CA2065425A1

CA2065425A1 - Macrocyclic compounds and novel method of treatment

Info

Publication number: CA2065425A1
Application number: CA 2065425
Authority: CA
Inventors: David K. Donald; Mark Furber; David N. Hardern; Paul Leff
Original assignee: Individual
Current assignee: Fisons Ltd
Priority date: 1989-09-14
Filing date: 1990-09-13
Publication date: 1991-03-15

Abstract

Compounds of formula (I), wherein R1 and R2 independently represent H or OH, or a second carbon-carbon bond; R3 represents optionally substituted methyl, ethyl or propyl; R4 represents H;
R5 and R6 represent a second carbon-carbon bond; R7 represents H or OH; R8 represents OCH3; R9 represents OH or OCH3; X and Y independently represent O or (H, OH); n represents 1 or 2; and in addition some of the substituents form rings with each other; with various provisos; are indicated in the treatment of immunodepression. A number of novel compounds of formula (I) are also provided.

Description

WO 91/04025 PCl /G B90/0 1412 2 ~ 65 ~2 i NOVEL MACROCYCLIC COMPOUNDS AND NOVEL METHOD OF TREATMENT
This invention relates to novel pharmaceutical uses of certain known macrocyclic compounds, and to novel - macrocyclic compounds which have the same novel utility.
European patent application No 184162 (to Fujisawa Pharmaceuticals Co Ltd) discloses several raacrocyclic compounds which are derivatives of 12-(2-cyclohexyl-l-methylvinyl)-13,19,21,27-tetr~methyl-11,28-dioxa-4-azatricyclo[22.3.1.04~9]octacos-18-ene (numbered 1~ FR-900506, FR-900520, FR-900523 and FR-900525) and which are isolated from microorganisms belonging to the genus Strep~omvces. The macrocyclic compounds and a numbPr of their derivatives are indicated as immunosuppressive agents.

International patent application No WO 89/05304 (also European Patent application No 323042, both to Fisons plc) discloses a large number of macrocyclic compounds which may ' :
be derived from those disclosed in European patent application No 184162. Again, the compounds are primarily 20 indicated as immunosuppressive agents.
European patent applications Nos 349049 and 349061 (both to Merck & Co Inc) each disclose a macrocyclic compound which is indicated as an immunosuppressive agent.
The compounds and methods disclosed in the patent 25 applications mentioned above may be used in the production of the novel compounds of the pr~osent invention.
Alternatively, the novel compounds may be produced by total synthesis.

.. ~ . ; : - . ~ :
t ', ' ' ' " ' '' .' ' ~ '. .' ', ' ' ' ' ` . . i " '' , ' ': ' ` '~ , : ' ~`

2 ~ - 2 - --, We have now found a novel group of macxocyclic compounds which act as antagonists of immunosuppressive compounds, particularly macrocyclic immunosuppressive compounds including derivatives of 5 12-(2-cyclohexyl-1-methylvinyl)--13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo~22.3.:1.04~9]octacos-1~-ene and rapamycin, as shown in the mixed lymphocyte reaction (MLR) (des~ribed in WO 89/05304, Exampl~ A). The novel group of compounds are therefore useful inter alia in the treatment 10 f immunodepression or. a._ disorder involving immunodepression.
Thus, according to the present invention, there is provided the use of a compound of formula I, H0~
fl9~ C~3 R2 (CH23~CH3~Y

N ~ ~R~ R
o~z5X R7~¦~R6 CH3,~<0H

I I ~ CH~

- CH3l1 3 "~
wherein Rl and R2 independently represent H or OH, or they may together represent a second carbon-carbon bond between :: . .
. . .
,. ~
', ' . ' . ~ ; ' ~ ' ." "'-., , ' , ' - : . .: .. , ,: ~. : : : , ~
.. . : .. . : -2a6~2~ 1 the carbon atoms to which they are attached;
R3 represents methyl optionally substitutPd by -CO2H or an ester or amide thereof; ethyl optionally substituted by O, OH or -CO2H or an ester or amide 5 thereof; propyl optionally substituted by OH or O; or allyl optionally substituted by OH;
R4 represents H;
R5 and R6 together represent a second carbon-carbon bond between the carbon atoms to which they 10 are attached; . _ R7 represents H or OH;
R8 repre~ents OCH3;
R9 represents OH or OCH3;
X represents O or (H,OH), Y represents O or (H,OH); and n represents 1 or 2;
in addition to their significances above Rl and R5 may together represent an oxygen atom, in which case R6 and R7 together rPpresent a second 20 carbon-carbon bond between the carbon atoms to which they are attached;
R7 and R8 may together represent an oxygen atom;
and R3, R4 and Y, together with the carbon atoms to which they are attached, may represent a methyl-substituted furanyl ring:
provided that i) when R2 represents H; R3 represents methyl, ethyl, . . ~ . . ,. : :
:: . '' '~ -' '':.. ~ .;.: , , ~ ,.. ,. , ':

WO 91/041)25 PCI/GB90/01412 2 ~ 2 5 propyl or allyl; R5 and R6 together represent a second carbon-carbon bond between the carbon atoms to which they are attached; R8 represents OCH3; and Y represents O;
then R7 represents OH; and 5 ii) when n is 1, then R3 is not methyl or ethyl;
in the manufacture of a medicament for the treatment of immunodepression or a disorder involving immunodepression.
Examples of disorders involving immunodepression ~ 10 include AIDS, cancer, senile dementia,~~trauma (including wound healing, surgery and shock), chronic bacterial infection, and certain central nervous system disorders.
The immunodepression to be treated may be caused by an overdose of an immunosuppressive macrocyclic compound, for 1~ example derivatives of 12-(2-cyclohexyl-1-methylvinyl)-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.04~9]octacos-18-ene such as FR-900506, or rapamycin. Overdosing of such medicaments by patients is quite co~mon upon their realising that they have forgotten 20 to tak2 their medication at the prescribed time, and can lead to serious side effects.
A further situation in which the compounds of formula I may be used to treat immunodepression is in vaccination.
It is sometimas found that the antigen introduced into the 25 body for the acquisition of immunity from disease acts as an immunosu;ppressive agent, and so antibodies are not produced by the body and immunity is not acquired. By introducing a compound of formula I into the body (for WO91/0402~ PCT/~B90/01412 - 5 - 2 0 65~

example in the vaccine) the und2sired immunosuppression may be overcome and immunity acquired.
The pr~sent invention further provides the novel compounds of formula I, as defined above, provided that 5 i) when Rl represents OH; R2 represents H; R5 and R6 together represent a sPcond carbon-c~rbon bond between the carbon atoms to which they are attached; R7 represents H; R9 represents OCH3; X and Y each represent O; and n represents 2; then R3 does not 10 represent 2-oxopropyl, 2,3-dihydroxypropyl or ethanalyl;
ii) when Rl rPpresents OH; R2 repres~nts H; R5 and R6 tosether repr~sent a second carbon-carbon ~ond between the carbon atoms to which they are attached; R7 represents OH; R9 represents OCH3; X and Y each 15 rePresent o and n represents 2; then R3 does not represent allyl or l-hydroxyprop-2-enyl; and iii) when Rl represents OH; R2 represents H; R~ and R6 together represent a s~cond carbon-carbon bond between the carbon atoms to which they are attached; R7 repre~ents H: R9 represents OCH3; X and Y each represent (H,OH); and n represents 2; then R3 does not represe~t allyl.
In the novel use or the novel compounds, we prefer R3 to be ethyl substituted by O or propyl substituted by 25 o.
Desirab].y, R7 is OH.
We prefer R2 to be OH, more preferably (S)-OH (ie to have S absolute stereochemistry at its point o~ ~ttachment :: -;.

WO91/04025 PCT/GB90/0l4l2 2~5~2~ - 6 -. to the molecule).
When R3 comprises an ester or amide group, we prefer the alcohol or amine moiety to contain from 1 ~o 10 carbon atoms, for example the alcohol moiety may be methanol.
Known compounds of formula I (as first defined a~ove) from WO 89/05304 include:
17-Allyl-1,14,20-trihydro~-12-[2-(4-hydroxy-3-methoxycyclohexyl)-l-methylviny~1]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo -- -10 [22.3.1.04~9]octacos-18-ene-2,3,10,16-tetraone, ~
17-(1-Hydroxyprop-2-enyl)-1,14,20-trihydroxy-12-[2-(4-hydroxy-3-mPthoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.04~9]octacos-18-ene-2,3,10,16-tetraone, 17-(2,3-Dihydroxypropyl)-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo ~22~3.1.04~9]octacos-18-ene-2,3,10,16-tetraone, 17-Ethanalyl-1,14-dihydroxy-12-[2~(4-hydroxy-3-20 methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.04'9]octacos-18-ene 2,3,10,16-tetraone, 17-(2-Oxopropyl)-1,14-dihydroxy-12-~2-t4-hydroxy-3-methoxycyclohexyl~ ethylvinyl]-23,25-dimethoxy-25 13,19,21,27-tetramethyl-11,28-dioxa-4 azatricyclo [22.3.1.04~9]octacos-18-ene-2,3,10,16-tetraone, 17-~lly:l-1,2,14,16-tetrahydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy-WO91/040~5 PCT/GB90/01412 _ 7 - 2 a ~

13,l9,21,27-tetramethyl-'1,28-dioxa-4-azatricyclo ~22.3.l.04~9]octacos-18-ene-3,lO dione.
According to the inv~ntion there is further provided a process for the production of a novel compound of formula 5 I, which comprisas:
a) producing a compound of formula I, in which R3 represents propyl substitutecl by O, by oxidation of a corresponding compound in which R3 rDpresents allyl;
b) producing a compound of formula I, which contains a ;icinal diol, by oxidation of a carbon-carbon double bond in a corresponding compound;
c) producing a compound of for~ula I, in which R3 represents ethyl substituted by O, by oxidative cleavage o~
a corresponding compound in which R3 represents 15 2,3-dihydroxypropyl;
d) producing a compound of ~ormula I, in which R3 represents methyl substituted by -CO2H or ethyl substituted by -CO2H, by oxidation o a corresponding compound in whi~h R3 represents ethanalyl or propanalyl;

20 ~) producing a compound of formula I, which contains two vicinal hydro~en atoms, by reduction o~ a corresponding compound which contains a carbon-carbon double bond;
f) producing a compound of ~ormula I, in which X or Y
represents (H,O~), by reduction of a corresponding compound 25 in which X or Y represents O;
g) producing a compound of formula I, in which R3, R4 and Y, together with the carbon atoms to which they are at.'ached, represent a methyl~substituted furanyl ring, by .:
- : . ,,:

~ WO91/04025 PCT/GB90/01412 206~25 - 8 - ~

the action o~ acid on a corresponding compound in which R3 repr~sents 2-oxopropyl, R4 represents H and Y
represents O;
h) producing a compound of formula I, in which Rl and 5 R~ together represent an oxygen atom and R6 and R7 together represent a second c,arbon-carbon bond between the carbon atoms to which they arP attached, by the action of acid on a corresponding compound in which Rl represents OH, R5 and R6 together represent a second carbon-carbon l0 bond between the carbon atoms to which they are attached, and R7 represents OH;
i) producing a compound of formula I, in which R7 and R8 together represent an oxygen atom, by the action of a dehydrating agent on a corresponding compound of formula I
lS in which R7 represents OH and R8 represents OCH3;
j) producing a compound of formula I, in which R7 represents OH, by allylic oxidation of a corresponding compound in which R7 represents H; or X) producing a compound of formula I, in which R3 20 represents allyl substituted by hydroxy, by allylic oxidation of a corresponding compound in which R3 represents allyl.
Esters and amides of carboxylic acids that R3 may represent may be produced by conventional methods.

Where clesired or necessary, hydroxy groups may ~e protected and deprotected using conventional protecting group chemistry [as described in "Protective Groups in Organic Chemistry", ed: J W F McOmie, Plenum Press (1973), - f . ::: , . .., . ~ . . . ..
.. ..

2 ~

and "Protective Groups in Organic Synthesis", T W Greene, Wiley-Interscience (1981)]. In addition, European patent application No 184162 describPs the use of protecting groups in macrocyclic compounds.
In process (a), suitable oxidizing agents include a palladium (II) halide, for example palladium (II) chloride, in conjunction with a cuprous halide, ~or example copper ~I) chloride. Suitable solvents include those that do not adversely affect the reaction, for example io ~dimethylformamide (DMF) and- water. The reaction is preferably carried out at a temperature of from 0 to 100 C, more preferably at or around room temperature.
In process (b), suitable oxidizing agents include osmium tetroxide, potassium permanganate, and iodine in 15 conjunction with silver acetate. Osmium tetroxide is preferably used in conjunction with a regenerating agent such as 4-methylmorpholine N-oxide. Suitable solvents includ2 those that do not adversely affect the reaction, for example diethyl ether or tetrahydrofuran (THF). In the 20 case of potassium permanganate, aqueous alkaline conditions are preferred. The reactisn is preferably carried out at a temperature of from 0 to 100-C, more preferably at or around room temperature.
In process (c), suitable reagents include lead 25 tetraacetate and phenyliodoso ac2tate. Suitable solvents include those that do not adversely affect the reaction, for example benzene and glacial acetic acid. The reaction is preferably carried out at a temperature of from 0 to ::

WO91/040~S PCT/~B90/01412 20~r!4~ - 10-100C, more preferably at or around room temperature.
In process (d), suitable oxidi~ing agents includesodium chlorite in conjunction with sodium hydrogen phosphate. Suitable solvents include those that do not 5 adversely affect the reaction, for example water. The rPaction is preferably carried out at a temperat11re of from 0 to l001C, more preferably at or around room temperature.
In process (e), the re.duction may be carries out catalytically using hydrogen. Suitable catalysts include l0 platinum catalysts (~or example platinum black) and palladium catalysts (for example palladium-on-carbon).
Suitable solvents include those that do not adversely affect the reaction, for example methanol and ethanol. The reaction may be carried out at or around room temperature.

In process (f), suitable reducing agents include borane (for example in the form of borane-a~monia complex) and sodium borohydride. Suitable solvents include those that do not adversely affect the reaction, for example diethyl ether and dichloro~ethane. The reaction may b~
20 carried out at or aro1~nd room temperature. Where desired or necessary, the (H,OH~ group may be oxidized back to O by the action of copper (II) acetate in acetic acid.
In processes (g) and (h), suitable acids include p-toluenesulphonic acid. Suitable solvents include those 25 that do not adversely affect the reaction, for example toluene. The reaction is preferably carried out at a temperature above room temperature, ~or example on a steam bath.

.

WO gl/0402~ PCltGB90/01412 - 11 - 2~ 12~

- In process (i), suitable reagents include Martin's sulphurane reagent. Suitabl,e solvents include those that do not adversely affect the reaction, for example dichloromethane. The reaction is preferably carried out at 5 a temperature below room temperature, for example -30 C.
In processes (j) and (k), suitable reagents include SeO2, preferably in the presence of tbutyl hydrogen peroxide. Suitable solvents include dichloromethane, and the reaction may be carried out at or around room -10 -temperature.
The invention further provides ~he use of the novel compounds cf formula I as pharmaceuticals, and a pharmaceutical composition comprising such a compound in association with a pharmaceutically acceptable adjuvant, 15 diluent or carrier.
For the above-mentioned therapeutic uses the dosage administered will, of course, vary with the compound employed, the mode of administration, the treatment desired (eg topical, parenteral or oral) and the disease indicated.
2~ However, in general, satisfactory results are obtained when the compounds are administered at a dosage of from 0.1 to 200mg per kg of animal body weight.
For man the indicated total daily dosage is in the range of ~rom lmg to lOOmg and preferably from lOmg to 25 SOOmg, which may be administered, for example twice weekly, or in divided doses from 1 to 6 times a day or in sustained release form. Thus unit dosage forms suitable for administration, eg oesophageally, comprise ~rom 2mg to .. , .. .. . -, wosl~o4o25 PCT/GB90/01412 2~ 6~ 42 ~ - 12 - `

SOOmg, and preferably lmg to 500mg of the compound preferably admixed with a sol:id or liquid phar~aceutically acceptable diluent, carrier or ~djuvant.
Suitable pharmaceutical compositions for 5 administration of compounds of formula I (as first defined above) comprise (preferably less than 80~, and more preferably less than 50~ by weight) of a compound of formula I (as ~irst defined above) in combination with a pharmaceutically acceptable adjuvant, diluent or carrier.
l~ Examples of suitable adjuvants, diluents or carriers are:
for tablets, capsules and dragees - microcrystalline cellulose, calcium phosphate, diatomaceous earth, a sugar such as lactose, dextrose or mannitol, talc, stearic acid, starch, sodium bicarbonate and/or gelatin; for 15 suppositories - natural or hardened oils or waxes; and for inhalation compositions - coarse lactose. The compound of formula I (as first defined above) is preferably in a form having a mass median diameter of from O.Ol to lO microns.
The compositions may also contain suitable preserving, 20 stabilising and wetting agents, solubilisers, sweetening and colouring agents and flavourings. The compositions may, if desired, ba formulated in sustained rel~ase form.
We prefer compositions which are designad to be taken oesophageally and to release their contents in the 25 gastrointestinal tract.
We have also found that tAe toxicity of immuno~uppreC;sive compounds, particularly i~munosuppressive macrocyclic compounds including derivatives of .: ; ' ,, ;: ~ ............ .'. ,: , ; ' ,-, , , .. ..

WO91/040~5 PCT/~B90/01412 - 13 - 2 Q ~

l2-(2-cyclohexyl-l-methylvinyl)-l3,l9,2l,27-tetramethyl-ll,28-dioxa-4-a~atricyclo[22.3.l.04~9]octacos-18-ene (for example FR-900506) and rapamycin, may be reduced by administering them in association with a compound of 5 formula I as first defined above.
Thus, according to a second aspect of the invention, there is provided a pharmaceutical mixture comprising a compound of formula I (as first defined above) and an immunosuppressive compound.
la Preferably, -- the greater proportion of active ingredient in such a mixture is the compound of formula I, for example the compound of formula I may be present at a ratio of greater than lO:l by weight, for example 99:l.
The invention also provides a method of treatment of 15 immunodepression or a disorder involvin~ immunodepression which comprises administaring a therapeutically efficacious ar~ount of a compound of fo~mula I, as first defined a~ove, t. ?. patient suffering from such a condition.
The compounds of formula I ~as first defined have the 20 advantage that they are less toxic, more efficacious, are longer acting, have a broader rangP of activity, are more potent, produce fewer side effects, are more easily a~sorbed or have other useful pharmacological propertles, than compounds previously us~d in the therapeutic fields 25 m~ntioned above.
The coMpounds of formula I (as ~irst defined above) have a n ~er of chiral centres and may exist in a variety of stereoisomers. The invention provides the use of all W091/0402s PCT/GB90/01412 20~5~21~

optical and stereoisomers, as well as racemic mixtures, andall optical and stereoisomers of the novel compounds of formula I per se.
However, the preferred stereochemistry of various 5 chiral carbon atoms are shown in formula Ia, H0~

R9 ~~ CH~ R2 _ _ _ _ _ (CH2~CH~ 1~Y

N--1-- R~ R3 ~ x R7 ~ cRH3 Ia CH3~ ) ""CH
/

wherein Rl to R8, X, Y and n are as de~ined above.
The invention is illustr~ted by the following 20 examples.
Example 1 17-t2-Oxopropvl)-1-h~droxy-12-~2-(4-hydroxv-3-methoxYcvclohexyl)-l-methvlvinvll-23.25-dimethoxv-13.19,21,27-tetramethYl-11,28-dioxa-4-azatricyclo r 22.3.1.04~9loctacos-18-ene-2.3.~0~16-tetraone a) ll-L2-Oxoeropv~ -hvdroxy-l2- r 2-14-hydroxy-3-methoxycvclohexvl)-l-methylvinYl1-23.25-dimethoxy-13.19,2L.27-tetramethvl-11.28-dioxa-4-azatricyclo '` ' : ' . , ., ' ' : ~ . . ' ' :~
:- .: ~ . ~ . : . .. . .

WO91~04075 PCT/GB90/0l4l2 - 15 - 2 ~5i~

r 22.3.1.04~910ctacosa 14.18-diene-2.3~10 16-tetraone To a solution of palladium (II) chloride (25mg) and copper (I) chloride (50mg) in DMF (dimethylformamide) (6ml) and water (lml~ which had been previously oxygenated by having 5 air bubbled through it for 30 minutes at room temperature was added a solution of 17-allyl-1-hydroxy~ [2~
hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.04~9]octacosa-14,18-diene-2,3,10,16-tetraone (the - 10 compound of Example~ 4-, W0-89/05304) (lOOmg) in DMF (2ml).
The reaction mixture was then stirred and oxygenated for 3 hours at room temperature after which it was diluted with diethyl ether. The organic extract was then washed with dilute aqueous hydrochloric acid (lM) and brine before 15 being dried (MgS04), filtered and evaporated to give the subtitle compound as an oil.
b) 17-(2-OxoproPyl)-l-hydroxy-12-~2-(4-hYdroxy-3-m~thoxycYclohexyl~ methvlvinYl!-23~25-dimeth 13.19,21,27-tetramethyl-11,28-dioxa-4-azatricYclo 20 r 22.3.1.04~91octacos-18-ene-2~3 10~1_-tetraone The crude product of step ~a) was then re-dissolved in dry methanol and was stirred with 10~ Pd-on-carbon (lOmg) under an atmo~phere of hydrogen for 4 hours. The reaction mixture wa~ then filtered, concentrated in vacuo and 25 chromatographed on silica eluting with acetone~hexane [2:3]
to give the title compound as a foam (84mg).

MS ~FAB): 889 [M+Rb]+: 827 [M+Na]+; 805 [M+H]+; 787 ~M-OH]+

.. . . . .

;,; :; ~
. : . . . ::: . ~ : : :: . : .
.:. . : :;
: ~ : , :. :

WO~1/04025 PCT/GB90/01412 2~5~2~ - 16 -3C N~R (CDCl~ 211.1 (C16); 207 (C41); 196.3 (C2); 169.1 (C10); 166.1 (~3); 138.7 (C19). 132 (C31); 131 (C29); 121.8 (C18); 97.3 (Cl); 84 tc34); 82.4 (C12); 75.1 (C23); 56.2 (C9); 48.9 (C20); 4'7.6 (C17); 13.3 (C39) Example 2 2-~1,14-Dihy~roxy-12 r 2-(4-hvdroxy-3-methoxycvclohexylL-l-methvlvinY11-23.25-dimethoxv-13,19,21,27-~etrameth~1-11,28-dioxa-4-azatricvclol22.3.1.04~9loctacos-18-ene-2,3.l0,16-tetraone-17-yl~-ethanoic acid solution of FR-900506 (1.2g), 4-methylmorpholine N-oxide (1.3g) and osmium tetroxide (0.7ml of a 4% solution in water) in THF (tetrahydrofuran) (25ml) and water (14ml) was stirred for 3.5 hours at room temperature. Solid sodium metàbisulphite was then added followed by ethyl acetate and Florisil (registered trade mark) and the combined mixture was filtered through celite. The separated organic extract (after washing with saturated aqueous sodium hydrogen carbonate solution~ was dried (MgS04), ~iltered and evaporated in v~cuo to give the crude 17-(2,3-dihydroxyprOpyl) compound as an oil. This was thendissolved in benzene (40ml~ and lead tetraacetate (1.46g) was added. A~ter stirring ~or 3 minutes at room temperature the reaction mixture was diluted with diethyl ether and was filtered, concentrated in Yacuo, redissolved in diethyl ether, re-filtered and re-concentrated in vacuo to give the crude 17-(ethanalyl) compound as an oil. This was then dissolved in tbutanol (25ml) and l-methyl cyclohex-l-ene (6ml) and to this was added portionwise over :- ;, :.. . , ,. : :. ~ -- ... ,. : .., . ;, ..
,, .: . .: -,, , .. :
., . : . ., -, ,; .

WO91/0402S PCT/GB~Otnl412 - 17 - 2~ 25 minutes solutions of sodium chlorite (0.8g) and sodium hydrogen phosphate (0.81g) each dissolved in 5ml of water.
After stirring ~or 0.5 hour~s at room temperature the reaction mixture was quenche~d by the addition of ethyl 5 acetate. Dilute aqueous hydrochloric acid (2M) was then added and the organic extracts (after washing with saturated aqueous sodium hydrogen car~onate solution) were dried (MgSO4), filtered and evaporated to an oil in vacuo. Chromatography on silica eluting with diethyl 10 ether/methanolJacetic acid - -[l90:9:1]---and then further chromatography on silica eluting with dichloromethane/
methanol/acetic acid [170:9:1] gave the title compound as a foam (402mg, 32~).
MS (FAB): 906 [M+Rb~+
1~ 13C N~R (CDC13) ~: 196.3 (C2); 177.7(C41); 168.8 (C10); 164.6 (C3); 139~7 (Cl9); 132.7 (C29~; 129.3 (C31);
120.6 (C18); 97.3 (Cl); 84.2 ~C3~); 70.5 (C14); 9.8 (C39) Exam~le 3 2- U~14-DihYdroxv-12- r 2-(~-hydroxy-3-methoxyc~clohex~l)-1-methylvinyll-23.25-dimethoxv-13.19,21,27-tetrameth~l~ 28-dioxa-4-azatricYclo r 22.3.1.04'91Octacos-18-ene-2 3 L 10 ~16-tetraon~.-17-vl~-ethanoic acid Methyl ester To a solution of the title compound of Example 2 (50mg) in dichloromethane (3ml) at room temperature was added a 25 solution of diazomethane in diethyl ether until no starting material remained. The reaction mixture was then concentrated in vacuo and chromatographed on silica eluting with acetone/hexane [1:2J to giYe the title compound as a WO91/0402~ PCT/GB90/0l412 2~4~ - 18 -foam (48mg).
MS (FAB): 920 [~+Rb]+; 858 [M+Na]+; 818 [M-OH]*
3C NM~ (CDC13) ~: 212.7 (C16); 196 (C2); 173 (C41);
168.8 (C10); 164.6 (C3); 1-~9.5 (C19); 132.9 (C29); 129.1 5 (C31); 120.6 ~C18); 97.2 (Cl); R4.2 (C34); 70.8 (C14); 14.5 (C30); 9.7 (C39) Example 4 2-Ll.14-Dih ~ oxy-12-r2-(4-hYdroxy-3-methox ~clohexYl)-1-methylvin~ll-23,25-dimethoxy~l3,19,21,27-tetramethyl-11l28-10 dioxa-4-azatricyclo~22.3.1.04~91octacos-18-ene-2,3.10,16-tetraone-17-yll-ethanoic acid N-Morpholine amide A solution of the title compound of Example 2 (63.1mg), morpholine ~20mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (28mg) and 15 4-dimethylaminopyridine (2mg~ in dry dichloromethane (4ml) was stirred at room temperature for 2.5 hours. Water was then added a~d the reaction mixture was extracted with dichloromethane. Aftar washing with dilute aqueous hydrochloric acid (lM~ and saturated aqueous sodium 20 hydrogen carbonate solution, the dichloromethane extracts were dried (MgSO4), filtered and evaporated to an oil in vacuo. Chromatography on silica eluting with hexane/acetone [1:1] then gave the title c~mpound as a foam ~58mg, a4%)~
25 MS (FAB): 975 t~+Rb]~: 891 ~M~H]+; 873 [M-OH]+.
3C NMR (CD~13) ~: 213.2 (C16); 195.9 (C2); 170.1 (C41); 168.8 (C10); 164.8 (C3); 139.1 (C19); 133.2 (C29);
128.9 (C31); 121.5 (C18); 97.4 (C1): 84.2 (C34); 66.3 and ., ,, s, -:. . . -. , ~ :: ..
.. . . . ...
- : , : - ;~,. , ~ , .: .

:. - :. - . , : :. . .. , : :,;

... . ~: ,, . . :
- .: . . . ~ . ,: : : .

WO91/04025 PC~/GB90tO1412 - 19 - 2~ 2 65.8 (Co of morpholine); 10.1 (C39).
Example 5 2-(1.14-Dihydroxy 12- r 2-(4-hvdroxY-3-methoxyc~clohexYl)~
methylvinyll-23,25-dimethoxy-13"19~21,27-tetramethyl-11,28-5 dioxa-4-azatr cvclo~22.3.1.04~9loctacQs-18-ene-2,3.10,16-tetraone-17-vl~-ethanoic acid N--Ethanolamide A solutio~ of the title compolmd of Example 2 (22.8mg) and 1,3-dicyclohexyl carbodiimide (6.7mg) in dry dichloromethane (2ml) was stirred at room temperature for 5 10 minutes. Ethanolamine (lOmg) was then added and the reaction mixture was stirred at room temperature for 1.5 hours. Dilute aqueous hydrochloric acid (lM) was then added and the reaction mixture was extracted with dichloromethane. The organic extracts, after washing with 15 saturated aqueous sodium hydrogen carbonate solution, were dried (MgS04), filtered and ev~porated to an oil in vacuo. Chromatography on silica eluting with dichloromethane/methanol/acetic acid [140:9:1] then gave tha title co~pound as a glass ~19.4mg, 81S).

20 ~ (FAB): 950 [M+Rb]t; 888 [M+Na]~; 865 [M+H]+; 848 EM-O}I]+
3C NMR (CDC13) ~: 198.7 (C2); 173.8 (C41); 169.2 (C10); 165.6 (C3), 140 (Cl9); 133.2 (C29); 129 (C31); 122.4 (C18~; 97.8 (Cl) 84.2 (C34); 61.2 (CO of ethanolamine);
25 44-0 (CN o~ ethanolamine); 16.2 (C47); 15.8 (C43); 14.7 (C30); 9.7 (I:39) Example 2-(1.14-Dihvdro~Y-l2-~2-L4-h~droxy-3-methox~cyclohexyl) ;

methvlvinyll-23,~5-dimethox~-13,19,21,2?-tetramethyl-11,28-dioxa-4-azatricyclo r 22.3.1.04~91octacos-18-ene-2,3~10,16-tetraone-17-yl)-ethanoic acid amide with qlYcine methyl ester 5 To a solution of the title compound of Example 2 (43.6mg) and methyl glycinate (3mg) :in dichloromethane (3ml) was added triethylamine (22~1) and ;2-chloro-1-methylpyridinium tosylate (24.2mg). After stirring at room temperature for 15 minutes dilute aqueous hydrochloric acid - 10 (2M) was added and-the-reaction mixture was extracted with dichloromethane. The organic extracts, after washing with saturat~d a~ueous sodium hydrogen carbonate solution, were then dried (MgSO4), filtered and evaporated to an oil in vacuo. Chromatography on silica eluting with hexane/
15 acetone [1:1] then gave the title compound as a foam (34.lmg, 72%).
MS (FAB): 977 [M~Rb]+; 915 [~+Na]+; 893 [M+H]+; 875 ~M-OH]+
3C NMR (CDC13) ~: 213 (C16); 195.9 (C2); 171.7 ~0 (glycine carbonyl);170.3 (C41); 168.7 (C10); 164.7 (C3);
139.~ (Cl9); 132.9 (C2g); 128.9 (C31~; 120.9 (C18); 97.2 (Cl); 84.1 (C34); 72.4 (C24~; 7~.1 (C14); 52.4 (OC~3 of glycine); 9.9 (C39) ExamDle 7 25 2-~1.14-DihYdroxv~-12-r2-(4-hvdroxv~-3-methoxy~cvclohexY~ll-l-methylvinvll~23,25-dimetho ~-13,~9 21.27-tetramethY1-11,28-dioxa-4-azatricyclor22.3~1.04~9loctacos-18-ene-2.3,10.16-tetraone-17-yl~ethanoic acid N-Piperidine amide .. . . . . :
. .: ~ . :,. . : . .

- . - . ' ~ :: :: ' : ..

WO 91/0402~ PCr/CB90/01412 - 21 - 2~5~23 To a solution of 2-chloro-1-methylpyridinium tosylate (24.9mg) in dry dichloromethane (lml) under nitrogen at room temperature was added a solution of the title compound of Example 2 (42.3mg), piperidine (lOmg) and triethylamine (22~1) in dry dichlorometham~ (1.5ml). After 3 hours at room temperature a further portion of 2-chloro-1~
methylpyridinium tosylate (15mg) and triethylamine (15~1) was add~d and stirring was continued for a further two hours at room temperature. Water was then `-10 added and the reaction mixture was extracted with dichloromethane. After washing with dilute aqueous hydrochloric acid (lM) and saturated a~ueous sodium hydrogen carbonate solut~on, the dichloromethane extracts ~ere dried (MgSO4), filtered and evaporated to an oil in 15 vacuo. Chromatography on silica eluting with dichloromethane/methanol [29:1] then gave t~e title compound as a foam (13.3 mg, 29%).
~S (FAB): 973 [M+Rb~+; 911 IM+Na]+; 889 [M+H]+
13C NMR (CDC13) ~: 213.4 (C16); 196 (C2); 169.4 20 (ClO); 1~8.6 (C41); 164.7 ~C3); 138.8 (C19); 133.3 (C29);
128.7 (~31); 121.7 (C18); 97.3 (~1); 84.1 (C34); lO.1 (C39) Example 8 2-L1,14-Dih~droxv-12- r 2-(4-hvdroxy-3-methoxvcvclohaxyl)-1-methylvinvl~23,25-dimethoxy~-13,19 21,27-tetramethyl-11.28-25 dioxa-4-azat~ricyclo r ~2.3.1.O4'91Octacos-18-ene-2.3 10~16-tet~aone-17-yl~-ethanoic a~d N-~en2ylamide A solution of the title compound of Example 2 (48mg3, 2-chloro-1-met~ylpyridinium tosylate (27.1mg), , r . . . ~: -WO9ltO4025 PCT/GB90/01412 2 ~ 22 -triethylamine (2~1) and benzylamine (lOmg) in THF (3ml)was stirred at room temperature for 1.5 hours. Dilute aqueous hydrochloric acid (,'M) was then added and the mixture was extracted with ethyl acetate. The organic 5 extracts, after w~shing wil:h saturated agueous sodium hydrogen carbonate solution, were dried (MgS04), filtered and evaporated to an oil .Ln vacuo. Chromatography on silica eluting with hexane/acetone [3:2] then gave the title compound as a foam (33.6 mg, 63~).
10 MS (FAB): 995 [M+Rb]+; 933--[M+Na]+; 911 [M+H]+; 893 tM-OH]+
3C NMR (CDC13) ~: 213.3 (C16); 196.1 (C2); 171.6 (C41); 168.9 (C10); 164.9 (C3); 139.3 (C19): 133.2 (C29);
1~8-9 (Car); 128 (Car); 121.4 (C18); 97.5 (C1); 84 3 15 (C34); 10.2 (C39) Example ~
2-~1.14-DihYdroxv-12- r 2-t4-hydroxy-3-methoxycYclohexyll-l-methylvinyll-23,25-dimethoxv-13l19.21~27-tetramethyl-11.28-dioxa-4-azatricyclo r 22.3.1.04~910ctaco5-18-ene-2 3 ! 10 ~ 16-2~ tetraone-17-yl~ethanoic acid N~Butylamid~
A solution of the title compound of Example 2 (57.8mg), 2-chloro-1-methylpyridinium tosylate (37.8mg~, triethylamine (11.5~1) and butylamine tl2mg) in THF
(2ml) was stirred at room temperature for 3 hours. A

25 further portion of 2-chloro-1-methylpyxidiniu~ tosylate (37.8mg) and triethylamine (11.5~1) were then added and stirring was continued for an additional 2 hours at room temperature, after which dilute aqueous hydrochloric acid . . ., , .. ~: .. ; . . :. . :, .. ~.;, . .

- 23 - 2~5/~

(2M) was added and the organic extracts (after washing with saturated aqueous sodium hydrogen carbonate solution) were dried (MySO4), filtered and evaporated to an oil in vacuo. Chromatography on silica eluting with 5 dichloromethane/methanol ~29:1] then gave the title compound as a foam (36.3mg, 59%).
MS (FAB): 961 [~+Rb]+; 899 [M+Na]+; 877 [M+H]+; 859 [M-OH]+
13c NMR (CDC13) ~: 213.4 (C16); 196.1 (C2); 171.7 10 (C41); 1~8.9 (C10); 165 (C3); 139.2 (Cl9); 133.3 (C29);
129.2 (C31~; 121.6 (C18); 97.5 (C1); 84.4 (C34); 10.2 (C39) ExamPl e 10 ~-~1.14-Dihvdroxv-12- r 2-(4-hydroxy-3-methoxycvclohexvl~-1-methvlvinvlL-23,25-dimethoxy-13.19.21,27-tetramethvl-11,28-15 dioxa-4-azatricvclo r 22.3.1.04~91Octacos-18-~ne-2,3.10.16-te~rao~e-17-vl~-ethanoic acid p-CyanoPhenYl ester A solution of the title compound of Example 2 (51mg), 2-chloro-1-methylpyridinium tosylate (33.4mg), triethylamine (29~) and p-cyanophenol (10.2mg) in THF
20 (2ml) was ~tirred at room temperature for 2 hours. Dilute aqueous hydrochloric acid (2M) was then added and th~
organic extracts (after washing with saturated ac~eous sodium hydrogen carbonate solution) were dried (~gS04), filtered ancl evaporated to an oil in vaouo. Chromatography 25 on silica e].uting with dichloromethane~methanol [39:1] then gav~ the tit}.~ compound as a foam (24.9 mg, 43%).
MS (FAB): 1007 [M+Rb]+, 905 [M-OH]+
13C NMR ~CDc13) ~ 212.4 (C16), 196 (C2), 170.1 ;~s .
-'~:, ,: , :"'' . ~ , . : ; , WO91iO402~ PCT/GB90/014l2 ~ 5^ - 24 -(C41), 168.8 (C10), 164.4 (C3), 153.8 (OCar), 140.4 (C19), 133~6 (Car), 132.7 (C29), 1~9.1 (C31), 122.6 (Car), 120.1(C18), 118.2 ~'CN of p-cyanophenol), 109.7 (CarCN), 97 (C1), 84.1 (C34), 14.4 (C30), 9.9 (C39) 5 Example 11 2-~1,14-Dihydroxy-12- r 2-(4-hydroxY-3-me~hoxycvclohexvl)-1-methylvinyll-23,25-dimethoxY-13.19,21,27-tetramethyl~llL28-dioxa-4-azatricyclQ~22.3.1.04~91Octacos-18-ene-2,3,10.16-tetraone-17-yl)-ethanoic acid Phenyl_ester - 10 A solution of the title compound -of---Example 2 (45mg), 2-chloro-1-methylpyridinium tosylate ~32.3mg), triethylamine (30~1) and phenol (28mg~ in dry dichloromethane (2ml) was stirred at room temperature for 2 hours. Dilute aqueous hydrochloric acid (lM) was then 1~ added and the organic extracts (after washing with saturated aqueous sodium hydrogen carbonate solution) were dried (MgSOd~, filtered and evaporated to an oil in vacuo. Chromatography on ~ilica eluting with hexane/
acetone ~3~2] then gave the title compound as a foam 20 (17-6mg, 36%).
MS (FAB3; 982 [M+Rb]+; 920 [M+Na]+; 898 [M+H]~; 880 tM_oH]
3C NMR (CDC13) ~: 212.4 (C16); 195.9 ~C2); 171.1 (C41); 168.8 (C10); 164.5 (C3); 150.5 (OCar); 140 (Cl9);
25 132.7 (C29); 129.4 (Car); 129.3 (C31); 12508 (Ca~);
121.4 (Car); 120.7 (C18); 9702 ~Cl); 84.1 (C34); 9.7 (C39) Exam~le 12 ., . . ,:

: . .: : . . .. r, .. ' ~' :. . ' '' '' ': , ' ''' . ;' . ,' WO91/0402~ PCT/GB90/01412 - 25 - 2a~3~2~

2-~1 14-DihydroxY-12-~2-L4-hvdroxv-3-methoxy~yclohexylL-l-methylvinvl~-23 25-dimethoxv-13 19.21.27-tetram2th~1-ll 28-dioxa-4-azatricyclo r 22.3.1.04~-910ctacos-18-ene-2.3 10.16-tetraone-17-yll-ethanoic acid p-NitrophenYl ester 5 A solution of tha title compound of Example 2 (150mg) 2-chloro-1-methylpyridinium tosylate (77mg), triethylamine (77~1) and p-nitrophenol (47mg) in dry dichloromethane (2ml) was stirred at room temperature for 2.5 hours. The reaction mixture was hen concentrated in vacuo and - 10 purified directly by chromatography on silica eluting with hexane/diethyl ether [3:2] to give the title compound as a ~oam (135mg 78~).
MS (FAB): 1027 [M+Rb]+; 925 [~-OH]+
13C NMR ~CDC13~ ~: 212.6 (C16); 196.2 (C2); 170.3 15 (C41); 169 (C10); 164.7 (C3); 155.4 (OCar); 145.5 (NCar); 140.6 ~Cl9); 132.9 (C29); 129.3 (C31); 125.4 (Car); 122.6 (Car); 120.3 (C18); 97.2 (Cl~; 84.3 (C34);
9.8 (C39) ExamPle 13 2a 17-Allyl-1 14.16-trihvdroxY-12- r 2-(4-hvdroxy-3-methoxycvclohexx~ methylvinyll-23 25-dimethoxy-13.19.~1.27-tetramethvl-11~28-dioxa-4-azatricYclo r 22.3.l.o4r9loctacos-l8-ene-2~3Llo-trione To a solution of FR-900506 (lOOmg) in diethyl 25 ether/dichloromethane was added excess borane ammonia complex (150mg). After stirrin~ for 2 hours at room temperature dilute aqueous hydrochloric acid (lM) was added and the el:hereal ex~ract was clried (Na2S04), filtered , ~ , : ~ ~, : - . -.;

- .: , -.. . .

WO ~1/0~025 PCI/GB90/01412 20~2a and evaporated in vacuo to gi~e the crude 1,2,14,16-tetrahydroxy compound as an oil. This was taken up in acetic acid and copper (II) acetate (lOOmg) was added.
After heating on a steam bath for 10 minutes the reaction 5 mixture was cooled to room temperature and water was added. The reaction mixture was then extracted with ethyl acetate and the organic extracts, after washing with saturated aqueous sodium hydrogen carbonate solution, were dried (MgS04), filtered and evaporated to an oil in ---10 vacuo. Chromatography on silica eluting - with acetone/hexane [1:2] then gave the title compound (30mg) with R-steraochemistry at C16 fsllowed by the title compound (24mg) with S-ster~ochemistry at C16.
~16R)-~tereoi~omsr 15 MS (FAB): 890 [M+Rb]+

3C NMR (CDC13) ~: 199.4 (C2); 169.1 (C10); 165.8 (C3); 136.9 (C41); 136.3 (C19); 132.6 (C29); ~28.2 (C31);
125.4 (C18); 115.9 (C42), 98.7 (C1); 84.2 (C34); 56.5 (C9);
49 (C20); 43.2 (C17); 10.4 (C39) 2~ ~168)-~t~rhoi30~r MS (FAB): 890 tM~Rb]+; 828 [M+NaJ+; 806 [M+H]+; 788 ~M-0~]+
3C NMR (CDC13) ~: 196.5 (C2); 169.3 (C10); 165.3 (C3): 137.8 (C41); 13~.2 (C19); 132.9 (C29); 128.5 (~31);

25 126.6 (C18); 115.7 (C42); 97 (~1); 84.3 (C34); 56.4 (C9); 9 (C39) Exam~le 14 1,14-Dihydro~y-12- r 2-(4-hydroxv-3-methox~c~clohexYl)-l-., ~,. ;... . .. . . . .

- 27 - 2 ~

methYlvinyll-26.28-dimethoxy-13.18,22.24,30-pentamethyl-11.17.31-trioxa-4-azatetracyclor25.3.1,04~9.01612 hentriaconta-16(20)~18.21-triene-2,3,10-trione A solution of 1,14-dihydroxy-12-[2-(4-hydroxy-3-5 methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-(2-oxopropyl)-13,19,21,27-tetramet:hyl-11,28-dioxa-4-azatricyclo [22.3.1.04~9]octacos-18~ene-2,:3,10,16-tetraone (the compound of Example 29, WO 89/05304) (lOOmg) in dry dichloromethane (25ml) containing p-toluenesulphonic acid 10 ~5mg) was heated under reflux for 30 minutes. .Volatiles were then removed in vacuo and the residue was chromatographed on silica eluting with acetone/hexane [3:2]
to give the title compound tl9mg) as a foam.
MS (FAB): 887 [M+Rb]+; 825 ~+Na]+;803 [M+H]+; 785 15 tM-H]
3C NMR tCDC13) ~: 195.9 (C2); 168.9 (C10); 164.9 (C3); 150.8 (C16); ~48.3 (C18); 106.3 (Cl9); 97 (C1); 84.1 (C37); 8.5 (C42 Exam~le 15 1.14-dihvdroxy-12- r 2-(4-h~drox~-3-methoxycvclohexYl~
methylvinYll-23.25-dimethoxy~-17-proPanalvl-13219.21~27-tetramethyl-11.,28-dioxa-4-azatricycl,o,~22.3.1.04~9]octacos- ,, 18-ene-2.3,10 16-tetr20ne A solution of FR-90050~ (3g) in DNF (9Oml) and water (15ml) 25 containing palladium (II) chloride (0.4g) and copper (I) chloride (1.8g) was stirr~d at room temperature while air was bubbled through the reaction mixture for 2 hours. The reaction mixture was then diluted with diethyl ether and WO91/0402~ PCT/~90/01412 2~ 28 -the organic extract (after washing with dilute aqueoushydrochloric acid (lM), water and brine) was dried (MgS04), filtered and evaporated to an oil in vacuo.
Chromatography on silica eluting with hexane in an 5 increasing acetone gradient then gave the title compound as a foam (3Omg).
MS (FAB): 821 [M+H]
3C NMR (CDC13) ~: 213~1 (C16), 202.1 (C42), 196.3 (C2), 169 (C10), 164.7 (C3), 139.8 (C19), 132.6 (C29), 10 129.6- (C31), 121.9 (C18), -97.1 (Cl), 84.2 (C34),-70.-3 (C14), 9.6 (C39) Exam~le 16 17-All~l-l-hydroxy-12-~2-(4-hydroxv-3-methoxycvclohexvl)-1-methylvinyll-23,25-dimethox~-13,19,21,27-tetramethyl-}~ 11.28.29-trioxa-4-azatetracyclo~22.3.1.114~13.04'9 nonacos-19-ene-2,3,10.16-tetraone A sample of 17-allyl-1,14,20-trihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl~-1-methylvinyl]~23,25-dimetho~y-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo ;20 ~22.3.1.04t9]octacos-18-ene-2,3,10,16-tetraone (as prepared in Example 13 of WO 89/05304, 60mg) was heated in ;toluene (lOml) containing p-toluenesulphonic acid for 10 minutes. Evaporation of the solvent in vacuo and chromatography on silica then gave the title compound as an 25 oil (30mg).
13~ NMR (CDC13) ~: (single rotamer) 210.22 (C16);
195.96 (C2); 168.96 (C10): 165.24 (~3); 135.92 (C20);
134.69 ~41); 131.13 (C29); 128.78, 128.65 (Cl9, C31~;

. : :: . . : ~ ,. . ~.. . ; . .
: . . ' :' `' ;''' ; . ':. .. ,, ,'; ' ' " ~.'; ;

; . : . ~.: : . .:

WO 91/04025 PCl/GB90/01412 2 ~

117.6~ (C42); 97.53 (Cl); 84.19 (C34); 80.79 (C18); 73.167(C14); 52.47 (C17); 44.638-(C15~; 29.99~ (C21) MS (FAB): 802.47 [M+H]+; 886.2 [M~Rb]~
Exam~le 17 5 17-Pro~l-l-hydroxv-12-~2-(4-hydroxy-3-methox~cyclohex~l)-1-methylvin~ 25-dimethoxy-13l19,~1,27-tetramethYl-11 28.29_ trioxa-4-azate~racvclo~22.3 1.12r23.04~9lnonacos-18-ene -2 3 ! 10 .16-tetraone A sample of 17-allyl-1,14,20-trihydroxy-12-[2-(4-hydroxy-3--methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy- -13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo ` t22.3.1.04~9]octacos~18-ene-2,3,10,16-tetraone (as prepared in Example 13 of WO 89/05304, 121mg) was dissolved in dry dichloro~ethane (10~1) and to this was added 1~ Martin's sulphurane reagent at -30C. After warming to O-C, volatiles were re~oved in vacuo and the residue was chromatographed on silica to give a ~oam. To a solution of this in dry methanol (5ml) was added 10~ Pd-on-car~on (lOmg) and the resulting suspension was stirred in an ;.
20 atmosphere of hydrogen for 7 hours at room temperature.
Volatiles were then removed in vacuo and the residue was purified by column chromatography on silica to yield the title compound as an oil (19mg).
MS (FAB): 770.76 ~M+H]+, 792.74 ~M+Na]~, 854.44 25 ~IRb]
13C NMR (CDC13~ ~: (single rotamer) 197.81 (C16);

196.11 (C2): 168.96 (C10); 165.~1 (C3); 146.16 (C1~);
139.46 (C19); 135.92 (C41); 134.63 (C31); 129.51 (C29);

". . . - . ......... ..... ..

.; . ~ , . . . .

WO9l/040~ PCr/GB90/01412 ~r ~

129.40 (C15); 125.31 (C18); 116.29 (C42); 97.33 (Cl); 92.07 (C20); 84.0~ (C34); 81.75 (Cl~); 80.03 (C23); 77.10 (C25);
74.67 (C24~; 73.43 (C35); 56.78 ~C9); 56-47 (OCH3); 55-95 (OCH3); 51.37 ~C17) 5 Example 18 17-All~l-l-hydroxy-12-[2-r4-h~drox~ _ethoxvcyclohexyl)-1-methylvinyl~-25-methoxy-13.19,21 27~tetramethvl-11 28.29-trioxa-4-azatetracyclor22.3.1.12~23 04'91nonacosa 14.18-diene-2,3 10,16-tetraone 10 A sample of 17-allyl-1,14,20-trihydroxy-I2-[2-(4-hydroxy-3-methoxycycloh~xyl)-l-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.04~9]octacos-18-ene-2,3,10,16-tetraone (as prepared in Example 13 of WO 89/05304, 80mg) was dissolved }5 in dry dichloromethane (5ml~ and to this was added excess Martin's sulphurane reagent at -30'C portionwise until all the s~arting material had disappeared (30 minutes). The reaction mixture was then diluted with ethyl acetate and washed with water and then brine. The organic 2xtract was dried (MgS04), filtered and evaporated in vacuo to an oil. Chromatography on silica th~n gave the title compound as an oil (2Omg).
13C NMR (CDC13~ S: (mixture of rotamer~) 212.05, 211.04 (C16): 197.47, 194.95 (C2~; 169.2~, 169.09 (C10);
25 165.21, 164.72 (~3); 136.85, 137.44 (C19); 131.43, 130.97 (C29); 131.25, 128.90 (C31), 125.02, 124.35 (C18); 98.82, 97.44 (Cl); 91.56, 91.29 (C20); 84.17 (C343 MS (FAB): 775.11 [M+H]+; 797.09 ~M+Na]~; 858.79 .

.
.,. .: :
- :

WO 91/04025 PCI/GB90/01~12 - 31 - 2~ 2~

[ M+Rb ]
ExamPle 19 12- r 2-(4-H~droxy-3-methoxvcyclohexYl~ methylvin~11-23,25-dimethoxy-17-pro~vl-13,19,21~27-tetramethvl-1,14,15-5 trihvdroxy-11,28-dloxa-4-azatricvcloL22.3.1.04~9 octacos-18-ene-2~3J~10.16-tetraone A sample of l-hydroxy-12-~2-(4-hydroxy-3-methoxycyclohexyl) -l-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetr~methyl-11,28-dioxa-4-azatricyclo[22.3.1.04~9]octacosa --14,18-diene-2,3,10,16-tetraone as an oil (the compound of Example 11 of WO 89~05304, 510mg) was dissolved in dry tetrahydrofuran (12ml) and N-methyl morpholine N-oxide (O.~g) and osmi~m tetroxide (3ml of a 4% aqueous solution~
- were added. After stirring for 1 hour at room temperature 15 solid sodium metabisulphite (lg) was added followed after 5 minutes by celite to produce a thick slurry. Th2 reaction mixture was then diluted with ethyl acetate and filtered.
The organic extract after washin~ with saturated aqueous sodium bicarbonate solution and brine was dried (magnesium 20 sulphate), filtered and concentrated in vacuo to an oil.
Column chromatogarphy on silica eluting with acetone/hexane t2:5] give two isc~rs of the title compound which differed in stereochemistry z. C14 and/or C15.
I~omer 1 (180mg) 25 13C NMR ~: (For the major rotamer) 211.82 (C16);
196.03 (C2);169010 (C10): 164.26 (~:3); 139.28 (Cl9);
132.12 (C29); 12~.22 (C31): 121.92 (C18); 95.65 (Cl); 83.94 (C34); 75.76(C~2); 74.81 (C23); 74.63 (C15): 73.26 ~C25);

. . .

' " ' , , ; '- ' ,. ~
, :~., ' ;., :

WO9l/0402~ PCT/GB90/01412 20~5/l~a 73.26 (C35); 72.22 (C24); 71.66 (Cl~); 56.02 (C9); 48.05 (C20); 47.29 (C17); 39.08 (C!;); 37.90 (C13); 34.92 (C33);
34.69 (C32); 34.47 (C27);33.~56 (C22); 32.78 ~C40); 32.63 (C26); 31.14 (C36); 30.57 (C37); 28.66 (C8); 25.78 (C21);
5 24.47 (C6); 21.24 (C7); l9.lB3 (C41); 19.83 (C44); 16.08 (C47); 15.66 (C43); 14.68 (C30); 14.07 (C42); ~.62 (C39) H NMR ~: (For the Major rotamer) 5.48 ~lH,brs,H~
5.15 (lH,d,J=9.6Hz,H-18); 5.08 (lX,d,J=8.8Hz,H-31); 3.79 (lH,-dd,J=1.5 and 8Hz,H-24); 1.66 (3H,brs,H-30); 1.60 10 (3H,brsjH-43); 1.02- ~3H,d,J=6.5Hz,H-47); 0.9-~ (3H,t,J=7.6 Hz,H-42); 0.90 (3H,d,J=7.6Hz,H-44); 0.83 (3H,d,J=7 Hz,H-39) MS: 823 [M+H]+; 845 ~M+Na]+; 906 [M+Rb]+
Isomer 2 (32mg) 13 C NMR ~: (For the major rotamer) 212.99 (C16);
15 194.39 (C2); 169.60 (C10); 1~4.85 (C3); 138.33 (Cl9);
131.44 (C29); 129.95 (C31); 123.16 (C18); 97.79 (Cl); 84.17 (C34); 76.54 (~12); 75.73 (C23); 75.68 (C15); 73.52 (C25);
73.52 (C35); 72.78 (C24); 71.88 (~14); 55.85 (C9); 49.21 (C17); 48.78 (C20); 39.3~ (C5); 38.83 (C13); 36.04 (C40);
- 20 35-34 (C32); 34.83 (C33); 34.46 (C27); 33.21 (C22); 32.35 (C~6); 31.21 ~C36); 30.58 (C37); 28.34 (C8); 26.33 (C21);
24.75 tC6~; 20.76 (~41); 20.63 (C~); 20.42 (C44); 16.31 (C47); 15.31 (C43); 1~.17 (~30); 13.95 (C42); 9.6~ (C39).
H NMR ~: (For the major rota~er) 5.24 (lH,brs,H-12?;
25 4-74 (lHlt~ur=3~4HzlH-9); 3.66 (lH,dd,J=1.4 and 9.6Hz,H-24);
1.63 (3HI,brs,H-30); 1.57 (3H,brs,H-43); 0.99 - (3H,d,J=6.4Hz,H-47); 0.94 (3H,d,J-7.3Hz,H-39); 0.92 (3H,d,J=4.5Hz,H-44); 0.90 (3H,t,J=7.5 Hz,H-42~

. - :, ,:,.. , , ...:..
,. :: . , -:: .: : : . ,:: . ~ : :

WO91/0402~ PCT/GB90/0141' 33 ~ r~ ~2 MS: 823 [M~H]+; 845 [M+Na]+; 906 [M+Rb] t Exam~le 20 In the mixed lymphocyte reaction (MLR) (described in Wo 89/05304, Example A), 17-(2-Oxopropyl)-1,14-dihydroxy-5 12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetrameth~yl-11,28-dioxa-4-azatricyclo t22.3.1.04~9]octacos-18-ene-2,3,10,16-tetraone, was found to have a PA2 value of 8.3 with agonist compound FR-900506. This means that a concentration of 5xlO 9~ of 10 the antagonist-- compound i5 required to occupy 50~ of the available receptor sites in the screen for which it competes with FR-900506.

.
: 15

Claims

CLAIMS:

1. The use of a compound of formula I, I

wherein R1 and R2 independently represent H or OH, or they may together represent a second carbon-carbon bond between the carbon atoms to which they are attached;
R3 represents methyl optionally substituted by -CO2H or an ester or amide thereof; ethyl optionally substituted by O, OH or -CO2H or an ester or amide thereof; propyl optionally substituted by OH or O; or allyl optionally substituted by OH;
R4 represents X;
R5 and R6 together represent a second carbon-carbon bond between the carbon atoms to which they are attached;
R7 represents H or OH;
R8 represents OCH3;
R9 represents OH or OCH3;
X represents O or (H,OH);
Y represents O or (H,OH); and n represents 1 or 2;
in addition to their significances above R1 and R5 may together represent an oxygen atom, in which case R6 and R7 together represent a second carbon-carbon bond between the carbon atoms to which they are attached:
R7 and R8 may together represent an oxygen atom;
and R3, R4 and Y, together with the carbon atoms to which they are attached, may represent a methyl-substituted furanyl ring;
provided that i) when R2 represents H; R3 represents methyl, ethyl, propyl or allyl; R5 and R6 together represent a second carbon-carbon bond between the carbon atoms to which they are attached: R8 represents OCH3; and Y represents O;
then R7 represents OH: and ii) when n is 1, then R3 is not methyl or ethyl;
in the manufacture of a medicament for the treatment of immunodepression or a disorder involving immunodepression.

2. A compound of formula I, as defined in claim 1, provided that i) when R1 represents OH; R2 represents H; R5 and R6 together represent a second carbon-carbon bond between the carbon atoms to which they are attached; R7 represents H; R9 represents OCH3; X and Y each represent O; and n represents 2; then R3 does not represent 2-oxopropyl, 2,3-dihydroxypropyl or ethanalyl;
ii) when R1 represents OH; R2 represents H; R5 and R6 together represent a second carbon-carbon bond between the carbon atoms to which they are attached; R7 represents OH; R9 represents OCH3; X and Y each represent O; and n represents 2; then R3 does not represent allyl or 1-hydroxyprop-2-enyl; and iii) when R1 represents OH; R2 represents H; R5 and R6 together represent a second carbon-carbon bond between the carbon atoms to which they are attached; R7 represents H; R9 represents OCH3; X and Y each represent (H,OH); and n represents 2; then R3 does not represent allyl.

3. The use of a compound of formula I, as defined in claim 1, or a compound of formula I as defined in claim 2, wherein R3 is ethyl substituted by O or propyl substituted by O.

4. The use of a compound of formula I, as defined in claim 1, or a compound of formula I as defined in claim 2, wherein R7 is OH.

5. The use of a compound of formula I, as defined in claim 1, or a compound of formula I as defined in claim 2, wherein R2 is OH.

6. The use of a compound of formula I, as defined in claim 1, wherein the compound of formula I is:
17-Allyl-1,14,20-trihydroxy-12-(2-(4-hydroxy-3-methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy-13 19 21 27-tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.04,9]octacos-18-ene-2,:3,10,16-tetraone 17-(1-Hydroxyprop-2-enyl)-1,14 20-trihydroxy-12-[2-t4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11-,28-dioxa-4-azatricyclo t22.3.1.04,9]octacos-18-ene-2,3 10,16-tetraone 17-(2,3-Dihydroxypropyl)-1 14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.04,9]octacos-18-ene-2,3,10,16-tstraone, 17-Ethanalyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa 4-azatricyclo [22.3.1.04,9toctacos-18-ene-2,3,10 16-tetraone, 17-(2-Oxopropyl)-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.04l9]octacos-18-ene-2,3 10,16-tetraone, or --Allyl-1,2,14,16-tetrahydroxy-12-t2-(4-hydroxy-3-methoxicyclohexyl)-l-methylvinyl]-23,25-dimethoxy-13,19,21,27--tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.04,9]octacos-18-ene-3,10-dione.

7. A compound of formula I, as defined in claim 2, which is:
17-(2-Oxopropyl)-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricycle [22.3.1.04,9]octacosa-14,18-diene-2,3,10,16-tetraone, 17-(2-Oxopropyl)-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl)-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone, 2-(1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl)-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone-17-yl)-ethanoic acid, 2-(1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone-17-yl)-ethanoic acid Methyl ester, 2-(1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone-17-yl}-ethanoic acid N-Morpholine amide, 2-(1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone-17-yl)-ethanoic acid N-Ethanolamide, 2-(1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone-17-yl)-ethanoic acid amide with glycine methyl ester, 2-(1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone-17-yl}-ethanoic acid N-Piperidine amide, 2-(1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone-17-yl)-ethanoic-acid-N-Benzylamide, 2-(1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone-17-yl)-ethanoic acid N-Butylamide, 2-(1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone-17-yl)-ethanoic acid p-Cyanophenyl ester, 2-(1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone-17-yl)-ethanoic acid Phenyl ester, 20(1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone-17-yl)-ethanoic acid p-Nitrophenyl ester, 17-Allyl-1,14,16-trihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.04,9]octacos-18-ene-2,3,10-trione 1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-26,28-dimethoxy-13,18,22,24,30-pentamethyl-11,17,31-trioxa-4-azatetracyclo[25.3.1.04,9.016,20]
hentriaconta-16(20),18,21-triene-2,3,10-trione, 1,14-dihydroxy-12-[2-t4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-propanalyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone, 17-Allyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28,29-trioxa-4-azatetracyclo [22.3.1.114,18.04,9]nonaco-19-ene-2,3,10,16-tetraone, 17-Propyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-25-dimethoxy-13,19,21,27-tetramethyl-11,28,29-trioxa-4-azatetracyclo[22.3.1.12,23.
04,9]nonacos-18-ene-2,3,10,16-tetraone, 17-Allyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-25-methoxy-13,19,21,27-tetramethyl-11,28,29-trioxa-4-azatetracyclo[22.3.1.120,23.
04,9]nonacosa-14,18-diene-2,3,10,16-tetraone, or 12-[2-(4-Hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-1,14,15-trihydroxy-11,28-dioxa-4-azatricyclo[22.3.1.04,9]
octacos-18-ene-2,3,10,16-tetraone.

8. The use of a compound of formula I, as defined in claim 2, as a pharmaceutical.

9. A pharmaceutical composition comprising a compound of formula I, as defined in claim 2, in association with a pharmaceutically acceptable adjuvant, diluent or carrier.

10. A pharmaceutical mixture comprising a compound of formula I as defined in claim l or claim 2, and an immunosuppressive compound.

11. A process for the production of a compound of formula I, as defined in claim 2, which comprises:
a) producing a compound of formula I, in which R3 represents propyl substituted by O, by oxidation of a corresponding compound in which R3 represents allyl;
b) producing a compound of formula I, which contains a vicinal diol, by oxidation of a carbon-carbon double bond in a corresponding compound:
c) producing a compound of formula I, in which R3 represents ethyl substituted by O, by oxidative cleavage of a corresponding compound in which R3 represents 2,3-dihydroxypropyl;
d) producing a compound of formula I, in which R3 represents methyl substituted by -CO2H or ethyl substituted by -CO2H, by oxidation of a corresponding compound in which R3 represents ethanalyl or propanalyl;
e) producing a compound of formula I, which contains two vicinal hydrogen atoms, by reduction of a corresponding compound which contains a carbon-carbon double bond;
f) producing a compound of formula I, in which X or Y

represents (H,OH), by reduction of a corresponding compound in which X or Y represents O;
g) producing a compound of formula I, in which R3, R4 and Y, together with the carbon atoms to which they are attached, represent a methyl-substituted furanyl ring, by the action of acid on a corresponding compound in which R3 represents 2-oxopropyl, R4 represents H and Y
represents O;
h) producing a compound of formula I, in which R1 and R5 together represent an oxygen atom and R6 and R7 together represent a second carbon-carbon bond between the carbon atoms to which they are attached, by the action of acid on a corresponding compound in which R1 represents OH, R5 and R6 together represent a second carbon-carbon bond between the carbon atoms to which they are attached, and R7 represents OH;
i) producing a compound of formula I, in which R7 and R8 together represent an oxygen atom, by the action of a dehydrating agent on a corresponding compound of formula I
in which R7 represents OH and R8 represents OCH3;
j) producing a compound of formula I, in which R7 represents OH, by allylic oxidation of a corresponding compound in which R7 represents H; or X) producing a compound of formula I, in which R3 represents allyl substituted by hydroxy, by allylic oxidation of a corresponding compound in which R3 represents allyl.

12. A method of treatment of immunodepression, or a disorder involving immunodepression, which comprises administering a therapeutically efficacious amount of a compound of formula I, as defined in claim 1, to a patient suffering from such a condition.