ) 2 ,CHCH2--CH=CH--CH=CH--CH--CI--CH CH--CO-f CH3 II

wherem R~ and R2 are both H or ~epr~sent an extra bond;
R3 is H;
R4 is -CO-CH3 or -CH(OH)-CH3 or R3 and R4 together ,epresenL a structure of formula m ,CH3 --,C-- I II

in free or protected form, or salt thereof.

Preferred macrolides in accordance with the invention are accor~ingly those comprising a macrocylic ring of formula IV

A
IV
X Y Z
wherein X, Y and Z are residues i), ii) and iii) as defined above and A is a hydroxy carboxylic acid residue as defined above in free or protected form, or salt thereof; in particular a l.la.,,u~;~rclic ring of formula V

W O 97~2285 rCTn~K~29S2 CO 'A) 3 ~ N,H ~ g lo 11 4 N - CO fH - NH-CO - CH - NH
HO ~ CH2 ~ CH

in free or protected form, or salt thereof Generally in the SqnE;IiLk~ , as in .Sqn~l;r h.;..c A to D, the ma~ .,lic ring is substituted at the carbon atom ~dj~e--~ the oxy moiety of the lactone bridge.Typically this substit~lent co...~ cs a 2-oxy-2'-aza-3'-oxo-spi obicycloh~YP~-3-yl residue, e.g. of the formula VI

Me s bJ~--6 a O2 VI
Me~N - R5 5' ~ O

Et wherein -a-b- is -(Me)C=CH- or -(Me)CH-CH(OH)- and R5 is H or Me (wL~,.c;n Me and Et re~.~,sent methyl and ethyl ~espee~ ,ly) in free or protected form, or salt thereof linked to the macrolide ring via a linker cGIIlpli~ing a linear scque~-ce of from 6 to 11, typically 9, carbon atoms b~h.~,e,ll the spiro residue and the macrolide ring.

The linker group may be ~l~bs~ cd or ~ uL.sl;n~llod and/or contain one or more unsalulated linkages in particular cl~m~ tive double bonds along its length.
Suitably the linker group may be methyl ~Lsl;lutud. e.g. by t~vo methyl groups.
Suitably the linker group may be further ~ub~l;lu~e~ by hydroxy, e.g. by 3 hydroxy WOg~102285 Pcr/Ers6~02ss2 s~bs~ ,e~ andlor rnay be ethylenically unsaturated, e.g. contain two carbon-carbon double bonds. More suitably the linker group c~ ..;ccs a l-methyl-7-methyl-nonanoyl-9-yl, espe~ 1y a 1-methyl-7-methyl-1-none,..u~ l-9-yl or a 1-methyl-7-methyl-1,3-nonadienoyl-9-yl, residue optionally su~ d~ e.g. in the 3, 4, and/or 8 pc cition E~cft,~ably the linker group is a group of formula VII

VII
1 6 ~7 Cl H3 c--CH2 CH(OH)--CH(CH3)--(CH2)2 CH--CH--CH C--d wherem c lep~senls linkage to the spiro residue;
d .~,pl~cse.lt~ linkage to the .~ xyclic ring and R6 and R7 are each OH or togethcr lc~ ,sc~lt an ~ ~lition~l bond, in free or p.ute.,lcd form.

The linker group will generally be attached to the rnacrocyclic ring at the carbon atom immptli~tely ~ cent to the lactone oxy group, i.e. when the the macrocyclic ring co,.lplises an ll-oxy-en~ec~nQyl-ll-yl residue, at the 11 position of thereof.

Accordingly the invention provides colllpounds of forrnula VIII

S--L~l vm wherein Sl~lcs~"ltS a spiro bicyclo residue as previously defined L l~lcSc~lt~i a linker group as previously defin~d and M lclJlcse.l~ a macrolide ring as previously defin~d in free or protected forrn, or salt thereof.

Particular colllpoullds of the invention are those of forrnula lX

WO g7/0228S rCTlErg6/02gS2 3b~27 '~h ~7 a~ o OH o~,o 169 o--R3 45 Me ~ 842N-R5 3 21 66~ 1~
4 ~N~ 2~o (CH2)2--R4 44 Me Ho~H /~ 66 62~ s7 wLe;elll -a-b- is as defined above;
-e-f- is -CH(OH)-CH(OH)- or -CH=CH-;
-g-h- is as defined above for-a-b-, and R3, R4 and R5 are as defined above, in free or p,ot~:led forrn or salt thereof The co~l.pounds of fonn~ I to IX contain ~.. ~t~;c carbon atoms and thus may exist in a number of ep;...r-;~ forrns. All of the pcs~ C~ as well as diastereoiso,..elic ~ lulcs thereof are enro-.-r~csed in the invention. However,comyounds of formulae VIII and lX in which the macrolide ring is in ring-closed form and which are of app,~yliate ~ oc1-r .~ y typically possess ~livilies which are characterisic of S~ng1ifio~rinc~ as hereinbefore referred to. ~illlCl~ which possess sanglifehrin char~rtencti-~ activities are ylcfell~,d. In general, e.g. for p h~ ",~r~ ;r~1 use in acco.dallce with the invention, e~i".cl~ which possess s~n~,]ir~h.;-- cha,~
activities in pure or ~ul~s~ l1y pure form (i.e. free or ~l,sl~ 1y free of epimers which lack san~ li.h~ cha, -- tcl;cI;c activities), e.g. I~Ill~ g at least 90%, e.g. at least 95% of active epimer (i.e. co..~ ;c;~g less than l0%, e.g. Iess than 5% of inactive epimer) will be p~cr._l~cd.

Preferably the 3~hL ~~ 7inyl carboxylic acid residue i) at positions 1 to 6 of the macrocyclic ring has the following COl,r -- ~ Qn W 0 97102285 r~ r./029S2 O H
--C _~N~N6 Preferably the aromatic amino acid ii) at po~i~ionc 7 to 9 of the ~I~;yclic ring has the L configuration, e.g. is of confi~ration O
~ NH
HO ~

~cf~,lably the ~lirh~ti- amino acid iii) at positions 10 to 12 of the ll~clocyclic ring has the L configuration, e.g. is of configuration ~NH~

When the rçrn~in-ler of the lllac~yclic ring cQrnrri~es a residue of formula Il, it plcfclably has the configuration CH3 ~CH2)2 O OH ~O O

or CH3 SCH2)2 When R3 and R4 together lcp~sent W 0 97/02285 P~l/~r,~JOtgS2 ,CH3 --,C--it pl~r~ bly has the configuration CH3~ ,~ OCH3 ~C

Preferably the 2-oxy-2'-aza-3'-oxo-s~ bic~clnh~Y~n-3-yl residue has the configuration Me b a~,~O
Mel~f N--R5 ~~

wherein when -a-b- is -(Me)CH-CH(OH)-, it preferably has the configuration HO~

When the linker is of formula VII, it is ~>lcfc.ably of confi~ration CH3 R, CH3 c ~--~d When R6 and R~ are each OH, the c~nfig-~ration at positions 26 and 27 is preferably either 26(S), 27(S) or 26(R), 27(R). When R6 and R7 tog~!h~r lep~ese.ll an additional bond, the configuration at positions 26 and 27 is pl~,fc.~bly CA 022247l5 l997-l2-l6 wo g7~0228~ r~ r,.~i/02952 Colllpounds of the invention of formula IX plcfe,àbly have the following conformation 47 ~ 49 b~g O--R3 45 Me ~ 2N-R5 ~ 65 1 ~
44 M~O ~o~(cH2)2--R;

wherein when -a-b- is -(Me)CH-CH(OH)-, it preferably has the configuration:
HO~

when -e-f- is -CH(OH)-CH(OH)-, it preferably has the (S),(S) configuration or the (R),(R) configuration;
when -g-h- is -(Me)CH-CH(OH)-, it preferably has the configuration:
~ OH

when -g-h- is -(Me)C=CH-, it plcfelably has the configuration:
\~H
,~

and when R3 and R4 are fused together they are plcfel~lbly of configuration WO 97/0228~ PCT/EP96/02952 CH3~ ~" OCH3 ~C

Preferably SangliÇ~,h"ns A to C have the following confi~l-rations OH OH Me Me Me Mle Me ~
e O ~ N ~ ~ Me Me Sanglifehrin A
OH

OH OH Me ~=o Me ~ ~o OH ~ ~ O ~ ~ o ~Me Me Sanglifehrin B
OH

Me-o ~ Me HO Me Me Me ~ } Me ~ ~
Me ~ OH ~ ~~

Sanglifehrin C OH

W 0 97/02285 PCT~EP96/02952 Me~O,,~Me OH O ~
Me Me Me ~,I~,J

1~--~ o Me ~O
Me~ O OH I Y
Me O~N~Me Sang 1 i f ehrin D
OH

The c~...po~ lc of the invention may be in free or protected forr4 e.g. in protected forrns as desrribe~ in "~O~ Groups in Organic S~ thcsis" by T. W.
Greene and P. G. M. Wuts, 2nd FAitiQ~, 1991, John Wiley & Sons Inc., New York. In particular OH groups may be in p.~,t~;t~ form, e.g. in the form of silyl ethers (for inct~nr,e as desc~;be~l in pages 68-86 of Greene and Wuts ibid.), esters (see e.g. pages 87-103 of Greene and Wuts ibid) and ca~ t~,s (see e.g. pages 104 111 of Greene and Wuts ibid). Such protected forms also include int~m~lly protected forms; for PY~ e in the case of m~rolides of formula IX, wherein -g-h- is -CH(CH3)-CH(OH)-, the protected fonn wherein the 14 to 17 positions of the I~ ,cyclic ring c~ .. ;ce a residue of formula X:

WO g7~2285 rCT~Erg6~29S2 ~ ~CH2)2 - CH-CH-CH - CH -e.g. of configuration O o Also for example, 1,3 diols present in Sanglifehrins may be plute~led as ap~l.)pliate ring structures, e.g. as desc~ibed on pages 118-142 of Greene and Wuts ibid.

Compounds of the invention also exist in salt form. Examples of suitable ph~...~re,JIically S~cept~hle salts for use in accordance with the invention include acid and base addition salts as ap~,lo~liate having regard to the particular substihlen present in the compound.

As previously int1ic~ the ll.acr~yclic ring of the col.l~oullds of the invention can be cleaved, in particular at the lactone oxy group, to provide co --~ow-ds wherein the ~ll~clo.;yclic ring is in ring-open form. Generally cleavage of the lactone oxy group plvc~ds by hydrolysis (solvolysis), e.g. to provide co --pounds of formula XI:

R6~X--Y--~A~H Xl e.g. of forrnula XII

W 0 97~2285 ~ 5~2gS2 CO XII

N~H

HO_~ CH2 l l CH3 CH3 e.g. a colll~uild of formula IX' 4~ 4~ 49 3b~ 26~,~h 36 a~~ OH 27 o OH21 19 169~~--R3 45 Me~3B 42N-R5 R 0~~ 65 14J~
39~ 6 3~,NH o o~ (CH2)2 R4 44 Me HO~/ H Me56 wherein X, Y, Z, A, R3, R4 and R5 are as defined above and R6 is H or C,~
alkyl, e.g. methyl.

Such ring-opened forms provide interm~ te means for m~lifi~-~ion of the basic Sanglifehrin macrocyclic ring system and are also part of the present invention.

Accordingly in a futher aspect the present invention provides:
- a macrolide as hereinbefore defined in ring-opened form. said ring-opened macrocycle being in free or protected forrn, or salt thereof;

- a compound R60 - X - Y - Z - A - OH as defined above, in free or protected form, or salt thereof;

CA 022247l5 l997-l2-l6 WO 97/02285 r~ ,5~29S2 - a CG.~ ~ O - X - Y - Z- A' - CH(OH) - L - S wL~.~;n - A' - CH(OH) -is a h~ Ay C~I~AY1iC acid residue, e.g. a residue of formula II, as defined above, and the other symbols are as defined above, in free or protected form, or a salt thereof;

a compound of formula XII' R60~
CO XII ' ,NH
N CO--ICH--NH--CO--CH--NH--A '--C, H L S
HO_~ CH2 in free or protected form, or salt thereof;

- a compound of formula IX"

b~27 ~h 17 4s Me~42N~Rs R O~~ 6s ~s 6 3~--NH o O ~ '~(CH2)2 R4 44 Me IX" ~HN Me56 in free or protected form or a salt thereof.

The invention also includes colllpoullds in which the 2-oxy-2'-aza-3'-oxo-3'yl-spirobicyclo hexane ring system is in ring-opened form, e.g. a colnrol-n~l of formula XII

WO 97/02285 PCT~EPg6/02gS2 Me b~L--M
a o x Me~><OH

wherein a, b, L and M are as defined above, in free or protected form, or a saltthereof.

The ring-opened compounds of the invention are p~ bly of conformation as the plefc.l~d conformations id~ntifi~d above for ring-closed ccill~ ndS. The ring-opened spiro bicyclo ring system of co.,.pounds of formula ~1 is p ~,fe,d~ly of conformation:
Me b~L--M
a~O
- Me~ OH

wherein when -a-b- is -(Me)CH-CH(OH)-, it pr~fcl~-bly has the configuration:

Ho~

Macrolides in acco..l~cc with the invention having a spiro bicyclo residue .-hed to the macrocyclic ring may also be ~ubj~;lcd to cleavage of the inlc~ ing WO 97102285 PCTlEP96/02gS2 linker group, e.g. in relation to foImula ~, in particular at the linkage ~t~ ,nresidues 26 and 27 to yield sep~tc novel spiro bicyclo col.l~ds and further macrolides. As also previously in~lir~d these colll~oullds too are useful as i~t~ f~ fS, the spiro bicyclic moiety of the S~ngl;r~ h,;.~c in particular having an integral filnrtion~l role in the biological activity of the Sq~gliff h. ;.~c as a class.

Accordingly the present invention provides:

- A 2-oxy-2'-aza-3'-oxo-3'yl-spirobicyclohexane, in free or protected fo~n, or a salt thereof, in particular a colllpo~llld of formula VI' Me b,~,R7 a o VI' Me~N - R5 ~0 Et whelc;n R~. is H, an optionally protected OH group, a l~.;~ e functional group, or a -CH2-CH(OH)-CH(CH3)-CH2-CH2-CHO group or the delta lactol equivalent thereof, in frce or protected form or salt thereof.

Preferably the compound of formula VI' has the following configuration Me bJ~
a~.~O
Mel~ f N--R5 O
Et wherein when -a-b- is -(Me)CH-CH(OH)-, it plcfe.ably has the configuration:

W 0 97/02285 rCT~W~/029S2 HO~

The invention also inrl~l"es a ring-open 2-oxy-2'-aza-3'-oxo-3'yl-syilvbi~ oh~y~n~ in free or protected form, or a salt thereof, in particular a cGlllyoLlnd of formula XII' Me b~R, ay O XII
Me~ OH

wherein a, b and R~ are as previously definç~1 in free or protected form, or a salt thereof. The ring-opened spiro bicyclo ring system of colllpoullds of formula XII' is preferably of conformation:
Me b~R7 a~,O
Me~l~ OH

wherein when -a-b- is -(Me)CH-CH(OH)-, it plcfelably has the configuration:

HO~

WO 97/02285 r~ /029S2 The invention also provides a _acrolide of formula xm XIII H,CO~M

wherein M is a m~~oli~e ring as defined above, in particular a macrolide of formual XIV

~ Me H~h ~q~~ 9~0--R3 XIV ~NH o o N~(cH2)2 R4 Ho~JH~Me , preferably of conformation ~ Me H~h ~q~~ 9~ ~--R3 ~NH o o NH--6)~ (CH2)2--R4 HoJ H~Me wlle~e;n when -g-h- is -(Me)CH-CH(OH)-, it preferably has the configuration:
~ OH

and when -g-h- is -(Me)C=CH-, it preferably has the confi~ation:

~H
J~ .

and when R3 and R4 are fused together they are plef~,...bly of configllration CH3~ ~ OCH3 ~C
in free or protected or ring-opened form, or a salt thereof.

In a further aspect the invention in~ludes the macrolides and c~ll~ou.lds of theinvention, in particular those which are natural products in substq-nti-q-lly purified form, e.g. at least 90%, preferably at least 95%, espe~iqlly at least 98% pure form.

In addition to the folcgo~g the present invention also provides a process for the p~d~lcLion of any compound of the invention as hereinbefore defineA. which process comprises:
i). for the production of any one of Sanglifehrins A, B, C or D, cultivating a Sanglifehrin A, B, C or D producing ~ ..o...~ e strain in a culture .I~fd;~
and isolating the desired Sanglifehrin A, B, C or D from the obtained culture broth;

ii). for the production of Sanglifc,h.iils C and D ~ubjecliilg Sq~lif~hnnc A andB to cyclisation at positions 15 and 16;

iii). for the production of Sanglifehrins A and B subjecting Sanglifehrins C
and D to ring opening of the lactol ring at posiliollslS and 16;

iv). for the production of a macrolide of formula IX or ~', wh~.c;l~ -g-h- is -C(CH3)=CH-, dehydrating a coll,pound of formula IX or ~' whe.c;n -g-h- is -CH(CH3)-CH(OH)- or a protected form thereof;

wos7/022ss rcrlErs6/02ss2 v). for the production of a macrolide of formula IX or IX' ~Lc.~l R4 is -CH(OH)-CH3, L~hu~l~ating a CO~ uu ~d of formula IX or IX' ~L~.,m R4 is -C(O)-CH3;

vi). for the pro~luc~ion of a macrolide of formula IX or IX' ~.l~.~;n the 14 to 16 pocitio~c of the macrolide ring CQ...l-. ;.ce a residue of formula X

O ' CH2 ) 2 o --CH--CH--CH--CH--causing a co~l,poulld of formula IX or IX' to u"dergo internal protection at positions 15 and 17;

vii). for the production of a macrolide of formula IX or IX', causing a compound of formula IX or IX' ~.h~ ;n the 14 to 16 pocitionC of the macrolide ring comprise a residue of formula X

O : CH2 ) 2 --CH--CH--CH--CH--to undergo reversal of internal protection at positions 15 and 17;

viii). for the production of a macrolide of formula IX or IX', in which R5 is methyl, subjecting a macrolide of forrnula IX or IX', in which R5 is H to methylation;

W O 97102285 ~ ,5/029S2 ix). for the production of a macrolide of forrnula IX or IX', in which R4 is in O-protected form, ~ubjecliug a rnacrolide of formula IX or IX', in which R4 is in O-u~ ot~,cl~,d forrn to O-protP~ion;

x). for the l~ludu-:~ion of a macrolide of formula IX or IX', in which R4 is in O-unprotected form, subjecting a macrolide of formula IX or IX', in which R4 is in O-protected forrn to O-deplot~ ;on;

xi) for the production of a macrolide of formula IX or IX', which comprices an O-~lule~,led hy~u~heny~ nine residue at pocitionc 7 to 10 of the macrocyclic ring, ~ub~e~;Ling a rnacrolide of formula ~ or IX', in which comprises an O-unprotected hy~uAyp}i~ nine residue at positions 7 to 10 of the macrocyclic ring to O-protection;

xii) for the production of a macrolide of formula IX or IX', which comprices an O-unprotected hydroxyphenyl~l~nine residue at positions 7 to 10 of the macrocyclic ring, s.~bjecling a macrolide of formula IX or IX' which comprises an O-protected hydroxyphenyl~l~nine residue at positions 7 to 10 of the macrocyclic ring to O-de~ul~;Lion;

xiii) for the production of a macrolide of formula IX or IX', in which -e-f- is -CH(OH)-CH(OH)-, ~u~je~ling a macrolide of formula IX or IX' in which -e-f-is -CH=CH- to oxidative hydrolysis;

xiv) for the production of a compound of formula V' or a compound of formula XII, subjecling a colllpolulld of formula IX or IX' to cleavage of the linker group bet~.een the spiro bicyclo group and the maclo.:yclic ring.

xv) for the production of a cGlll~ulld of formula R60 - X - Y - Z - A - OH or of formula R60 - X - Y - Z - A' - CH(OH) - L - S, ~ubj~Liilg a ~l~l~ycle of formula IV or the macrocyclic ring of a compound of formula vm to ring-opening at the lactone bridge thereof;

W 0 97102285 r~ ,51~029S2 xvi). for the p~duc~ion of a macrolide of formula IX or XII in ring-closed form, ~ubjecting a c~l,l~u,.d of formula R60 - X - Y - Z - A - OH or of formula R60 - X - Y - Z - A' - CH(OH) - L - S to closure of the ll~&~l~yclic rmg.

xvii) for the production of a coll.~,ou..d of formula X~ or XII', subjecting a colll~ou.ld of formula IX or VI' to ring-ope ;ng within the spiro bicyclic ring system, and xix) for the productior~ of a col,.~u..d of formula IX or VI', ~ul~;ecth~g a co.l.pound of formula XII or XII' to ring-closure within the spiro bicyclic rin system ~ locesses of the invention may be pe.rolll-ed, e.g. as described in the examples As will be appreciated the ~,.ocesses defined above rnay be applied in any app,op.iate seque~re or combination to obtain other macrolides in free, I,lot~ .ted rig-open and ring-closed forrn as hereinbefore described The macrolides of the invention, e.g Sanglifehrins A to D, are, or are derived from, natural compounds typically obtained from ...r . ~.~ of the family Streptomycet~reae.

Microorg~nicrnc capable of producing macrolides as he~i"befole defined not previously been identifi~d Accordingly in a yet further aspect the present invention ~lo~ides:
- a macrolide producing i~c~ cetc strain ~he,~ the macrolide is a macrolide in which i) positions 2 to 6 inclusive of the l..ac,c.~;yclic ring are provided by a piperid~7inyl carboxylic acid residue; and/or WO 97/0228s PcT/Ers6/029s2 ii) po~citionc 7 to 9 inclusive of the ~ lic ring are provided by an aromatic a-amino acid residue; andlor iii) positions 10 to 12 inclu~ of the lllaclu~;yclic ring are provided by an qliphqtir o~-amino acid residue, in particular - a Sqnglifohrin A, B, C or D producing n l;..~....~cct~, strain.

Suitably the a~ G~ ete strain is of the family Sl~ toll-y~t~ e, more suitably of the genus SL,cplolll.yces, in particular the strain Sll~i~tol..~es sp. A92-308110 as hereinafter described, or is derived th~,lcfl~,lll, e.g. inrll-fling ~ ;.nlc, variants, fusants, r,col..hi-- ~ntc or m~ifiPd forms thereof.

Suitably the strains of the invention are in the form of biologically pure isolates.

For example Sll~p~l,lyces sp. A92-308110 may be mllt~ or m~ified into dirr~ t forllLc by conventional te~hni-lues, e.g. by UV radiation or by tlCdtlllC~lt with a chPmir~l mllt~ge~ such as N-methyl-N'-nitro nitrosoguanidine. RP~ h~ .l clonesmay be obtained by protoplast fusion. All such mlltq~ntc or l~o...h;,.~-~tc or m~ifiP~
forms, capable of producing s~glif~ s, in~lurling mlltqntc and l~cclllbi~ lt~ capable of producing increased yields of, sqnglif~hrinc are in~h~de~ within the scope of the present invention.

In a particular e.llbor~ -.l of the invention Sa.,~lif.,lhills A, B, C, and D, ong~l others, are isolated from the novel S~ nolll~s sp. A92-308110. Sqmples of S~ lol"yces sp. A92-3081 lO were de~ositcd with the Del-l~lle Sqrnml~lng von Mi~oolg.~ ....~n und 7~l1hlltllren GmbH, Masch~ r Weg lb, D-38124 Bla~llsellv~eig, Germany on the 3 May 1995 under the terms of the Bud~st Treaty and have been qcci~P,d deposidon number DSM 9954. S~mp'es of SI1G~)IO1~ ;eS SP. A92-308110 may also be obtailled from Sandoz Ltd. CH4002 Basel, S~

WO g7/02285 rCT/Erg6/02952 Nodce is hereby given that access to samples of DSM 9985 is lirnited in accc".lallce with the provisions of Rule 28 (4) and (5) EPC.

The i~o~;On of S ~gl;r h.;~c A, B, C, and D from cultures of St~ to.ll~s sp.
A92-308110 is ~ec~ in Example 2.

Sl,eptc~l"yces sp. strain A92-308110 belongs to the genus Sl,c~)lul~ cs acco~ing to the ~c~ in ~C.E,~ Manual (Volume 4, 1989, Williams and WiL~cins, Roltimore) and The Prokaryotcs (1992 Srrin~er Verlag, New York). The cell walls contain LL,tliorninopimp-lir acid. The fatty acids are iso- and anteiso-~,~,cl,ed, straight and ~ t t~d The sugar ~t,~ is non .1;~ ,. The ~e~tdLi./e mycelium does not break down into r.~6,..~ The aerial mycelium forms long chainsof spores.

According to the lef,.~,nce books cited above, the strain de-sign-o-t~l A92-308110 is a new Streptomyces. A92-308110 grows on various organic and illu16~hliC media and in most cases forms aerial mycelium. The primary slll~L.dte ~ ~lhilll grows as hyph~
and is generally beige to greyish-brown. The color of the ~rial mycelium belongs to the grey series, number 4, and this l,ly~liu,,, forms lûng chains of spores which belong to the type spira b.

The ability of St,~,p~lll~s ~. A92-308110 to grow on usual bi~ l media, its carbon utili7~tion, and its phy~iologi~ ch~ s are pl~;se.lted in the following tables.

Table 1 . Growth on various birl~ 91 media Culture .. ~di~ll Culture chal.t~ ;cs yeast extract/ growth: good malt agar sul,~Ll~e Ill,~liulll: brownish aerial mycelium: grayish-brown soluble pi~n~.nt none WO 97/02285 rCTnEP96/02952 oalmeal agar growth: good subs~ate lll~ ll; dark brown aerial mycelium: greyish-brown soluble pi~m-nt- ~u~. IliSh glucose-~ala~le growth: moderate substrate mycelium: bl~Jw,Lish aerial ",~.icliu",; grcyish-brown soluble pi~n~nt- none Lol~; c salts/ growth: moderate starch agar subst~ate ",~eliu",: grey aerial mycelium: greyish-brown soluble pigJ~ I~r~

Sucrose/ growth: very poor nitrate agar s~ l.,t~ mycelium: whitish aerial mycelium: poor, greyish brown soluble pigrn~nt- none Glyceroll growth: moderate as~J~aginc agar substrate mycelium: b~llish aerial mycelium: greish-brown solublepigrn~nt none Nutrient agar growth: moderate substrate mycelium: beigc aer al mycelium: none soluble pigrn~nt brown Table 2: carbon utilization WO 97/02285 PCT/EPg6/02g52 moderate or good: gll~rose~ Luulose, al~k; lose, ~cylose, .. ~ ~o.~
poor .1. .. -o~ sucrose, ,~rril~ose~ cellulose, salicin ne d~ m-inositol Table 3: PhYsiolo~ical characteristics nitrate reduction: positive starch hydrolysis: moderate on ino.2;~,c salts-starch agar, negative on oatmeal agar.
tyrosine deg, ~ inn ne~;~.e milk peptonic~tion positive mrl~nin formation: positive growth te."~.~ .es: 18-37~C.
Ve~y poor growth at 13~C.
No growth at 45~C.

pH-range: rich growth at pH 5 and 7, good gro vth at pH 9 NaCI recict~nre: up to 6%, but reduced growth already at a 2%
CQl-r~ dtion.

Macrolides of the invention including S~lglif I ;i-c A B, C and D may be produced by cultivating SL,ept~""~ces sp. A92-308110 or a mutant, ~~col. h; lant or m~lifird folm thereof on an ap~"~,p,iate culture ...~ Y~mplr 1 desr~ihs. by way of illustration only of the invention a p,oce~lu.~e for the cultivation of Sl,ci~tc ",~ces sp A92-308110.

Thus in further aspects the invention inrlllrirs WO g7/02285 ~ /02gS2 a)a bic'~rqlly pure isolate of stTain SLc~Jtùm~ces ~2. A92-308110 (DSM
9954) or a mutant, ~...h;~ 1 or ",~1;rr~ form thereof which is capable ~f yl~~J~ e a macrolide of the invention, and b) a process for the ylud~ ;on of a macrolide of the invention, which cornrricf~s cultivating strain S~pto"~ ;es sp. A92-308110 (DSM 9954) or a mutant"ecc-..k;..~-l or ,~ul;rlpd form thereof in an app,upfi~t~
culture ...~ .... and optionally ~u~ g the sar.gl;f~ k. ;--Macrolides in accordance with the invention, e.g. colllyoullds of formula ~;for example, Sanglifehrins A, B, C and D, and their rhqrrn~~eutirqlly ~CC~Jt; '~le salts, hereinafter genrricqlly "agents of the invention", exhibit sqn~lifrhrin ch~,-~t~ l;r activities, i.e. the following cornhinqtiQn of activities:
- have cyclophilin binding activity;
- have i.. ~ros~ ylc;~si~/e activity;
- inhibit proliferation of both B-cells and T-cells;
- but do not have FK binding protein binding activity, and - do not inhibit c-q-lcin~ in activity.

These activities and assays to ~ete ...inf these activities are described hereinafter in greater detail. Biological activity of macrolides of the invention, e.g. of forrnula IX, e.g of Sanglireti,ins A to D, may be demonstrated in s~,da~ in vitro and ~n vivo test methods, e.g. as follows.

1. Primary Humoral ~mmllne l~r~ e to Sheep Red Blood Cells (MD, Mishell-Dutton) Mouse spleen cells (OF l, female, 8-10 weeks, 1 x 107) are co-cultured with sheep erythrocytes (SRBC, 3 x 107) for 3 days in 1 ml final volume in 24 well plates.
Lymphocytes are harvested, washed and plated at a density of 1 x 106 cells onto soft-agar with fresh antigen (SRBC). Co~ e .~f nt (guinea pig serum) is added after a 60-90 minute inrub~lion period and ;n~ bA~;O~ is co.~ for a further 60 minu~
after which the test is evaluated by co----ti.-g (lllic,uscope) the pl~ques During the 3 day W 0 97/02285 r~ gr~/02952 ion, the ly"l~ho~;~s are 3~ ;1;7~ to the antigen (SRBC). When incubated with antigen again, B~ Jhoc~tes se~rcte specific antibody which binds to the antigen in the vicinity of t_e sec.etuly l~ hG.;y~. ~tlitinn of complement causcs Iysis of the antibody-coated e ~ tcs yidding a plaque. Each plaque l~,~C~ a single antibody-~,~u~ .g cell.

Tnhihition of plaque forrn ~ion is indicative of pk~ ;r~l utility. C'~ pu....~c of the i"~ tion, e.g. S~n~;fi h. ;nc A to D, are active in this assay at a ~ n~-.l.ation of about..?... toabout....?....nM.

Ref~l.,nces.

R.I. Mishell & R.W. Dutton (1966) T....~ l;on of normal mouse spleen cell hnCions in vitro. Science 153: 1004 1006 R.I. Mishell & R.W. Dutton (1967) T.. ;~,ation of dissociated spleen oell cultures from normal mice. J.Exp.Med. 126:423-442 2. Proliferative Response Of L~u,l)ho~s to Allogenic Stim~ tion Two-way MLR (Murine Mixed Lymphocyte Reaction):

Spleen cells (2 x 105) from Balblc mice (female, 8-10 weeks) are co-inrub-~d for 4 days with 2 x 105 spleen cells from CBA mice (female, 8-10 weeks). The ~llog~ni~ cells induce a proliferative ~.,.c~nce in the r~spon~er spleen cell population which is measured by l~ or inco,~o,dtion into the DNA. Macrolides of the invention, e.g. co..~l~o~ .fls of formula IX and their ph~ y ~- pt~ salts, e.g. Sanglif.,h,i"s A, B, C and D, have ICsos in the range from about 30 up to about 2û0 nM as colll~.,d with an ICso of about 20 nM for cyrlQsrrin A when tested in thisassay.

P~eference:

T. Meo (1979) The MLR in the mouse. In: "Tmmlln~D~ M~thods", L. L~kovi~, and B. Pernis, Eds., ~~ Içmir Press, N.Y. pp. 227- 239 3. LPS- stimulated murine B-cells Spleen cells (2 x 105) from CBA mice are incubated for 48 hours with 50 ~u~/ml LPS plus test co,ll~ul,d. F~ulif~.ation is measured by l-~e~l~ pl~ul2,or ~lcol~olalion into DNA. Macrolides of the invention, e.g. CCilll~ UII~S of formula ~f and their ph~....7.~ 711y ur~fp~ le salts, e.g. S~lglifi~hrinc A, B, C and D, inhibit B-cell proliferation and have Icsos in the range from about40 up to about 100 ~M.

References:

Greaves, M. and J. Janossy, 1972, F.li~it~tinn of sele~ T and B Iymphocyte ~ onse by cell surface binding ligands, Tr~ncpl ~nt Rev., 11: 87 Janossy, G. and M. F. Greaves, 1971, Ly~ hoc~t~ activation, L 12fspol-~e of T and B
lymphocytes to phytomitogçnc, Clin. Exp. Tmml~nol. 9: 483498 4. Cytotoxic and cytostatic activity in vitro usin~ the THPl cell line Cytotoxicity is ~tel.llined using the human monocytic cell line THPl (5 x 104 cellslwell) which are ;-u-,JI~ d in the ~ ence of IFN~ (100 U/ml)and LPS (5 ~ m~) plus test colllpound (up to 10 ~M) for 24 to 72 h at 37~C. Living cells are ,~ ;r.çd using the col~ read-out MTT which ulea~ul~s ...;I.x~ ri~l dehy~u~nase enzymatic activity in living cells (Moscm~r 1983). Macrolides of the i~v~,n~ion, e.g.
~Illpounds of formula lX and their ph7 ~.7-,~ ly ~ e salts, e.g. S~nglifehrinc A, B, C and D, have IC50s of about 1000 5000 nM after 24 h il.uub~iQn in this assay.

Reference:

I~Q.ccmq~l T. J. (1983), Rapid C~ ''h;~A' assay for cellular growth and survival:
aMli~ ion to prolife A-~inn and cytoto~cic assays, J. Imm. M~ Ih~lc, 65, 55-63.

5. TNF release by human p ~ ;ph~al blood l-~- n~ cells Monor~ Ale~r cells are ~,cpa~d from the ~..;l.h..~l blood of healthy vollmt~rs using Ficoll-Hypaque density separation according to the method of Hansell et al.
(1991). Cells (105 cellslwcll in 20û ~1 RP~ 10% FCS by volumc) are incubatcd ~vith serial ~lil--tionc of the test co...~ c for 30 min at 37~C prior to the ~1rlitinn of stim-lhlc. In~.r~,luil y (100 U/ml) and LPS (5~g/ml) arc uscd as stimuli to induce Tumour Necrosis Factor (lNE) a rclease by ~ ".l blood ...f ~ k~r cells. After 3 h ;nA~l,d~ion, the cells are cPntrifugP~d (120û rpm for 10 min) and the s ~ are collP~AtP~l The amount of TNF prcsent in the ccll ~u~ .~Atants is d~e....i~-~l using a c~.. r-.cially available enzyme-linked ;~ n~nosû~ t assay kit. Macrolides of the invention, e.g. colllpou.lds of formula lX and their ph~,..~ ly --r~pl-~lA salts, e.g. Sanglir~hlills A, B, C and D, have IC50 in the range from about 200 nm to about 1000 nm when tested in this assay.

6. Cyclophilin Binding Assay A suitable cyclophilin binding assay is the co...l~ti~ , ELISA test described byQu~PcniA- Is in Eur. J. ~.. lll--ol. 1987 17 1359-1365. In this test, the CQ~ n~l tO be tested is added during the inA.~I.Ation of e~;lophilin (human ,~G-..b;.lant cyclophilin A) with coated BSA-cyclosporin A and the cQn~ . ~ion lcquued to give a 50% inhihi~ion of the control reaction without CQ-ul~ri~ol is then calculated (ICso). An Al~b... ~ assay is the co~ ;l;ve binding test describecd by Schn~ r et aL in Biorh ~ l.y (1994), 33, 8218-8224, which involves ~lition of test COlll~U. d during the ;..~ ion of biotinylated cyclophilin (human l~colllbir,~hlt cyclophilin A) with coated BSA-cyclosporin A. The arnount of biotinylated cyclophilin bound in the PICS~IICe and ~hs~nre of a test co~ i is ~t.~ ;nf~ by il,.~ n with sllcpta~idin~ hr'ed AncAlin~- pho5~.hA~;~ce Macrolides of the invcntion, e.g. co--l~u--ds of forrnula lX, e.g.

W 097r02285 1~ 5/029S2 ;r k.;.. A, B, C and B, have ICs~s in the range from about 10 to about 100 nM, compared with an Icso of about 80 nM for ~ lo~ . A when tested in these assays.

Further in vitro assays which may be used to de-~n .cl.dte the biological activity of Sangl;Lh.;~.c are IL,2 ~ gene assays and ConA-stimuhted spleen cell assays (indicative of effect on T~ell a~:liv~Lio~

olides of the invention, e.g. cc....pol~ ~1c of formula lX, e.g. San~l;f k.;.. A, B, C and B, do not have FK binding protein binding activity and do not inhibit activity when tested in standard tests for these activities.

7. T ~ iced Graft-versus-Host (GvH) E2~ction in the rat [Ford et aL, TRANSPL. PROC. 10 (1979) 258].

Spleen cells (1 x 107) from 6 week old female WistarlFurth (WF) rats are injected P4UCIY on day O into the left hind-paw of female (F344 x WF)FI rats weighing about 100g. Animals are treated for 4 cc...~ , days and the popliteal lymph nodes are removed and weighed on day 7. The dirr~.e"ce in weight ~ h.~n the two lyrnphnodes is taken as the p~dlll~,t,l for ev~ ting the reaction.

Inhibition of GvH reaction in the above test is indicative of ph~ r~ utility.
Macrolides of the invention, e.g. cQ...l ou..ds of forrn~ IX and their pha,...q~..l;r~lly ~ccep1~ble salts, e.g. S~ngJ;f. h . ;..c A, B, C and D, are able to inhibit the GvH reaction by up to about 30% when ~ d at a dose of about 1 mg~g s.c..

8. DTH in~uced by SRBC-T~ cells Fifty microliters of a 1:1 (v/v) rnixture of a TH (sheep red blood cell primed) cell clone (2 x 106) and a 10 % sheep red blood cell (SRBC) ~ ~ n~iol~ are injected s.c. into the right hind footpad of female C57 BIJ6 mice (6-12 weeks old). 50 ~1 of the SRBC
cell s ~cncioll (diluted 1:1 v/v with PBS) is injerte~ s.c. into the left hind footpad (to W O 97102285 1~ /029S2 ~u,~ non specific in~;l~e in footpad swelling due to the ;..j~l;m~ l,l~lu,~,). Right and left hind footpad th ~- ~.rCc is ~cid 24 hours later.

The percent in,_l~ in 1~ rl~ C~ of t_e dght footpad over the left footpad (z) isc~lr~ L..~cs of right footpad = x; ~ c~ of left footpad = y; % specific ir,.,,~se = z z=((x-y)/y). 100 M~rolides of the invention, e.g. colllpounds of formula ~ and their ph~ lir~lly ~rcep~ salts, e.g. S~nglifrhrinc A, B, C and D, reduce swelling of the DTH mouse by up to about 50% at doses of the order of 5 mg~kg s.c..

References:

A.T.J. Bianchi, H. Hooijkaas, R. Brenner, R. Tees, AA. Nordin & M.H. Schreia (1981) Clones of helper T-cells mediate antigen spe~ific, H-2 ...~ DTH. Nature 290:62-63 P. Herrrnann, M.H. Schreier, J.-F. Borel & C. Feurer (1988) Mast cell de~ inn as a major event in the effector phase of delayed-type lly~A ~n~ y in~luc~d by clonedhelper T cells. In~. Archs Allergy appl. ~nunun. 86: 102- 105 9. Rat/Mouse Heart Allotr~nc~l~nt~tiol-The in vivo efficacy of macrolides of the invention is ~ sc,d in rat and mouse heart allotr~ncpl~nt~tiQn using Alzet o~ ;r ...;..;l.u..ls for s.c. a(~ t.~tion. In mouse heart allol- ~cplAnt~tion (BALB/c to C3H), macrolides of the invention, e.g.
compounds of formula IX and their ~.k~ r~.~l ;r~lly ~ ~r~ salts, e.g. San~; r~ k. ;nc A, B, C and D, prolong grah survival at doses of the order of 30 mg/kg/day. In rat heart allotr~ncpl~nt~~ion (DA to Lewis) ~ with SUllO~ doses of cyrlnsp- rin A in co...hin~lion with Macrolides of the invention, e.g. c~...po~ lc of formula IX and their W O 97102285 P~ 6~29S2 pha . ~ul;r~lly r- rt-~lr salts, e.g. San~l;f-k.;.-c A, B, C and D, prolonged graft survival as e~e-mrlifiP~ in the table below.

C~clospo.i" A Sanglife~i.l A Graft survival (mg/kg) (mg/~g) (days) - 12,12,12,13,13,14 29, 30, 45, 48, >51, >46 Control (Placebo)Control (Placebo) Control (Placebo) Agents of the invention are useful as ph~rn ~eutir~lc, e.g. as in...... o~u~l"Gs~i~_ as well as an anti-infl~ ....~A~ agents.

They are, in particular, useful for the pl~ ion of acute andlor chronic organ or tissue allo- and xenot~n~ nt rejecti~n~ e.g. for the t~ rnP.nt of recipients of heart, lung, combined heart-lung, liver, kidney, panc..,àlic, skin or comeal transplants. They are also inAir~tP,d for the p~ htion of graft-versus-host dise~, such as following bone rnarrow transplants.

Agents of the invention are also useful for the t~ t of anl~.;...... ~nf disease and of infl~mm~tory conditions, in particular infl~ conditions with an aetiology inrluAing an autollllll~unr co--lpone-lt such as arthritis (for Py~mpl~ oid arthritis, arthritis chronica progrediente and arthritis defoll..ans) and ,I,. u",; -;r AiCP~CeS Specific auto-immnne Aicç~cçs for which the agents of the invention may be employed include autoimm~.nP h~pm~tological disord~ (innluAing e.g. h~molytic anaemia, aplastic ~n~Pmi~ pure red cell ~ mi~ and idiopathic ~u..~boc~rtopcnia), systemic lupus eryll.. -.. ~lo~c, pol~. hor,d ilis, scl.,.udo-~-a. Wegener gr~n~ csiC~ de .. ~t.. ~o~ilis, chronic active hPp~~itic my~cthPni~ gravis, psc.. ;~cis, Steven-Johnson syndrome, idiopathic sprue, ~ltoi.. r infl~.~.. ,~o~y bowel disease (innlllAing e.g. ulc~
colitis and Crohn's disease) enA~rinp ophth~l...oF.~11.y, Graves disease, sarcoidocic, mnltirle sclerosis, primary biliary cirrhocic~ juvenile Ai~bPtes (.' ~ s mrllihlc t~pe I), uveitis (anterior and posterior), keratoconju..ctivitis sicca and vernal and/or allergic k.,.atoconjuncti.~itis, int~ lung fibrosis, psoriatic arthritis, glomer~ .n~.lJ}i. ;l;c (with WO g7/02285 r~ 02ss2 and without nep~.~Lic S~ u..e, e.g. ;..~lu~ e i~ h;r, nCphlOtiC s~ uule or minim~l change ~el)~û~ ) and asthm~

For these and ûther uses agents of the i~ ioL~ may be al1...;nict~ d on their ûwn or toe~thPr with other ;~ o~ ,l~s~t ûr antiinflam~natory agents, innl~ ne ~,lo~yulills, l~Ull~;ills, FK 506, and steroids.

For the above ;-~ n.C the appropriate dosage will, of course, vary ~CpC'If~;Q~, and the agent of the invention chosen, for example, on the subject to be treated, the mûde of ~."inic~ ion and the nature and severity of the con-lition being treated. However, in general, s~ticf~tory results in animals are obta ned at daily ~osa~,c of from about 0.01 to 10 mg/kg/day p.o.. In larger ,,.~."",~lc, for PY~mrl~P humans, an indicated daily dosage is in the range of from about 0.5 to about 500 mg of san~l;f~ k~ ;n ~timinict~Pred orally once or, more suitably, in divided dosages two to four times/day.

In organ transpl~nt~tion in hllrn~nc, oral doses of 0.1 to 100, preferably 0.3 to 30, more preferably 0.5 to 10, mg/kg of a cc~lllpound of agent of the invention. When an agent of the invention is given along with Other;-~ nQ~ lcsS~lt~ (e.g. with coficosteroids or with co,l,pounds of the cyclQs~ or ~ C~I class as part of a double, triple or quadruple drug therapy) lower doses (e.g. 0.1 mg/~cg/day i.v.; 3 mg/kg/day oral initially) may be used. In particular agents of the invention may be given with other non-steroidal immnl-o~p~,ss&lts, e.g. with cyclosporin A, l~a~ r or FK 506, with a view to thepartial or complete repl~rc ..~ .1 of steroids.

Agents of the invention may be ~ l.,,;ni~ ,d by any conve ~I;on~l route, in particular enterally, e.g. orally, for el~mr'e in the form of sollltionc for .I.;,.~ g tablets or carsnlPs or ~..uc.rte.~lly, for exarnple in the form of injectible solllti~nc or ~.."~ nc Normally for systemic atlminictration oral dosage forms are p ~,f,.-.,d, ~hholl~h for some conrlitionc, for ex~mrle for p.c~l~,"tion of rPjection of liver transplants, an intravenously inje~ble form is desudble. t'omro!lntlC may also be r l.~ d topically or dermally, e.g. in the form of a dermal cream or gel or like preparation or, for the pu~l)oses of application to the eye, in the form of an occular cr~am, gel or eye drop p.~- ~ion.

W 0 97/02285 r~ ,.S/029S2 Sl~itiqhl~ unit dosage forms for oral ~ 1. t;~n CC~ ;~ e.g. from 0.5 to 100 mg of the U~ .fi per dosage.

In accol.l~oe with the fol~go.ng the present invcntion also provides in a further series Of Pr.~ho~];~,.. .~t~

A. A method of çffP~~-ting i~ o~sjion in a subject in need of such llc~-mrnt which method ~...1.. ;~s administering to said subject an erf~ amount of an agent of the in~lention.

B. A m,~-thod 1) for the pl~ tion of acute andlor c ronic organ allo- or ~PnO~
rejectinn for example for t_e treatment of recipients of organ transplants of any of the particular types listed above; or 2) for the ~lc~ ntion of graft-versus-host disease, for exqmrlr in lr ~r -ntc of bone marrow ~ , or 3) for the ~ nt of ~1~;.. - .~ disease or for the L~ .. ~1 of any such disease or cor ~lition listed above; or 4) for the tle~ r-~l of asthma in a subject in need of such llrA~m~nl~ which method co...l..;ces ~ tl ;n~ to said subject an effecti~e amount of an agent of the invention.

C. An agent of the invention for use as a ph~ --I;r~l. e.g. for use as an immllno~V~ ssant or for use in the ll~ ,t~". nt of any disease or cQntlition as set forth under B above.

D. A ph ... ~ l co~ o~ilion co...l.. ;cing an agent of the invention in ACSo.~~ inn with a pharm~~M~ti~~qlly ~ 1A diluent or carrier.

W O 97/02285 1~ 5/02952 E. The use of an agent of the invcntion.for the preparation of a medicament for use as an ;.. - --r~ ;,sant or for use in the ~ of any disease or cQn~liti~n as set forth under B above.

In addition rn~rolides of the il,~ tion which possess ~ ophi1in binding ~tivity, may be useful as lcage.ll~ in ~~icr1 ~r~ nl ;.. ~ .r~csqvys for cyclGs~lms and other cyclorhi1in binding cc~ ls, for example in the assay pl~hllC d<~;h~ in our copçn~ling patent ~ fiQn WO 95/07468. This patent application relates to an assay procedure for d~ t~. - ...;ni~-g the conr~..1.dtion of an ;~ ~hilin-binding ~h~ArciLI;ç-q-l e.g. Ciclosporin, in blood; the pl~dulci c~...l..;~;..g adding a binding CQ,.,I~ilor that ~licrl~es the ~.hr~ -lirql from ;~ ns~ ci~sant~ ophilin cornpleYss in the blood; adding a l~JIor that binds to the ph~ ,"'--~,I;rq1 but not ci~ifirqntly to the binding c4l.~ ;l0l, separating the lcceptol-ph~ )fi~ complexfrom the sample; and detl ....;ni~g the amount of the ph~ ",~ 1 Ss-~gliLh.;--c may be used as the binding co---l~t;ln- in such assays; for i~ r~, to ~ p1~ cyclosporins from cyclophilins, thereby releqcing the cyclosporin for q..~ ;on, e.g. by a monoclonal antibody which is specific for the cyclosporin The invention is further ~çsçribed by way of illustration only in the following FY.~mplç which refers to the ~~c....p~ .jing Figures:
in which Figure 1 shows the Mass SpCCIlulll of the ~lll~oulld of Sqnglif~hrin B;Figure 2 shows the Mass ~ lll of the co~ of SAng1;r~k. ;-. A;
Figure 3 shows the Mass s~L~llll of the colll~u,.d of c~ .gl;r.,hlill D;
Figure 4 shows the Mass Sp~LIulll of the CO~1~U .~ of S~n~;r. k. ;n C;
Figure 5 shows the IR S~:tl~llll of the cc.. . ~ l of San~l;fi k. ;. . B;
Figure 6 shows the ~ S~:tlulll of the ~ n~l of San~l;r~k~ ;.- A;
Figure 7 shows the IR ~LI.llll of the colll~uund of S~agl;f~h. ;n D;
Pigure 8 shows the IR Sp~tlulll of the colll~ound of S~ l~lir. k. ;.. C;
Figure 9 shows the NMR Sp~:llUlll of the colll~,uu.ld of formula S~ir~h. ;n A;
Figure lO shows the N~ ~llulll of the co...~ of SA~glifi k. ;.- D.
Figure l l shows the N~ S~:tlulll of the colllpoLnd of S~n~lif~hrinR~ and WO g7102285 rCT/EP96102gS2 Figure 12 shows the NMR ~:l,~ of the coLu~ulld of ~s-1~,1if~ h. ;i- C.

O 97/02285 P~l/rl,~0 ~ 2 EXA~LES
Culture conditions SIlG~tulll~cGs sp. A 92-308110 may be cultured at suitable temperatures on various culture media using al~luyliate ~ ;e~t~ and mineral substances, using aerobic ori... ~;OI~ culture procedures. The f~ ;on media typically c~ S inc a utilisable source of carbon, sources of ~ ug_n and mineral salts inr~ i~ trace e~ , aLI of which can be added in the form of wcll defined pl~lu~:~ or as complex l~ ul~S, for as are found in biological products of various origins.

Fl~mpl~ 1 desr ;kc the original con~ u~ under which co~ ils of formula I
were obtained. I,~ u~cd yields rnay be obtained by opt;~ nn of the culture conditions (aeration, t~ C.dlu,c, pH, quality and yu~ltily of the carbon and nillu~.l sources, quantity of the mineral salts and of the trace ek".~ ) and by contTolling the fe.".enldtion conditions in bio~

ExamPle lCulture of stMin A 92-308110.

a. Agar starting culture Agar slant cultures of the strain A 92-308110 are grown for 10 to 14 days at 27~C on the following agar medium:
Glucose lO.Og Soluble starch 20.0g Yeast extract S.Og (Gistex, Gist Brocades) NZ-Amine,Type A (Sheffield) S.Og C~ um c~l,onate l.Og Agar (Bacto) lS.Og Dc~lin~" alised water to lOOOml WOg7/0228S PCrtEP96/02gS2 The .. ~.1;.. is ~ to pH 6.6-6.8 with NaOH/H2SO4, then st~ili~A for 20 min. at 121~C.

The cultures can be stored at -25~-70~C. A S~ f nc;~n in glycerol-peptone can be stored under liquid nil,ogell.

b Preculture Spores and mycelium of 10 starting cultures are ~u~lf l~-led in 100 ml of a 0.9% salt solution. Two 2 Liter-r.l~ ,. flasks each CQnl;~;n;~g 1 liter of preculture ,..,.1;.,...
are inoclll~t~d each with 50 ml of this ~sp~l.C;c~n The co,~ ion of the preculture m~Ail-m is as follows:

(~lueose techn 7.50g Glycerin 7.50g Yeast extract (BBL) 1.35g Malt extract liquid (Wander) 7.50g Starch soluble 7.50g NZ-Amine,Type A (Sheffield) 2.50g Soya protein 2.50g L(-) Asparagine l.OOg CaCO3 O.O50g NaCI 0.050g KH2PO4 0.250g K2HPO4 0.500g MgSO4.7H20 O. lOOg Trace el~rn~ont solution A lml Agar (Bacto) lg Dc.~ eldlised water to lOOOml The ".~ ,... is adjusted to pH 6.8-7.2 with NaOH/H2SO4 and st~-ilicçd for 20rn at 121~C.

WO 97hl22~AS PCTlEpy6lo2gs2 The co...l o~ on of the trace cl~ .. n~ snllltifm A is as follows:

FeS04.7H20 5.0g ZnS04.7H20 4.0g MnCI2.4H20 2.0g CuS04.5H20 0.2g CoCl2.6H20 2.0g H3B03 0. lg KI 0.05g H2S04 (95%) lml Dc.llin~,lalised water to 1000 ml The precultures are f~ - n~ ~ d for 24 hr. at 27~C on a rotary shaker at 200 rpm with an eccentricity of 50mm.

c First intermediate culture Two 75 Liter bioreactors cont~ining each 50 liters of preculture ."~ 1;..." are inQc~ ted each with I liter of the preculture and fe ... ~-~ed for 96 hr. at 27~C. The f~.",f~.~t~. is rotated at 150 rpm. Air is introduced at a rate of 0.5 liter per minute per liter ...~.1;.....

d Second h~cl...f~ te culture Two 750 liter fe.~ nt~iQr' vessels each co~ i..g 500 liters of the ~ ".~.1;.....
are each inoc~ .e~ with 50 liter of the first j"t~ ",~ A culture. The second h~te~ A;~tfe cultures are ;..r~lb~l~ for 70 hr at 27~C. The fermenters are rotated at 100 rpm and air into~uced at a rate of 0.8 liter per minute per liter ...f.1;.~ ..

W 0 97/0228S rCTn~6/02952 e. Main culture Two 5'000 Liter biolca~lol~ each u~ g 3'000 liters of the main ...~ .. are inoculated l~ ely with 250 and 300 liters of the second int~ Ai~~~ cultures. Themain cultures are incubated during 96 hr at 24~C. The biorcactors are rotated at 45 Ipm and air introduced at a rate of 0.5 liter per minute per liter ...~ 1;...., The c~ on of the main culture ...~.1;...., is as follows:

GlucQse techn 20g Malt extract liquid (Wander) 2g Yeast extract (Bacto) 2g Soytone (Bacto) 2g KH2PO4 0.2g K2HPO4 0.4g Mgso4~7H2o 0.2g NaCI 0.05g CaCk.6H2O 0.05g Trace ele .. r nt solution B lml Agar (Bacto) lg rl~ ,.;n-,~alisedwatertol000ml.

The pH is adjusted to 6.3 with KOH/HCI. The ~ .1;n~" is st~riliced for 20 min at 121~C.

The co...l os;l;on of the trace ele-..~n~ sollltion B is the following:

FeSO4.7H20 5.0g ZnSO4.7H20 4.0g Mncl2.4H2o 2.0g CuSO4.5H20 0.2g (NH4)6 Mo7024 0.2g W O 97/02285 1~1/r~ J029S2 CoCl2.6H20 l.Og H3BO3 0. lg KJ O.O5g H2SO4 (95%) lml iced water to 1000 ml An op~im;ce~ culture ".~.1;.. for the main culture is as follows:

Soybean meal 20.0g Glycerol 40.0g MES O.lM
nemin~ised water to 1000 ml at pH 6.8 WO 97~2285 PCT~Erg6~2952 ExamDle 2 - Isolation of ~;~n~lifehlins A. B. C and D from StreDtom~vces sP. A92308110 The first i~ol~inn and cl~ -- t~ t;on of the 4 new CBA active metabolites was done from two 3000 I tank r~.ll.c~ltations by activity guided rl ~I;mr~';nn and HPLC and thinlayer chlu~ o~la~Jhic analysis. CBA (cy~lophilin binding assay) as described above was used to test for biol~r~l activity.

The two 3000 liter fe-". ~1 t;nn~ are pr~xessed s~ ately. 1500 liter from each fe.~rnt~iûn is stirred with 2000 l ethyl acetate in 4000 liter stainlPss steel vessel for 20 hûurs. The separation of the organic phase is done with a Wçstf~ Sep~alor typ SA-20. The ethyl ~etate extr~ts are washed twice with 80 liters of water and e~,a~l.~t~,d to dryness under reduced pl~Ul~i to give 1.64 and 2 kg eYtr1çtc~ The two crude extr~ts are dPfattPd by a three step extraction with 40 liter ",.lh a-)l/water 9:1 and 40 liter of hexane. Ev~ Lion to dryness under reduced pl~ U~, gives 1.34 kg extr~t.

The def~ttP-d extr~t is c}~ o~a~hed in two portions (670 g) on a column of 10 kgSeph~lPY H in III..IhAnOl Solution E~h portion is dissolved in 3.3 liters of mPthAnol when added to the column After collection of the first 15 liters eluate as fr~tion 1 the chromatography is co~tinued by collP~ting 2 liter fr~tions.
The most ~tive fr~tions were 2,3 and 4 and are the.elbl~; col..hil-~A to give 146 g.
This sample is further cll~ o~ ~l.h~d on 1 kg Silir~lgel Merck 0.04 0063 mrn with methyl-tertiary-butyl-ether (M'I~E), MTBE/5 % mrth~ns~l and MTBE / 10 %
mPth~nol. Fr~tions of 2 liters are CQllP~ctçA Fractions 5 to 9 are the most ~tive ones and are combined to give a sample of 43.8 g. This sample is further sepalated on a column of 1 kg Silir~gel (Merck) 0.0~0.063 mm with a gl~.die,lt of hexane/a~etone 7:3 to ac~onp~ From this c~lllâtO~hy fraction 6 (7.0 g) is further sepal~ ted on a column of 3 kg Lich,~l~ RP 18 (Merck) 40-63 um with mPth~nol/water 94:6 (fr~tion 4-7 2.16 g), then on a colurnn of 100 g Silicagel H with methyl~nPrhkridp and 3 %
mPth~nol (733 mg), a column of 3 kg Licl~o~l~p RP18 with l... lh~nol/water 9:1 (621 mg) and then on 100 g Liclhoyl~ RP18 with ~reto..;l.;lP/water 1:1 to yield 324 mg of W 0 97~2285 rCTnEr96~29S2 pure Sqn~;r k-;-. A (mp 142-145~ C (~lol~ho~), (a)D25--67.30 (c=1}988, Fractions 5 and 7 from the hex~c/acelùmc column are c~...k;..r~l (7.1 g) and further purified on a column of 3 kg Licluul.l~ RP18 4~63 ~n with methanol/water 9:1 (769 mg), on a column of 100 g ~iliragt !l H with MTBE 3 % ~ ol (309 mg) and finally on 100 g ~ r:lgt'l H with lll~,lh~ r~l-lnri~e and 3 % methanûl to yield 90 mg pure San~l;fuh. ;.. B (mp. 117-121~ C (~ollJhous), (a)D25=-52.80 (c=1-128 in ...- Ih~
Fractions 9 and 10 (2.147 g) of the chromatograrnm with ..,~lh~nol/water 94:6 on 3 kg Licl~o~lep RP18 are further pulified on 100 g Silicagel H with lll~ yl~.n~l.lnri~ 5 %
m~ thqnol (800 mg) and finally on 3 kg Lichlupl~ RP18 with ... I~ .ol/water 9:1 to give 480 mg of S~-glir~h.;.- C (mp. 165-170~ C, (a)D25=-35.60 (c~736 in ~ !h~nol).

Fractions 11 and 12 (835 mg) of the ch~ alogramm with m~.th~nol/water 94:6 on 3 kg Licl.,up.ep RP18 are purified on 100 g Silicagel H with MTBE/5 % m~th~-~ol to give 140 mg of Sanglifehrin D (mp. 137-142~ C, a,llull,huu~).

Sanglifehrin A, B, C and D were then char~rteri~ by UV, IR, Mass and N~
S~;lruSCOpy. The results obtained are given in Table 4 below and in the ~~rc....pP~,jillg figures.

Table 4 S~n~lifi-k.;~ A
molecular formula: C60HglN5OI3 (1090.4) W (MeOH): 275 (1962), 242 (54500), 197 (75755) H+: 275 (1635), 242 (51884), OH-: 292 (1973), 242 (60495) IR-spectra: Figure 6 Mass-spectra: FAB 1096[MH+Ii]+: Figure 2 NMR spectra: Figure 9 Sanglifehrin B
m~'ecnl~r formula: C60H89NsOI2 (1072.4) UV (MeOH): 273 (4395), 242 (50600), 197 (78577) IR-spectra: Figure S
Mass-spec~a: FAB 1098[MH+Li]+: Figure 1 NMR spec~a: Figure 11 Sanglifehrin C
molecular formula: C6~Hg3N5O~3 (1104.4) W (MeOH): 275 (1876)), 242 (51557), 197 (72643) H+: 275 (1391), 242 (50120) OH-: 292 (1832), 242 (57960) IR-spectra: Figure 8 Mass-spectra: FAB 1110[MH+Li]+: Figure 4 NMR spectra: Figure 12 PCT~02gS2 WO g7/02285 SDr~1; f~h~ D
~ D-~fonn~a: C~IHglN5012 (1086.4) W (MeOH~: 273 (3194),242 (47584),197 (73766) H~: 273 (3237),242 (46389) OHr: 285 (2600),242 (52907) IlR-spec~: Figuue 7 Mass-spec~a: FAU31092[MH+Li]~: Figure 3 N~DR spec~a: Figure 10 WO g7/02285 rCT/EP96/029S2 47 ~ 49 --~H

H~
Sangl i f ehrin A 57 ExamPle 3 - Transformation of ,C~nvljfehrjn A into ~CAnvlifehrjn C

To a stirred, cooled (0~C) solution of 20 mg (18.3 llmol) of Sanglir.,h,in A in 0.5 mL
of methanol is added one crystal of par~tohlçnesulfonic acid monoh~yd,dte. The res~lltine yellow solution is stirred for one hour and the reaction is quçn~h~d with saturated aqueous sodium bic~l~onate solution. The res~ ine Illi~lUlG iS extracted twice with ethyl acetate. The organic solution is washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered and co~ ..,t~,d under reduced pressure. The residue is purified by column ch,oll,dtography on silica gel (95:5methyl-tert.-butyl eth~ h~nol) to yield S~nglifehrin C as a white amorphous powder. The latter consisted of a 4:1 mixture of Sanglifehrin C and its C53 epimer, Sanglifehrin C having the (S) configuration as ~lepictçd below (R = Me).

4:1 mixture of ~ tereG, llera HO ~ OH
O OH O~, O ~' ~o ~ R

~o ~NH o o HOJ~

n ~ively, this tran~Ç ~ ion can be carried out by using other protic acids (suchas pynrlinillm paratolueneslllfonate~ h~dlu-,hloric acid or sulfuric acid) or Lewis acids ~0 W O g7/02285 r~ r5~29S2 (such as zinc rhlnri~e, ~ ;--.-- blul~ide or çhlnri~e, ~ iSoplopoAide or borûn trifluoride) in ...~lh~nl Use of other ~lcQholir solvents or cosolvents like ethqnol, isopropanol, butanol, allyl ~lrohol ~,u~a,E~l ~lcQhnl~ benzyl alcohol lead in the same rnanner to analogues where R in the structure above is l~s~~ ,ly ethyl,isoplupyl, butyl, allyl, prop~;~l, benzyl.

In the same manner as described above, ~q~gli~h.;.. B can be transÇ~ d into Sanglifehrin D.

ExamPle 4 - Transformation of ~n~lifehrin C ;nto ~ Jl;rehrin A

A solution of 550 mg (0.50 rnmol) of sangliÇuhlin C in 5 rnL of 4:1 THF-water istreated with 0.5 rnL of 2N ~queol~s sulfuric acid and stirred for 1.5 h. The reaction is quenchPd with s~ul~t,d aqueous sodium bic~l~nate and the resulting llliAlUl~; isextracted twice with ethyl acetate. The organic solution is washed with saturated aqueous sodium bic~l,onate solution and twice with saturated brine, dried over anhydrous sodium sulfate, filtered and co~e~ dted under reduced pl~S~ . The residue is purified by column chç~lllalography on silica gel (90:10 methyl-tert.-butyl ether:lnPth-q-nol) to yield sqngl;f~k. ;n A as a white amo~phous powder.

Other inorganic or organic acids can be used in a ~"P~ -." contqining water and optionally an organic cosolvent. Suitable acids include hydrochloric acid, paratoluenPs--lfonic ~id or other sulfonic ~ids, pyriAinillm par9tohlPnP~slllfonate~
acetic ~id, trifluoluaceLic acid, forrnic ~id. S~itqkle organic cosolvents are acetonitrile, dhn~ lr.J....qmi~e, dimethylsulfoxide"lioYqnP

These reactions are accc pr- ~ by the formation of varying ~mollntC of the compound of formula XV, ~epen~linp among others on the reaction time (for a better procedure leading to the compound of formula XV see Example 5 below).
Analogously, sqnglifehrin D can be ll~lsÇol",ed into sanglifehrin B.

ExamPle 5 - Trhl.~r~ ...ation of .~..Jl;fehr;n A into the comPound of formula XV

To a stirred, cooled (0~C) soh~tion of 50 mg (46 ~mmol) of s~n~l;r~ k ;i- A in 1.9 mL
of ~ to..;l.;le is added 0.1 mL of l~c.g~" fln~ri-le - pyridine. The rçslllting yellow soll~tion is stirrcd for 1 hour and thc rcaction is qu~ n~'h~ with saturated ~4u~uS
sodium bica L--~te . The res~lting ll~ih~lulc is c~l. led twice with ethyl acetate. The organic solution is washed twicc with saturatcd brine, dried over anh~li.uus sodium sulfate, filtered and co~ e .I dtcd under l~,duced p.~ u.e. The rcsidue is purified by column cl~lllatography on silica gel (95:5 methyl-tcrt.-butyl eth~.lll~lhanol) to yield the cc. ~ of formula XV as a whitc &llUl~oUs powdcr.
HO ~ ~ 1 I _ ~ rO
~; O OH O~ O ~ ~7 ~~ CN~ ~ O
HO ~H
xv ~

In an analogous manner s~nglifçhrin B can be ll~sÇ~,...,ed into the compound of formula XVI. These subs~n~çc exist as a single epimer at C53, but the absolute co~fignration has not been unambiguously d.,tu~lined.

J J~ ~0 1~ O OH O O
11 ~ NH r HO~ ) XVI

Formula XV: MS rnlz 1078 [M+Li]' (rel. il~te.~ily 100); ~H NMR (DMSO) (only characteristic signals listed) ~ 0.40 (3H, d, H-S0), 1.20 (3H, s, H-54), 1.69 (3H, s, H-WO g7/02285 1~11r1~6~2952 49),4.20(1H,t,H-15),4.58(1H,dd,H-17),5.19(1H,dd,H-18),5.28(1H,dd,H-23), 5.62(1H,m,H-21),5.67(1H,m,H-27),5.99(1H,d,H-25),6.03(1H,dd,H-19),6.14 (lH, dd, H-20), 6.22 (lH, dd, H-26).

ExamPle 6 - Transformation of the comPound of formula XV into .cnn~lifehrin A

To a stirred solution of 54 mg (50 ~mol) of the co...~ of formula XV in 0.5 mL
of 4: 1 THF-water is added 50 IIL of 2N aqueous sulfuric acid. The rer~1tir~ SOIIltiQn is stirred at ~llbient tc~ dtule for 12 hours and the reaction is qu~ hrd with saturated aqueous sodium bic~l~oliate solution. This I~U~tUlC is cA~l~Led twice with ethyl acetate. The cG...hin~d organic solution is washed with saturated aqueous sodium bic~l,onate solution and brine, dried over anhydluus sodium s~-lf~~P~ filtered and concen~ ed under educed ples~ul~. Colllmn ch~l..~t~, ~h~ of the residue on silica gel gel (90: 10 methyl-tert.-butyl elhc~ ..-- -!I.P~-O1) yields san~lif. L.l~ A as a white amorphous solid.

Analogously the compound of formual XVI can be ~ sÇul,lled into s ~ngli r~ ~. ;.. B.

The procedures described in examples 3 to 6 can be used as selectivc intramolecular protection-deprotection sequences. Thus, by the reaction ~lesnrih~ed in eY~mple 5, the hydroxyl at position 15 can be selectively protçcte~l which allows the selec~-~_manipulation of the rern~ining free hydroxyls. The procedure in ex~ 5 allows forthe selective protection of both the hydroxyls in the 15 and 17 positionc Both procedures can also be used as an intr~mok ~ protectirn of the C53 ketone. The hydroxyls and the ketone can be l. ~nel~t~d by the re~ctionc described in 4 and 6.
Sanglifehrins C and D, as well as the co~ ounds of forTnl~ XV and XVI are therefore hll~l~ c"..P.~i~tes for the ~e~ dtion of further sanglifehrins.

W 0 97/02285 rCT~6/02952 ExamPle 7 ~.,ar..tion of 16-D~h~.lr., 17-D~h~d~..A~-~nvlifehrin A (Formula XVII) HO_~, J

'~ NH ~ ~, OH
~o ~NH o o XVIIHO~

A solution of 54 mg (50 llmol) of the colll;)ou-,d of formula XV and a crystal of paratolue~s~llfonic ~id monohydrate in 1 mL of 4~ reton;~ ç-water is heated to 80~C for 1.5 hours. The re~tion is q-,cn~l-rA by the ~ itinn of saturated aqueous sodium bic~bonate solution The resulti~ l~ lul~ is e~ tud twice with ethyl acetate. The organic layer is washed with saturated ~queo~ sodium bica,l,onate and brine, dried over anhydrous sodium sulfate, filtered and co~ehl-ated. The residue is purified by column clho..~tography on silica gel (90:10 methyl-tert.-butyl ether:mçth~nol), followed by reverse phase chlOn~o~la~)lly (RP18, 50:50 acetonitrile-water to açetonitrile over 45 ...;...ll~s) to yield the pure title co~,i)uulld as a white amorphous solid.

MS mlz 1078 [M+Li]+ (rel. intensity 100); ~H NMR (DMSO) (only ch&~ct~ ;ctic signals listed) ~ 1.58 (3H, s, H-50), 1.71 (3H, s, H~9), 2.08 (3H, s, H-54), 4.03 (2H, d, H-15 and C31-OH), S.57 (2H, m, H-21 and C35-OH), 5.72 (lH, dt, H-27), 5.96 (lH, d, C15-OH), 6.03 (lH, d, H-25), 6.09-6.28 (4H, m, H-18, H-l9, H-20 and H-26), 6.37 ( lH, d, H- 17).

WO 97/02285 rCTn~6/02g52 ExamPle 8 - PreDaration of 42~N-methvl ~ _li~f~hr~.~ A (Fonnula 2~VIII) HO_ I J~ J~, ~OH
~ O OH O O ~ OH
~ r N-Me ~ I
~0 CNNH~o o~

XVIII HO~H

To a stirred, cooled (-15~C) soludon of 109 mg (0.1 mmol) of san~l;rlk.;~ A and 67 ~L (0.3 mmol) of 2,6-di-tert.-butylpyridine in 1 mL of methylene chloride is added 16.5 ~L of methyl triflate. The llu~lulc is allowed to wann to room tc,lll~.atul~ and stirring is co..li...~ed for six hours, after which the reacdon is ~ en~ d by ~rliti~n of saturated ayue~,us sodium bica.lonate soludon. The res~ ing ll~iAIUl~ is e~ll~t~,d twice with ethyl acetate. The organic layer is washed with brine, dried over anhydrous sodium sulfate, filtered and concer,t,aled. The residue is purified by two successh,~
c}~omatographies on silica gel (90:10 methyl-tert.-butyl ethel ... ~ ol then 9S:5 methyl-tert.-butyl eth~,, .... Ih~nol) to yield the pure title cG,ll~ou,ld as a white amorphous solid.

MS m/z 1110 [M+Li]' (rel. inle.,si~ 100); IH NMR (DMSO) (only ch~--t~
signals listed) o 1.70 (3H, s, H49), 2.06 (3H, s, H-54), 3.53 (3H, s, 42 N-Me), 3.98 (lH, d, C31-OH), 4.50 (lH, d, H-65), 4.77 (lH, d, C17-OH), 5.43 (lH, d, C15-OH),5.49(1H,d,C35-OH),7.50(1H,d,H-12),8.11 (lH,d,H-9),9.22(1H,s,C61-OH).

WO g7/02285 rCT/EP96/02gS2 ExamPle 9 - PreParation of S3 Dihvdro ~ l;f~h~ in A (Formuh XIX) HO ~ ~ ~ OH
~, O OH o~,O ~ OH
~~ CN ~) - ~H

XIx HO~,~J

To a stirred, cooled (0~C) sollltion of 54 mg (50 ~ol) of sanglir~h.in A in 0.5 mL of m~.th~nol iS added 2.8 mg (75 ~lmol) of sodium bo.u},~ide. Stirring is continl~ed for one hour and saluldt~,d ~1ueouc sodium bic&l,on,it~, is added. The ~ tUlC is e~tracted twice with ethyl acetate. The organic solution is washed with brine, dried over anhy~uu~ sodium sulfate, filtered and conçe~ ated. The . The residue is purified by cl,~ l,atography on silica gel (95:5 methyl-tert.-butyl c~ h~nol followed by90:10 methyl-tert.-butyl ell,~ ol) to yield the pure title co",~und as a white amorphous solid. The icol-~ product co,,csl,onds to a ca. 1:1 mixture of diactereoisomers at C-53) MS m/z 1098 [M+Li]+ (rel. intensity 63), 1104 [M+2Li-H]+ (rel. intensity 100); IH
NMR (DMSO) (only cl~ re.;,~lic signals listed) ~ 0.62 (3H, d, H-50), 1.02 (3H, d, H-54), 3.55 and 3.59 (lH, 2m, H-53).

WO g7r02285 r~ 2gS2 Example 10 - PreParation of ~3- Tosvll.~d~ ~or.e, ..-lifehrin A (formula ~X) HO ~, ~ ~ ~ ~OH
O OH O O ~ OH
NH
~N~) XX HO~0~ o~o A mixture of 55 mg (50 ~umol) of ~n~lifehrin A and 23 mg (125 lumol) oftosylhydrazide in 0.5 mL of methylene chlon~e is stirred at room temperature for six hours. The solvent is ,~;llloved and the residue is p-- ifi~d by chromatography on silica gel (90: 10 methyl-tert.-butyl ethe..,..- ~h~nol) to yield the title co.-l~oll"d as a white amolphous powder.

MS m/z 1264 [M+Li]~ (rel. intensity 100); ~H NMR (DMSO) (only ch&act~islic signals listed) o 1.70 (3H, s, H-49), 1.77 (3H, s, H-54), 2.37 (3H, s, -NSO2C6H4CH3), 6.51 (lH, s, H-60), 6.59 (2H, 2d, H-62 and H-64), 7.06 (lH, dd, H-63), 7.35 (2H, d, tosyl meta protons), 7.73 (2H, d, tosyl para protons).

ExamPle 11 - ~r~"arat;on of 26S.27S-Dihvdroxv-c~n~lifehrin A (Formula XXI) and 26R~27R-D:h~JruA~ lirehrin A (Formula xxm HOJ~ ~ ~ OH
~,0 OH OH ~~,,O ~I~,OH
NH
~O ~ ,NH~

XXI HO~ H

W O 97/02285 P~ 9~1/02952 OH I
HO ~ ~ OH
~, O OH OH O~,O ~ OH
,.... ' NH

XXII HO~HN
W

To a stirred, cooled (0~C) solution of 49S mg (1.5 mrnol) of pOtAC~;I.... ferricyanide, 207 mg (1.5 mmol) of potassium c~l,onale, 19.5 mg (0.02S mmol) of (DHQ)2PHAL, 65 ~L (0.005 mmol) of 0.08 M O~ .. tetroxide in t-butanol and 95 mg (1 mmol) of methyl sulror~ le in 2.5 mL of t-butanol and S mL of water is added a solutiQn of 545 mg (0.5 mmol) of sanglifellrin A in 2.5 mL of t-butanol. The resllltin~ b;l)h~'c u,e is allowed to warm to room te.l,~.dlu.c and stirred for three hours. Then 1.08 g (8.6 mmol) of sodium sulfite is added, followed by ethyl acetate and water, and the mixture is vigorously stirred for 15 ~ tl S, The layers are se~aldtt,d and the aqueous layer is e~l,~led twice with ethyl acetate. The coll.bined organic layer is washed with sdlul.~t~d ~ queous sodium bic~l~nate and brine, dried over anl~ sodium sulfate,filtered and con~e-~ t~d. The residue is p~l~ifiecl by reverse phase chromatography (RP18, 30:70 acetor.it ile-water to ~~eto~i~rile over 60 ...;~.u~.,s) yielding the 26S,27S-diol as an ~-,oll,hous powder.

The co"~s~,onding 26R,27R-diol is ob~h,ed by the above p,ocedu,~, but using (DHQD)2PHAL instead of (DHQ)2PHAL.

26S,27S-diol: MS m/z 1130 rM+Li]+ (rel. h~tel~sily 100); lH N~ (DMSO) (only charact~ tir signals listed) o 1.64 (3H, s, H49), 2.06 (3H, s, H-54), 3.20 (lH, broad m, H-27), 3.45 (lH, broad m, H-31), 3.94 (3H, m, H-17, H-26 and C31-OH), 4.30 (lH, d, C27-OH), 4.57 (lH, d, C26-OH), 5.20 (lH, t, H-23), 5.33 (lH, d, H-25), 5.57 (3H, m, H-18, H-21 and C35-OH), 6.03 (lH, dd, H-l9), 6.14 (lH, dd, H-20).

WO g7~2285 1~ 6,~29S2 26R,27R-diol: MS m/z 1130 rM+Li]+ (rel. illte~ily 100); IH N~ (DMSO) (only charact~"islic signals listed) ~ 1.64 (3H, s, H-49), 2.06 (3H, s, H-54), 3.16 (lH, broad m, H-27), 3.48 (lH, broad m, H-31), 3.94 (3H, m, H-17, H-26 and C31-OH), 4.30 (lH, d, C27-OH), 4.57 (lH, d, C26-OH), 5.20 (lH, dd, H-23), 5.35 (lH, d, H-25), 5.57 (3H, m, H-18, H-21 and C35-OH), 6.03 (lH, dd, H-l9), 6.14 (lH, dd, H-20).

ExamPle 12 - Cleav~oe of the diol in 26S.27S-Dih~,.lru.~y-c~n~lifehrin A

To a solution of 90 mg (79 ~mol) of the 26S,27S diol in 0.9 mL of 2: 1 THF-water is added 33.7 mg (157 llmol) of sodium periodate. Stirring is col.l;n~ed for one hour and saturated aqueous sodium bicall,onate is added. The ~uAtule is extracted twice with ethyl acetate. The organic solution is washed with brine, dried over dnliy~u~ sodium sulfate, filtered and col-~e ~I.dted. Purification of the residue on silica gel (95:5 methyl-tert.-butylethc. ~.~rlhsnol) yields the co~l~ul.ds of formula X~ (foam) and XXIV (powder).

~W~
J~o o J
~ f NH ~ XXIII
~o OH

1~~ ~ _~OH
0~,0 ~,OH
XXIV ~ ,NH o o 1;

HO~C~H

formula X~lI: MS m/z 366 [M+H-H2O] ' (rel. iute~ Sily 100); IH N~ (DMSO) (2: 1 mixture of OH~ OH~ C-~ at the anomeric center) (only ch&~h- ;cl;c signals W O 97/0228S 1~ 1,5~29S2 listcd) ~ 3.54 and 4.08 (lH, 2m, H-31), 3.57 (lH, broad r4 H-35), 3.66 (lH, m, H-33), 4.38 (0.67H, ddd, H-27,,), 4.95 (0.33H, broad m, H-27eq), 5.40 (0.33H, d, C27-OHeq), 5.59 (0.33H, d, C35-OH), 5.61 (0.67H, d, C35-OH), 5.96 (0.67H, d, C27-OH~,~), 7.89 (0.67H, s, NH-42), 7.91 (0.33H, s, NH-42).

formula X~V: MS mlz 745 [M+Li]~; IH NMR (DMSO) (only ch~ signals listed) o 0.64 (3H, d, H-50), 0.81 (6H, d, H-56 and H-57), 2.06 (3H, s, H-54), 2.17 (4H, s, H-14 and H49), 3.80 (lH, broad m, H-15), 3.94 (lH, dd, H-17), 5.33 (lH, broad d, H-23), 5.62 (2H, m, H-18 and H-21), 6.89 (lH, d, H-25), 6.10 (lH, dd, H-19), 6.18 (lH, dd, H-20), 10.0 (lH, d, H-26).

ExamPle 13 - AcetYlation of S~n~lifehrin A to ~ve 61-O-Acet~l-.canvlifehrin A
(Formula XXV) HO ~ ~H

~C ~ ~H ~~~ ~' ~H
-~ NH

~ ~ ~N~ ) O
XXV ~~O~ H

To a stirred, cooled (0~C) solution of 54 mg (50 ~Lmol) of sA l~lif~hrin A and 50 IIL of pyridine in 0.5 mL of methylene chloride is added 5.2 ~IL (55 ~nol) of acetic anhydride. The reaction is kept at 0~C for one hour, then allowed to warm to room te~ all~re and stirring is cor~tinl-ed for twelve hours. Satulated aqueous sodium bic~l,onate is added and the res~lltine rnixture is extracted with ethyl acetate. The organic layer is dried over anhydrous sodium sulfate, filtered and con~çn~atred. The residue is purified by reverse phase chlulllàlO~Ia~hy (RP18, 40:60 ~r~lo~ -waterto acetonitrile over 45 ...~ Gs) yielding the title colllpoLI~d as an &llol~holls powder.

MS m/z 1132 [M+H]+ (rel. intc~ y 100); lH NMR (DMSO) (only chal~
signals listed) o 1.68 (3H, s, H-49), 2.06 (3H, s, H-54), 2.25 (3H, s, CH3CO2), 4.04 CA 0222471~ 1997-12-16 W O 97/0228S 1~ 5/02952 (lH, d, C31-OH), 4.67 (lH, d, C2-NH), 4.76 (lH, d, C17-OH), 5.42 (2H, m, H-8 andC15-OH), 5.57 (3H, m, H-18, H-21 and C35-OH), 6.85 (1H, s, H-60), 6.98 (1H, d, H-62),7.06(1H,d,H-64),7.31 (lH,dd,H-63),7.51 (lH,d,H-12),7.89(1H,s,H-42), 8.23 (lH, d, H-9).

Claims

1. A macrolide in which i) positions 2 to 6 inclusive of the macrocyclic ring are provided by a piperidazinyl carboxylic acid residue; and/or ii) positions 7 to 9 inclusive of the macrocyclic ring are provided by an aromatic .alpha.-amino acid residue; and/or iii) positions 10 to 12 inclusive of the macrocyclic ring are provided by an aliphatic .alpha.-amino acid residue, in free or protected form, or a salt thereof.

2. A macrolide according to claim 1 comprising two, or especially all three of the characteristic structural features i), ii) and iii).

3. A macrolide according to claim 1 or 2, in which the remainder of the macrocyclic ring comprises a hydroxy carboxylic acid residue which has a chain length of from 6 to 20, preferably 11 carbon atoms.

4. A macrolide according to claim 3, in which the hydoxy carboxylic acid residue is a residue of formula II.

wherein R1 and R2 are both H or represent an extra bond;
R3 is H;
R4 is -CO-CH3 or -CH(OH)-CH3 or R3 and R4 together represent a structure of formula III

in free or protected form, or salt thereof.

5. A macrolide according to any one of claims 1-5, comprising a macrocyclic ring of formula IV

wherein X, Y and Z are residues i), ii) and iii) as defined in claim 1 and A is a hydroxy carboxylic acid residue as defined in claim 3 or 4, in free or protected form or salt thereof.

6. A macrolide according to claim 5 comprising a macrocyclic of formula V

wherein A is as defined in claim 5, in free or protected form, or salt thereof.

7. A macrolide according to any one of the previous claims, is substituted at the carbon atom adjacent the oxy moiety of the lactone bridge by a 2-oxy-2'-aza-3'-oxo-spirobicyclohexan-3-yl residue.

8. A macrolide according to claim 7 in which the spirobicyclohexanyl residue is of the formula VI

wherein -a-b- is -(Me)C=CH- or -(Me)CH-CH(OH)- and R5 is H or Me in free or protected form, or salt thereof linked to the macrolide ring via a linker comprising a linear sequence of from 6 to 11, typically 9, carbon atoms between the spiro residue and the macrolide ring.

9. A macrolide according to claim 8, in which the linker between the spiro residue and the macrolide ring is a group of formula VII

wherein c represents linkage to the spiro residue;
d represents linkage to the macrocyclic ring and R6 and R7 are each OH or together represent an additional bond, in free or protected form.

10. A compound of formula VIII

S~L~M VIII
wherein S represents a 2-oxy-2'-aza-3'-oxo-spirobicyclohexan-3-yl residues;

L represents a linker comprising a linear sequence of from 6 to 11, typically 9,carbon atoms, and M represents a macrolide ring as defined in any one of claims 1 to 6, in free or protected form, or salt thereof.

11. A compound according to claim 10 of formula IX

wherein -a-b- is as defined above;
-e-f- is -CH(OH)-CH(OH)-or-CH=CH-;
-g-h- is as defined above for-a-b-, and R3, R4 and R5 are as defined above, in free or protected form or salt thereof.

12. A compound according to claim 11 the following conformation wherein when -a-b- is -(Me)CH-CH(OH)-, it preferably has the configuration:

when -e-f- is -CH(OH)-CH(OH)-, it preferably has the (S),(S) configuration or the (R),(R) configuration;
when -g-h- is -(Me)CH-CH(OH)-, it preferably has the configuration:

when -g-h- is -(Me)C=CH-, it preferably has the configuration:

and when R3 and R4 are fused together they are preferably is of configuration 13. A Sanglifehrin selected from the group consisting of Sanglifehrin A,B,C and D.
14. A macrolide according to any one of claims 4 to 9 or a compound according toany one of claims 10 to 12 wherein the 14 to 17 positions of the macrocyclic ring comprise a residue of formula X:

e.g. of configuration 15. A compound of formula XI:

e.g. of formula XII

e.g. a compound of formula IX' wherein X, Y, Z, A, R3, R4 and R5 are as defined above and R6 is H or C1-4 alkyl, e.g. methyl in free or protected form, or a salt thereof.

16. A macrolide as defined in cany one of claims 1 to 9 in ring-opened form, said ring-opened macrocycle being in free or protected form, or salt thereof;

17 A compound R6O-X-Y-Z-A-Ohwherein X,Y,Z and A are as defined in claim 5 and R6 is H or C1-4 alkyl, in free or protected form, or salt thereof.

18 A compound R6O-X-Y-Z-A'-CH(OH)-L-S wherein -A'-CH(OH)-is a hydroxy carboxylic acid residue as defined in claim 3 and the other symbols are as defined in claim 17, in free or protected form, or a salt thereof.

19 A compound of formula XII' in free or protected form, or salt thereof;

20 A compound of formula IX"

in free or protected form or a salt thereof.

21. A compound in which the 2-oxy-2'-aza-3'-oxo-3'y1-spirobicyclo hexane ring system is in ring-opened form, e.g. a compound of formula XII

wherein a,b,L and M are as defined above, in free or protected form, or a salt thereof.

22. A 2-oxy-2'-aza-3'-oxo-3'y1-spirobicyclo hexan, in free or protected form, or a salt thereof, in particular a compound of formula VI' wherein R7 is H, an optionally protected OH group, a reactive functional group, or a -CH2-CH(OH)-CH(CH3)-CH2-CH2-CHO group or the delta lactol equivalent thereof, in free or protected form or salt thereof.

23. A ring-open 2-oxy-2'-aza-3'-oxo-3'y1-spirobicyclo hexan, in free or protected form, or a salt thereof, in particular a compound of formula XII' wherein a,b and R7 are as previously defined in free or protected form, or a salt thereof.

24. A macrolide of formula XIII

wherein M is a macolide ring as defined above, in particular a macrolide of formual XIV

in free or protected form, or salt thereof.

A process for the production of any compound of the invention as hereinbefore defined, which process comprises:
i) for the production of any one of Sanglifehrins A,B,C or D, cultivating a Sanglifehrin A,B,C or D producing actinomycete strain in a culture medium and isolating the desired Sanglifehrin A,B,C or D from the obtained culture broth;

ii) for the production of Sanglifehrins C and D subjecting Sanglifehrins A and B to cyclisation at positions 15 and 16;

iii) for the production of Sanglifehrins A and B and subjecting Sanglifehrins C and D to ring opening of the lactol ring at positions 15 and 16:

iv) for the production of a macrolide of formula IX or IX', wherein -g-h- is -C(CH3)=CH-, dehydrating a compound of formula IX or IX' wherein -g-h- is -CH(CH3)-CH(OH)- or a protected form thereof;

v) for the production of a macrolide of formula IX or IX' wherein R4 is -CH(OH)-CH3, hydrogenating a compound of formula IX or IX' wherein R4 is -C(O)-CH3;

vi) for the production of a macrolide of formula IX or IX' wherein the 14 to 16 positions of the macrolide ring comprise a residue of formula X

causing a compound of formula IX or IX' to undergo internal protection;

vii) for the production of a macrolide of formula IX or IX', causing a compound of formula IX or IX' wherein the 14 to 16 positions of the macrolide ring comprise a residue of formula X

to undergo reversal of internal protection.

viii) for the production of a macrolide of formula IX or IX', in which R5 is methyl, subjecting a macrolide of formula IX or IX', in which R5 is H to methylation;

ix) for the production of a macrolide of formula IX or IX', in which R4 is in O-protected form, subjecting a macrolide of formula IX or IX', in which R5 is in O-unprotected form to O-protection;

x) for the production of a macrolide of formula IX or IX', in which R4 is in O-unprotected form, subjecting a macrolide of formula IX or IX', in which R5 is in O-protected form to O-deprotection;

xi) for the production of a macrolide of formula IX or IX', which comprises an O-protected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring, subjecting a macrolide of formula IX or IX', in which comprises an O-unprotected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring to O-protection;

xii) for the production of a macrolide of formula IX or IX', which comprises an O-unprotected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring, subjecting a macrolide of formula IX or IX' which comprises an O-protected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring to O-deprotection;

xiii) for the production of a macrolide of formula IX or IX', in which -e-f- is -CH(OH)-CH(OH)-, subjecting a macrolide of formula IX or IX' in which -e-f- is -CH=CH- to oxidative hydrolysis;

xiv) for the production of a compound of formula V' or a compound of formula XII, subjecting a compound of formula IX or IX' to cleavage of the linker group between the spiro bicyclo group and the macrocyclic ring.

xv) for the production of a compound of formula R6O-X-Y-Z-A-OH or of formula R6O-X-Y-Z-A'-CH(OH)-L-S, subjecting a macrocycle of formula IV or the macrocyclic ring of a compound of formula VIII to ring-opening;

xvi) for the production of a macrolide of formula IX or XII in ring-closed form, subjecting a compound of formula R6O-X-Y-Z-A-OH or of formula R6O-X-Y-Z-A'-CH(OH)-L-S to closure of the macrocyclic ring.

xvii) for the production of a compound of formula ????, subjecting a compound of formula ???? to ring-opening within the spirobicyclic ring system, and xix) for the production of a compound of formula ????, subjecting a compound of formula ???? to ring-closure within the spirobicyclic ring system.

26. a macrolide producing actinomycete strain wherein the macrolide is a macrolide in which i) positions 2 to 6 inclusive of the macrocyclic ring are provided by a piperidazinyl carboxylic acid residue; and/or ii) positions 7 to 9 inclusive of the macrocyclic ring are provided by an aromatic .alpha.-amino acid residue; and/or iii) positions 10 to 12 inclusive of the macrocyclic ring are provided by an aliphatic .alpha.-amino acid residue.

27. A biologically pure isolate of strain Streptomyces sp. A92-308110 (DSM 9954) or a mutant, recombinant or modified form thereof which is capable of producing a macrolide of the invention.

28. A process for the production of a macrolide of the invention, which comprises cultivating strain Streptomyces sp. A92-308110 (DSM 9954) or a mutant, recombinant or modified form thereof in an appropriate culture medium and optionally recovering the sanglifehrin.

29. A method of effecting immunosuppression in a subject in need of such treatment which method comprises administering to said subject an effective amount of an agent of the invention.

30. A method:

i) for the prevention of acute and/or chronic organ allo- or xenotransplant rejection, for example for the treatment of recipients of organ transplants of any of the particular types listed above; or ii) for the prevention of graft-versus-host disease, for example in recipients of bone marrow transplants; or iii) for the treatment of autoimmune disease or for the treatment of any such disease or condition listed above; or iv) for the treatment of asthma in a subject in need of such treatment, which method comprises administering to said subject an effective amount of an agent of the invention.

31. An agent of the invention for use as a pharmaceutical, e.g. for use as an immunosuppressant or for use in the treatment of any disease or condition as setforth under B above.

32. A pharmaceutical composition comprising an agent of the invention in association with a pharmaceutically acceptable diluent or carrier.

33. Use of an agent of the invention for the preparation of a medicament for use as an immunosuppressant or for use in the treatment of any disease or condition as set forth under B above.

34. Use of a compound according to any one of claims 1-6, 8-10, 15 or 16 as a reagent in a displacement immunoassay for a cyclosporins or other cyclophilin binding compounds