IL96268A - Substituted cycloalkyl tricyclic macrolides containing heteroatoms their preparation and pharmaceutical compositions containing them - Google Patents

Description

96268/3 αηΝ e >D)an nmpn 1) vy3ni orusn , >p 0i>j7>a Substituted cycloalkyi tricyclic macrolides containing heteroatoms, their preparation and pharmaceutical compositions containing them Sandoz A.G.

C.81947 It concerns -2- 900-9581 either R1 is a group (a) of formula 7.XT.90 wherein R5 is chloro, bromo, iodo or azido and except the R, is hydroxy or methoxy; compounds R. is oxo and there is a single bond in 23,24 position; optionally on pages 3 and 4 protected hydroxy and there is a single or a double bond in 23,24 position; or absent and there is a double bond in .23,24 position; and R4 is hydroxy and there is a single bond in 10,11 position; or absent and there is a double bond in 10,11 position; or R: is a group (b) or (d) of formula wherein R 6, is as defined above; R is as defined above; and R4 is hydroxy and there is a single bond in 10,11 position; and methyl, ethyl, n-propyl or allyl; in free form and, where such forms exist, in salt form, hereinafter referred to as "the compounds of the invention".

As is evident from formula I and the definition of the substituents when there is a single bond in 10,11 position the carbon atom to which the methyl group in 11 position is attached has the β-configuration and there -3- 900-9581 is a hydrogen atom with the a-conf guration attached to the carbon atom in 11 position; when there is a double bond in 10,11 position this methyl group lies in the plane of the paper and there is no hydrogen atom in 11 position. When R. is oxo no hydrogen atom is attached to the carbon atom in 24 position.

R2 preferably is unprotected hydroxy and there is a single bond in 23,24 position. R. preferably is ethyl or allyl. R4 preferably is hydroxy. R5 preferably is chloro. Rfi preferably is methoxy.

Protected hydroxy preferably is hydroxy protected by a conventional hydroxy-protecting group such as formyl, tert-butoxycarbonyl, or trialkylsilyl; it especially is tert-butyldimethylsilyloxy.

A compound of the invention preferably is in free form. It preferably is in unprotected form.

The compounds of the present invention have a chemical structure related to that of macrolides of the FK 506 type, e.g. as disclosed in Fisons EP 323042. There is no specific disclosure in the art of substituents at position 33 (R5) having the unusual a-configuration nor of the compounds of the present invention wherein R-^ is a group (a), and the presence of a cyclopentyl moiety (d) (as group R-^) in position 29 is also highly unusual. Further, the compounds of formula I of the present invention apart from immunosuppressant activity also possess antiinflammatory activity and low overall toxicity.

"~ A subgroup of compounds of the invention is the compounds lp1 , i.e. the compounds of formula I wherein R: is a group (a) wherein Rfi is methoxy and either R. is chloro or bromo and R4 is hydroxy and there is a single bond in 10,11 position or R. is azido and R4 is hydroxy and there is a single bond in 10,11 position or absent and there is a double bond in 10,11 position; R2 is optionally protected hydroxy and there is a single or a double bond in 23,24 position; and R3 is as defined above under formula I; in free form and, where such forms exist, in salt form. -4- 900-9581 A further subgroup of compounds of the invention is the compounds Ip3 , i.e. the compounds of formula I wherein Rx is a group (b) wherein Rfi is methoxy, R. is optionally protected hydroxy and there is a single bond in 23,24 position; or absent and there is a double bond in 23,24 position; R4 is hydroxy and there is a single bond in 10,11 position; and R. is as defined above under formula I; in free form and, where such forms exist, in salt form.

A further subgroup of compounds of the invention is the compounds Ip4 , i.e. the compounds of formula I wherein R1 is a group (d), R_ is optionally protected hydroxy and there is a single bond in 23,24 position; or absent and there is a double bond in 23,24 position; R4 is hydroxy and there is a single bond in 10,11 position; and R3 is as defined above under formula I; in free form and, where such forms exist, in salt form.

A preferred subgroup of compounds of the invention is the compounds of formula I wherein Rl, is a group (a) wherein Rb. is as defined above under formula I and is methoxy; R2 is optionally protected hydroxy and there is a single bond in 23,24 position; R4 is hydroxy and there is a single bond in 10,11 position; or absent and there is a double bond in 10,11 position; and j is ethyl or allyl.

A further preferred group of compounds of the invention is the compounds of formula I wherein Rx is a group (b) wherein Rfi is methoxy; R. is optionally protected hydroxy and there is a single bond in 23,24 position; or absent and there is a double bond in 23,24 position; R4 is hydroxy and there is a single bond in 10,11 position; and R3 is ethyl or allyl. -5- 900-9581 A further preferred subgroup of compounds of the invention is the compounds of formula I wherein Rx is a group (d), R. is optionally protected hydroxy and there is a single bond in 23,24 position; or absent and there is a double bond in 23,24 position; R4 is hydroxy and there is a single bond in 10,11 position; and R3 is ethyl or allyl.

A further subgroup of compounds of the invention is the compounds Iq, i.e. the compounds of formula I wherein either Rx is a group (a) wherein Rg is chloro, bromo, iodo or azido and Rg is hydroxy or methoxy, R2 is oxo and there is a single bond in 23,24 position; optionally protected hydroxy and there is a single or a double bond in 23,24 position; or absent and there is a double bond in 23,24 position; and R4 is hydroxy and there is a single bond in 10,11 position; orR^ is absent and there is a double bond in 10,11 position; or Rx is a group (b) wherein Rg is hydroxy or methoxy or (d); R2 is as defined above for this subgroup; and R4 is hydroxy and there is a single bond in 10,11 position; and R3 is methyl, ethyl, n-propyl or allyl, in free form and, where such forms exist, in salt form.

A further subgroup of compounds of the invention is the compounds Ir, i.e. the compounds of formula I wherein either Rx is a group (a) as defined above under formula I; and R2 and R4 have the significance indicated above under group (a); or Rx is a group (b) or (d) as defined above under formula I; and R2 and R4 have the significance indicated above under groups (b) and (d); and R3 is as defined above under formula I; in free form and, where such forms exist,, in salt form.

In a subgroup of compounds Ir R2 is other than absent. -6- 900-9581 A further subgroup of compounds of the invention is the compounds of formula Is OCH. wherein either R Is is a group (a) wherein R5 is chloro, bromo, iodo or azido and Rg is methoxy; R 2s is hydroxy optionally protected by tert-butyldimethylsilyl and there is a single bond in 23,24 position; and R 4 s is hydroxy and there is a single bond in 10,11 position; or R^s is absent and there is a double bond in 10,11 position; or 1 s is a group (b) wherein Rg is methoxy, or a group (d); 2 s is hydroxy optionally protected by tert-butyldimethylsilyloxy and there is a single bond in 23,24 position; or absent and there is a double bond in 23,24 position; and 4 s is hydroxy and there is a single bond in 10,11 position; and R 3s is ethyl or allyl, in free form and, where such forms exist, in salt form. -7- 900-9581 A compound of the invention can be obtained by a process comprising for the preparation of a compound of formula I wherein Rx is a group (a) as defined above under formula I, R. and R3 are as defined above under formula I and R4 is hydroxy (i.e. a compound la) , replacing under simultaneous epimerization the hydroxy group by chlorine, bromine, iodine or azido in a corresponding compound having unprotected hydroxy in 33 position (i.e. a compound Ila, of formula Ila 0CH3 wherein R2 and R. are as defined above under formula I and 6 is hydroxy or methoxy); -8- 900-9581 for the preparation of a compound of formula I wherein Rx is a group (b) as defined above under formula I, R2 and R3 are as defined above under formula I and R4 is hydroxy (i.e. a compound lb), treating a corresponding compound Ila with cyanogen bromide in the presence of a base or treating a corresponding compound Ila with thiophosgene, reacting the resultant product with an inorganic azide and allowing the resultant unstable intermediate having a group c) for the preparation of a compound of formula I wherein R: is a group (d) as defined above under formula I, R2 and R3 are as defined above under formula I and R4 is hydroxy (i.e. a compound Ic) , treating a corresponding compound lb with an acid having a non-nucleophilic anion; and - when a resultant compound of formula I has a protected hydroxy group, optionally splitting off the protecting group(s) to give a corresponding compound of formula I having one or more unprotected hydroxy group(s) (i.e. a compound Ij), whereby when R is a group (a), a water molecule may be simultaneously split off and a compound of formula I is obtained wherein Rj is a group (a) as defined above under formula I, R is unprotected hydroxy and there is a single or double bond in 23,24 position; and R4 is absent and there is a double bond in 10,11 position (i.e. a compound Ii); or -9- 900-9581 - optionally protecting an unprotected hydroxy group in a resultant compound of formula I as appropriate to give a corresponding compound of formula I having one or more protected hydroxy groups(s) (i.e. a compound Ik), and recovering the resultant compound of formula I in free form and, where such forms exist, in salt form.

The process variants of the invention can be effected in a manner analogous to known procedures.

Process variant a) is a substitution reaction under simultaneous epimerization. It is preferably effected in an inert solvent such as tetrahydrofurane or toluene. Preferably for the substitution by halogen the reaction is effected with tetrachloro-, tetrabromo- or tetraiodomethane in the presence of triphenylphosphine, and for the substitution by azido with azodicarboxylic acid ester, preferably diethyl ester, and hydrazoic acid. A hydroxy group in 24 position may be in protected form. As protecting group known hydroxy protecting groups such as tert-butyldi-methylsilyl may be used. A protecting group may subsequently be split off in accordance with known procedures, e.g. with hydrofluoric acid in acetonitrile. Upon deprotection a water molecule may, depending on the reaction conditions chosen, simultaneously be split off in position 10,11 and a double bond formed. The individual compounds can be separated from such a resultant mixture in conventional manner, e.g. chromatographically .

Compounds la may be further processed by e.g. oxidation or dehydration to corresponding compounds wherein is absent; for example, oxidation of compounds la wherein R. is hydroxy leads to corresponding compounds wherein R 4„ is absent and R 2, is oxo.

Process variant b) is a cyanidation reaction. It preferably is effected in an inert solvent such as a chlorinated hydrocarbon, e.g. dichloromethane. The temperature preferably is about room temperature. The base is e.g. 4-dimethylaminopyridine.

A compound of formula I obtained accordance to process variants a) and b) above may be isolated from the reaction mixture and purified in accordance with known methods. When R. is hydroxy and there is a single bond in 23,24 position a water molecule may be simultaneously split off. -10- 900-9581 A corresponding mixture of compounds lb is obtained wherein either R2 is hydroxy and there is a single bond in 23,24 position or R∑ is absent and there is a double bond in 23,24 position. The individual compounds can be separated from such a resultant mixture in conventional manner, e.g. chromatographycally .

The second procedure according to process variant b) is effected by reaction with thiophosgene, preferably in the presence of an acid scavenger such as 4-dimethylaminopyridine. Preferably an inert solvent such as acetonitrile is used. The temperature preferably is about room temperature. The subsequent reaction with an inorganic azide is preferably effected with sodium azide. The resultant compounds lib are unstable and decompose already at room temperature to compounds lb, under splitting off of nitrogen and sulfur. This reaction step preferably is effected in an inert solvent such as an aromatic hydrocarbon, e.g. benzene. Temperature preferably is elevated, e.g. about 50°C.

In process variant c) a ring contraction takes place. Protecting groups which are present may be simultaneously split off. Preferably an inert solvent such as acetonitrile is used. Preferably hydrofluoric acid is used as acid having a non-nucleophilic anion. Temperature preferably is about room temperature.

The optional deprotection process variant may also be effect in conventional manner. For splitting off of e.g. tert-butyldimethylsilyl it is effected by treatment with e.g. hydrofluoric acid in a solvent such as acetonitrile. Depending on the reaction conditions selected (duration, temperature, etc.) the splitting can be steered in such a manner that either all or only some protecting group are removed. Partial deprotection is particularly indicated where a definite hydroxy group is to be subsequently reacted in a later reaction.

The optional protection step variant may also be effected in conventional manner along similar lines.

Thus for subsequent reactions involving a hydroxy group, particularly a hydroxy group in position 24 and/or 33, selective protection of only one of the two free hydroxy groups or selective deprotection of only one of the two protected hydroxy groups may be effected in such a manner that reaction occurs only at the desired position. Mixtures of end products may be obtained thereby; such mixtures can be separated in conventional manner, -11- 900-9581 e.g chromatographically . Resultant end products still containing protecting groups can be subsequently deprotected. Reaction conditions may alternatively be selected such that simultaneously vith or immediately after reaction the protecting groups are removed (one pot process).

The compounds of formula I may be isolated and purified from the reaction mixture in conventional manner.

Insofar as their preparation is not specifically described herein, e.g. in the Examples, the compounds used as starting materials are known or can be obtained in conventional manner from known compounds, e.g. starting from appropriate Streptomyces strains such as Streptomyces tsukubaensis No. 9993 described in e.g. Fujisava EP 184162. Samples can be obtained from the Fermentation Research Institute, Tsukuba, Ibaraki 305, Japan under provisions of the Budapest Treaty under deposit No. FERM BP-927. This strain has been redeposited on April 27, 1989 e.g. as disclosed in Sandoz EP 356399, vith the Agricultural Research Culture Collection International Depository, Peoria, Illinois 61604, USA under the provisions of the Budapest Treaty under deposit No. NRRL 18488. -12- 900-9581 The following Examples illustrate the invention and are not limitative. All temperatures are in degrees Centigrade. All NMR spectra are in CDCl, , ppm. The abbreviations have the following meanings: BOC: tert-butoxycarbonyl; cfr: colourless foamy resin: db: double bond; Et: ethyl; FK 506: the compound of formula -13- 900-9581 i.e. 17a-allyl-ie, 14a-dihydroxy-12- [ 2 ' -(4" (R)-hydroxy- 3" (R)-methoxycyclohex-l"(R)-yl)-l' -methyl-trans-vinyl ]- 23a, 25 -dimethoxy-13a, 19 , 21a, 27 β-tetramethyl- ll,28-dioxa-4-azatricyclo[22.3.1.0 ' Joctacos- 18-trans-ene-2,3,10,16-tetraone (according to the atom numbering in EP 184162; however, in the Examples the atom numbering of formula I is used throughout) ; FR 520: as FK 506, but with ...CH2CH. (ethyl) in place of allyl in position 21 in the formula; iBuoyloxy: isobutanoyloxy [ (H3C)2CHC00-] ; iPr: isopropyl; na: not applicable; N : azido; 0¾e (or MeO) methoxy; OtBDMS: tert-butyldimethylsilyloxy; sb: single bond; tBu: tert-butyl.

Examples 15 to 63 illustrate the preparation of compounds no longer encompassed by the scope of the present invention but useful as intermediates for the preparation of compounds falling under the scope the present invention. They are of formula X OCH, -14- 900-9581 wherein Rx is a group (c) of formula wherein Rr is as defined above and R7 is oxo; optionally protected hydroxy; methoxy; methylthiomethoxy; isobutanoyloxy; arainooxalyloxy ; R 8„R09 CHC00- wherein RBD is· optionally protected hydroxy or optionally protected amino and Rg is hydrogen or methyl; or p-tolyloxythiocarbonyloxy; R2 is oxo and there is a single bond in 23,24 position; absent and there is a double bond in 23,24 position; or is optionally protected hydroxy, methoxy, methylthiomethoxy, isobutanoyloxy, aminooxalyloxy or R 8'Ro9CHC00- wherein R„8 and Rn9 are as defined above, and there is a single or a double bond in 23,24 position; whereby for that group (c) 1) when R7 is oxo, unprotected hydroxy or methoxy then R. is other than absent and other than unprotected hydroxy or methoxy, and there is a single bond in 23,24 position; 2) when R 6, is methoxy and R.7 is methylthiomethoxy then R2 is other than absent and other than unprotected hydroxy; 3) when Rfi is methoxy and R7 is protected hydroxy then Rj is other than optionally protected hydroxy; and 4) when R, is hydroxy then R7 is other than optionally protected hydroxy; and R4 is hydroxy and there is a single bond in 10,11 position; and is as defined above; in free form or, where such forms exist, in salt form. -15- 900-9581 Example 1: 24-tert-Butyldimethylsilyloxy-33-epi-33-chloro-FK506 [Formula I: R = a group (a) wherein R$ = chloro, Rfi = OMe; R = OtBDMS, single bond in 23,24 position; R3 = allyl; R4 = OH, single bond in 10,11 position] [Process variant a), replacement with epimerization] «. 0.092 g 24-tert-butyldimethylsilyloxy-FK506 is heated for 15 hours under refluxing with 0.037 g triphenylphosphine in 4 ml of tetrachloromethane. The solvent is evaporated to dryness 1 under reduced pressure and the residue is purified by column chromatography over silicagel using a mixture of hexane and acetic acid ethyl ester (2:1) as the eluant. The title compound is obtained (colourless foam): l ! H-NMR: about 2:3 mixture of conformers: ; main conformer: 4.56 (m, v = 7 Hz, H-33).

I The starting material is obtained as follows: a) 20 g FK 506 is dissolved in 400 ml of dry dimethylformamide, 5.08 g imidazole and 11.25 g tert-butyldimethylchlorosilane is added in portions and the mixture is stirred for 110 hours at room temperature. The reaction mixture is diluted with acetic acid ethyl ester and washed five times with water. The organic phase is dried over sodium sulfate and the solvent distilled off under reduced pressure. The resultant crude product is purified by chromatography over silicagel using hexane/acetic acid ethyl ester 3:1 as the eluant. 24,33-Bis-(tert-butyldimethylsilyloxy)-FK 506 is obtained: 13C-NMR: main conformer: 69.7 (C-24); 75.1 (C-33); 84.1 lcC-32) ; 164.6 (C-8); 168.9 (C-l); 196.4 (C-9); 209.3 (C-22); ; minor conformer: 70.9 (C-24); 75.3 (C-33); 84.1! (C-32); 165.8 (C-8); 168.2 (C-l); 191.2 (C-9); 210.0 (C-22); , b) 0.5 g 24,33-bis-(tert-butyldimethylsilyloxy)-FK506 is dissolved at 0° under stirring into a mixture of 10 ml of acetonitrile and 0.5 ml of 40 X hydrofluoric acid. After 2 hours at that temperature the reaction medium is diluted with dichloromethane. The solution is washed successively with saturated aqueous sodium bicarbonate solution and water and the organic phase dried over sodium sulfate, and the solvent is evaporated* ■16- 900-9581 under reduced pressure, the resultant residue is purified by column chromatography over silicagel (eluant: dichloromethane^methanol 9:1). 24-tert-Butyldimethylsilyloxy-FK 506 is obtained as a colourless foam: 13C-NMR: main conformer: 69.7 (C-24); 73.6 (C-33); 84.1 (C-32); 164.6 (C-8); 168.9 (C-l); 196.4 (C-9); 209.2 (C-22); | minor conformer: 70.7 (C-24); 73.6 (C-33); 84.2 (C-32); 165.8 (C-8); 168.2 (C-l); 191.4 (C-9); 209.2 (C-22). i I I Example 2 : 24-tert-Butyldimethylsilyloxy-33-epi-33-azido-FK506 [Formula I: Rx = a group (a) wherein R5 = azido, Rg = OMe; R2 = OtBDMS, single bond in 23,24 position; R3 = allyl; R4 = OH, single bond in 10,11 position] [Process variant a)] To a solution of 0.092 g 24-tert-butyldimethylsilyloxy-FK506 and 0.08 g triphenylphosphine in 2 ml of dry tetrahydrofuran'e is added at 0° 0.047 ml of azodicarboxylic acid diethyl ester, followed by 0.15 ml of a 2 M solution of hydrazoic acid in toluene. The solution is brought to room temperature and stirred for 18 hours. The solvent is evaporated to dryness under reduced pressure and the residue purified as described above under Example 1. The title compound is obtained (colourless foam): XH-NMR: 4.07 (m, H-33).

I The following compounds of formula I are obtained in analogous manner in accorda Example to R5 Re R7 R2 Position R3 No. Ex. No. 23,24 1 ) 3 (a) CI OHe na OtBDHS sb Et 1 ) 4 (a) Br OMe na OtBDHS sb Et 5 Br OHe na OtBDHS sb ally 1 ) (a) l 6 (a) 3 OHe na OtBDHS sb Et 1 ) 6a (a) I OMe na OtBDHS sb Et NMR: Example 3: H-NMR: 4.56 (m, H-33); Example 6a: C-NMR: mixture of conformers: 210.33 (C-22); 168.91 (C-1) 78.90 (C-32); 25.81 (tBu); 1 ) The starting material is obtained from FR 520 in a manner analogous to 24-te (see Example 1): j a) 24,33-bis-(tert-butyldimethylsilyloxy)-FR 520: H-NMR: about 2:1 mixture o main conformer: 4. eq.); 4.05 (txt, J= J=1.5 Hz and 10 Hz, and 11 Hz, H-32); _ minor conformer: 4 " J=2 Hz and 10 Hz, H 11 Hz, H-32); b) 24-tert-butyldieethylsilyloxy-PR 520: H-NMR: about 2:1 mixture of 2 confor main conformer: 4.44 (b, H 4.05 (dxt, J=l,5 Hz and 6 H and 10 Hz, H-14); 3.01 (dx minor conformer: 4.24 (H-2 H-14); 3.01 (dxdxd, J=4 Hz, -19- 900-9581 Example 7; 2 -tert-Butyldimethylsilyloxy-33-cyanoxy-FR 520 [Formula I: Rx = a group (b) wherein R6 = OMe; R∑ = OtBDMS, single bond in 23,24 position; R. = Et; R4 = OH, single bond in 10,11 position] [Process variant b), treatment with cyanogen bromide] A solution of 2 g 24-tert-butyldimethylsilyloxy-FR 520 and 0.94 g 4-dimethylaminopyridine in 100 ml of dichloromethane is rapidly reacted at room temperature with a solution of 0.4 g cyanogen bromide in 15 ml of dichloromethane and the mixture is stirred at room temperature for 20 minutes. The mixture is filtered over silicagel (eluant: n-hexane/acetic acid ethyl ester) and the solvent is removed from the relevant fraction ί under reduced pressure. The title compound is obtained as a colourless foamy resin: 1H-NMR: mixture of conformers: 4.3 (m; H-33). j Example 8: 24-tert-Butyldimethylsilyloxy-33-cyanoxy-FR 520 [Formula I: as for Example 7] i [Process variant b), treatment with thiophosgene and sodium azide] A solution of 2 g 24-tert-butyldimethylsilyloxy-FR 520 and 2 g 4-dimethylaminopyridine in 50 ml of acetonitrile is carefully reacted with 0.4 ml of thiophosgene and the mixture stirred for 3 hours at room temperature. The reaction mixture is poured onto a well-stirred mixture consisting of 150 ml of acetic acid ethyl ester, 40 ml of saturated aqueous sodium chloride solution and 50 ml of 2 N sodium azide solution, vigourous stirring is continued for 5 minutes and the organic phase is separated.

The organic phase is then successively washed with water,' 1 N hydrochloric acid solution, water, and saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue is taken up in about 100 ml of benzene and heated at 30-40° for 2 hours. The benzene is removed under reduced pressure and the title compound is recovered from the residue as a colourless foamy resin by column chromatography over silicagel (eluant: n-hexane / acetic acid ethyl ester): 1H-NMR: see Example 7.

The following compounds of formula I are obtained in analogous manner in accord Analogous Example to Position No. Ex. No. 23,24 NMR: Example 9: H-NMR: mixture of conformers: 4.3 (m, H-33); Example 10a: H-NMR: mixture of conformers: 5.34 (H-26); 4.63 (db, J=4 Hz, H-2); 4.44 (db, J J=5 Hz, 8 Hz and 11 Hz, H-33); 3.01 (tb, J=13 Hz, Example 10b: H-NMR: 6.81 resp. 6.75 (dxd resp. dxd, J=5 Hz and 15 Hz resp. 6.3 (dxd resp. dxd, J=2 Hz and 15 Hz resp. 5.23 (d resp. d, J=3 Hz resp. 3 Hz, H-26); 4.3 ( 1 ) 2J_ A mixture of both compounds is obtained; they can be separated chromatog n-hexane/acetic acid ethyl ester). -21- 900-9581 Example 12; 29-Des-( -hydroxy-3-methoxycyclohexyl)-29-(3-formylcyclo- pentyl)-FR 520 [Formula I: Rx = a group (d); R = OH, single bond in 23,24 position; R3 = Et; R4 = OH, single bond in 10,11 position] [Process variant c), treatment with a non-nucleophilic anion] 0.5 g 24-tert-butyldimethylsilyloxy-33-cyanoxy-FK 520 (compound of Examples 7 and 8) or 33-cyanoxy-FR 520 (compound of Example 10a) is dissolved into a mixture of 50 ml of acetonitrile and 2 ml of 40 X wt. aqueous hydrofluoric acid and the mixture is stirred for 2.5 hours at room temperature. The reaction mixture is then distributed between acetic acid ethyl ester and saturated aqueous sodium bicarbonate solution, the aqueous phase is discarded and the organic phase is washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered and concentrated under reduced pressure. The title compound is obtained as a colourless foamy resin from the residue by column chromatography over silicagel (eluant: n-hexane / acetic acid ethyl ester): 1H-NMR: mixture of conformers: 9.64 (d, J=2 Hz, CH0); 2^87 (m, H-32); 2.67 (m, H-30).

The following compounds of formula I are obtained in analogous manner in accord Analogous Example to Rt Position No. Ex. No. 23,24 13 12 14 12 NMR: Example 13: H-NMR: mixture of conformers: 9.65 (d, J=2 Hz, CHO); 2.86 (m, H-32); 2.15 (dxdx H-31a); 1.45 (dxt, J=12.5 and 9 Hz, H-31b); 2.67 ( Example 14: 1H-NMR: about 5:3 mixture of conformers: 9.66 (d, J=2 Hz, resp. 6.77 (dxd, J=15 and 7.5 Hz) H-24; 6.19 (dxd, J=15 and 1 Hz) H-23; 1 ) Starting from the compound of Example 9 or 11a; 2 ) Starting from the compound of Example 10b. a) 2 -tert-Butyldimethylsilyloxy-33-oxo-FK 506 and b) 24-tert-Butyldimethylsilyloxy-33-methylthiomethoxy FK 506 [Formula X: Rx = a group (c) wherein Rfi = OMe, R? = oxo and, respectively, methylthiomethoxy; R = OtBDMS, single bond in 23,24 position; R3 = allyl; R4 = OH, single' bond in 10,11 position] 1 g 24-tert-Butyldimethylsilyloxy-FK 506 is dissolved at room temperature into a mixture of 20 ml of acetic anhydride and 30 ml of dimethylsulfoxide and stirring is effected for 5 hours atiroom temperature. The reaction mixture is poured onto a mixture of acetic acid ethyl ester and potassium carbonate solution, stirred for 20 minutes, the phases are separated and the organic phase is repeatedly washed with water, dried over sodium sulfate, filtered and concentrated under reduced pressure.

Following column chromatographic fractionation of the residue over silicagel (eluant: acetic acid ethyl ester / n-hexane 2:1) the title compounds are obtained as colourless foamy resins: compound a): 13C-NMR: about 2:1 mixture of conformers: i 209.3/209.9 (C-22); 208.3/208.5 (G-33); 196.4 (C-9); 168.9/168.2 (C-l); 164.6/165.9 (C-8); 138.5/139.4 (C-19); 135.6/136.1 (C-37); 133.4/134.1 (C-28); 131.8/127.6 (C-29); 123.1/122.3 (C-20); 116.5/116.1 (C-38); 97.6/98.9 (C-10); 83.0 (C-32); l 69.6/70.6 (C-24); compound b): H-NHR: about 2:1 mixture of conformers: 4.82/4.79 (AB; JAB=12 Hz; -0-CH.-$); 2.19 resp. 2.18 (s resp. s, -SCH, ) ; The following compounds of formula X are obtained in analogous manner: Analogous Example to R¾ R6 R7 R2 Position R3 No. Ex. No. 23,24 1 ) 16a 15 OtBDMS OCH2SCH3 sb ally 1 ) (c) na OHe l 16b 15 OtBDHS o 2 ) (c) na OMe xo sb allyl 16c 15 OHe oxo O BDHS sb Et 3 ) (c) na 16d 15 (c) na OHe O BDHS oxo sb Et 1 ) Starting from 33-tert-butyldimethylsilyloxy-FK 506 (compound of Example 16 eluant: toluene / acetic acid ethyl ester 9:1; 2 ) Starting from 24-tert-butyldimethylsilyloxy-FR 520; 3 ) Starting from 33-tert-butyldimethylsilyloxy-FR 520 (DOS 39 38 754); NMR: Example 16a: H-NMR: about 2:1 mixture of conformers: 4.36 (s, -0-CH2-S) and 2.16 (s, -SCH3) resp. 4. (s, -SCH ) Example 16b: H-NMR: about 1:1 mixture of conformers: 5.29 and 5 •59 (s, H-23); 13C-NMR: about 1:1 mixture of conformers: 200.77197.7 (G-22) ; 195.3/194.9 (C-24); 193.2/1 164.4/165. 2 (C-8); 137.5/137.9 (C-19); 135.1/13 130.1/129. 3 (C-28) ; 123.9/123.7 (C-20); 116.7/1 96.3/97.8 (C-23). -25- 900-9581 Example 17; 33-p-Tolyloxythiocarbonyloxy-FK 506 [Formula X: Rx = a group (c) wherein R = OMe, R? = p--tolyloxythio- carbonyloxy; R. = OH, single bond in 23,24 position; R3 = allyl; R4 = OB, single bond in 10,11 iposition] A solution of 2 g F 506 in 70 ml of acetonitrile is successively reacted with 0.46 g 4-dimethylaminopyridine and 1.8 g p-tolyloxythio-carbonyl chloride and the mixture is stirred for 15 hours at room temperature. The reaction mixture is then diluted with acetic acid ethyl ester and successively washed with saturated aqueous sodium bicarbonate solution, 0.5 N hydrochloric acid and water, dried over isodium sulfate, filtered and concentrated under reduced pressure. The title compound is isolated from the residue as a light yellow foamy resin by column chromatography over silicagel (eluant: acetic acid ethyl ester / n-hexane 1:1): ^-NMR: 7.22 and 7.01 (AABB-syst . , ar-H); 5.35 (d, J=l Hz, H-26); 5.18 (dxdxd, J=5 Hz, 9.5 Hz and 11 Hz, H-33); 3.475,13.47, 3.41, 3.40, 3.355 and 3.32 (each s, -0CH ) ; 2.38 (s, ar-CH.); 23 Example 18: 33-Aminomethylcarbonyloxy-Δ -FK 506 [Formula X: R = a group (c) wherein Rfi = OMe, R7 = R8R9CHCO0- (R8 = amino; R9 = H); R = absent, double bond in 23,24 position; R. = allyl; R^ = OH, single bond in 10,11 position] 2 g N-BOC-glycine, 1 g dicyclohexylcarbodiimide, 0.5 g Δ23-ΡΚ 506 (second compound of Example 17 in EP 184162) and 1 g 4-dimethylaminopyridine are successively taken up at room temperature in 70 ml of acetonitrile and the mixture is stirred for 20 minutes at room temperature. The reaction mixture is filtered, the filtrate diluted with acetic acid ethyl ester and successively washed with I hydrochloric acid, aqueous half-saturated sodium bicarbonate solution and water, the organic phase is -26- 900-9581 dried over sodium sulfate, filtered, concentrated, and the residue is taken up in 50 ml of acetonitrile.

In order to split off the protecting group 0.5 g p-toluenesulfonic acid monohydrate is added and the mixture heated to refluxing for 5 minutes, the solution is cooled off, diluted with acetic acid ethyl ester, washed to neutrality with water, the organic phase is dried over sodium sulfate and concentrated. From the residue the title compound is obtained as a colourless foamy resin after column chromatography over silicagel (eluant: acetic acid ethyl ester / methanol 20:3): 1H-NMR: about 6:5 mixture of conformers: 6.81 (dxd, J=5 Hz and 15 Hz) resp. 6.76 (dxd, J=7.5 Hz and 15 Hz) H-24; 6.18 (dxd, J=l Hz and 15 Hz) resp. 6.29 (dxd, J=l Hz and 15 Hz) H-23; A.77 (m, H-33); Example 19: 24-tert-Butyldimethylsilyloxy-FR 520- 33-[(tert-butyldimethylsilyloxy)-(S)-lactate] [Formula X: R = a group (c) wherein Rfi = OMe, R? = R8R9CHC00- (Rg = OtBDMS, 9 = Me, S-conf guration) ; R. = OtBDMS, single bond in 23,24 position; R. = Et; R4 = OH, single bond in 10,11 position] To a solution of 450 mg 24-tert-butyldimethylsilyloxy-FR 520 and 120 mg tert-butyldimethylsilyloxy-(S)-lactic acid in 10 ml of dichloro-methane are added at room temperature 120 mg N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and 23 mg dimethylaminopyridine . After 60 hours the reaction mixture is diluted. with acetic acid ethyl ester, washed successively with 0.5 N hydrochloric acid and then water, dried over sodium sulfate, filtered and the solvent is evaporated under reduced pressure. The residue is chromatographed over silicagel (eluant: n-hexane / acetic acid ethyl ester 2:1). The title compound is obtained as a colourless foam: 1 H-NMR: 1.41 (d, J=7 Hz); 4.34 [q, J=7 Hz, -C0CH(CH_ )0Si ] ; 4.75 (m, H-33). -27- 900-9581 Example 20; FK 506-33-glycolate [Formula X: βχ = a group (c) wherein Rfi = OMe, R = R8R9CHCOO- (Rfl = OH, R9 = H); R? = OH, single bond in 23,24 position; R. = allyl; R4 = OH, single bond in 10,11 position] To a solution of 300 mg tert-butylditnethylsilyloxymethylcarboxylic acid in 5 ml of dichloromethane are added under stirring at 0° 0.67 ml of oxalyl chloride and one drop of dimethylformamide. The mixture is brought to room temperature and is stirred for 1 hour. The reaction mixture is concentrated under reduced pressure. The residue is taken up in 5 ml of dichloromethane and this soution is added dropwise at 0° to a solution of 600 mg FK 506, 0.28 ml triethylamine and a catalytic quantity of 4-dimethylaminopyridine. After 18 hours at 0° the solution is diluted with acetic acid ethyl ester, successively washed with 0.1 N hydrochloric acid and water, the organic phase is dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue is taken up in 20 ml of acetonitrile, reacted with 0.5 ml of 40 X wt. aqueous hydrofluoric acid and stirred for 20 minutes at room temperature. The mixture is diluted with acetic acid ethyl ester, washed with saturated aqueous sodium hydrogen carbonate solution, dried over sodium sulfate, filtered and concentrated under reduced pressure. The title compound is obtained as a colourless foamy resin from the residue by chromatography over silicagel (eluant: n-hexane / acetic acid ethyl ester): XH-NMR: 4.13 (s, -C0CH OH); 4.41 (d, br, J=13 Hz, H-6e); 4.60 (d, br, J=4 Hz, H-2); 4.78 (m, H-33); 5.16 + 5.30 (H-26).

The following compounds of formula X are obtained in analogous manner: Analogous Example to R4 R¾ Rfi R? R2 Position R3 No. Ex. No. 23,24 21 17 to 20 (c) na OMe BOC-NHCH COO- OtBDMS sb allyl 22 17 to 20 (c) na OMe tBDMS-OCB C00- OtBDMS sb allyl 23 17 to 20 (c) na OMe B0C-NHCH C00- OtBDHS sb Et 24 17 to 20 (c) na OMe tBDMS-OCH C00- OtBDMS sb Et 25a 17 to 20 (c) na OMe B0C-NHCH2C00- OH sb allyl 25b 17 to 20 (c) na OMe 0H B0C-NHCH2C00- sb allyl 25c 17 to 20 (c) na OMe BOC-NHCH C00- B0C-NHCH2COO- sb allyl 25d 17,19,20 (c) na OMe B0C-NHCH2COO- absent db allyl 26a 17 to 20 (c) na OMe B0C-NHCH2C00- OH sb Et 26b 17 to 20 (c) na OMe OH BOC-NHCH2COO sb Et 26c 17 to 20 (c) na OMe BOC-NHCH2COO- BOC-NHCH2COO- sb Et 26d 17 to 20 (c) na OMe BOC-NHCH COO- absent db Et 27a 17 to 20 (c) na OMe NH2C0C00- OH sb allyl 27b 17 to 20 (c) na OHe NH2COCOO- NH2COCOO- sb allyl 27c 17 to 20 (c) na OMe NH2C0C00- absent db allyl 28a 17 to 20 (c) na OHe NH2C0C00- OH sb Bt 28b 17 to 20 (c) na OHe NH2C0C00- NH2C0C00- sb Et 28c 17 to 20 (c) na OHe NH2C0C00- absent db Et 29 17 to 20 (c) na OHe NH2C0C00- OtBDHS sb Bt 30 17 to 20 (c) na OHe NH2C0C00- OtBDHS sb allyl 31 17 to 20 (c) na OHe p-tolyloxy- OH sb Et thiocarbonyloxy 32 17 to 20 (c) na OHe HOCHjCOO- OH sb Et Analogous Example to Position No. Ex. No. 23,24 (S) 33 17 to 20 (c) na OHe tBDHS-0CH(CH3)C00- 0B sb Et 34 17 to 20 (c) na OHe iBuoyloxy OtBDHS sb Et 35 17 to 20 (c) na OHe iBuoyloxy OtBDHS sb allyl 36 17 to 20 (c) na OHe iBuoyloxy OH sb allyl 37 17 to 20 (c) na OHe iBuoyloxy OB sb Et 38 17, 18,20 (c) na OHe tBDHS-OCH(CB )C00- OtBDHS sb Et (S) * H-NMR: Example 21: mixture of conformers: 4.85 (m, H-33); 3.93 (s, 0=C-CH2-N-); 3.22 (m, H-32); H-NMR: Example 22: 4.25 (s, -C0CH20Si); 4.76 (m, H-33); H-NMR: Example 24: 4.26 (s, -C0CH OSi); H-NMR: Example 25a: 5.7 (m, H-37); 4.75 (dxdxd, J=5 Hz, 9 Hz and 10 Hz, H-33 H-NMR: Example 25b: mixture of conformers: 5.69 (m, H-37); 4.52 (H-2); 4.44 (H-6 eq.); 3.87 (m, -N- H-NMR: Example 25c: 5.7 (m, H-37); 4.76 (dxdxd, J=5 Hz, 8 Hz and 10 Hz, H-33 (s, tBu); H-NMR: Example 27b: about 2:1 mixture of conformers: 7.15-7.0 and 6.1-6.2 (b, each 2H, 0=C-NH2); 5.28 and 5.4 (m, H-33); H-NMR: Example 28a: mixture of conformers: 7.01 and 5.98 (-C0NH ); 5.35 (d, J=l Hz, H-26); 4.85 (m, 4.44 (db, J=13 Hz, H-6 eq.); LH-NMR: Example 29: mixture of conformers: 7.03 and 6.12 (C0NH ); 4.85 (m, H-33); 4.45 (H-2); 4.42 (H-6 eq.); 0.88 (s, tBu); 0.04 (s, Si-CH3); H-NHR: Example 31 mixture of conformers: H-NMR: Example 32: [H-NMR: Example 34: H-NMR: Example 37: H-NMR: Example 38: see Example 19. -31- 900-9581 Example 39; 24-tert-Butyldimethylsilyloxy-33-aminooxalyloxy-FK 506 [Formula X: Rx = a group (c) wherein Rg = OMe, R? = aminooxalyloxy; R2 = OtBDMS, single bond in 23,24 position; R3 = allyl; R4 = OH, single bond in 10,11 position] A solution of 24,33-bis-(tert-butyldimethylsilyloxy)-FK 506 in 70 ml of acetonitrile is reacted at 0° to 5° with 1 ml of oxalyl chloride and stirred at 0 to 5° for AO minutes. The reaction mixture is stirred with a mixture of acetic acid ethyl ester and satured aqueous ammonia solution, any precipitate formed is sucked off, the phases are separated, the organic phase is washed successively with 1 N hydrochloric acid and then water, dried over sodium sulfate, filtered and concentrated under reduced pressure. From the residue the title compound is obtained as a colourless foamy resin following column chromatography over silicagel (eluant: n-hexane / acetic acid ethyl ester 1:1): 1H-NMR: about 2:1 mixture of conformers: 7.04 and 6.17 (b, each 1 H, H2NC=0); 4.86 (m, H-33).

The following compounds of formula X are obtained in analogous manner: Analogous Example to Rj R. R.

No. Ex. No. 23,24 40 39U (c) na OHe NH2C0C00- NH C0C00- sb allyl 41 39,40 (c) na OMe NH2COCOOOtBDHS sb Et 42 39,40 (c) na OMe NSCOCOO- NH2C0C00- sb Et Stirring is effected for 1 hour at room temperature; column chromatography is of 3:1 to 1:3; * XH-NMR: Example 40: see Example 27b; Example 41: see Example 29. -33- 900-9581 Example A3; 24-Methoxy-33-tert-butyldimethylsilyloxy-FK 506 [Formula X: R = a group (c) wherein R¾ = OMe, R? = OtBDMS; R = OMe, single bond in 23,24 position; R3 <= allyl; R^ = OH, single bond in 10,11 position] 1 g 33-tert-butyldimethylsilyloxy-PK 506 is dissolved into a mixture of 50 ml of dichloromethane and 0.04 ml of borotrifluoride etherate previously cooled to 0° to 5°. A solution of 20 ml of an approximately 1 N solution of diazomethane in methylene chloride is then added dropwise in such a manner that the yellow coloration of the solution which initially forms persists for as shortly as possible. The reaction mixture is diluted with acetic acid ethyl ester, successively washed with saturated aqueous sodium hydrogen carbonate solution and water, dried over sodium sulfate, filtered and the solvent is removed under reduced pressure. The title compound is obtained as a colourless foamy resin from the residue following column chromatographic purification over silicagel (eluant: acetic acid ethyl ester / n-hexane): 1H-NMR: about 3:1 mixture of conformers: main conformer: 5.25 (d, J=8 Hz, H-29); 5.17 (d, J=7 Hz, H-26); 4.79 (d, J=10 Hz, H-20); 3.82 (dxd, J=9 Hz and 1.5 Hz, H-14); 3.42, 3.40, 3.33 and 3.24 (4xs, 0CH3); 2.68 (dxd, J=13 Hz and 8 Hz, H-23); minor conformer: 3.90 (dxd, J=9/2,5 Hz, H-14); The following compounds of formula X are obtained in analogous manner: Analogous Example to Rj R5 Rfi R7 2 Position R3 No- Ex. No. 23,24 44 431' (c) na OMe OMe OtBDMS sb allyl * 1 H-NMR: about 2:1 mixture of conformers: main conformer: 5.22 (d, 1.5Hz, H- H-23); 0. minor conformer: 4.26 (m, U Starting from 24-tert-butyldimethylsilyloxy-FK 506. -35- 900-9581 Example 45; 24- ert-ButyIdimethylsilyloxy-33-oxo-FR 520 [Formula X: R = a group (c) wherein R$ = OMe, R = oxo; R = OtBDMS, single bond in 23,24 position; R3 = Et; R4 = OH, single bond in 10,11 position] 2 g 24-tert-butyldimethylsilyloxy-FR 520 and 1 g N-methylmorpholin-N-oxide are dissolved in 100 ml of methylene chloride, reacted with 5 g molecular sieve (Molsieb 4A) and the mixture is stirred for 15 minutes at room temperature. 0.15 g tetrapropylammonium perruthenate is added and stirring is continued for 3 more hours at room temperature. The mixture is concentrated, the residue is taken up in acetic acid ethyl ester and the solution successively washed with saturated aqueous sodium hydrogen sulfite solution, saturated aqueous sodium chloride and saturated aqueous copper sulfate solution, the organic phase is dried over sodium sulfate, filtered and concentrated under reduced pressure. The title compound is obtained from the residue following column chromatography over silicagel (eluant: n-hexane / acetic acid ethyl ester).

The following compounds of formula X are obtained in analogous manner: Analogous Example to Rj Posi tion No. Ex . No . 23,24 46 (c) na OHe OtBDHS oxo sb Et 46a (c) na OHe oxo OtBDHS sb allyl 46b 45 (c) na OHe OtBDHS oxo sb allyl 13 Example 46a: 1 C-NMR: see Example 15a; Example 46b: H-NMR and C-NMR: see Example 16b; Starting from 33-tert-butyldimethylsilyloxy-FR 520 (DOS 39 38 754); Starting from 24-tert-butyldimethylsilyloxy-FK 506; Starting from 33-tert-butyldimethylsilyloxy-FK. 506; -37- 900-9581 Example 47; 24-Oxo-FK 506 [Formula X: Rx = a group (c) wherein Rg = OMe, R. = OH; R. = oxo, single bond in 23,24 position; R. = allyl; R4 = OH, single bond in 10, 11 position] 3.6 g 24-oxo-33-tert-butyldimethylsilyloxy-FK506 (compound of Example 16b) is dissolved at room temperature into a mixture of 110 ml of acetonitrile and 3 ml of 40 X wt. aqueous hydrofluoric acid and the mixture is stirred at room temperature for 45 minutes. The reaction mixture is diluted with acetic acid ethyl ester, successively washed with saturated aqueous sodium bicarbonate solution and then water, dried over sodium sulfate, filtered and concentrated under reduced pressure. The title compound is obtained as a colourless foamy resin following chromatographic purification of the residue over silicagel (eluant: acetic acid ethyl ester / n-hexane 3:2): 1H-NMR: about 1:1 mixture of conformers: 5.80 and 5.60 (s, H-23); 3.44, 3.41, 3.39, 3.38 and 2x3.275 (0CH3).

The following compounds of formula X are obtained in analogous manner: Analogous (ample to R5 R6 R7 R, Position R3 No. Ex. No. 23,24 The following compounds of formula I are obtained in analogous manner i deprotection: Analogous Example to Rt R5 R6 R7 R2 Position R3 No. Ex. No. 23,24 1 7 ) 64 47 1 8 ) (a) I OHe na OH sb Et 65a 47 1 8 ) (a) CI OHe na OH sb allyl 65b 47 1 9 ) (a) CI OHe na OH sb allyl 66a 47 1 9 ) (a) CI OHe na OH sb Et 66b 47 2 0 ) (a) CI OHe na OH sb Et 67a 47 2 0 ) (a) Br OHe na OH sb Et 67b 47 na OH sb Et 2 1 ) (a) Br OHe 68a 47 na OH sb ally 2 1 ) (a) 3 OHe l 68b 47 2 2 ) (a) OHe na OH sb allyl 69a 47 2 2 ) (a) Br OHe na OH sb allyl 69b 47 OH sb 2 3 ) (a) Br OHe na allyl 70a 47 t 2 3 ) (a) N3 OHe na OH sb E 70b 47 (a) OHe na OH sb Et 1 Starting from the compound of Example 25a; 2 Starting from the compound of Example 25d; 3 Starting from the compound of Example 46 (= 4 16d); Starting from the compound of Example 23; 5 6 Starting from the compound of Example 26d; 7 Starting from the compound of Example 22; Starting from the compound of Example 24; 8 ) m the compound of Example 9 ) Starting fro 19 or 0 ) Starting from the compound of Example 26b; Starting from the compound of Example 26c; 1 ) Starting from the compound of Example 25c; 2 ) Starting from the compound of Example 25b; 3 ) Starting from the compound of Example 25a; 4 ) Starting from the compound 5 ) of Example 26a; 6 ) Starting from the compound of Example 34 7 ) Starting from the compound of Example 35 8 ) Starting from the compound of Example 6a Starting from the compound of Example 1 9 ) 0 ) Starting from the compound of Example 3 Starting from the compound of Example 4 1 ) Starting from the compound of Example 2 2 ) 3 ) Starting from the compound of Example 5 Starting from the compound of Example 6 NMR: Example 48: H-NMR: about 2:1 mixture of conformers: 5.33 and 5.20 (d/d, J=l Hz and 1 Hz, H-26); 4.84 Hz, H-33); 3.44 (s, 2H, 0=C-CH2-N-); 3.22 (dxdxd, H-32); Example 49: 1H-NMR: see Example 18; Example 50: H-NMR: mixture of conformers: 5.8 and 5.6 (s/s, H-23); 5.69 (H-26); 4.38 (d, J 3.80 (dxd, J=9 Hz and 2 Hz, H-14); Example 51: H-NMR: about 2:1 mixture of conformers: 5.34 (d, J=2 Hz, H-26); 4.75 (dxdxd, J=5 Hz, 9 H Hz, H-2); 4.44 (db, J=13 Hz, H-6e); 3.45 (s, -CH Example 53 H-NMR see Ex Example 54 H-NMR see Ex Example 55 H-NMR mixtur J =7 H (ddd, Example 60 H-NMR see Ex Example 61 H-NMR see Ex Example 62 H-NMR see Example 37 Example 65a H-NMR 4.59 (m, H-33) C-NMR: about 2:3 mixture of conformers main conformer: 59.1 (C-33; 79.2 (C-32); 97.5 (C (C-20); 135.6 (C-37); 138.4 (C-19); 164.6 (C-8); (C-22); Example 66a: H-NMR: 4.56 (m, H-33); Example 66b: H-NMR: 2.09 (s, 11-CH3); 4.5 (bm, H-33); C-NMR: about 2:1 mixture of conformers: main conformer: 56.2 (C-33); 80.6 (C-32); 116.4 ( (C-11); 129.5 (C-29); 131.9 (C-28): 135.8 (C-37); 166.7 (C-8); 168.7 (C-1); 188.0 (C-9); 212.4 (C-2 minor conformer: 56.1 (C-33); 80.6 (C-32); 116.5 (C-11); 128.5 (C-29); 131.8 (C-28); 135.6 (C-37); 166.5 (C-8); 169.5 (C-1); 184.8 (C-9); 213.3 (C-2 Example 67a: H-NMR 4.44 (d, J=13 Hz, H-6 eq.); 4.60 (d, J=4 Hz, H-2); Example 68a: H-NMR 4.07 (m, w Example 68b: H-NMR about 2:1 Example 70a: H-NMR about 5:4 5.66 (d, J 3.80 (dxd, J=9 Hz and 2 Hz, H-14).

Iodine analyis: theor. : 14.06 %; found: 13.57 %.

The compounds of Examples 10a, 11a, 12 and 13 may be prepared in analogous variant deprotection. -42- 900-9581 Example 71; 24-tert-Butyldimethylsilyloxy-29-des-(4-hydroxy-3-methyl- cyclohexyl)-29-(3-formylcyclopentyl)-FR 520 [Formula I: Rx = a group (d); R = OtBDMS, single bond in 23,24 position; R3 = Et; R4 = OH, single bond in 10,11 position] [Process variant protection] A solution of 1.2 g 29-des-(4-hydroxy-3-methylcyclohexyl)-29- (3-formylcyclopentyl)-FR 520 (compound of Example 12), 1.5 g tert-butyl-dimethylsilyl chloride and 0.8 g imidazole in 20 ml of dry dimethylformamide is stirred for 15 hours at room temperature and thereafter partitioned between 1 N hydrochloric acid solution and acetic acid ethyl ester. The organic phase is separated, washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered and concentrated under reduced pressure. The title compound is obtained from the residue as a colourless foamy resin following column chromatography over silicagel (eluant: n-hexane / acetic acid ethyl ester): 1 H-NMR: mixture of rotamers: 9.65 (d, J = 2 Hz, CH0); 5.39 (d, J = 9 Hz, H-29); 5.01 (d, J = 7.5 Hz, H-26); 4.81 (d, J = 10 Hz, H-20); 3.82 (dxd, J = 9/2 Hz, H-24).

The compounds of Examples 1 to 9 may be prepared in analogous manner according to process variant protection. -43- 900-9581 The compounds of the invention possess pharmacological activity. They are indicated for use as pharmaceuticals.

In particular they possess antiinflammatory, and immunosuppressant and antiproliferative activity.

Antiinflammatory activity may e.g. be determined in the following test methods : 1. Oxazolone allergic contact dermatitis in the mouse in vivo upon topical application: the test method is as described in F.M. Dietrich and R. Hess, Int. Arch. Allergy 38 (1970) 246-259.

The compounds elicit in this test an activity between about 15 % and about 68 % upon topical administration at a concentration of about 0.01 %. 2. DNFB allergy (swine): the test method is as described in e.g.

EP 315978.

Topical application of a 1.2 X formulation of the compounds repeated twice results in from about 36 % to about 40 X inhibition of the inflammatory reaction.

Immunosuppressant and antiproliferative activity may e.g. be determined in the following test methods: 1. Proliferative response of lymphocytes to allogen stimulation in the mixed lymphocyte reaction (MLR) in vitro: T. Meo, "The MLR in the Mouse", Immunological Methods, L. Lefkovits and B. Pernis, Eds., Academic Press, N.Y. (1979), 227-239.

The compounds elicit in this test (IC50) suppression of mixed lymphocytes at a dosage of from about < 0.0008 yg/ml to about 0.09 ug/ml. -44- 900-9581 2. Inhibition of the primary humoral immune response to sheep erythrocytes in vitro: the test method is as described in R.I. Mishell and R.V.

Dutton, Science 153 (1966) 1004-1006; R.I. Mishell and R.V. Dutton, J. Exp. Med. 126 (1967) 423-442.

The compounds are active in this test with an IC50 of from about 0.0024. g/ml to about 0.32 vg/ml. 3. Inhibition of proliferation of human keratinocytes; the test method is as described in e.g. EP 315978.

The compounds are active in this test at concentrations of from about 1 ug/ml to about 10 pg/ml resulting in an inhibition of from about 30 % to about 90 X.

The compounds of the invention in free form and where such forms exist in pharmaceutically acceptable salt form are therefore indicated as antiinflammatory and as immunosuppressant and antiproliferative agents for use in the prevention and treatment of inflammatory conditions and of conditions requiring immunosuppression, such as a) the prevention and treatment of - resistance in situations of organ or tissue transplantation, e.g. of heart, kidney, liver, bone marrow and skin, - graf t-versus-host disease, such as following bone marrow grafts, - autoimmune diseases such as rheumatoid arthritis, systemic Lupus erythematosus, Hashimoto's thyroidis, multiple sclerosis, Myasthenia gravis, diabetes type I and uveitis, - cutaneous manifestations of immunologically-mediated illnesses; b) the treatment of inflammatory and hyperproliferative skin diseases, such as psoriasis, atopical dermatitis, contact dermatitis and further eczematous dermatitises, seborrhoeic dermatitis, Lichen planus, Pemphigus, bullous Pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides , erythemas, cutaneous eosinophilias , Lupus erythematosus and acne; and c) Alopecia areata. -45- 900-9581 The compounds may be administered systemically or topically.

For these indications the appropriate dosage will, of course, vary depending upon, for example, the host, the mode of administration and the nature and severity of the condition being treated. However, in general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.15 mg/kg to about 1.5 mg/kg animal body weight. An indicated daily dosage in the larger mammal is in the range from about 0.01 mg to about 100 mg, conveniently administered, for example, in divided doses up to four times a day or in retard form.

For topical use satisfactory results are obtained with local administration of a 1-3 % concentration of active substance several times daily, e.g. 2 to 5 times daily. Examples of indicated galenical forms are lotions, gels and creams.

The compounds of the invention may be administered by any conventional route, in particular enterally, e.g. orally, e.g. in the form of tablets or capsules, or topically, e.g. in the form of lotions, gels or creams.

Pharmaceutical compositions comprising a compound of the invention in association with at least one pharmaceutical acceptable carrier or diluent may be manufactured in conventional manner by mixing with a pharmaceutically acceptable carrier or diluent. Unit dosage forms contain, for example, from about 0.0025 mg to about 50 mg of active substance.

Topical administration is e.g. to the skin. A further form of topical administration is to the eye, for the treatment of immune-mediated conditions of the eye, such as: auto-immune diseases, e.g. uveitis, keratoplasty and chronic keratitis; allergic conditions, e.g. vernal conjunctivitis; inflammatory conditions and corneal transplants, by the topical administration to the eye surface of a compound of the invention in a pharmaceutically acceptable ophthalmic vehicle. -46- 900-9581 The ophthalmic vehicle is such that the compound is maintained in contact with the ocular surface for a sufficient time period to allow the compound to penetrate the corneal and internal regions of the eye, e.g. the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary, lens, choroid/retina and sclera.

The pharmaceutically acceptable ophthalmic vehicle may be e.g. an ointment, vegetable oil, or an encapsulating material.

Whilst the antiinflammatory and immunosuppressant and antiproliferative activity is the main activity of the compounds of the invention they also possesses some degree of activity in increasing sensitivity to, or in increasing the efficacy of, chemotherapeutic drug therapy.

This activity may e.g. be determined according to the test methods described in EP 360760.

The compounds of the invention are therefore indicated for use in reversing chemotherapeutic drug resistance of varying types, e.g. acquired or innate, or in increasing sensitivity to administered drug therapy, e.g. as a means of reducing regular chemotherapeutic dosage levels, for example in the case of anti-neoplastic or cytostatic drug therapy, as a means of decreasing overall drug toxicity and, more especially, as a means of reversing or reducing resistance, including both inherent and acquired resistance, to chemotherapy.

' ' Preferred in the above indications are the following compounds of :the invention: - 29-des-(4-hydroxy-3-methoxycyclohexyl)-29-(3-formylcyclopentyl)-FR 520 (compound of Example 12); and - 33-epi-33-chloro-FR 520 (compound of Example 66a).

Claims (1)

1. -47- 900-9581 C L A I M S 96268/4 -48- 900-9581 y is 96268. -49- 900-9581 4. A compound according to claim 1 which is a compound Ιρχ , i.e. a compound of formula I wherein Rx is a group (a) wherein Rg is methoxy and either R5 is chloro or bromo and R4 is hydroxy and there is a single bond in 10,11 position or R5 is azido and R4 is hydroxy and there is a single bond in 10,11 position or absent and there is a double bond in 10,11 position; R. is optionally protected hydroxy and there is a single or a double bond in 23,24 position; and R3 is as defined in claim 1; in free form or, where such forms exist, in salt form. 5. A compound according to claim 1 which is a compound Ip3 , i.e. a compound of formula I wherein Rx is a group (b) wherein Rg is methoxy, R. is optionally protected hydroxy and there is a single bond in 23,24 position; or absent and there is a double bond in 23,24 position; R is hydroxy and there is a single bond in 10,11 position; and R3 is as defined in claim 1; in free form or, where such forms exist, in salt form. 6. A compound according to claim 1 which is a compound Ip4 , i.e. a compound of formula I wherein Rx is a group (d), R. is optionally protected hydroxy and there is a single bond in 23,24 position; or absent and there is a double bond in 23,24 position; R4 is hydroxy and there is a single bond in 10,11 position; and R3 is as defined in claim 1; in free form or, where such forms exist, in salt form. 7. A compound according to claim 1 of formula I wherein K1 is a group (a) wherein R5 is as defined in claim 1 and Rg is methoxy; R2 is optionally protected hydroxy and there is a single bond in 23,24 position; R4 is hydroxy and there is a single bond in 10,11 position; or absent and there is a double bond in 10,11 position; and 96268/ -50- 900-9581 R3 is ethyl or allyl; in free form or, where such forms exist, in salt form. 8. A compound according to claim 1 of formula I wherein Rx is a group (b) wherein Rg is methoxy; R. is optionally protected hydroxy and there is a single bond in 23,24 position; or absent and there is a double bond in 23,24 position; R4 is hydroxy and there is a single bond in 10 , 11 position; and R3 is ethyl or allyl; in free form or, where such forms exist, in salt form. 9. A compound according to claim 1 of formula I wherein Rx is a group (d) , R2 is optionally protected hydroxy and there is a single bond in 23,24 position; or absent and there is a double bond in 23,24 position; R4 is hydroxy and there is a single bond in 10 , 11 position; and R3 is ethyl or allyl; in free form or, where such forms exist, in salt form. 10. A compound according to claim 1 which is a compound Iq, i.e. a compound of formula I wherein either R1 is a group (a) wherein Rg is chloro, bromo, iodo or azido and Rfi is hydroxy or methoxy, R2 is oxo and there is a single bond in 23,24 position; optionally protected hydroxy and there is a single or a double bond in 23,24 position; or absent and there is a double bond in 23,24 position; and R4 is hydroxy and there is a single bond in 10 , 11 position; or R4 is absent and there is a double bond in 10 , 11 position; or Rx is a group (b) wherein Rg is hydroxy or methoxy or (d) R2 is as defined above in this claim; and R4 is hydroxy and there is a single bond in 10 , 11 position; and R. is methyl, ethyl, n-propyl or allyl; in free form or, where such forms exist, in salt form. 96268. -51- 900-9581 11. A compound according to claim 1 which is a compound Ir, i.e. a compound of formula I wherein either Rx is a group (a) as defined in claim 1; and R2 and R4 have the significance indicated in claim 1 under group (a); or Rx is a group (b) or (d) as defined in claim 1; and R, 2 and R4„ have the significance indicated in claim 1 under groups (b) and (d); and R. is as defined in claim 1; in free form or, where such forms exist, in salt form. 12. A compound according to claim 11 wherein R∑ is other than absent. compound according to claim 1 which is a compound of formula Is wherein 96268/4 -52- 900-9581 either Rls is a group (a) wherein R5 is chloro, bromo, iodo or azido and R6 is methoxy; R2a is hydroxy optionally protected by tert-butyldimethylsilyl and there is a single bond in 23,24 position; and R. is hydroxy and there is a single bond in 10,11 position; or Rfle. absent and there is a double bond in 10,11 position; or Rls is a group (b) wherein Rg is methoxy, or a group (d); R2s is hydroxy optionally protected by tert-butyldimethylsilyloxy and there is a single bond in 23,24 position; or absent and there is a double bond in 23,24 position; and R4s is hydroxy and there is a single bond in 10,11 position; and R3s is ethyl or allyl; in free form or, where such forms exist, in salt form. 14. A compound according to claim 1 wherein Rx is a group (a). 15. A compound according to claim 1 wherein Rx is a group (b). 16. A compound according to claim 1 wherein Rx is a group (d). 17. A compound according to claim 1 wherein R. is unprotected hydroxy and there is a single bond in 23,24 position. 18. A compound according to claim 1 wherein R3 is ethyl. 19. A compound according to claim 1 wherein R is allyl. 20. A compound according to claim 1 wherein R 4, is hydroxy, 21. A compound according to claim 1 wherein R is chloro. : XL.90 22. A compound according to claim 1 wherein R 6, is hydroxy. 23. A compound according to claim 1 wherein protected hydroxy is OtBDMS. 24. A compound according to claim 1 wherein Rg is methoxy. 96268/4 -53- 900-9581 25. A compound according to claim 24 wherein is a group (a), R. is OtBDMS and there is a single bond in 23,24 position, R4 is hydroxy and there is a single bond in 10,11 position and either R5 is chloro, azido or 7. XI.90 bromo and R. is allyl or R5 is chloro, bromo, azido or iodo and R3 is ethyl. 26. A compound according to claim 24 wherein R1 is a group (b), R4 is hydroxy and there is a single bond in 10,11 position, R. is ethyl or allyl and either R2 is OtBDMS or hydroxy and there is a single bond in 23,24 position or R2 is absent and there is a double bond in 23,24 position. 27. A compound according to claim 1 wherein Rx is a group (d), R4 is hydroxy and there is a single bond in 10,11 position, R3 is ethyl or allyl and either R2 is hydroxy and there is a single bond in 23,24 position or R. is absent and there is a double bond in 23,24 position. 28. A compound according to claim 1 wherein Ri is a group (d), R. is hydroxy and there is a single bond in 23,24 position, R3 is ethyl and R4 is hydroxy and there is a single bond in 10,11 position. 29. A compound according to claim 24 wherein R^^ is a group (a) wherein Rg is chloro, bromo or azido, R2 is hydroxy and there is a single bond in 23,24 position, R. is allyl or ethyl and either R4 is hydroxy and there is a single bond in 10,11 position or R4 is absent and there is a double bond in 10,11 position. 30. The compound according to claim 24 wherein R5 is chloro, R3 is ethyl and R4 is hydroxy and there is a single bond in 10,11 position (33-epi-33-chloro-FR 520) (compound of Example 66a). 31. The compound according to claim 1 wherein R1 is a group (d), R2 is OtBDMS and there is a single bond in 23,24 position, R3 is ethyl and R4 is hydroxy and there is a single bond in 10,11 position. 96268/4 -54- 900-9581 ■7. XI.90 32. A process for the preparation of a compound according to claim 1 comprising except a) for the preparation of a compound of formula I wherein for the R: is a group (a) as defined in claim 1, compounds of claims R2 and R3 are as defined in claim 1 and 4,5 and 6 R4 is hydroxy (i.e. a compound la), replacing under simultaneous epimerization the hydroxy group by chlorine, bromine, iodine or azido in a corresponding compound having unprotected hydroxy in 33 position (i.e. a compound Ila, of formula Ila 0CH3 wherein 2 and R3 are as defined in claim 1 under formula I and R6 is hydroxy or methoxy); 96268/4 -55- 900-9581 b) for the preparation of a compound of formula I wherein . XI.90 Rx is a group (b) as defined in claim 1, xcept R2 and R3 are as defined in claim 1 and or the R4 is hydroxy ompounds (i.e. a compound lb), f claims treating a corresponding compound Ila with cyanogen bromide in the ,5 and 6 presence of a base or treating a corresponding compound Ila with thiophosgene, reacting the resultant product with an inorganic azide and allowing the resultant unstable intermediate having a group N N C 0 II I in 33 position (i.e. a compound lib) N S to decompose to a corresponding compound lb; c) for the preparation of a compound of formula I wherein Rx is a group (d) as defined in claim 1, R2 and R3 are as defined in claim 1 and 4 is hydroxy (i.e. a compound Ic) , treating a corresponding compound lb with an acid having a non-nucleophilic anion; and - when a resultant compound of formula I has a protected hydroxy group, optionally splitting off the protecting group(s) to give a corresponding compound of formula I having one or more unprotected hydroxy group(s) (i.e. a compound Ij ) , whereby when R1 is a group (a), a water molecule may be simultaneously split off and a compound of formula I is obtained wherein Rx is a group (a) as defined in claim 1, R2 is unprotected hydroxy and there is a single or double bond in 23,24 position; and R4 is absent and there is a double bond in 10,11 position (i.e. a compound Ii); or 96268/ -56- 900-9581 - optionally protecting an unprotected hydroxy group in a resultant 7. XI.90 compound of formula I as appropriate to give a corresponding except compound of formula I having one or more protected hydroxy groups(s) for- the (i.e. a compound Ik) ; compounds of claims and recovering the resultant compound of formula I in free form and, where 4,5 and 6 such forms exist, in salt form. 33. A pharmaceutical composition comprising a compound according to any one of claims 1 to 31 in free form or, where such forms exist, in pharmaceutically acceptable salt form, together with a pharmaceutically acceptable carrier or diluent. 34. Use of a compound according to any one of claims 1 to 31 in the preparation of a pharmaceutical composition by mixing with a pharmaceutically acceptable carrier or diluent. 35. Process for the preparation of a pharmaceutical composition comprising mixing a compound according to any one of claims 1 to 31 in free form or, where such forms exist, in pharmaceutically acceptable salt form, with a pharmaceutically acceptable carrier or diluent. For th jplicants, 6300/VA5732