CA2093429A1 - Process for biophosphorylating organic compounds - Google Patents

Abstract

Described is a new microbial biophosphorylation process for regiospecifically phosphorylating the hydroxyl group, in a ''phosphate active'' hydroxyl containing organic compound, under biotransformation conditions utilizing the microorganism, Rhizopus oryzae) ATCC No. 11145. The phosphorylated compounds produced by the process include e.g., macrolide FK-506 type immunosuppressants which are useful in preventing human host rejection of foreign organ transplants, e.g. bone marrow, liver, kidney, lung and heart transplants. Other hydroxyl containing compounds, applicable in this process include rapamycin, echinocandins, HIV protease inhibitors, simvastatin and zearalenone.

Description

Wog~OCsg2 PCT/US9l~l6 2~93~29 , TITLE OF T~E INVENTI~N
NEW P~OCESS FOR BIOPE~OSP~IO~YLATING ORGANIC COMPOI~NDS

CRQss-~EERENcE TO R~LATE~ AR~LICATIONS
The present application i8 a combined continuation-in-part application of SN 594,500 (Case 18238) filed October 9, 1990; SN 594,214 (Case 18240) filed October 9, 1990; SN 595,894 (Case 18208) filed October ll, 1990; SN 691,606 (CaQe 18261) filed ~April 26, 1991; SN 691,607 (Case 18382) filed April 26, 1991; S~ 701,387 (Case 18340) filed May 16, 1991; and SN 735,963 (Case 18462) filed July 2S, 1991.
BAC~G~O~ND OF TEE INVENTION
l. Field of the Invention - This invention relates to a new regio-specific biophosphorylation proceæs for producing 30 phoæphorylated deri~atives of "pho~phate active"
hydroxyl containing organic compounds utilizing the i ~ -; , ,, . . ~ , , .

, - . .; , . .

W092/06992 PCT/US91/~16 -microorganism Rhizopus oryz~, ATCC No. 1114~. The process involves contacting the microorganism and an organic compound containing a free phosphate acti~e hydroxyl group, under biotransformation conditions, which phosphorylate the hydroxyl group. The process in~olves either resting RhizQpus cells or culturing the microorganism in the presence of the organic compound.
Rhizopus oryzae ATCC No. 1114~ is known in 10 the art primarily as a hydroxylating agent. It also -degrades insect moulting hormones (J.C.S. Chem. Comm.
1974: 656-657, 1974>; participates in hydro y lation of steroids (Can, J, Chem. 57:436-440 and 1585-1587, 1979; ibid., 59; 1651-1655, 1981; ibid., 63: 1127-1131, 1985; ~ J. Peppier, ed., Microbial Technology Reinhold, New York, p. 288-297, 1967; U.S. Patent 2,646,370); is involved with transformations of sesquiterpene lactone costunolide (J.C.S, Perkin 1:
3022-3028, 1979): produces 16-hydroxyverrucarin A
and B and 3'-hydroxyverrucarin A by tranæformation of verrucarins A and B (Appl. Envir~n, Microbiol, 46:480-483, 1983); and produces OH-products of imipramine (J. Pharmaceut. Sci. 70: 151-153, 1981).
~owever, in the above references, there is no description of its ability as a regiospecific hydro~yl phosphorylating agent. --Rhizo~5 i8 a genus of fungi which commonly occurs on mature fruits, grain and vegetables, as well as soil. Typically they are eaprobes and facultative parasitee, and for~ a branched, aæeptate mycelium.
Rh~Qp~ spp are used commercially, e.g., in the preparation of carboxylic acide or steroids, or in the metabolism of hydrocarbons.

- - . .

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

~092/06~2 PCT/US91/~16 2 0 9 3 ~
~.

Culture ~r fermentation of Rhizo~us arrhizus ATCC 11145 is straightforward, conventional and.well known. See, for example, U.S. 4,410,629. Adapta-tions, modifications and variations of the protocol are given in the Examples are within the routine skill of the fermentation micro~iologist.
Regiospecific biophosphorylating aEents are desired in the art since ~tandard chemical phosphorylation using, e.g., POCl3 or PC15, are 10 generally non-regiospecific and do not produce selectively phosphorylated compounds, which can lead to improved water solubility and pharmacokinetics.

ATCC DE~Q~I~
A sample of MF 4974 has been deposited at the American Type Culture Collection (ATCC), 12301 Par~lawn Drive, Rockville MD 20352. The culture access designation is ATCC No. 11145.

20 BRIEF DESCBI~ION OF ~E_EIÇ9~ES
Figure l is an 1~ nuclear magnetic resonance (NMR) spectrum taken at 400 M~z of C-32 phosphorylated FR-900520 in CDC13.
~ igure 2 exhibit~ the as~igned molecular structure for C-32 phosphorylated FR-900520.
Figure 3 is 1~ nuclear magnetic resonance (NMR) Epectsum taken at 400 M~z of the C-43 methylated phosphate ester of the phosphorylated rapamycin macrolide in MeO~
Figure 4 exhi~its the as~ig~ed molecular structure for the C-43 phosphorylated rapamycin macrolide.

- -, ,, ......... ~,: . .. ; '.' ' , , ~ ., .
. . ,., . ,' . ' .. ' ` ,' ., ~ ' ' : . ~ ' ,.
- ., . . . : . .. ... : ~ .

W092/06992 PCT/US91/~16 ,,~
.J ~ ~ ~

Figure 5 is lH nuclear magnetic resonance spectrum of echinocandin IIIA.

SUMMARY OF T~ ~NV~NTION
We have discovered a new overall process for selectively biophosphorylating an organic compound containing ~phosphate active" hydroxyl groups. The or~anic compound is su6pended in an a~eous medium with a strain of ~hiæopus orvzae ATCC No. 11145 for a sufficient time to produce the phosphorylated organic compound.
By the term ~'phosphate active hydroxyl con-taining organic compound~ as used herein is meant a compound containing a hydroxyl group which can stereo-specifically interact with the ~hi~QEy~ microorganismto undergo phosphorylation unter biotran~formation conditions. A simplç test with the organic compound - under the biotransformation conditions described herein will determine, without undue eæperimentation, 20 if the hydroxyl group in the organic molecule is phosphate active.
The process involves contacting `resting Rhizo~uæ cellæ in a phosph2te buffered medium con-taining e.g. glyceroI, as a carbon nutrient, or by 25 the fermentation of the microorgani5m BIL~U~Ia oryzae, ATCC No. 11145 together, in the presence of the hytroxyl containing organic compound, ~.g., an FK-506 type macrolide immunosuppressant i.e. F~-520, under su~merged aerobic conditions in an aqueous carbohydrate medium, containing a nitrogen nutrient, said conditions being contucted at a p~ of about 7 for a sufficient time, e.g. 24 hours at 27-C, to selectively biophosphorylate the phosphate active ~92/069g2 PCT/US91/~16 !

2~3~

. , hydroxyl group. Either process can be utilized but preferred i~ the process using resting cell6.
It will be recognized by those skilled in the art that phosphorylation by R~Lzopus oryzae ATCC 11145 is not limited to that particular Etrain.
Rather, other RhizQp~ orvzae strains can also be expected to be capable of acting to perform hydroxyl phosphorylation of hydroxy containing organic compounds.

DETA~LED DESCRIPTI~N 0~ T~E
INVENTION AN~ PREFERRED EMBO~IMENTS
- In accordance with this invention there is provided a process for producing a biophosphorylated L5 hydroxyl containing organic compound, wherein said hydroxyl group is phosphate reactive, comprising the step of contactin~ a strain of Rhizo~us oryzae ATCC
No. 11145 microorganism together with the hydroxyl containing organic compound in an aqueous medium containing a carbon nutrient at ambient temperature for a sufficient time to produce the biophosphory-lated hydroxyl containing organic compound.
The present invention involves a biotrans-formation process, which involves the contacting of resting cells, or the fermentation of, the micro-organism, Rhizopus oryzae, together with an organic compound containing a free hydroxy group, to produce the phosphorylated derivative. The microorganism is currently on depoæit with the American Type Culture Collection, 12301 ~arklawn Dri~e in Rockville, Maryland as ATCC No. 11145.

: . ~ " - , , . .. ~, ii ., . .. ' ' ' ' ' ' '' ' ' W092/06g92 PCT/USs~ 16 The scope of the compounds included within the l'phosphate reactive hydroxyl containing organic compounds" includes a C-32 hydro~y-containing macro-lide as described in Fuji~awa~ USP 4,894,366 of the formula:

~ 3 ~
R0 ~ CH3 ~CHz~

H3C ~ H C

W

25 wherein R is ~, Cl-C4 alkyl, R2 is hydrogen, hydroxy or lower alkanoyloxy, R3 is methyl, ethyl, propyl or allyl, n is an integer of 1 or 2, and the symbol of a line and dotted line i8 a single bond or a double bond, and a pharmaceutically acceptable basic salt thereof. '.

W092/o6992 PCTIUS91/~16 Specifically included is where the compound FK-506 is where R is methyl, R2 is hydroxy, R3 i8 allyl, n i~ 2 and the dou~le bond is absent; and the compound FK-520 is where R is methyl, R2 is hydroxy, R3 is ethyl, n is 2 and the double bond is ab6ent.
Also included are phosphate reactive hydroxyl containing compounds including the following FK-506 type compounds (from ~SP 4,894,366 to Fujisawa):

7-Allyl-1,14-dihydroxy-12-~2-(4-hydroxy-3-methoxycyclohexyl)-l-methylvinyl~-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.04~9]octacos-18-ene-2,3,10,16-tetraone, 15 1,14-Dihydroxy-12-[2-(4-hydroxy-3-metho~ycyclohexyl)-l-methylvinyl]-23,25-dimethoxy-13,19,17,21,27-penta-methyl-11,28-dioxa-4-azatricyclo~22.3.1.0 4~9]octacos-18-ene-2,3,10,16-tetraone, 16-Allyl-1,13-dihydroxy-11-~-2-(4-hydroxy-3-metho y cyclohexyl)-l-methylvinyl]-22,24-dimethoxy-12,18,20,26-tetramethyl-10,27-dioxa-4-azatricyclo-[2l.3.1.04~83heptacos-17-ene-2,3,9,15-tetraone.
Further included within the term "FK-506 type macrolide" a8 used herein are ~pecifically the compounds disclosed in ~i~on's FP0 0 323 042 ~f the formula:

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

Wog2/o6ss2 PCT/US91/~16~-~9 "

HO~

R20 ~ R R22 ~3 lS Rl7 Rl6 II

wherein each ~icinal pair of gubstituents tRl and 20 R2], ~R3 and R4], ~R5 and R6] independently:
a) represent two vicinal hydrogen atoms, or b) form a second bond between the vicinal carbon atoms to which they are attached;
in addition to it significance abovel ~
~:
R2 can re~resent an Cl-ClO alkyl ~roup;
R7 represents ~, 0~ or 0-Cl-ClO al~yl, or in conjunction with Rl it may represent =0;
R8 and R9 independently represent ~ or 0~; :
RlO represents ~; Cl-ClO alkyl, wherein ~aid alkyl .. :: . ,- ;; . - . . . .. .. . . .

W092/06~2 PCT/US91/~16 2~9~.2J' _9_ can be substituted by one or more hydroxyl groups;
Cl-C10 alkenyl, which can be substituted by one or more hydroxyl groups, or Cl-C10 alkyl substituted by =O;
X represents 0, (~, 0~), (H,H) or -C~20-;
Y represents 0, (H, OH), (~,H), N-NRllR12 or N-oR13 wherein, Rll and R12 independently represent ~, Cl-Cl0alkYl. Cl-C10aryl or tosyl, and R13 ~14 R15, R16, R17, R18, R19, R22 and R23 lo independently represent H or Cl-C10 alkyl; R20 independently represents 0, or it can independently represent (R20a,~); wherein R20a independently represents O~, O-Cl-C10 alkyl or OCH20C~2CE20C~3;
n is 1,2, or 3;
in addition to their significances above, Y, R10:and R23, together with the carbon atoms to which they are attached, can rep~esent a 5- or 6- membered N-, S- O-containing heterocyclic ring, which is saturated or unsaturated, and which can be su~stituted by one ~r 20 more groups selected from Cl-C10 alkyl, hydroxyl, Cl-C10 al~yl substituted by one or more hydro~yl groups, O-Cl-C10 alkyl, benzyl and -C~2SeCC6~5);
provided that when ~ ant ~ both represent O; R9 represents O~; R14, RlS, R16, R17, R18, R19 and R22 2s each represent methyl; R20a represent6 OC~3; R8 and R23 each represent ~; ~R3 and R43 and ~R5 and R63 each -represent a carbon-carbon bond;
and pharmaceutically acceptable salts thereof, which includes acid addition salts of any amine groups pregent-' ': . ............................. . -' W092/06992 PCT/US91/~6--q ~3~9 -lo-Preferably when R2, R7, ~11, R12 R13 R14 R15 R16 R17, R18, R19, R20a, R22 and R23 comprise carbon-containing groups, those groups contain up to 10 carbon atoms, more preferably from 1 to 6, e.g., methyl or methoxyl.
Also preferred is wherein each of R14, R15, R16 R17, R18, Rl9 and R22 represents methyl.
The alkyl groups: R2, R7, Rll, R12 R13 R14 R15 R16, R17, R18, Rl9, R20a, R22 and R23 can 10 comprise straight chain, branched and cyclic groups.
Alkyl groups substituted by =0, include R10 which can represent include ketone and aldehyde groupæ.
Preferably, R10 is allyl (i.e., 15 prop-2-enyl), propyl ethyl or methyl.
Preferably, n is 2;
R7 is H or 0~;
Rl and R2 both represent ~;
~ is preferably O or (~, OH);
R20a represents 0~ or OCH3; and when ~, R10 and R23 together represent a N-,S- or 0- containing heterocyclic ring, preferred is where that ring is fi~e-membered, more preferably a pyrrole or tetrahydrofuran ring.
Preferred embodiments as products are C-32 phosphorylated F~-506, . ;. ;
. I ................ ~ ~ . . ..
:.,,, ~ . . :
. . . .
. . .: ;

~vo 92~06992 PCr~US91/06816 2~93~2~

HO--P{~",3~34 CH30~ 2~ CH3 10 ~=

27 ~

and C-32 phosphorylated ~-520, 2s :, - ~ . .,.... . . . . . . - . , .

WO 92/06992 9 PCI/US91/068l6 ~
.
~9 HO 7 o",3.~3~ H

CH3~ 2B CH3 30 ,~H

~ ~ .

~D ~20 CH3~ C~1B -Z~ ~ :
~ 14 .

a nd C_32 PhO8PhOrY1ated Fg 523;

: .
. "'.
- .

:

.

~092/06992 PCT/US91/~16 2~93'~

HO-P~"3h34~

CH3~ 2~ CH3 ~
CH3 21~ H3 ~D \~20 CH3~00 C~1B
~J 1 6 ~ 14 .

and the correspondin~ C-32 phosphorylated C-31 desmethyl anal~gs, and also the following ~pecific compounds from EP0 323,042 to Fisons:

25 1,14-Dihydroxy-12-t2-(4-hydroxy-3-methoxycyclohe~yl)-l-methylvinyl]-18-[(phenylseleno)methyl~-16,26,28-trimethoxy-13,22,24,30-tetramethyl-11,17,31-trio~a-4-azatetracyclo~25.3.1.04~9. ol6 ~ 2]hentriàcont-21-ene-2,3,10-trione, .
17-Allyl-1,2,14-trihydroxy-12-~2-(4-hydroxy-3-methoxycyclohexylj-l-methyl~inyl3-23,25-dimetho~y- -.
13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-~22.3.1Ø4~9]octacos-18-ene-3,10,16-trione, ~ ' .. . ~ . . , ............ . ., , . . . . . . ~ . - .

wO92/06g92 PCT/US91/~16 -~ .,;.;

17-Allyl-1,2,14,16-tetrahydroxy-12-t2-(4-hydroxy-3-methoxycyclohexyl)-l-methylvinyl]-23,2~-dimetho y -13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.04~9]octaco~-18-ene-3,10-dione, 17-Propyl-1,14-dihydroxy-12-~2-(4-hydro~y-3-methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-t22.3.1.04~9]octacosane-2,3,10,16-tetraone, 17-Propyl-1,14-dihydrogy-12-[2-(4-hydro~y-3-methoxycyclohexyl)-l-methylethyl3-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioæa-4-azatricyclo-~22.3.1.04~9]octacos-18-ene-2,3,10,16-tetraone, 17-Propyl-1,14-dihydroxy-12-t2-(4-hydroxy-3-methoxycyclohexyl)-l-methylethyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-r22.3.1.04~9~ctaco~ane-2,3,10,16-tetraone, 17-Propyl-l-hydr~xy-12-t2-(4-hydroxy-3-methoxycyclo-hexyl)-l-methylvinyl-23,25-dimetho2y-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclot22.3.1.04~9~-octacosa-14,18-diene-2,3,10,16-tetraone, 17-Propyl-l-hydro~cy-12-t2-(4-hytroxy-3-methoxycyclo-hexyl)-l-methylvinyl~-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo~22.3.1.04~93-octacos]-18-ene-2,3,10,16-tetraone, . . .
. ~ , . ~ ` ' ' , .. ,, .. ~
, . , W092/06992 PCT~US91/~16 ~0 ~3~29 17-Allyl-1,14,20-trihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl~-l-methyl~inyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-~22.3.1.04~930ctacos-18-ene-2,3,10,16-tetraone, 17-(1-Hydroxyprop-2-enyl~-1,14,20-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-2,3,10,16-lO tetraone, 17-Allyl-1,2-dihydroxy-12-t2-(4-hydro~y-3-methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-15 t22.3.1.04~9]octacos~-18-ene-3,10,16-trione, 17-Allyl-1,16-dihydro~y-12-~2-(4-hydroxy-3-metho~y-cyclohexyl)-l-methylvinyl~-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-20 [22.3.1.04~930ctacosa~-14,18-diene-2,3,10-trione, 17-Allyl-l-hydroxy-12-~2-(4-hydroxy-3-methoxy-cyclohexyl)-l-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-25 {22.3.1.04~9~ octac08 ~ -1 8-ene-2,3,10,16-tetraone, 17-(2,3-Dihydroxypropyl)-1,14-dihydroxy-12-~2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl-23,25-di-methoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatri-30 cyclo~22.3.1.04~9~octacos-18-ene-2,3,10,16-tetraone, , .. . . .. . . . . . .................. ... . . . .
~,.,' : ... ,, '.' - '. '' ',:; ' , ' ' . ' ',,~ . ' ' .. :

W092/06992 PCT/US91/~6 ~
C~
~' ~ ~ -16-17-Ethanalyl-1,14-dihydroxy-12-~2-(4-hydroxy-3-metho~ycyclohexyl)-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, 1,14-Dihydro~y-12-t2-(4-hydro~y-3-methoxycyclo-hexyl)-l-methylvinyl]-26,28-dimetho~y-13,22,24,30-tetramethyl-11,31-dioxa-4,17-diazatetracyclo-[25.3.1.04~9.016~2~hentriaconta-16(20),18,21-10 triene-2~3~lo-trione~

1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-l-methylvinyl~-26,28-dimethoxy-17-(2-hydro~yethyl~-13,22,24,30-tetramethyl-11,31-dio~a-4,17-diazatetra-15 cyclot25.3.1Ø4~9.016~2]hentriaconta-16(20),18,21-triene-2,3,10-trione, 1,14-Dihydroxy-12-t2-(4-hydroxy-3-methogycyclohexyl)-l-methylvinyl~-26,28-dimethoxy-13,22,24,30-tetra-20 methyl-17-phenylmethyl-11,31-dioxa-4,17-diazatetra-cyclo-r25.3.1.04~9. ol5 ~ 2]hentriaconta-16(20),18,21-triene-2,3,10-trione, 1,14-Dihydro y -12-~2-(4-hydroxy-3-methoxycyclohe~yl)-25 1-methyl~inyl~-26,28-dimethoxy-13,22,24,30-tetra-methyl-17-phenylmethyl-11,31-dioxa-4,17-diazatetra-cyclot25.3.1.04~9.016~2]hentriaconta-l6(20),18,21- .
triene-2,3,10-trione, . ~,. .. , ~ , . . , , . , ~ .

. , . . : . , . , :
- . . , ~ -WOg2/06992 PCT/US91/~16 2~3~2~

17-Allyl-1,14-dihydroxy-12-~2-(4-hydroxy-3-methoxy-cyclohexyl)-l-methyl~inyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dio~a-4-azatricyclo-~22.3.1.04~9~octacos-18-ene-2,3,10,16-tetraone C16 o~ime, 17-Allyl-1,14-dihydroxy-12-~2-(4-hydrogy-3-metho~y-cyclohe~yl)-l-methylvinyl~-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 C16 oxime 0-methyl ether, 17-Propyl-l-hydroxy-12-[2-(3,4-dihydrogycyclohexyl)-1-methyl~inyl]-23,25-dimethogy-13,19,21,27-tetramethyl-15 11,28-dioxa-4-azatricyclo~22.3.1.04~9]octacos-18-ene-2,3,10,16-tetraone, 1,14-Dihydroxy-12-~2-(4-hydroxy-3-metho~ycyclohe~yl)-l-methylvinyl]-23,25-dimetho~y-13,19,21,27-tetra-20 methyl-17-(2-oxopropyl)-11,28-dioxa-4-azatricylo-~22.3.1.04~9~octacos-18-ene-2,3,10,16-tetraone, 17-Allyl-1,14-dihydroxy-12-[2-(4-hydrogy-3-methoxycyclohe~yl)-l-methylvinyl~-23,25-timetho~y-25 13,19,21,27-tetramethyl-11,28-dioxa-4-aza-spiro-ttricyclo~22.3.1Ø4~9~octacos-18-ene-2,2' -02i rane,-3,1~,16-trione, 17-Ethanalyl-1,2,14-trihydroxy-12-t2-(4-hydro~y-3-30 methoxycyclohe y l-1-methyl~inyl-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-~22.3.1.04~9~octacos-18-ene-3,10,16-trione, .: : . .......... . . ~. , - . . . . .

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

W092~069s2 PCT/~S9~ 6 -' 1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-l-methylvinyl~-26,28-dimethoxy-18-~(phenylseleno)-methyl-13,22,24,30-tetramethyl-11,17,31-trio~a-4-aza-tetracyclo[25.3.1Ø4~90.16~2]hentriaconta-16(20),21-diene-2,3,10-trione, 4-Methyl-~17-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohe~yl-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-lO ~22.3.1.04~9]octacoæ-18-ene-2,3,10-trione-16-ylidene]-hydrazide, or a pharmaceutically acceptable salt of any one thereof, In general, the phoæphorylated organic com-pound can be produced by either contac~ing resting cells of Rhizoyus, in an aqueous buffered phosphate medium containing a carbon nutrient at ambient temperature, or by culturing (fermenting) the abo~e-20 described microorganism, Rhizo~us oryzae, in thepresence of the hydro~y containing organic compound in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, preferably under submerged aerobic conditions ~e.g. ~haking culture, 25 submerged culture, etc.). The agueous medium is preferably maintained at a pH of about 7 at the initiation and termination (harvest) of the fermen-tation process. A higher p~ leads to su~stantial and/or total 108s ~f product. The desired p~ may be 30 maintained by the use of a buffer guch as morpholino-ethanesulfonic acid (MES), morpholinopropanesulfonic acid (MOPS), and the like, or by choice of nutrient materials which inherently possess buffering - . . , ., -~ . . . . . .. . . . .

~092/06~2 PCT/US91/~6 20q3~2~ -properties, such as production media descri~ed hereinbelow.
The preferred 60urceæ of carbon in the nutrient medium are carbohydrates such as glucose, xylose, galactose, glycerin, starch, de~trin, and the like. Other æources which may be included are maltose, rhamnose, raffinose, arabinose, manno~e, salicin, sodium succinate, and the li~e.
The preferred ~ources of nitrogen are yeast lO extract, meat extract, peptone, gluten meal, cotton-seed meal, soybean meal and other vegetable meals (partially or totally defatted), casein hydrolysates, soybean hydrolysates and yeast hydrolyæates, corn steep liquor, dried yea~t, wheat germ, feather meal, 15 peanut powder, distiller'æ æolubles, etc., as well as inorganic and organic nitrogen compounds ~uch as ammonium saltE (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids, and the like.
The carbon and nitrogen sources, though advantageously employed in combination, need not be used in their pure form, because less pure materials which contain traces of growth factors and consider-able quantities of mineral nutrients, are also 25 suitable for use. When desired, there may be added to the medium mineral ~alts such as sodium ~r calcium carbonate, sodium or potaæsium phosphate, sodlum or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, cobalt saltæ, and 30 the li~e. If neceææary, especially when the culture medium foams seriously, a defoaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone may be added.

- . ~ ... . .
,, ;, W092/06~2 ~ PCT/US91/~16 ~

c~ .

As one of the starting materials in the process, the FK-520 starting material can be obtained by the fermentation of S. hygroscopic~
var. ascomyceticus, ATCC No. 14891, as described S in U.S. Patent 3,244,592, and by the fermentation of S. hyg~osso~icus subsp. y~kushimaensis No. 7278, (to produce ~R-900520, or "F~-520", and the other F~-506 type macrolides can be obtained ~y the processes) as described in EPO Publication No. 0184162 to Fujisawa, 10 and PCT WO 89/05304 to Fisons, said above references hereby incorporated by reference for this particular purpose.
As to the conditions for the production of phosphorylated organic compound in ~a~si~e amounts, 15 submerged aero~ic cultural conditions are preferred therefor. For the production in small amounts, a shaking or surface culture in a flask or bottle is employed. Furthermore, when the growth iB carried out in large tankæ, it is preferable to u~e the 20 ~egetative form of the organism for inoculation in the production tanks in order to avoid growth lag in the proces6 of production. Accordingly, it i8 desirable first to produce a vegetative inoculum of the organi~m by inoculating a relatively small 25 quantity of culture medium with spores or mycelia of the organism produced in a "slant" and culturing ~aid inoculated medium, also called the "seed medium", and then to transfer the cultured vegetative inoculum aseptically to large tanks. The fermentation medium, 30 in which the inoculum is protuced, is substantially the same as or different from the medium utilized for the production of the phosphorylated organic compound and is generally autoclaved to sterilize the medium .
- : :
.
:, :

.

W092/06g92 2 ~ ~ 3 ~ PcT/US91/~16 prior to inoculation. The pH of the medium is gener-ally adjusted to about 7.0 prior to the autocla~ing step by suitable addition of an acid or base, preferably in the form of a buffering solution.
Agitation and aeration of the culture mixture may be accomplished in a variety of ways.
Agitation may be provided by a propeller or similar mechanical agitation equipment, by revol~ing or shaking the fermentor, by various pumping equipment 10 or by the passage of sterile air through the medium.
Aeration may be effected by passing sterile air through the fermentation mi~ture.
The fermentation is usually conducted at a temperature between about 20C and 40C, preferably 15 25-35C, for a period of about 10 hours to 24 hours, which may be Yaried according to fermentation con-ditions and scales. Preferably, the production cultures are incubated for about 24 hours at 27C
on a rotary shaker operating at ~20 rpm, wherein 20 the p~ of the fermentation medium i8 maintained at 7.0 to harvest.
Preferred culturing/production media for carrying out the fermentation include the following media: -Seed and Transformation Mediu~_ ~LL
Blucose 20.0 Soy Flour (Sigma) ~.0 Yeast ~xtract (Fidco) 5.0 NaCl 5.0 -AdjuBt pX to 7.0 W092/069s2 ~ .~ PCT/US91/~16-~
'~' The produced phosphorylated organic compound can be recovered from the culture medium by conven-tional means which are commonly u~ed for the recovery of other known biologically active substances. The phosphorylated substance produced is found in the cultured mycelium and filtrate, and accordingly can be isolated and purified from the mycelium and the filtrate, which are o~tained by filtering or centrifuging the cultured broth, by a conventional 10 method such as concentration under reduced pres~ure, lyophilization, ex*raction with a conventional sol-vent, such as methanol and the like, pH adjustment, treatment with a conventional resin (e.g. anion or cation exchange resin, non-ionic adsorption resin, lS etc.), treatment with a conventional adsorbent (e.g.
activated charcoal, silicic acid, æilica gel, cellu-lose, alumina, etc.), crystallization, recrystalliz-ation? and the like. A preferred method is solvènt extraction, particularly using methanol.
The phosphorylated organic compound obtained according to the resting cell or fermenta-tion proces~es as explained above can be isolated and purified in a conventional manner, for example, extraction, precipitation, fractional crystalliza-2S tion, recryætallization, chromatography, and the li~e.
Suitable formulations of the material may also include conYentional pharmaceutically acceptable biolabile esters of phosphorylated organic compound, formed via the hydroxy groups on the molecule, such 30 as the acetate.
The phosphorylated organic compound, andparticularly that of FK-520, of the present invention . .
- ~ , ~,. . .
., ' " ':~ .

., ~v092/06~2 PCT/US91/~16 2~93~

are water soluble possesses pharmacological activity such as immuno6uppressive activity, antimicrobial activity, and the like, and therefore are u~eful for the treatment and prevention of the transplantation rejection o~ or~ans or tissues ~uch as heart, ~idney, liver, medulla ossium, s~in, etc., graft-versus-h~t diseases by medulla osæium transplantation, autoi~mune diseases such as rheumatoid arthritis, ~ystemic lupus erythematosus, ~ashimoto~s thyroiditis, multiple lO sclerosis, myasthenia ~ravis, type I diabetes, uveitis, and the like.
The pharmaceutical composition of this invention can be used in the form of a pharmaceu- -tical preparation, for example, in solid, semisolid 15 or liquid form, which contains the instant invention compounds, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications.
The active ingredient may be compounded, ~or example, 20 with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, supposi-tories, æolutions, emulsians, æuspensions, and any other ~orm suitable for u8e. The carriers which can be used are water, glucose, lactose, gum acacia, 2S gelatin, mannitol, starch paste, magnesium trisili-cate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carrier~ suitable for use in manufacturing prepasations, in solid, sem~- -solid, or liquid form, and in addition auxiliary, ~tabilizing, thickening and coloring agents and perfumes may be used. The active object compound is included in the pharmaceutical composition in an wo92/o69s2 ~ PCT/US91l~16_ amount sufficient to produce the desired effect upon the proces~ or condition of diseases.
For applying this composition to a human, it is preferable to apply if by parenteral or enteral administration. While the dosage of therapeutically effective amount of the C-32 phosphorylated FK-506, varies from, and also depends upon the age and c~n-dition of each individual patient to be treated, a daily dose (calculated on the basis of a 70 kg man) lO of about 0.01-1000 mg, preferably 0.1-500 mg and more preferably 0.5-10~ m~, of the acti~e ingredient is generally gi~en for treatin~ diseases, and an average sin~le dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, ~0 mg, 100 mg, 250 mg and 500 mg is generally 15 administered.

Rapamycin It has been further found that a new immunosuppressant, a phosphorylated rapamycin 20 macrolide, can be obtained via the present process by contacted resting Rhizopus cells in a phosphate buffered medium containing glycerol as a carbon nutrient, or ~y the fesmentation of the microorganism Rhizopus oryzae, ATCC No. 11145 together, in the 25 presence of th-e macrolide rapamycin, under submerged aerobic conditions in an aqueous carbohydrate medium, containing a nitrogen nutrient, said conditions being conducted at a p~ of about 7 for a sufficient time, e.g. 24 hours at 27C, to selectively C-43 phosphory-30 late the rapamycin type macrolide. Either processcan be utilized but preferred is the process usin~
resting cells.

.. . . . .. .. . . . . .

wos2~06gs2 PCT/US91/~16 2B93~9 The resultant C-43 phosphorylated macrolide exhi~its immunosuppressant activity, similar to rapa-mycin i.e., inhibition of T-cell proliferation of mouse T lymphocytes 6timulated with the combinati~n of interleukin-2 plus PMA. A positive sample in this assay will inhibit T-cell proliferation, as indicated by reduced tritiated thymidine uptake.
Also, in accordance with this invention, there is provided a process for producing an 10 immunosuppressant, identified as a phosphorylated macrolide comprising the step of contacting a strain of a RhizQp~ microorgani~m capable of phosphorylat-ing free hydroxy groups, e.g., Rhizopus orvzae, and specifically Rhiz~ orvzae ~TCC No. 11145, togethe~
15 with rapamycin macrolide (See U.S. Patent 3,929,992 for its preparartion), in an aqueous medium, containing a carbon nutrient, preferably at a pE
below about 8.0, for a sufficient time to produce the C-43 phosphorylated macrolide.
Also provided is the unfiltered broth produced by the above process, which e~hibits positive inhibition of T-cell activation.
Also provided is a new immunosuppressant being a phosphorylated macrolide which exhibits 25 a proton nuclear magnetic resonance spectrum as identified in Figure 3, and a molecular weight of 993 as obtained by (FAB) mass-spectrometry.
Also provided is a pharmaceutical composition for the treatment of immunoregulatory 30 disorders and diseases containing a therapeutically effective amount of the phosphorylated macrolide in combination with a pharmaceutically acceptable, substantially non-toxic carrier or excipient.

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

W092/06992 c~; PCT/US91/~16 In addition, there is provided a method of uæe for treating a human host to prevent organ transplantation rejection, i.e., heart, kidney, liver, lung, bone marrow, or for treating autoimmune diseases i,e., juvenile diabetes mellitus comprising administering to said host a therapeutically effec-tive amount of the phosphorylated macrolide.
The present invention involves a biotrans-formation process, which involves the contacting lO of resting cells, or the fermentation of, the micro-organism, Rhizopus orvzae, together with a rapamycin macrolide containing a free hydroxy group, to produce the phosphorylated macrolide derivative. The micro-organism is currently on deposit with the American 15 Type Culture Collection, 12301 Parklawn Drive in Rockville, Maryland as ATCC No. 11145.
By the term "phosphorylated macr~lide" as used herein is meant the compound having a proton NMR
spectrum as illustrated in Figure 3 (of the methylated 20 derivative), a mass spectrum molecular ion of 993, and an assigned molecular structure of the formula;

~ . .
.

. .

~0 92/06g92 PCrtUS91/06816 2~3~2~

, ,OR
~5 ~3~{)CH3 47 ~ ~z ~ .
CH3 ~9~1 41 CH3 lD

CH~, 3CH3 .

ll :
~ere R= [ HO] 2-P-In general, the C-43 phosphorylated macrolide can be produced by either contacting resting cells, of Rhizo~us in an aqueous buffered phosphate medium containing a carbon nutrient at ambient temperature, or by culturing (fermenting) the above-described microorganism, Rhiz~ oryza~, in the presence of the known rapa~ycin macrolide, 25 described in U.S. Patent 3,9~9,992, in an aqueous nutrient medium containing sourceæ of assimilable carbon and nitrogen, preferably under submerget aerobic conditions (e.g. shaking culture, submerged culture, etc.). The aqueous medium is preferably 30 maintainet at a p~ of about 7 at the initiation and termination (harvest) of the fermentation :

.

W092/06992 ~ PCT/US91/~16 -~
r .) process. A higher p~ leads to substantial and/
or total loss of product. The desired pH may be maintained by the use of a buffer such as morpholinoethanesulfonic acid (MES), morpholino-propanesulfonic acid (MOPS), and the like, or bychoice of nutrient materials which inherently possess buffering properties, such as production media described hereinbelow.
The preferred sources of càrbon in the 10 nutrient mediùm are carbohydrates such as glucose, xylose, galactose, glycerin, ætarch, dextrin, and the like. Other sources which may be included are maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, and the like.
The preferred sources of nitrogen are yeast extract, meat extract, peptone, gluten meal, cotton-seed meal, soybean meal and other vegetable meals (partially or totally defatted), casein hydrolysates, soybean hydrolysates and yeast hydrolysates, corn 20 steep liquor, dried yeast, wheat germ, feather meal, peanut powder, distillerls solubles, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.-), urea, amino 25 acids, and the like.
The carbon and nitrogen sources, though ad~antageously employed in com~ination, need not be used in their pure form, because less pure materials which contain traces of growth factors and consider-30 able quantities of mineral nutrients, are alsosuitable for use. When desired, there may be added to the medium mineral ~alt~ such as sodium or calcium , . ........................ , .: , . , , . . ,, . . : , : .. :

~092/069g2 PCTJUS91/~16 2a93~29 carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, cobalt salts, and the like. If necessary, especially when the culture medium foams æeriously, a defoaming agent, such aæ
liquid paraffin, fatty oil, plant oil, mineral oil or silicone may be added.
The rapamycin starting material can be ob-tained by the known fermentation of S. hy~Q~copicus, 10 NRRL No. 5491, as described in U.S. Patent 3,929,992.
As to the contitions for the production of the phosphorylated macrolide in maæsive amountæ, submerged aerobic cultural conditions are preferred therefor. For the production in small amounts, a 15 6haking or surface culture in a flask or bottle lS
employed. Furthermore, when the growth iB carried out in large tanks, it iB preferable to use the vegetative form of the organism for inoculation in the production tanks in order to avoid growth 20 lag in the proceæs of production. Accordingly, it is desirable firæt to produce a vegetati~e inoculum of the organism by inoculating a relatively Rmall quantity of culture medium with sporeg or mycelia of the organism produced in a "slant" and culturi~g ~aid 25 inoculated medium, also called the "seed medium", and then to transfer the cultured vegetative inoculum aseptically to large tankæ. The fermentation medium, - ;
in which the inoculum is produced, is æubstantially the æame as or different from the medium utilized for 30 the production of the phosphorylated macrolide and iæ
generally autoclaved to sterilize the medium prior , -to inoculation. The p~ of the medium is generally - - .. , .. ~ . , : , , . . ~ -., .. ~ . .. ,. - -- .. . : i ~ .; . . . . .:
.
.,. , . ., , ~

W092/06g92 c~ PCT/US91/~6 adjusted to about 7.0 prior to the autoclaving step by suitable addition of an acit or base, preferably in the form of a buffering solution.
Agitation and aeration of the culture mixture may be accomplished in a variety of ways.
Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment or by the pasæage of ~terile air through the medium.
lO Aeration may be effected by passing sterile air through the fermentation mi~ture.
The fermentation is usually conducted at a temperature between about 20C and 40C, preferably 25-35C, for a period of about 10 hours to 24 hour6, 15 which may be varied according to fermentation con-ditions and scales. Preferably, the production cultures are incubated for about 24 hours at 27C
on a rotary shaker operating at 220 spm, wherein the pH of the fermentation medium is maintained at 20 7,0 to har~e~t.
Preferred culturing/production media for carrying out the fermentation include the following media:
Seed and T~an~forma~ion Mediu~ ~Lk De~trose 20.0 Soy Meal (Sigma) 5.0 Yeast E~tract (Fitco) 5.0 NaCl 5.0 K2~P04 5.0 Adjust pH to 7.0 ~-- - - . . - . -. . .
.

. ~ '' .

W092/06992 PCT/US91/~16 ~ A;~ ~

The produced phosphorylated macrolide can be reco~ered from the culture medium by conventional means which are commonly used for the recovery of other known biologically active substances. The phosphorylated macrolide produced is ~ound in the cultured mycelium and filtrate, and accortingly can be isolated and purified from the mycelium and the filtrate, which are obtained by filtering or centri-fuging the cultured broth, by a conventional method lo such as concentration under reduced pressure, lyo-philization, extraction with a conventional solvent, such as methanol and the like, p~ adjustment, treat-ment with a conventional resin (e.g.anion or cation exchange resin, non-ionic adsorption resin, etc.), 15 treatment with a conventional adsorbent (e.g. acti-vated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, recrystallization, and the like. A preferred method is solvent extraction, particularly using methanol.
The product phosphorylated macrolide from the fermentation exhibits positive immunosuppressi~e activity by the "T-cell proliferation assay" and possesses utility on this basis and exhibits the followi~g physical characteristics:

1. White amorphouQ powder 2. Solubility in methanol 3. Molecular weight of 993, as determined by FAB mass spectroscopy and is consistent with the assigned molecular structure in ~igure 3.

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

. ~

WO92/06s92 PCT/US91/~16 c~ .

The phosphorylated macrolide obtained accord-ing to the resting cell or fermentation processes as explained above can be isolated and purified in a conventional manner, for example, ex*raction, precipi-tation, fractional crystallization, recrystallization,chromato~raphy, and the like.
Suitable formulations of the material may also include conventional pharmaceutically acceptable biolabile esters of the phosphorylated macrolide, 10 formed via the hydroxy groups on the molecule, such as the acetate~
It is to be noted that in the aforementioned fermentation reactions and the post-treatment of the fermentation mixture therein, the tautomeric and 15 conformational isomer(s) of the phosphorylated macrolide, including those due to rearrangement of the hemiketal ring system are also included within the scope of the present invention.
The phosphorylated macrolide of the present 20 invention possesses pharmacological activity such as immunosuppressive activity, antimicrobaal activity, and the like, and therefore are useful for the treatment and prevention of the transplantation rejection of organs or tissues ~uch as heart, ~idney, 25 liver, medulla ossium, ~kin, etc., graft-versus-host diseases by medulla 088ium transplantation, autoimmune diseases such as rheumatoit arthritis, systemic lupus erythematosus, ~ashimotois thyroiditis, multiple --sclerosis, myasthenia gravis, type I diabetes, 30 uveitis, and the li~e.
: The pharmaceutical composition of this invention can be used in the ~orm of a pharmaceutical ~092/06~2 PCT/US91/~16 2a93~

preparation, for example, in solid, semisolid or liquid form, which contains the instant invention compounds, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for e~ternal, enteral or parenteral applications.
The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsuleæ, supposi-tories, solutions, emulsions, suspensions, and any lO othel form suitable for use. The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisili-cate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for 15 u6e in manufacturing preparations, in solid, semi-solid, or liquid form, and in addition ausiliary, stabilizing, thickening and coloring agents and perfumes may be used. The active object compound i6 included in the pharmaceutical composition in an 20 amount sufficient to produce the desired effect upon thè process or condition of diseases.
For applying this composition to a human, it is preferable to apply if by parenteral or enteral administration. While the dosage of therapeutically 25 effective amount of the phosphorylated macrolide, varies from, and also depends upon the age and con-dition of ~ach individual patient to ~e treated, a daily dose (calculated on the basis of a 70 kg man~
of about 0.01-1000 mg, preferably 0.1-500 mg and more 30 preferably 0.5-lO0 mg, of the active ingredient is generally given for treating diseases, and an a~erage single dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, ~0 .
,. - . , , , . ,,. . ~ .~
.: , .

W092/~6gg2 ~ PCT/~S91~16 mg, lO0 mg, 250 mg and 500 m~ is generally administered.

Echinocand~
The present invention process also is applicable in a process for selectively biophosphorylating a cyclic lipopeptide related to echinocandins and ha~ing a peptide skeleton bearing se~eral hydroxy groups wherein in said phosphorylated cyclic lipopeptide, the phosphate group is attached to the hydroxy group of the 4-hydroxyproline component of the lipopeptide.
Echinocandins or echinocandin compounds are cyclohexapeptide compounds ha~ing a lipophilic side 15 chain and ha~in~ antifungal properties. Many are natural products ~ut many compounds are semi-synthetic. The natural products are described in the literature by such names as echinocandin~, aculeacins, mulundocandin, by number designations or 20 by structure. Many have been ~nown a long time and the structure and properties may be found summarized in the CRC ~andbook of Antibiotic Compounds, Vol IV, Part I, pp 355-367, CRC Press, Inc., Boca Raton, Fla.
1980. Still others include a more recently 25 discovered compound such as that described in U.S.
Patent 4,931,352.
~ he ~resent invention is especially directed to a compound having the formula (III):

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

- . ..... , .. -.

WO 92/06592 PCI/US~/06816 2~3~2~

HO OH
~IO~ O

1 1 ~N
NH2CCH2~ HN~H3 HO NH o=~~OH
HO~, o R ( III) ~o OH

wherein R is -P~0~)2 or a cation ~alt thereof.
By "cation salttl is meant a salt of Li, ~, Mg, Na, Ca, and (Cl-C4 alkyl)ammonium.

When R i~ -P(0~)2, the compound may be represented by formula (IIIA), and hereinafter referred to as Com~ound IIIA.

HO OH
IOI ~

NH2CCH2 ~ HN ~ H3 ~ C~92 (III~) HO OH

: . .
- . . . . . .. . ..

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

W092/06~2 PCT/US91/~16 _ c~

BRI~F DES~RIPTION OF T~E DRAWING
Fig. 5 is nuclear magnetic resonance o spectrum of Compound III in which R is -P(~)(O~).

MASS SPECTRAL~n~E~
Comp~und IIIA has a molecular weight of 1144 by FAB-MS (observed (M ~ Na)+ of 1167).

1~ MMR Spectra of the compound isolated as a monopotassium salt in CD30D at 400 M~z is seen in Fig. 5; and :
13C NMR chemical shifts of the ~ame isolate 15 in CD30D at 100 M~z are a~ follows: -~c 177.2, 175.9, 174,3, 173.2, 172.~, 172.58, 172.57 169.0, 158.4, 133.1, 129.6, 116.2, 77.0, 75.8, 75.2 (doublet, JCP=4-0 ~ertz), 74.3, 73.9, 70.6, 70.5, 69.7, 6B.3, 62.7, 58.3, 56.3, 56.1, 55.6, 51.2, 47.0, 20 45.9, 39.4, 38.1, 37.6, 36.7, 35.0, 34.6, 32.9, 31.24, 31.20, 30.8, 30.6, 30.3, 28.1, 27.0, 20.7, 20.2, 19.8, 11.6.
On the basis of the~e and other data, Compound IIIA i8 belie~ed with con~iderable certainty 25 to have the 8tructure indicated.
Compound IIIA is a white solid soluble in water and polar solvents such as lower al~anols and in dilute alkali metal, ~agnesium, calciu~, ant tetra (lower alkyl)a~monium bases. From the ba~es, salt~ ~
30 in which R is a cation salt of phosphate may be ~ -obtained.

. ~, . .. . . . . . .

~092/06gg2 PCT/US91~16 20~3~29 The compound of this invention ha6 similar antibiotic properties as the non-phosphorylated compound and thu~ would be u~eful a~ an antibiotic for the control of parasites, especially Pneum~y~is S carinii, the causative agent of pneumocystis pneumonia, a particular problem with immune compromised patients, and for the control of fungi.
Its antifungal properties are particularly u~eful against certain strains of yeast, such as Cand i a ~lbican~ and Candid~ tropicalis.
o The compound in which R i~ -P(0~)2 is conveniently produced by incubating a compound having the formula (Z) (Compound Z) HO OH

2 0 1 1 2N~H ~
NH2CCH2 ~) HN~H3 HO NH o ~ OH

~ ~ N
HO OH

.
- ~ .. . . . .

'' ' "'' ; ~ ' '.`

: - , -.
.
.

WO92/06ss2 ~ PCT/US91/~16 with induced resting cells of Rhizopus arrhizus ATCC
11145 maintained in the Merck Culture Collection as MF 4974. The culture was originally obtained from the American Type Culture Collection at 12301 Parklawn Drive, Rockville, MD 20852.
Compound Z maybe produced by cultivating Zal~rion arboricola ATCC 74030 i~ nutrient medium as hereinafter described as well as by methods described in copending applications S.N. 492,025, filed lo March 12, 1990 and S.N. 492,026, also filed March 12, 1990.
The microorganism Rhizopus arrhiz~s ATCC
11145 is also known a~ R~i~o~us oryzae (J.J. Ellis, 1985, Mycologia 77: 243-247). The species has also 15 been described under the names Rhizopus nodos~s and Rhizopu~ tritici. The strain ME 4974, ATCC 11145, exhibit6 all the eæsential features of ~. arrhizus described by M.A.A. Schipper under the name ~.
oryzae, CBS Studies in Mycology 25:1-19 (1984).
20 During a recent re~rowth of MF4974 ATCC 11145, the following diagnostic characteristics were observed.
The ~train is apparently heterothallic because zygoepore spore formation was not observed.
Colonies grow on most standard mycological media, but 25 on cornmeal agar (Difco) are extremely fast-growing, reaching 35 mm in diameter in 36 hQurs at 20OC, reaching >90 mm in 36 hours at 37-C, with ~porangiophores completely filling Petri dishes, hyaline at fir6t but soon becoming pale yellowish ~--gray to light gray, brownish gray, finally dark gray. Sporangiophores 200-lO00 ~m tall, 7.5-19 ~m wide, aseptate, 6traight to curved at base, flared at ' "' ' ' ' '" ''; ` ' .'. ' "- :'' '. .' ..~
- . , . . .. , , ~ .

W092/06992 PCT/US9l/~16 20~3~ 3 apex, sometimes furcate, with walls slightly thickened, with minutely granular ~urface, pale yellowish brown or yellowish gray, arising from rhizoidal hyphae. Rhizoidal hyphae consisting of 3-10, thick, often contorted branches.
Sporangia globose to subglobo6e, slightly flattened on underside, 100-230 ~m in diameter, opaque, with surface spiny, dark gray to black.
Columellae 18-50 ~m in diameter, hemispherical to subglobo~e, smooth, often collapsing, without adhering sporangia remnants. Sporangiospores 5-8 X 2~5-5 ~m, subglobose to irregularly elliptical, or angular in sideview, with faint to prominent longitudinal ~triations, hyaline to pale yellowish 15 brown.
Although the invention is di~cussed hereinbelow principally with respect to a 6pecific 6train, not only the strain described above, but varieties and mutantæ, whether obtained by natural selection, produced by the action of various mutating agents such as ionizing radiation or chemical agents such as nitrosoguanidine are contemplated within the scope of the present invention.
Compound IIIA may be produced by cultivating 25 Rhizop~ arrhi~g~ ATCC 11145 in a guitable nutrient medium containing Compound Z under condition~
hereinafter described and thereafter recovering from the product medium by extracting the desired protuct from the fermentation medium with a suitable 801~ent, concentrating the component containing the desired compound, and then subjecting the concentrated material to chromatographic separation. --... . . .

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

W092/06992 ~ PCT/US9~
~,,' The cultivation i8 carried out in a medium containing sources of carbon and nitrogen assimilable by the microorganiism.
The sources of carbon include glycerol, sugars, sugar alcohols, starches and other carbo-hydrateæ, or carbohydrate deivatives isuch as de~tran, cerelose, as well as complex nutrients such as oat flour, corn meal, millet, corn and the like. The exact quantity of the carbon source which is utilized lo in the medium will depend, in part, upon the other ingredients in the medium, but it is usually found that an amount of carbohydrate between 0.5 and 40~/. by weight of the medium is satifactory. These carbon sources can be used individually or several æuch 15 carbon sources may be combined in the same medium.
The sources of nitrogen include amino acids such as glycine, arginine, threonine, methionine and -the like, ammonium salt, and comple~ sources such as yeast hydrolysates, yeast autolysates, yeast cells, tomato paste, soybean mea~, casein hydrolysates, yeast extracts, corn steep liquors, distillers solubles, cottonseed meal, meat extract, and the like. The various ~ources of nitrogen can be used alone or in combination in amountis ranging form 0.2 2~ to 10 percent by weight of the medium.
- In addition, the medium should contain a phosphate salt. The phosphate salt ~hould be at least about 10 percent by weight of the solid components.
It i~ preferably from about 12 to about 15 percent. -30 A particularly suitable medium is soy-glucoæe medium of the following composition which may be employed both as a seed medium and a culture medium:

- . - i.. ~, . . .
. . . , ~., ~

~092/D6992 PCT~US91/ ~ 16 2~3~`2~

.
5Qy-Glucose Medl~m ~Ll Glucose 20.0 Soya meal 5.0 Fidco yeast extract* 5.0 NaCl K2EP04 5.0 Adjust pH to 5 _______ *Fidco yeast extract is a nitrogen source, product of Difco Laboratories, Detroit MI.
The fermentation may be carried out by first preparing a seed culture. In preparing a seed l~ culture, spores of Rhizo~s arrhizus are obtained from oatmeal agar slants of MF 4974 maintained in the Merck Culture Collection and dispersed in water to obtain a ~pore suspension containing about 7 x 109 spores per milliliter.
The spore ~uspension of MF 4974 is inoculated into a seed flask containing the ~oy - glucose broth and the inoculated suspension incubated on a rotary shaker in the temperature range of from about 15C to about 30C, preferably 25 to 28-C.
2S The agitation may be up to 400 rpm but generally about 2Z0 rpm is preferred. The incubation is carried out over a period of at least 24 hours to about two days.
When growth is abundant, the mycelia are harvested by filtering throu~h a nylon mesh. ~or biophosphorylation, the mycelia are suspended in a phosphate buffer containing 3 percent glycerol or some other simple carbon ~ource. Compound Z then is .. . . .

~ ' ~ , . 1 , W092/06992 ~C~3 PCT/US91/~

added at a concentration of about 50 ~g/ml in dimethylsulfoxide (DMSO). The pH of the production medium is important. The flasks are incubated, pre-ferably with ~haking at 220 rpm at 27C for 24 to 48 hours to produce Compound IIIA. It is critical that it be maintained in the range of about 6.0 to 6.3.
After completion of the incubation period the contents of all the flasks are pooled and filtered through a nylon mesh filter. The mycelial lo cake on the filter is slurried with aqueous methanol and filtered. The procedure is repeated with the filter cake and the filtrate loaded onto a styrene/
divinylbenzene column. The column is then washed -with water and the phosphorylated product eluted with 20 percent aqueous acetonitrile and the remaining metabolite and substrate eluted with 70 percent aqueous acetonitrile.
After elution, the fractions may be assayed by HPLC. The fractions determined to have the desired 20 product as indicated by a retention time of 12.8 min~.
are combined and concentrated under reduced pressure to obtain the product as residue.
The salts, i.e., where R is a cationic salt of the phosphate, may be prepared by intimately 25 contacting a base corresponding to the cation in an alcoholic or other polar solvent, then concentrating to initiate crystallization of the salt. Thereafter, the salt is recovered by filtration.
One method of preparing salts is to apply an aqueous solution of the acid onto a non-functional-ized resin column. Representative resins include "AMB~RC~ROM"-161 (divinylbenzene/polysyyrene resin, : ,.- . - . . -. . ~
- : ,: . . . . . , . , ,. , . : :. .

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

~92/06gg2 PCT/US9l/~16 obtainable from TosoHaas; trademark name registered by Rohm and ~aas), "DIAION" ~P-20 and SP-207 (croæs-linked styrene-di~inylbenzene and brominated ~tyrene-divinylbenzene, respectively, products of Mitsubi6hi Chemi~al). The column is then washed with aqueou6 MH2P04 or M (~2P04)2 where M and M are monovalent and divalent cations respectively, thereby converting the acid to a mono-cation ~alt form. The column is washed with water to remove excess inor~anic phosphate lo salt. The product M or M~ salt is then removed from the column by applying an aqueous eluant having greater than 50 percent organic content. Useful eluants are 80 percent acetonitrile, 80 percent ethanol or 80 percent methanol. The product is isolated by concentration to tryness and/or lyophilization of the eluate.
This procedure also may be employed to prepare one salt from another.
Alternati~ely, the acid iB dissolved in an aqueous mobile phase containing low amount of organic solvent such as acetonitrile and containing phosphate salt thereby forming a solution of the ~alt of the acid. The ~olution is subjected to reduced pressure to remove the acetonitrile, then applied to a C-18 extraction column to retain the salt of the product on the column. The salt of the product then may be removed as above described.
As previously noted, the phosphate is a compound which is active against certain yeast fungi such as ~. al~lcan~ and C. tropicali~. The activity may be seen in a microbroth dilution assay employing a Yeast Nitrogen Base (Difco) with 1% dextrose - : . .: -~ , . -.~ . . . . .

, . . . .. . . . . . . . .

W092/06992 ~ PCT/US91/~16 ~3 (YNBD~. In carrying out the assay, Compound IIIA was solubilized in lO percent dimethyl sulfo~ide (DMS0) and diluted to 2560 ~g/ml. The compounds were further diluted to 256 ~g/ml in YNBD. Then 0.15 ml of the suspension was dispensed to the first row of a 96-well plate (each well containing 0.15 ml of YNDB) resulting in a drug concentration of 128 ~g/ml. Two-fcld dilutions were then made to obtain final drug concentrations ranging from 128 to 0.06 ~gtml.
lOThe yeast cultures, maintained on Sabouraud dextrose agar were transferred to YM broth (Difco) and incubated overnight at 35C with shaking (250 rpm~. After incubation, each culture was diluted in -sterile water to yield a final concentration of 1-5 x 106 colony forming units (CFU)/ml.
96-well microplates were inoculated using a MIC-2000 (Dynatech? which delivers 1.5 ~1 per well yielding a final inoculum per well of 1.5-7.5 ~ 103 cells. The microplates were incubated at 35C for 24 hours. The minimum inhibitory concentrations (MICs) were recorded as the lowest concentrations of drug showing no visible growth.
After recording ~he ~IC, the plates were shaken to resuspend the cells. Thereafter, 1.5 ~1 samples from the wells in the 96-well microplate were transferred to a single well tray containing Sa~ouraud dextrose agar. The inoculated trays were incu~ated 24 hours at 28C and then read. The MEC is defined as the lowest concentration of drug ~howing no growth or less than 4 colonies per spot.

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

~?92/06g92 PCT/US91/06816 2 ~ ,a 3 d~

Minimum Fungicidal Concentration Fungus ~lml) St~ain No. Compound IIIA
Candida albicans -S MY 1~ 64 Candida tropicalis The fore~oing illustrates particular suitability for treating mycotic infections.
The compounds of the present invention may be employed in inhibiting or alleviating pne~mQsy~
carinii infections. In such use, Compound III/IIIA
or a composition containing Compound III/IIIA may be administered in a therapeutically effective or inhibitory amount to subject~ infected with or susceptible to being infected with ~a~gmQCvsti~
~arinil.
The 8U i tability of the compounds of the present invention sor therapeutic or anti-infective purposes may be determined in studies on immuno-suppressed rats when Sprague-Dawley rats (weighing 2s approximately 200 grams) are immuno6uppres~ed with dexasone in the drin~ing water ~2.~ mglL) and maintained on a low ~rotein diet for five weeks to induce the deYelopment of pneumocystig pneumonia from a latent infection. Before drug tseatment, tw~ sat6 are sacrificed to confirm the presence of Pne~mocystiæ
carinii pneumonia (PCP). Siæ ratæ then are injected twice daily for four days intra~enously (I.V.) ~ia . ' .... , , ~ ' ., ~ ` ' , , , . :

," `." " . ' ' ' ' ' ' , ' ~ "`, ' . ' , ` ~ "' `` ` ', -W092/06992 PCT/US91/~

the tail ~ein with Compound III in 0.25 ml of vehicle (distilled water). A vehicle control is also carried out. All animals continue to receive dexasone in the drinking water and low protein diet during the treatment period. At the completion of the treat-ment, all animals are sacrificed, the lungs are removed and processed, and the egtent of diæease determined by microscopic analysis of stained slides.
A similar egperiment may be carried out in lO which the rats are injected intraperito~eally (I.P.) twice daily for four days and then sacrificed, the lungs removed and processed, and the extent of disease determined by microscopic analysis of stained slide~.
The outstanding properties are most ef$ec- -15 tively utilized when the compound is formulated into -novel pharmaceutical compositions with a pharmaceu- -tically acceptable carrier according to conventional pharmaceutical compounding techniques.
The novel compositions contain at least a therapeutic antifungal or antipneumocystis amount of the active compound. Generally, the composition contains at least 1% by weight of Compound III.
Concentrate compositions suitable for dilutions prior to use may contain 90% or more by weight.
25 The compositions include compositions ~uitable for rectal, topical, parenteral (including gubcutaneous, intramuscular, and intravenou~), pulmonary (naæal or buccal inhalation), nasal administration, or inæufflation. The compcsitions may be prepacked by intimately mixing Compound III with the components æuitable for the medium desired.

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

~V092/06992 PCT/US9l/~16 2Q ~3~

When the compound is for antifungal use any method of administration may be used. For treating mycotic infection, oral administration is frequently preferred. When oral administration is to be employed, it may be with a liquid composition or a solid composition. For liquid preparations, the therapeutic agent is preferably formulated with water or aqueous compositions, but if desired, may be formulated with glycols, oils, alcohols, and the like. For solid preparations such as capsules and tablets, solid carriers such as starches, sugars, kaolin, ethyl cellulose, calcium and sodium carbonate, ealcium phosphate, kaolin, talc, lactose, generally with lubricant such as calcium stearate, together 15 with binders, disintegrating agents and the like.
Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage form. It is especially advantageous to formulate the compositions in unit dosage form (as 20 hereinafter defined) for ease of administration and uniformity of dosage. Composition in unit dosage form constitutes an aspect of the present in~ention.
The Compound III is preferably formulated in aqueous therapeutic compositions for intravenous or - 25 intraperitoneal injection or aerosol when use against ~n~s~n~ystis ~Linii i8 contemplated, and may be presented in unit dosage form in ampoules or in multidose containers, if necessary with an added preservative. The compositions may al80 take æuch 30 ~orms as solutions in aqueous ~ehicles such as 0.85 ~ : -percent sodium chloride or 5 percent de~trose in water, ànd may contain formulating agents æuch a~ -- : . : . . .:, . , , ., . ~, .

... . . . . . . . .

W092/W992 ~ PCT/US9l/~l6f-.~ ., stabilizing and/or disper~ing agent~. Buffering agents as well as additives æuch as saline or glucose may be added to make the solutions isotonic. The drug also may be solubilized in alcohol/propylene glycol or polyethylene glycol for drip intravenous administration. Alternatively, the active ingre-dients may be in powder form for reconstituting with a suitable vehicle prior to administration.
The term "unit dosa~e form~ as used in the specification and claims refer to physically discrete units, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceu-tical carrier. Examples of such unit dosage forms are tablets, capsules, pills, powder packets, wafers, measured units in ampoules or in multidose containers and the like. A u~it dosage of the present invention may contain from 100 to 1000 milligrams of one of the compounds.

~IV Protease In~i~itor~
The present invention is also concerned with a compound which inhibits the protease encoded by human immunodeficiency virus (~IV). The compound, 25 or pharmaceutically acceptable salt thereof, is of ~ :
value in the prevention of infection by ~IV, the treatment of infection by ~IV and the treatment of the resulting acquired immune deficiency syndrome (AIDS). The present invention also relates to pharmaceutical compositiong containing the compounts, and to a methot of use of the present compound~ with or without other agents for the treatment of AIDS
viral infection by HIV.

- . . .
, . : . ', ' . , : .

~92/06992 PCT/US91/~16 2~93~

A retrovirus designated human immunodefi-ciency virus (H~V~ is the etiological a~ent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system This virus was previously known as LAV, XTLV-III, or ARV. A common feature of retro~irus replication is the e~tensive post-trans-lational processing of precursor polyproteins by a virally encoded protease to generate mature ~iral proteins required for viruæ aæsembly and function.
Interruption of this proceæsing appearæ to prevent the production of normally infectious virus. For example, Crawford, S. et ~1-. J. Virol., 53, 899, 1985, demonstrated that genetic deletion mutations of the proteaæe in murine leukemia virus which prevent processing of precursor structural proteins re~ult in non-infectious ~iral particles. U~processed ~truc-tural proteins also have been observed in clones of non-infectious ~IV strains isolated from human patients. These results suggest that inhibition of the ~IV protease represents a viable method for the treatment of AIDS and the prevention or treatment of infection by EIV.
Nucleotide sequencing of ~I~ shows the presence of a EQl gene in one ope~ reading frame ~L. Ratner, et ~l., Nature, ~l~. 277(19853~. Amino acit sequence homology provides e~idence that the pQl sequence encodes reverse tran~cripta8e, an endo-nuclea8e and an ~IV protease t~. Tol, et ~1., EMB0 J.4, 1267 ~1985); M. D. Power, et ~1., Science, ~
1567 (1986); L. ~. Pearl, et ~ ature 329, 351 (1987)].

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

.

wO92~06g92 ~ PCT/US91/~16--9~7 Applicants demonstrate that the compound of this invention is an inhibitor of ~IV protease. The compound of this invention provides a prodrug for the inhibition of ~IV protease.
A biotransformed compound as herein defined is disclosed. This compound is useful in the inhibition of ~IV protease, the pre~ention of infection by HIV, the treatment of infection by ~IV
and in the treatment of AIDS and/or ARC, either aæ
a compound, pharmaceutically acceptable salt (when appropriate), pharmaceutical composition ingredient, whether or not as a prodrug or as a combination with other anti~irals, anti-infecti~es, immunomodulators, antibiotics or vaccines. Methods of treating AIDS, 15 methods of preventing infection by ~IV, and methods of treating infection by ~IV are also disclosed.
This invention is also concerned with the use of a compound given below, or pharmaceutically acceptable salts thereof, in the inhibition of ~IV
20 protease, the prevention or treatment of infection by -- ~IV and in the treatment of the resulting acquired immune deficiency syndrome <AIDS). The biotransformed compound is the product of the incubation of RhizQ~s arrhi~us (A~CC 11145) in the presence of L-702,083, 25 an ~IV protease inhibitor. It is tefined as follows:

.

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

W092/06g92 PCT/US91/~16 2 ~

OH ~ N

(CH3~3ct~n~N ~ N"" ~
~/ ~

or pharmaceutically acceptable ~alts, hydrates or 15 esters thereof.

The pharmaceutically-acceptable salts of the compound of the present invention (in the form of water- or oil-~oluble or dispersible products) include the conventional non-toxic salt~ or the quaternary ammonium salts of this compound, which are formed, e.g., from inorganic or organic acids or bases. Eæamples of 8uch acid addition 6altg include acetate, adipate, alginate, aspartate, benzoate, 25 benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethane~ulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, 30 hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, . .
. . . ~ . , . ~ .. . ~ .
- - , .,. ~ , . , ~ . ...

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

W~92/Oh992 ~ PCT/US91/~16~
G~

lactate, maleate, methanesulfonate, 2-naphthalene-sulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium ~alts, alkali metal salts such aR sodium and potassium salts, alkaline earth metal ~alts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dial~yl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aral~yl halides like benzyl and phenethyl bromites and others. ~ydrates or ester~ are also encompassed by the present invention. Such hydrates or esters are thoæe which would readily occur to the skilled artisan, and include, for example, Cl_4alkyl e3ters.
The compound of the present invention i8 useful in the inhibition of ~IV protease, the 2S prevention or treatment of infection by the human immunodeficiency ~irus (~IV) and the treatment of consequent pathological conditions such as AIDS.
Treating AIDS or pre~enting or treating infection by ~IV is defined as including, ~ut not limited to, treating a wide range of states of ~IV infection:
AIDS, ARC (AIDS related complex), both symptomatic .
. , . . . ~

.

. ;

W092/06~2 PCT/US9~ 16 2~93~3 and asymptomatic, and actual or potential exposure to ~IV. For e~ample, the compound of this invention i6 useful in treating infection by ~IV after suspected past exposure to ~IV by e.g., blood tran~fusion, accidental needle stic~, or exposure to patient blood during surgery.
For these purpose~, the compound of the present invention may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infu6ion techniques), by inhalation 6pray, or rectally, in dosage unit formulations containing conventional non-to~ic pharmaceutically-acceptable carriers, adjuvants and vehicles.
Thus, in accordance with the present inven-tion there is further provided a method of treating and a pharmaceutical compo8ition for treating ~IV
infection and AIDS. The treatment in~olves admini-6tering to a patient in need of such treatment a pharmaceutical composition comprising a pharmaceu-tical carrier and a therapeutically-effective amount of the compound of the present invention.
The6e pharmaceutical compo~itions may be in the form of orally-administrable suspensions or tablets; nasal sprays; sterile injectable prepara-tions, for example, as sterile injectable aqueous or oleagenou~ suspensions or suppositories.
When admini6tered orally as a suspension, these~compositions are prepared according to techniques well-known in the art of pharmaceutical ,. - . - , . ... ,.. , . . ,., . . :

W092/06g92 ~ PCT/US91/~16_ ~;

formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or ~odium alginate as a suspenting agent, methylcellulo~e as a viscosity enhancer, and swee~nerslflavoring agents known in the art. As immetiate relea~e tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other e2cipients, binders, extenders, disintegrants, diluents and lubricants known in the art.
When administered by nasal aerosol or inhal-ation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
The injectable solutions or suspen6ions may 20 be formulated according to known art, using ~uit-able non-to~ic, parenterally-acceptable diluents or -solvents, such as mannitol, 1,3-butanediol, water, Ringer~s solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agentg, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
When rectally admi~istered in the form of suppo6itorie6, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, ~uch as cocoa butter, synthetic glyceride . .~ . , - ~ ........ . . . ,.............. .. , ......... . . :

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

W092/06~2 PCT/US91/~16 2 0 ~ 3 ~

esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.
Dosage levels of the order of 0.02 to 5.0 or 10.0 grams-per-day are useful in the treatment or prevention of the above-indica~ed conditions, with oral doses two-to-five times higher. For example, infection by ~IV is effectively treated by the administrati~n of from 10 to 50 milligrams of the compound per kilogram of body weight from one to three times per day. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the acti-15 vity of the specific compound employed, the metabolicstability and length of action of that compound, the age of the patient, body weight, general health, se~, diet, mode and time of administration, rate of excre-tion, drug combination, the severity of the particular 20 condition, and the host untergoing therapy.
The present invention is also directed to combinations of the HIV protease inhibitor compound with one or more agents useful in the treatment of AIDS. For esample, the compound of this inven~ion 2S may be effecti~ely administered, whether at periods of pre-exposure and/or post-exposure, in combination with effective amounts of other AIDS antivirals, immunomodulators, anti-infecti~es, or vaccines.

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

W092/069g2 PCT~US9l/~16_ ~ - 56 -HIV Protease Inhibitor - II
The present invention is also further con-cerned with another compound which inhibits the protease encoded by human immunodeficiency virus (~IV). The compound, or pharmaceutically acceptable salt thereof, is of value in the prevention of infection by ~IV, the treatment of infection by HIV
and the treatment of the resulting acquired immune deficiency syndrome (AIDS). The present invention lo also relates to pharmaceutical compositions containing the compound, and to a method of use of the present compound with or without other agents for the treatment of AIDS & viral infection by ~IV.
A retrovirus designated human immunodefi-15 ciency virus (~IV) is the etiological agent of thecomplex disease that include~ progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system. This virus was previously 20 known aæ LAV, ~TLV-III, or ARV. A common feature of retrovirus replication is the extensive po~t-tran~-lational processing of precursor polyproteins by a virally encoded protease to generate mature viral proteins required for virus assembly and function.
25 Interruption of this processing appears to prevent the protuction of normally infectiouæ virus. ~or example, Crawford, S. et ~1.. J. Virol., ~, 899, 1985, demonstrate,d that genetic deletion mutations of the protease in murine leukemia virus which prevent 30 processing of precursor structural proteins result in non-infectious viral particles. Unprocesset struc-tural proteins also have been observed in clones of non-infectious ~IV strains isolated from human . .. . . . . .

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

,. , . . . : : ~ , .

W092/06992 PCT/US9l/~16 2~3d~3 - ~7 -patients. These results æuggest that inhibitio~ of the HIV protease represents a viable method for the treatment of AIDS and the prevention or treatment of infection by HIV.
Nucleotide sequencing of ~IV 6hows the presence of a ~Ql gene in one open reading frame ~Ratner, L. et al., Nature, 313, 277(1985)]. Amino acid sequence homology provides evidence that the ~Ql sequence encodes reverse transcriptase, an lO endonuclease and an EIV protease ~Toh, ~. et al., EMBO J. 4, 1267 ~1985); Power, M.D. et al., Science, , 1567 (1986); Pearl, L.~. et ~1., Nature 329, 351 (1987)], Applicants demonstrate that the compound of 15 this invention is an inhibitor of HIV protease. The compound of this invention provides a prodrug for the inhibition of HIV protease.
A biotransformed compound as herein defined is disclosed. This compound is useful in the inhibi-20 tion of EIV protease, the prevention of infection byEIV, the treatment of infection by EIV and in the treatment of AIDS and/or ARC, either as a compound, pharmaceutically acceptable salt (when appropriate), pharmaceutical composition ingredient, whether or 25 not as a prodrug or as a combination with other antivirals, anti-infectives, immunomodulators, antibiotics or vaccines. Methods of treating AIDS, methods of preventing infection by ~IV, and methods of treating infection by EIV are alæo disclosed.
This invention is concerned with the use of a compound given below, or pharmaceutically accept-able salts thereof, in the inhibition of EIV protease, the prevention or treatment of infection by EIV and .. : ~ , . . . . .

W092/06~2 PCT/US91/~16_ C~.3 1.
in the treatment of the resulting acquired immune deficiency ~yndrome (AIDS). The biotransformed !
compound is the product of the incubation of Rhizop~s ar~hizus (ATCC lll4~) in the presence of L-689,502, a ~ -~IV protease inhibitor. It is defined as follows:

~/c) ~0-C~ ,~ OH

(3~
O
R=--P--OH
OH

or pharmaceutically acceptable salts, hydrates or esters thereof.

The pharmaceutically-acceptable salt6 of the compound of the present invention ~in the form of water- or oil-soluble or dispersible products) include the conventional non-toxic ~alts or the quaternary ammonium salts of this compound, which are formed, e.g., from inorganic or organic acids or 3~ bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, - . - , . . ~ , .. . . .
- . .

. '. , . . ~ . , :
,: . : , W092/06g9~ PCT/US91/~16 2~n3~
_ ~9 _ benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochlorite, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalene-culfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium ~alts, salts with organic bases ~uch as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and BO forth. Also, the ba~ic nitrogen-containing groups may ~e ~uaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halideg such as decyl, lauryl, myristyl and stearyl chloride6, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others. ~ydrates or esters are al~o encompassed by the present invention. Such hydrates or ester~
are those which would readily occur to the s~illed artisan, and include, for-example, Cl_4alkyl esters.
The compound of the present invention~
30 i8 useful in the inhibition of ~IV protea~e, the prevention or treatment of infection by the human immunodeficiency viru~ (~IV) and the treatment of . .
~- .

W092/06992 ~ PCT/USgl/~16 c~ .

consequent pathological conditions such as AIDS.
Treating AIDS or pre~enting or treating infection by HIV is defined as including, but not limited to, treating a wide range of ætates of EIV infection:
AIDS, ARC (AIDS related comple~, both symptomatic and asymptomatic, and actual or potential e~posure to HIV. ~or example, the compound of this invention i6 useful in treating infection by HIV after suspected past e2posure to HI~ by e.g., blood transfusion, accidental needle ~tick, or e~posure to patient blood during surgery.
For these purposes, the compound of the present in~ention may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion technigues), by inhalation spray, or rectally, in dosage unit formulationæ containing conventional non-toxic pharmaceutically-acceptable carrieIs, adjuvants and vehicles.
Thus, in accordance with the present inYen-tion there i8 further providet a method of treating and a pharmaceutical composition for treating ~IV
infection and AIDS. The treatment involves admini-stering to a patient in need of such treatment a phar-maceutical composition comprising a pharmaceuticalcarrier and a therapeutically-effective amount of the compound of the present inventio~.
These pharmaceutical compositions may be in the form of orally-administrable suspensions or tablets; na6al spray6; sterile injectable preparations, for example, as sterile injectable aqueous or olea~enous suspensions or suppoæitories.

.. ,, , .,, . , . . , , . ,, ~.... . ........ . . .

,~ , , .
.

W092/OC992 PCT/~S9l/06816 2~3~9 When administered orally as a suspen-sion, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may contain microcrystalline cel-lulose for imparting bulk, alginic acid or sodiumalginate as a suspendi~g agent, methylcellulose as a viscosity enhancer, and sweetners/fla~oring agents known in the art. As immediate release tablets, these compositions may contain microcrystalline o cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other e~cipients, binders, extenders, disintegrants, diluents and lubricants known in the art.
When administered by nasal aerosol or inhal-lS ation, these compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable pre~er~atives, absorption promotèrs to enhance bioavailability, fluorocarbons, andJor other solubilizing or dispersing agents ~nown in the art.
The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringeris solution or isotonic g,odium chloside solution, or suita~le dispersing or wetting and suspending agents, such as sterile, bland, fixed oilæ, including ~ynthetic mono- or digiycerites, and fatty acids, including oleic acid.
When rectally administered in the form of suppositories, these compositions may be prepared by ' ' ~. ,' . ~ ', -.

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

W~9~/~g92 ~ PCT/US91/~16,_ mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic ~lyceride esters or polyethylene glycols, which are ~olid at ordinary temperatures, but liquidify and/or diss~lve in the rectal cavity to release the drug.
Dosage level~ of the order of 0.02 to ~.~
or 10.0 grams-per-day are useful in the treatment or prevention of the above-indicated conditions, with oral doses two-to-five times higher. For example, infection by ~IV is effectively treated by the administration of from lO to 50 milligrams of the compound per kilogram of body weight from one to three times per day. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied a~d will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stabi-lity and length of action of that compound, the age of the patient, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular contition, and the host undergoing therapy.
The present invention is also directed to combinations o~ the ~IV protease inhibitor compound with one or more agent8 u8eful in the treatment of AIDS. For example, the compound of this invention may be effecti~ely administered, whether at periods of pre-exposure and/or post-e~pogure, in combination with effective amounts of other AIDS anti~irals, immunomodulators, anti-i~fectives, or vaccines.

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

W092/06992 PCT/US91/~16 2`~ 9;~

Simvastatin Analo~s.
Also included by the process of this invention are two phosphorylated derivatives of simvastatin, whose chemical name is 6~R)-~2-~8' (S)-2",2"-dimethylbutanoyloxy-2'(S~,6'(R)-dimethyl-1',2',6',7',8',8'a (R~-hexahydronaphthyl-l'(S)-ethyl]-4(R>-hydroxy-3,4,~,6-te~rahydro-2~-pyran-2-one. See EP0 Publication No. 0033 358 and The Merck Inde~, Eleventh Edition, for a description of the compound, its synthesis and utility as an anti-hypercholesterolemic agent in interfering with cholesterol biosynthesis.
Contacting simvastatin with the Rhizo~us microorganisim under the conditions described herein results in two phosphorylated derivatives:

': ~ ', ' . . i , " ,, " ,,, , ", , WO 92/06992 C~ PCI/US91/06816 C) I
HO- ~H

H3~H3 - L-706, 526 lS :

H3C/ / ~-P- O- H

3 CH3~--~H3 H3C"`

L- 7 06, 527 -~
~.

Wog2~o69s2 PCT/US91/~16 2~3~9 - 6~ -The above two compounds of thi~ invention are u~eful a6 antihypercholesterolemic agents for the treatment of atherosclerosi~, hyperlipemia and like diseases in humans. They may be administered orally or parenterally in the form of a cap~ule, a tablet, an injectable preparation or the like. It is usually desirable to use the oral route. Doses may be varied, depending on the age, severity, body weight and other conditions of human patients but daily dosage for adults is within a range of from about 2 mg to 2000 mg (preferably 10 to 100 mg) gi~en in three or four divided doses. ~igher doses may be favorably applied as required.
The compounds of this invention also haYe useful anti-fungal activities. For e~ample, they may be used to control strains of Penicillium sp., Aspe~r~illg~ ni~e~, Cladosporium ~p., Cochliobslus miyabea~ and ~elmiD~h~LE~}iLm cynodnotis. For those utilities they are admixed with suitable formulating agents, powders, emulsifying agents or solvents such as aqueous ethanol and ~prayed or duæted on the plants to be protected.
The pharmaceutically acceptable salts of --this invention include those formed from cations ~uch aæ sodium, potassium, aluminum, calcium, lithium, magnesium, zinc and tetramethylammonium as well as those 8alts formed fsom amines such as ammonia, ethylenediamine, N-methyl~lucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzyl-phenethylamine, l-p-chlosobenzyl-2-pyrrolidine-1~-yl-methylbenzimidazole, diethylamine, piperazine, and tris(hydroxymethyl)aminomethane.

", . . , . , . , ,, , , . . . , . :, : . .. . ., . . , .: . :-: . .

W092/06992 PCT/US91/~16_~ 1 ~' .,~ .

Zearalen~e Also included by the process of thi~
invention is the biotransformation product: alpha zearalenol 6'-phosphate: ¦

OH CH
Ho ~

o~- zear alenol- 6' - phos phat e The present invention also relates to the abo~e new compound and an object of the present invention is to provide compounds whi~h eghibit estro~enic activity or aid in increasing the rate of growth in meat-producing animal6, e.g. cattle, lamb and æwine.
The compound can be administered to animals by any suitable method including oral and parenteral administrations. ~or example, the compound can be blended with ordinary ~eed containing nutritional values in an amount sufficient to produce the desired rate of growth and can thus be fed directly to the .

.... ,; .. i , , ............... ,.",,"".. , :,. . ..
. ~ ., , :. , ~92~06g92 PCT~US91/~16 2093 ~2~

animals, or the compound can be æuspended in a suit-able injection suspenæion medium æuch as peanut oil and injected parenterally. The amount of compound fed to an animal, of course, varies depending upon the animal, desired rate of growth and the like.
When the new products are to administered in feeds, an animal feed composition may be prepared con-taining the usual nutritionally-balanced quantities of carbohydrates, proteins, vitamins and minerals, together with the compounds of the preæent in~en-tion. Some of these usual dietary elements are grains, such as ground grain and grain by-products;
animals protein substances, such a~ those found in fish meal and meat æcraps; vegetable proteins li~e æoybean oil meal or peanut oil meal; vitaminaceous materials, e.g. vitamin A and D comples members; and bone meal and limestone to pro~ide mineralæ. A type of conventional feed material for u~e with cattle includes alfalfa hay and ground corn cobs together with supplementary vitaminaceous substances is desired.
The starting material racemic (+)-zearalenone, a potent anabolic agent useful in the raising of ~eat-producing animalæ, originally waæ
preparet by fermenting the microorganism, Gib~re zeae (Gordon), on a suitable nutrient medium according to the techniques described in ~.S. Patent No. 3,169,019. More recently there haæ been described the total chemical ~yntheæis of racemic (+)-zearalenone and the 2,4-dimethyl ether derivative : ':

, .. . . . . . . .
. , - . ... . . .. . . .

W092/06992 PCT/US91~16r~
c~ .

~ - 68 -thereof (Taub et al., Chemical Communications, 1967, p. 225~. The proce~s described in U.S. Patent No.
3,551,455 and in U.S. Patent No. 3,239,545 afford6 new and more direct routes to the total synthesis of ~+)-zearalenones.

kx~m~le I~

A spore ~and culture containing GibberelLa zeaç (Gordon) NRRL-2830 was aseptically placed in a sterile tube containing 15 milliliter~ of Czapek's-Dox solution and a small amount of agar. This medium was then incubated for about 168 hours at appro~i-mately 25C. At the end of the incubation period, the medium was waæhed with 5 milliliters of sterile deionized water and transferret to a sterile tube containing 45 milliliters of Czapek's-Dox solution.
The contents of the tube were then incubated for about 96 hours at about 259C after which the material was available for use in inoculation of a fermentation medium.
The following example illustrates the fermentation of the organism Gib~ella ~a~ (Gordon) NRRL-2830 to produce zearalenone.

Ex~le II~
To a 2 liter flask were added 300 grams of finely divided corn. The flask and its contents ¦ -were then sterilized and after sterilization 150 milliliters of sterile deionized water were added.

~092/06992 PCT/US91/~16 2093 ~`%~ j To the mixture in the flask were then added 45 milliliters of the inoculum prepared by the process of Example IA and the material was thoroughly mixed.
The mixed material was then incubated for about 20 days at 25C in a dark room in a water-saturated atmosphere.
The following example illustrates the recovery of the zearalenone from the fermentation medium.

F.xample IIIA

A 300 gram portion of fermented material produced by the method of Example IIA was placed in lS 500 milliliters of deionized water and slurried.
The slurry was then heated for a~out 15 minutes at 75C, 300 grams of filter aid were then added and the material was filtered. The golid filtered material containing the anabolic subgtance was then air dried, and 333 grams of the dried cake were then extracted with 500 milliliters of ethanol. This procedure was repeated three more times. The ethanol extract was e~aporated to dryness under vacuum to gi~e 6.84 grams of solid material. This solid material was then dis-25 sol~ed in 20 milliliters of chloroform and extracted ~;
with 30 milliliters of an aqueous solution co~taining ~Z by weight of sodium carbonate ha~i~g an adjusted p~ of about 11.2. The extraction process was repeated se~en more time~. The p~ of the sodiumcarbonate e~tract was then adjusted to 6.2 with hytrochloricacid, to yield an anabolic ~ubstance-containing precipitate. The precipitate and the a~ueous sodium ,. .. .. . . ... ...
. , .;; . . , :
.

W092/06gg2 ~ PCT/US91/~16~-_ 70 -carbonate extract were then each in turn extracted with 75 milliliters of ethyl ether. This procedure was repeated three more times to yield a light yellow ethereal Qolution, which was then e~aporated to yield 116 milligrams of solid anabolic substance. This material was then subjected to multiple transfer countercurrent distribution using 100 tubes and a xolvent system consisting of two parts chloroform and two parts carbontetrachloride as the lower phase and four parts methanol and one part water as the upper phase, all parts by ~olume. The solid material obtained from the multiple transfer countercurrent distribution was zearalenone.
The following examples are given for the purpose of illustrating the present invention and should not be construed as being limitations on the scope or spirit of the instant invention.

~gA~LE
C-32 Phos~hory~ d FK-5~Q
Microor~anism a~d~ylt~ o~ o~s Spores of Rhizo~us oryzae MF4974 (~TCC
No. 11145) cultivated on an oatmeal agar ~lask were inoculated into 50 ml Soy-Glucose medium in a 250 ml Erlen~er flask and ~haken at 27-C on a rotary shaker at 220 rpm for 24 hours. The second ~tage flask (50 ml in a 250 ml Erlenmeyer flas~) were inoculated with 2.5 ml of seed culture and incubated on a rotary sha~er (220 rpm) at 27-C for 24 hours. Following incubation, each flask was harvested by . . . ~. . .
- . .. . : . . .. . . . ..

W092/06992 PCT/US91/~16 ~93 ~

centrifugation, washed once with sterile water, and resuspended in equal volume of 100 m~ pH 7.0 PO4 buffer containing 3% glycerol. EK-520 was added to achieve a final concentration of 0.2 mg/ml. The flasks were then incubated on a rotary æhaker (220 rpm) at 27C for 48 hours. Followin~ incubation, the whole broth waæ e2tracted as described in the Isolation/Purification Section below.
Media Soy Glucose Medium ~Ll De~trose 20.0 Soy Meal 5,0 Fido yeast e~tract 5.0 NaCl 5.0 K2~P04 5;0 Adjust p~ to 7.0 ISOLATIQn AND Pv~IEl5~IQ~
The whole broth ~400 ml) waæ adjusted to p~ 3 5 and e~tracted three times with methylene chloride ~3 x 400 ml). Methylene chloride eætracts were combined and evaporated to dryne6s under reduced pre~sure at 30~C. The resulting oil was dissolved in methanol and subjected to EPLC purification. ~PLC
was carried out on Whatman Parti~il 10 ODS-3, 9.4 mm x 25 cm at 50C and monitoret at 205 nm. The column was developed at 3 ml/minutes with a linear gradient from 35% acetonitrile in 0.1% phosphoric acid to 80%
in 0.1% phosphoric acid in 60 minutes. The compound was collected duIing repeated injections of the above described extract. The fractions at retention time, 32 minutes, were pooled, adjusted to p~ 3 and . .. . . . . . .
. , . . . ~
- .'............... : ' ' ' ~ . '' . ~
. .
; ' ~ ' ,: . ' .
. .

W092~0C~2 ~3~ PCT~US91/~16-~

evaporated to remove acetonitrile. The compound was further purified using C18 Sep Pak ~Water Associates) and methanol-water elution ~olvent to yield 4.5 mg pf product.

Cha~acteriZa~Qn C-32 Phosphorylated FK-520 (FK-900520) was characterized by FAB mass spectrometry ~nd FK-520 via NMR spectrometry yielding the proton NMR ~pectrum of Figure 1, which confirms the assigned molecular structure in Figure 2.

~X~PL~
T-C~ll Pr~lif~L~ion ~æsay 1. Sample Pre~aration Purified C-32 phosphorylated ~K-520, as prepared by ~PLC above, was dissolved in absolute ethanol at 1~ mg/ml.
2. Assay Spleens from C~7Bl16 mice were ta~en unter sterile conditions and gently dis60ciated in ice-cold RPMI 1640 culture metium (GIBCO, Grand Island, N.Y.) supplemented with 10% heat-inacti~ated fetal calf serum (GTBCO). Cells were pelleted ~y centrifugation at 1500 rpm for 8 minutes. Contaminating red cells were removed by treating the pellet wi~h ammonium chloride lysing buffer (GIBCO) for 2 minutes at 4-C.
Cold medium was atded and cells were again centrifuged at 1500 rpm for 8 mi~utes. T lymphocytes were then isolated by separation of the cell su~pension on nylon wool columns as follows: Nylon wool columns ~ -.
. . ~ ; . .

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

W092/06gg2 PCT/US91/~16 2~3l~

were prepared by packing approximately 4 gra~s of washed and dried nylon wool into 20 ml plastic syringes. The columns were sterilized by autoclav-ing at 250F for 30 minutes. Nylon wool columns were wetted with warm (37C) culture medium and rinsed with the same medium. Washed spleen cells resuspended in warm medium were slowly applied to the nylon wool. The columns were then incubated in an upright position at 37C for 1 hour. Non-adherent T lymphocytes were eluted from the columns with warm culture medium and the cell suspensions were spun as above.
Purified T lymphocytes were resuspended at 2.5 x 10~ cells/ml in complete culture medium cDmpo~ed of RPMI 1640 medium with 10% heat-inacti~ated fetal calf serum, lO0 mM glutamine, 1 mM sodium pyru~ate, 2 x 10-5 M 2-mercaptoethanol and 50 ~g/ml gentamycin.
Ionomycin was added at 250 ng/ml and PMA at 10 ~g/ml.
The cell suspension was immediately distributed into 96 well flat-bottom microculture plates (Costar) at 200 ~l/well. The control, being the medium without test drug, and various below-indicated dilutions of the above sample of purified C-32 phosphorylated FK-520 to be tested were then added in triplicate wells at 20 ~l/well. ~-520 was used a6 a etandard.
The culture plates were then incubated at 37C in a humidified atmosphere of ~ C02-95~ air for 44 hours. The proliferation of T lymphocytes wa8 assessed by measurement of tritiated thymidine incorporation. After 44 hours of culturing, the cells were pulse-labelled with 2 ~Ci/well of tritiated thymidine (~ÆN, Cambridge, .

W092/06992 ~63 PCT/USglJ~16,--MA). After another 4 hours of incubation, cultures were harvested on glass fiber filter~ using a multiple ~ample harvester. Radioactivity of filter discs corresponding to individual wells wa6 mea~ured by standard liquid scintillation counting methods (Betacounter). Mean count~ per minute of replicate wells were calculated and the results e~pressed as percent inhibition of tritiated thymidine uptake (proliferation) as follows:

rMean_cpm ~am~le ~ested % Inhibition = 100 - ~ean cpm control medium ~ lO0 The results of % inhibition at ~arious:
15 concentrations of C-32 phosphorylared FK-520 (C-32-~
FK-520) are pre~ented in the following table:

In~iki$io~_Q~ T-Cell Prolife~ation bv C-32-P FK-520 C-32-P-FK-520 (ng/ml) % Inhi~ition lO0 97 2550 95 :
89.6 12.5 71.3 6.2 2~.7 3.1 0.0 . . .

~V092/06g92 PCT/US91/~16 ~93~

Notes: i. Mouse T cell cultures were pulæed with 3H-thymidine for 4 hours prior to harvesting at 48 hours.
2. Standard FK-520 (4 ng/ml) gave 97%
inhibition.
3. The mean IC50 for C-32-P F~-S20 was determined to be: 15.0 + 2.1 ng/ml (17.2 + 2.4 nM) in 3 independent experiments.
4. Inhibition of T-Cell proliferation was reversed by the addition of 100 units/ml of recombinant human IL-2 at the initiation of culture.

EXAMPL~ 3 C-32 Phos~h~rylated FK-506 Ferment~io~
Spores of Rhizopus oryzae MF 4974, cu?ti~ated on oatmeal agar, were inoculated into 50 ml Soy-Glucose medium in a 250 ml ErleDmeyer flask and 6haken at 25C on a rotary shaker at 220 rpm for 24 hours. The second stage flasks (50ml in a 250 ml Lrlenmeyer flask) were inoculated with 2.5 ml of seed culture and incubated on a rotary shaker ~220 rpm) at 27OC for 24 hour~.
Following incubation, each flask was harvested by centrifugation, washed once with sterile water, and resuspended in equal volume of lO0 mM ~04 buffer containing 3% glycerol, Fg506 was added to achieve a final concentration of 0.2 m~/ml. The W092~069g2 ~ PCT/US91/~16 _ charged flasks were incubated on a rotary ~haker (220 rpm) at 27C for 24 hours. Following incubation, the whole broth was worked up as described below.

Med~ Sov Glucose Medium_~L~
De~trose 20.0 Soy meal 5 0 Fido yeast egtract 5.0 NaCl 5.0 K29P04 5.o - Adjust pH to 7.0 ISQL~5ION AND Pu~IElcATIoN

The whole broth t200 ml> was maintained at p~ 6.8 and centrifu~ed. The mycelial cake was washed with water, then discarded. The clear filtrate and washings were pooled and passed thru a Spe-ed octadecyl cartIidge ~14% carbon load, Applied Separations) under vaccum. The column was washed with lOO ml of water. Colum~ effluent and wash did not contain microbial transformation product when tested with HPLC, The cartridge was elu~ed with 200 mL methanol. Methanol was evaporated to dryness-under reduced pressure at 30-C. The resulting oil wa~ dissol~ed ~ ~ethanol and subjected to ~PLC
purification.
~PLC was carried out on Whatman Magnum 20 Partisil l- ODS-3 Column (Cl8,22.l mm ID ~ 25 cm) -~t 50-C and monitored at 205 nm. The colum~ was developed at 7 mL/min with linear gradient from .; ....... . . ........ ., . , . , , , , , , ~, . , . ~ . .

w092/06g92 PCT/US91/~16 2~3~

35Z acetonitrile in 0.1% phosphoric acid to 80%
acetonitrile in 0.1% phosphoric acid in 70 minute6.
The compound was collected during repeated injections of the abo~e described extract. Fractions of retention time 50 minutes were pooled, adjusted to pH 3.0 and evaporated to semove acetonitrile. The compound was desalted using a C18 Sep Pak (Water Associate) to yield 20 mg of product.

CHARACTERIZATIQ~
The compound was characterized by MS and confirmed by NMR as the C-32 phosphate ester derivative of FK 506.

C-32 Phosphorylated C-31-desmethyl F~ 0 The fermentation procedure of Example 3 was carried out substantially identical except that C-31 desmethyl F~-520 (available as for example, by the procedure in ~P0 Publication 0 349 061, published January 3, 1990) was used in place of FK-506.
The iæolation/purification procedures were virtually identical to those descri~ed in Example 3.
The C-32 phosphorylated C-31 desmethyl FK-520 was characterized by mass ~pectrometry and.
proton nuclear magnetic resonance in which the obtained spectra wa8 con~istent with the assigned 8tructure.

- . . . . ' .
, ~ :, . .
: ' ' :'' . ' ' ' WO92/06~2 PCT/US91/~16--.
~' .

EX~L~ 5 Carrying out the above IL-2 as~ay described above in Example 2 for C-32 phosphorylated FK-506 S (P-FK-506) and C-32 phosphorylated C-31 de~methyl FK-520 (P-31-desMe F~-520) yielded the following reæults:

Inhibition of T cell proliferation by P-FK-506 Concentration % of of P-FK-506 inhibition (mg/ml) 50 98.5 25 97.7 12.5 93.3 6.2 80.3 3.1 42.9 IC50 = 3 4 ng/mL (3.9nM) Inhibition of T cell proliferation bv P-31-desMe F~-520 Concentration % of of P-31-desMe ~K-520 inhibition (mg/ml) 250 95.2 125 g 3 62.5 ` 79 5 31.2 ~3 8 1~.6 ~2 IC50 = 31.3 ng/mL (36.7 nM).

:

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

~'~92/06992 PCT/US91/~16 2Q93~- ?,~

E~
EhQ~phQ~ylated Rao9~5~ 9li~e Microor~anism and Culture Condi~ions Sporeæ of Bhi~Qp~s oryz~e ME4974 (ATCC
No. 11145) culti~ated on an oatmeal agar flask were inoculated into 50 ml Soy-Glucose medium in a 250 ml Erlenmer flask and shaken at 27DC on a rotary shaker at 220 rpm for 24 hours. The second stage ~lask (500 ml in a 1000 ml Erlenmeyer flask) were inoculated lo with 25 ml of seed culture and incubated on a rotary shaker (220 rpm) at 27C for 24 hours. ~ollowing incubation, each flask was harvested by centrifuga-tion, washed once with sterile water, and reæuspended in egual ~olume of 100 m~ p~ 7.0 phosphate buffer con-taining 3% glycerol. Rapamycin macrolide was addedto achieve a final concentration of 0.2 mg/ml. The flasks were then incubated on a rotary 6haker (220 rpm) at 27C for 24 hours. Following incubation, the whole broth was extracted as described in the Isolation/Purification Section below.

Media Soy Gluc~se Medium ~Ll Dextrose 20.0 Soy Meal 5.0 Fido yeast extract 5.0 NaCl 5.0 ~2~P4 5.0 Adjust p~ to 7.0 ... ... .

.

'. ,: . '; ' '' ," ,, '. , :
. .

W092/06992 PCT/US9l/~

~t`

~ - 80 -ISQ$ATION AND PURIFICA~IO~
The whole broth (500 ml) was maintained at p~ 6.8 and centrifuged. The mycelial cake wac washed with water, then dicarded. The clear filtrate and washings were pooled and passed through a Speed octadecyl cartridge (14% carbon load, Applied Sepa-rations) under vacuum. The column was washed with 100 ml of water. Column effluent and wash did not contain microbial transformation product when tested with HPLC. The cartridge was eluted with 200 ml methanol. Methanol was evaporated to dryness under reduced pressure at 30C. The resulting oil was dissolved in methanol and subjected to ~PLC puri-fication. ~PLC was carried out on Whatman ~agnum 9 Partisil 10 ODS-3, 9.8 mm i.d. x 25 cm at 25DC and monitored at 225 nm. The column was developed at 3 ml/minutes with a linear gradient rom 35% to 80%
acetonitrile in 0.1% phosphoric acid in 30 minutes.
The compound was collected during repeated injections of the above described extract. The fractions of retention time 17.3 minutes, were poolet, adjusted to p~ 3 and evaporated to remove acetonitrile. The compound was further purified using C18 Sep Pak (Water Associates) and methanol-water elution solvent to yield 14 mg pf product.

CharacterizatiQn The C-43 phosphorylated macrolide was charac-terized by FAB mass spectrometry and NMR spectrometry as the methyl phosphate eæter deri~ative yielding the proton NMR spectrum of Figure 3, which confirms the assigned molecular structure in ~igure 4.

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

: i . . ', . .. :
. .

~92/069g2 PC~/US91/~16 2 ~

Methylation to produce the phosphate ester was necessary to minimize the considerable line broad-ening which characterized the proton NMR spectrum of the free acid. The ~ey features were the downfield S displaced ~-42 and H-43 signals (which are H-31 and ~-32 in F~-506 nomenclature) at 3.87 ppm and 4.12 ppm, respectively, and the additional fine structure of ~-43 resulting from couplin~ with the phosphorus.
The downfield shift of H-43 is a normal consequence o of replacing the active hydrogen with an electron withdrawing substituent. H-42 is also displaced downfield by proximity to the new substituent. The obtained spectrum of the phosphorylated macrolides as ~he phosphate methyl ester with assignments of H-42, ~_43, and the methyl ester peaks is shown in Figure 3.

T-Cell Proliferation Assay 1. Sample Prepa~ion Purified phosphorylated macrolide, as prepared by ~PLC above, was dissolved in absolute ethanol at 1 mg/ml and serially diluted in culture medium prior to addition to the cultures.
2. Assay The assay was carried out as described in J. Immunol. Vol. 144, pp.251 (1990~ ~y F. Dumont, et al.
Spleens from CS7B116 mice were taken under sterile conditions and gently di~ociated in ice-cold RPMI 1640 culture medium (GIBC0, Grand Island, N.Y.) : ~ , ~: ' ..

.
.

. ~.....

W092~06992 ~ PCT~US91/~1f-c~

supplemented with 10% heat-inactivated fetal calf serum (GIBCO). Cells were pelleted by centrifugation at 1500 rpm for 8 minutes. Contaminating red cells were removed by treating the pellet with ammonium chloride lysing buffer (GIBCO~ for 2 minutes at 4C.
Cold medium was added and cells were again centri-fuged at 1500 rpm for 8 minutes. T lymphocytes were then isolated by separation of the cell ~uspension on nylon wool columns as follows: Nylon wool columns 1~ were prepared by packing approximately 4 grams of , washed and dried nylon wool into 20 ml plastic syringes. The columns were sterilized by autoclav-ing at 250F for 30 minutes. Nylon wool columns were wetted with warm (37C) culture medium and rinsed with the same medium. Washed spleen cellæ resuspended in warm medium were slowly applied to the nylon wool.
The columns were then incubated in an upright posi-tion at 37C for 1 hour. Non-adherent T lymphocytes were eluted from the columns with warm culture medium and the cell suspension~ were spun as above.
Purified T lymphocytes were resuspended'at 2.5 x 105 cells/ml in complete culture medium composed of RPMI 1640 medium with 10% heat-inactivated fetal calf serum, 100 mM glutamine, 1 mM sodium pyruvate, 2 x 10-5 M 2-mercaptoethanol and 50 ~g/ml gentamycin.
~uman recombinant interleu~in-2 (~0 ~g/ml) and PMA at 10 ng/ml. $he cell su8pension wa~ immediately di~tributed into 96 well flat-bottom microculture plates (Co~tar) at 200 ~llwell. The control, being the medium without test;drug, and various below-indicated,dilutions of the above sample of purified phosphorylated macrolite to be tested were then .
, ....................... . . ..

. . ~ , . .

~92/06g92 PCT/US91/~16 2~3~ J' added in triplicate wells at 20 ~l/well. Rapamycin (~.S. Patent 3,929,992) was used as a stantard. The culture plates were then incubated at 37C in a humi-dified atmosphere of 5% C02-95Z air for 44 hours.
The proliferation of T lymphocytes was assessed b~
measurement of tritiated thymidine incorporation.
After 44 hours of culturing, the cells were pulse-labelled with 2 ~Ci/well of tritiated thymidine (NEN, Cambridge, MA). After another 4 hours of incubation., cultures were harvested on glass fiber filters ueing a multiple sample harvester. Radioacti~ity of filter tiscs correæponding to individual wells was measured by standard liquid scintillation counting methods (Betacounter). Mean counts per minute of replicate wells were calculated and the results expressed as percent inhibition of tritiated thymitine upta~e (proliferation) as follows:

rean cpm samDl~ te~ted % InhibitiOn = l00 -I ~ l00 ~ean cpm control medium Proliferation was assessed at 48 hours of culture~by tritiated thymidine upta~e.
The re~ults, e~pre~sed a~ IC50 ~alues of three independent e2periments, are presented in the following table:

. . - --, , -, .
' - ' ~ 1 ; ' W~92/06992 PCT/US9l/~lr-`
.

Inhi~ition of T-Cell Proliferation I~50 (~/ml) Phosphorylated Expt. # Ra~amycin Ra~Amy~in 1 7.7 0.6 2 7.3 0 4 3 7.9 0 3 -.

Notes: 1. Mouse T cell cultures were pul~ed with 3~-thymidine for 4 hours prior to harvesting at 48 hour~.
2. The mean IC~o for the C-43 phosphory- :
lated rapamycin was determined to be:

7.6 + 0.2 ng/ml and 0.4 i 0.1 ng/ml for rapamycin in 3 independent experiments.

-~'~92/069g2 PCT/US91/~16 ~ ~ ~ 3 ~ 9 ~

E~ LE

S~N~
The following preparation and synthesis follows, in general, U.S. Patent 4,661,473; Evan6, B.E. et ~1, J. Or~. Che~, 50, 4615, (1985) and Evans, ~.E. ~ ~1., "A Stereocontrolled Synthesis of ~ydroxyethylene Dipeptide Isosteres," Proc. Am. Pept.
Symp., 9, 743-6(1985), and Luly, J.R. ~ ~1. J. Org.
Chem, 52, 1487 (1987).

N(2(R)-~ydro~y-l(S)indanyl)-5(S)-((l,l-dimethylethoxy-carbonyl)amino)-4(S)-hydroxy-6-phenyl-2(R)-3-(4-(2-(morpholino)ethoxy~phenyl)~ro~-2-en=1=yl~hexanamide:

St~p ~: Preparation of 4-tert-butyldimethylsilyloxy-- phenyl~ro~-2-en-1-yl bromide:
To a 1 L round bottomed flask with a stirring bar and an ar~on inlet was added 26.25 g (160 mmol) of p-hydroxycinnamic acid, 50.62 g ~335 mmol) of tert-butyldimethylsilyl chloride, 32.68 g (480 mmol~ of imidazole, and 250 mL of dry DMF. This mixture was stirred at room temperature for 24 hours. The DMF
was removed Ln vacuo and the residue was partitioned between EtOAc and 10% aqeous citric acid. The layers were separated and the organic phase was washed with water (4x) and brine. Drying ~MgS04), filtration and removal of the solvent ~ ~acuo ga~e 62.3 g of the ~issilylether-ester. This ester was placed in an oven dried 2 L round bottomed, 3 necked flask with a mecha~ical ~tirrer, argon inlet, and addition funnel. Ether (460 mL) was added and the solution .. , . . . ... - ,- - .. , ~ ~ ~ .

W092~069s2 PCT/US91/~

was cooled in an ice bath to 0C. To this solution was added a ~olution of Dibal-~ 397 mL of a 1.0 solution in hexanes), dropwise over 1 hour. The mixture was stirred for 1 hour and the reaction was then quenched by careful addition of 1 L of saturated aqueous sodium potassium tartrate solution. This viscous mixture was stirred for 20 hours at ~oom temperature. The mixture was filtered through a celite pad, the filtrate layers were separated and the or~anic phase was extracted with 2 portions of ~tOAc. The com~ined organic phases were washed with brine, dried (MgS04), filtered and concentrated in vaCU~ to give the crude alcohol. This material was chromatographed on 500 g of silica gel using 20%
EtOAc in he~anes as eluant. There was obtained 34.9 g of 4-tert-butyldimethylsilyloxycinna~yl alcohol as a viscous oil. This material crystallized on cooling to -15-C. A portion of this alcohol (18.5 g, 69.96 mmol) was placed in a 300 mL round bottomed flask 20 with a stirring bar and 125 mL of dry ether. This solution was cooled to O~C ant 20.83 g (76.95 mm~l>
of PBr3 was added dropwise with a syrin~e over 5 minutes. This solution was maintained at O-C for 20 minutes, diluted with hexanes <lL) and washed with aqueous Na~C03. Thi~ solution was washed with brine, tried (MgS04), filtered throuEh a pad of silica gel, and concentrated in vacuo to give 17.53 g of 4-tert-butyldimethylsilylo y penylprop-2-en-1-yl bromide as a colorless oil.

.: . , ........ : . , .
. . .: -,,, ' , , ,, .' . ' ' ~92/06992 PCT/US91/~
~9~d~

~p B: Preparation of 5(S)-(l,l-dimethylethoxycar-bonyl)amino-4(S)-(l~ -dimethylethyl-l,l-dimethylsilyloxy)-6-phenyl-2(R)-(4-(1',1'-dimethylgilylo~{y)phenylprop-2-en-yl)hex-anoic acid: _ To a 500 mL, 3-necked, oven dried round bottomed flask with an argon inlet, stirring bar, low temperature thermometer, and a jacketed addition fun-nel was added ~5 mL of dry T~F and 1~.39 mL (109.78 mmol) of diisopropylamine. This solution was cooled to -200C and n-butyllithium (42.84 mL, 107.11 mmol of a 2.~M sol~tion in hexanes~ waæ added slowly. The resulting solution was cooled to -780C and a solution of (5S,1~S)-5-((1,l-dimethylethoxycarbonyl)amino)-2-phenylethyl)dihydrofuran-2-(3~)-one (16.36 g, 53.55 mmol) in 75 ~L of dry T~F was added at such a rate that the temperature of the solution did not rise above -70-C (ca. 40 minute~ required for this addi-tion). The resulting solution was aged at -78C ~or 1 hour and the dropping funnel was charged with a solution of 4-tert-butyldimethylsilyloxyphenylprop-2-en-1-yl bromide (17.53 g, 53.55 mmol~ in 7~ mL of dry TEF. The bromide solution was cooled to -78-C
and was then added to the enolate solution dropwise o~er 45 minutes, keeping the tempe~ature below -70C. When the addition was complete the solution was aged at -78C for 1 hour, warmed to -50-C and guenched with a ~olution of Na~S04 (33 g) in 250 mL
of water. The mixture was diluted with EtOAc and the layers were separated. The organic phase was washed with Na~C03 solution and brine. Drying (MgS04), filtration, and removal of the solvent i~ Yacuo . .~ ,,, ~ : . ., ~ . - : ,. -.:

W092/06~2 PCT/US91/~

2~93~2~ - 88 -provided the crude alkylation product as an oil.
This material was chromatographed on 1000 g of silica gel using 20% EtOAc in hexanes as eluant.
There was obtained 23.6 g of pure (55,3R,l'S)3-(4-tl',l'-dimethylethyl-1,1-dimethysilyloxy)phenyl-prop-2-en-1-yl)-5-(1-((1,1-dimethylethoxycarbonyl)-amino)-2-phenylethyl)dihydrofuran-2-(3~)-one as a colorless foam. This material was dissolved in 32~ j mL of DME and a solution of LiO~ (6.99 g, 291.97 lo mmol) in ~2 (325 mL) was added. This solution was stirred at room temperature for 24 hours. The DME
was removed in vaCUQ and the aqueous residue was acidified with 10% aqueous citric acid. This milky suspension was extracted with several portions of LtOAc and the combined extracts were washed with wa~er and brine, dried (MgS04), filtered, and concentrated in va~uo. To the crude a-hydroxy acid was added imidazole (56.79 g, 834 mmol), tert-butyl-dimethylsilyl chloride (62.87 g, 417 mmol) and dry DMF (325 mL). This mi~ture was stirred at room temperature for 24 hours. The mi~ture was then treated with methanol (325 mL) for 4 hours at room temperature. The solvents were removed in vacuo at 60C, 20 torr. The residue was dissolved in lL of ~tOAc and washed with lOZ àqueouæ citric acid, water and brine. Drying ~MgS04), filtration and removal of the solvent gave 28.53 g of 5(S)-(l,l-dimethylethoxy-carbonylamino)-4(S)-(l',l'-dimethylethyl-l,l-dimethyl-silylo y )-6-phenyl-2(R~-(4-(1',1'-dimethylethyl-1,1-30 dimethylsilyloxy~phenylprop-2-en-1-yl)hexanoic acid !
as a colorless foam.

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

.

W092/OC~i2 PCT/US9l/~16 2~93d~ ~9 Ste~ C: Preparation of N-(2(R~-hydroxy-l(S)-indanyl)-~(S>-(l,l-dimethyletho~ycarbonylamino)-4(S)-(l~,l'-dimethylethyl-l,l-dimethylsilylo~y)-6-phenyl-2(R)-((4-(hydroxyphenyl)prop-2-en-1-yl~hexa~a~id~
To a 2L round bottomed flask with a stirring bar and an argon inlet was added 5(S)-(l,l-dimethyl-ethylethoxycarbonylamino)-4(S)-(l',l'-dimethylethyl-1,1-dimethylæilyloæy)-6-phenyl-2(R)-~4(1~ -dimethyl-ethyl-1,1-dimethylsilyloxy)phenylprop-2-en-1-yl) hexanoic acid (28.53 g, 41.71 mmol) from Step ~, 2(R)-hydro~y-l(S)-aminoindane (6.85 g, 45.88 mmol), 3-(N,N-dimethylaminopropyl)ethylcarbodiimide hydrochloride (8.7~ g, 45.8 mmol), l-hydroxybenztriazole hydrate (6.20 g, 45.88 mmol), and dry DMF (300 mL). When all of the solids had dissolved, triethylamine (12.79 mL, 91.76 mmol) was added and the mixture was stirred at room temperature for 18 hours. The mixture was partitioned between EtOAc (1500 mL) and 10% aqueous citric acid (1500 mL). The layers were separated and the organic phase was washed with water (3 x lOOO
mL), and brine. Drying (MgS04), filtration and removal of the solvent Ln vacuo gave 32 g of a yellow foam. Thiæ material was dissolved in 500 mL of methanol and LiO~ (4.99 g, 208.~5 mmol) was added.
This solution was stirred at room temperature for 1 hour. The Bolution was acidified with aqueous citric acid and ~he methanol wa~ removed in Yacuo. The resulting aqueous residue was extracted with EtOAc (lL). The EtOAc extract was dried (MgS04), filtered, and concentrated in vacuo. This material was chromatographed on 1 Kg of silica gel using 8L of 40%

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

WO92/06s92 ~ PCT/US91/~16 EtOAc in hexanes as eluant. There was obtained 20.07 g of N-(2(R)-hydroxy-l(S)-indanyl)-5(S)-(l,l-dimethyl-ethoxycarbonyl)amino-4(S)~ dimethylethyl-l,l-dim ethylsilylo~y)-6-phenyl-2~R)-((4-hydroxyphenyl~prop-2-en-l-yl)hexanamide as a colorlesg foam.

Step_D: Preparation of N-(2(R)-hydroxy-l(S~-indanyl~-5(S)-(l,l-dimethylethoxycarbonylamino)-4(S)-hydroxy-6-phenyl-2(R)-(4-(2-(4-morpholino)-ethc~y~phe~yl~ro~-2-en-1-vl)he~an~amide:
To a lL round ~ottomed flask with a stirring bar and an argon inlet was added N-(2(R)-hydroxy-l(S)-indanyl)-5(S)-(l,l-dimethylethoxycarbonyl)-4(S)-(l',-l'-dimethylethyl-l,l-dimethylsilyloxy)-6-phenyl-2(R)-(4-(1',1'-dimethylethyl-1,1-dimethylsilyloxy)phenyl-prop-2-en-1-yl)hexanamide (19.49 g, 28.1 mmol) 1,4-dioxane (400 mL), 4-(2-chloroethyl)morpholine (12.61 g, 84.3 mmol), and powdered cesium carbonate (27.5 g, 84.3 mmol). This mixture was heated at 80~C with ~igorous stirring for 3 hour6. The cooled reaction mixture was filtered through a celite pad and the 1,4-dioxane was removed ~n vacgQ. To this residue was added a solution of tetrabutyla~monium fluoride in T~F (280 mL of a lM solution, 280 mmol) and a stirring bar. This ~olution was stirred at room temperature under argon for 28 hours. The reaction mixture was poured into 3L of ~2~ with ~tirri~g.
The white solid was collected by filtration a~d dried Ln YàCU~o overnight. The crude product was chromatographed on ~ilica gel u6ing 5Z MeOH in chloroform as eluant. The chromatographed material was recrystallized from boiling ~t~Ac-hexanes to ~ive .
.: .

- - - - , . . .. .. . . . ......... .

.

: . . . .

!:'; ' ' ' ' ' W092/06992 PCT/US91/~16 2 ~ s~ 9 13 g of analytically pure N-(2(R)-hydroxy-l(S)-indanyl)-5(S)-(l,l-dimethylethoxycarbonylamino)-4(S)-hydroxy-6-phenyl-2(R)-(4-(2-(4-morpholino)ethoxy)-phenylprop-2-en-l-yl)hexanamide as a white solid, mp:178-179C.

EX~M~k~_~

Preparation of N-(2R)-hydroxy-l(S)-indanyl)-5(S)-(1,1-dimethylethyloxycarbonylamino)-4(S)-hydroxy-2(R)-(4-(2-(4-morpholino)ethoxy)phenyl)prop-2-en-1-vl-6-c~clohexylhexanamide. ~arent compound L-702.083 Ste~ A: Preparation o~ (5S,l'S)-5-(1'-((1,1-di-methyletho~ycarbonyl)amlno)-2'-cyclo-hexylçthyL2-4~ ihydro~an=2=~3~ ne:
A solution of the (5S,l'S)-5-(1'-((1,1-di-methylethoxycarbonyl),amino)-2'-phenylethyl)-4,5-dihydrofuran-2-(3~)-one was dissol~ed in ethyl acetate and rhodium on alumina was added. This mixture was Ehaken under a hydrogen atmosphere (50 psi) at 50~C overnight. Filtration and evaporation of the ~olvent afforded the title compound as a viscous oil, which solidified as a hard glas6.

S~ep B: Steps B, C and D of Example 1 are repeated except that (5S, l'S)-5-((1,l-dimethylethoxy-carbonyl)amino)-2-p~enylethyl)dihydrofuran-2-(3~)-one in Step B i~ substituted with its cyclohe~yl analog, (5S, 1'S)-5-((1-dimethyl-etho~ycarbonyl)amino~-2-cyclohexylethyl)-4,5-dihydrofuran-2-(3~)-one. The title compound, L-702,083, is obtained.

. ~ ~.. ' .. :.

W092/06~2 PCT/US9l/~16 ~' . , .

EXAM~LE 10 ~IOTRANSFORMATIQ~ OF L-702.083 Spores of Rhi~opus arrhizus MF 4974, cultivated on oatmeal agar, were inoculated into 50 ml of seed medium [containing(in gramæ per liter) dextrose 20.0, soy meal 5.0, Fidco yeast e~tract 5.0, NaCl 5.0, K2~PO4 5.0, pH 7.0 before autoclaving~ in a 1~ 250 ml 3-baffle Erlenmeyer flask. The flask was incubated for 24 hours on a rotary shaker (220 rpm) at 27C. The 2.5 ml of the seed cultures was used to inoculate 50 ml of the same medium in a 250 ml Erlenmeyer flask and incubated on a ro~ary shaker (220 rpm) at 27C for 24 hours. Following incubation, each flask was harves~ed by centrifugation, washed once with sterile saline solution, and resuspended in equal ~olume of 100 mM
phosphate buffer (p~ 7.0) containing 3Z glycerol.
L-702,083 in DMSO (20 mg/ml) was then added to achieve a final concentration of 0.1 mg/ml, and cultivation was continued at 27C on a rotary shaker at 220 rpm. After 12 hours incubation the whole broth was extracted as tescribed in Example 16.

~ .

ISOL~ION AND P~RI~ICA~ION
Two æhake flasks were combined to yield 100 ml of broth. The broth was centrifuged. The mycelial cake was washed with water and discarded.
The clear filtrate and the wash were combined and ., : , ~ ,. . . . ..

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

W092/06g92 PCT/US91/~16 ~as3~2~ .

passed thru a Spe-ed oc~adecyl 14~/D cartridge (Applied Separations) under vacuum. The column was washed with 100 ml of water and then eluted with 200 ml methanol. Methanol e~tract was concentrated in vacuo to a slightly oily residue. The residue was taken up in methanol and subjected to preparative ~PLC
chromatography on a Whatman Magnum 20 Partisil 10 ODS-3,22.1 mm x 25 cm at room temperature at a flow rate of 7 ml/minute. The effluent stream was monitored by U.V. absorption at 215 nm. Elution was achieved with a linear gradient from 30% acetonitrile in 0.1% phosphoric acit to 80% acetonitrile in 0.1%
pho~phoric acid in 80 minutes, The fractions at retention time of 35 minutes were combined, adjusted to p~ 4.0 and evaporated to remove acetonitrile; The compound was then desalted using C-l~ Sep Pak (Waters Associate) to afford 3 mg of product L-706,579.

NMR C~ARACT%RIZ~TIQ~

The product of Example 11, L-706,579, waæ
characterized by MMR as the phosphate ester derivative at C-4 of L-702,083. The key observation was the 0.6 ppm downfield displacement of ~1 in the Formula :

~- , , . . :.
.
-. . ~ ~ , , ~ - . , W092/069g2 PCT/US91/~lC_ HOY~ ~~h H O i ~Hb OH 9 ~
( CH3) 3C~N~r1~NY~

Compared to its chemical shift in the parent L-702,083, the neighboring methine (m) is slightly displaced downfield and is barely discernible as a shoulder at the base of the morpholine C3i20 at 3.74 ppm. Also perturbed i8 the nearby HK which now appears underneath the lower field ~j 6ignal at 3.10 ppm. Its presence i8 re~ealed only by the additional area of the Hj signal, compared with the ~j double .:
doublet in L-702,083.

ORGANIC SYNTHESIS OF L-706,579, PHOSPEATE ~STER .
OF L-702.083 .

A. ~ Ylæti~n of ~lan N-~(2R)-Acetoxy-l(S)-indanyl]-5(S)-(l,l-dimethyl-ethoxycarbonylamino)-4(S)-hydro y -2(R)-~(4-(2-(4-~ ~ - , - . .. .; . , : . ,.

W092/06g92 PCT/US91/~16 2 ~

morpholino)ethoxy)phenyl)prop-2-en-1-yl]-6-cyclo-hexanamide, L-702,083, is prepared by the protocol of Examples 1 and 2, except that 2(R)-hydro~y-l(S)-aminoindan is substituted with 2(R)-acetoxy-l(S)-aminoindan in Example 1, Step C.

B. L-706.~79 The product of step A i5 reacted with monophenyl phosphorodichloridate according to the principles and practice of Chambers, ~.W. and ~.G.
Khorana, J. Am. Chem. Soc. 80, 3749 (1958).
Subsequent treatment with excess ammonia, followed by removal of the acetyl groups under basic conditions, yields L-706,579.

C. Alternative Syntheti~ Routes Another phosphorylating agent is dibenzyl phosphorochloridate. An extensive discussion of phosphorylation and phosphorylating agents can be found in Y. Mizuno, Studies in Or~. Chem, 24, 171-175 (1986) and references cited therein.

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

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

WO 92/06992 PCI~/US91/06816_ ~r,~ 9 6 EXAI~LE 1 4 Assay for Inhibition of Recombinant ~IV Protease Inhibition studies were performed on the reaction of the ~IV protea~e e~pressed in Escherichia coli with a tritiated peptide substrate r3H~-ace~yl-~al-Ser-Gln- Asn-(beta-napthyl-Ala)-Pro-lle-Val-Gln-Gly-Arg-Arg-NH2(MW 1800). The two arginine residues at *he car~oxyl terminus give this peptide an overall positive charge at acidic p~ and enable it to bind to the H+ form of DOWEX AG-SOW-X8 resin and ~imilar resins. The HIV protease cleaves between the ~-napthyl-Ala and proline residues to yeild a product (3~-acetyl-val-ser-aen-~beta-napthyl-ala) that is either neutral or slightly negatively charged and does not bind to the cation exchange re6in. It is therefore po~sible to conveniently eeparate the labelled product from the ~ubstrate.
Aliquots of 25 ~l containing 6.0-8.0 nM ~IV
protease in assay buffer (100 mM sodium acetate, pE
5.5 and O.lZ BSA) are placed in assay tubes. The reaction i8 initiated by addition of 25 ~l aliquots of 4.2 ~M tritiated peptide substrate in lOOmM eodium acctate, p~ 5.5. After incubation for 60 min at 37C, the reaction i~ stopped-with 100 ~1 of 5Z ~3P04, then analysed by application of column chromatography.

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

W092~06992 PCT/US91/~16 2 ~

Results are as follow~:

C~n~. L-706.579(~M~ %Inh ~ tion .l 3 0.01 0 O.001 EXAM~LE 15 S~NI~ESIS OF~L-689,~
The preparation and 6ynthesis follows, in general, U.S. Patent 4,661,473; Evans, B.~. et ~1.
J. O~. Che , 50, 4615, (1985) and ~vans, B.E. et - -~1-. "A Stereocontrolled Synthesis of ~ydro~yethylene Dipeptide I~ostere~," Proc. Am. Pept. Symp., 2.
743-6~1985), and Luly, J.R. ~ ~1. J. Org. Chem, ~2.
1487 (1987), all herein incorporated by reference.
All temperatures are in degrees centigrade, unless indicated otherwise.

Preparation of N-(cis-2(R)-hydrogy-l(S)-indanyl)- -5(S)-(l,l-dimethylethoxycar~onylamino)-4~S>-hydroxy-6-phenyl-2(R)-~(4-(2-~4-morpholinyl)ethoxy)phenyl) methy~-hexa~m~ , L_689~502 _ -Step A: Preparation of N-3(5)-~(1,1-Dimethylethoxy-carbonyl)amino]-2(RS)-hydroxy-4-phenyl-l-tri-methvlsilyl butan~: -To a stirred 6uspension of magnesium turnings W092/06g92 ~ PCT~US9l/~16_ - 9& -(9.79 g, 403 mmol) in dry diethyl ether (200 mL) under nitrogen was added chloromethyltrimethylsilane (50 mL, 358 mmol). The reaction was initiated by gentle warm-ing and then was cooled in an ice bath to maintain gentle reflux. After e~otherm was complete the reaction was stirred at room temperature for 1 hour then cooled to -78~C in a dry ice/acetone bath. To the s~lution of the ~rignard was added dropwise with stirring a solution of N-2(S)-(1,1-dimethyl-ethoxycarbonyl)amino]-3-phenyl propionaldehyde (19.3 g, 77.4 mmol) in dry diethyl ether (250 mL) dropwise such that the temperature of the reaction remained below -55C. The resultant gray suspension was allowed to warm to room tempèrature where it was lS stirred for 30 minutes then was quenched by pourin~
into a mixture o~ ice (500 g) and 10% citric acid (500 mL). The organic phase was collected and the aqueou~ phase was e~tracted with diethyl ether (3 X
300 mL). The combined or~anics were washed with 10% citric acid (1 X 300 mL) and brine (1 ~ 200 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to give crude N-3(S)-t(l,l-dimethyl-ethoxycarbonyl)amino]-2(RS)-hydroxy-4-phenyl-1-tri-methylsilyl butane (26.6 g, quantitative crude yield) as a yellow oil. An analytical sample was obtained by low pre~sure chromatography (silica gel, 230-400 mesh; diethyl ether: hexanes, 30%:70%) followed by recrystallization from heptane. mp = 91-95C;
elemental analysiS- Calcd- for C18~31N3Si (337-53) C = 64.05, ~ = 9.26, N = 4.15;
Found: C = 64.05, X = 9.13, N = 4.22; ta]D20 = -40-0-. . ~ .

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

WO 92/06992 PCI`/US91/06816 2~93 ~ ~

Ste~ B: Prepa~a~ion of ~(S2-~mi~o-4-phenyl-1-butene.
To a stirred solution of the product of Step A (22.8 g, 67.5 mmoL) in dry methylene chloride (400 mL) cooled in an ice bath and under nitrogen wa3 added in a fine stream boron trifluoride etherate (43 mL, 345 mmol). The solution was allowed to warm to room temperature where it was ætirred for 4 days.
Reaction was cooled in an ice bath and quenched by the dropwise addition of 10% sodium hydroxide lo (400 mL). The organic phase was collected and the aqueous phase was extracted with methylene chloride (2 X 250 mL). The combined organics were washed with brine (1 X 200 mL), tried over anhydrous magnesium sulfate, filtered, and concentrated to give crude lS 3(S)-amin~-4-phenyl-1-butene (14.2 g) as a yell~w oil.

Step C: Preparation of N-3(S)-~(l,l-Dimethyl-ethoxy~arbonvl~aminol-4-~henyl-1-butene:
A solution of the product of Step B (14.2 g) and di-tert-butyl dicarbonate ~31.0 g, 142 mmoL~ in try methylene chloride (200 mL) was stirred at room temperature for 18 hours, washed with 10% citric acid (3 X 100 mL), water (1 X 100 mL), sat'd. sodium bicarbonate (3 X 125 mL), and brine (1 g 250 mL), dried over anhydrous magnesium sulfate, filtered and concentrated to yield crude N-3(S)-l~(l,l-dimethyl-ethoxycarbonyl)amino~-4-phenylbutene (34.6 ~) as a yellow oil. Crude product was purified by low pres~ure chromatography (silica gel, 230-400 mesh, 10 ~ 20 cm column; diethylether: he~anes, 20X: 80%) to yield N-3(S)~ dimethyletho y lcarbonyl)amino]-4-phenyl-1-butene (16.3 g, 97.6% yield) as a white ,,: ; . .. ; . . ..
.. . .. . . ..

W092/06992 PCT/US91/~16 _ i'1~

solit. An analytical sample was obtained by recrystallization from heptane. mp = 67.5-68.5oC;
elemental analysis, Calcd. for C15~21N02 (247.34):
C = 72.84, H = 8.56, N = 5.66.
~ound: C = 72.78, ~ = 8.76, N = 5.64.

Ste~ D: Prepara~ion of l(R)-~l'(S)-(l,l-Dimethyl-ethQxvcarbonyl~amino-~-ghenyl~hyll5~ilane:
To a solution of the product of Step C
lo (9 4 g, 38 mmol) in dry methylene chloride (100 mL) cooled in an ice bath and under nitrogen was added 3-chloroperoxybenzoic acid (technical grade, 80-a5%;
41 g, 200 mmol). The mixture was stirred at 0C for 18 hours and 25C for 23 hours, then diluted with diethyl ether (300 mL), and poured in ice cold aqeous 10% sodium sulfite ~1 L). The or~anic layer was collected ant the aqueous layer was extracted with diethyl ether (3 ~ 100 mL). The combined organics were washed with 10% sodium sulfite (3 X 100 mL), satd. sodium bicarbonate (3 X 100 mL), and brine (1 X 100 mL), dried over anhydrous sodium gulfate, filtered and concentrated to give a white ~olid.
Crude product was purified by low pressure chroma-tography (silica gel 230 - 400 mesh, 8 X 15 cm column; ethyl acetate: hexanes, 25%:75%) to yield l(R)-~l'(S)-(l,l-dimethylethoxycarbonyl)amino-2-phenylethyl~oxirane (7.0 g, 70% yield) as a clear oil which crystallized upon standing. An analytical ~ample was obtained by recrystallization from heptane.
mp = 51.5-52-C; elemental aDalysis, Calcd. for ClsE21N02 (263.34):
C = 68.42, ~ = 8.04, N = 5.32.
Found: C = 68.22, E = 8.26, N = 5.29; ~a~D20 _ 1.34.

~ . .. .... ..

W092/069g2 PCT/US91/ ~ 1~
20 a3 ~2~

~tep E: Preparation of(5S,1'S)-3-carboethoxy-5-(1-((1',1'-dimethylethoxycarbonyl)amino)-2-phe~ylethyl~-dihydrofuran~ oa~
The product from Step D, 9.93 g, was dissolved in 100 mL of absolute ethanol and added to a ~olution of 2.6 g of sodium and 20.1 mL of diethyl malonate in 170 mL of absolute ethanol. After stirring overnite, the reaction wa~ acidified to pH 4 with lOZ citric acid and extracted with 2 X 500 mL of ether. The combined organic ex*racts were washed 1 X
~00 mL ~2~ 1 ~ 500 mL ~at'd Na~C03, 1 X 500 mL sat~d brine and dried over MgS04. The solvents were removed and the crude product purified by low pressure chroma- -tography on silica gel eluting with 50% ether/hexanes (or EtOAc/hexane6). The yield of semi-solid product was 10.6 g. The later fractions contained 2.5 g of the undesired 5 R ,isomer as a white solid.

Step F: Preparation of ~5S,l'S)-3-carboethoxy-3-(4-benzyloxyphenylmethyl)-5-~1-(1,1-dimethyl-etho~ycarbonyl)amino)-2-phenylethyl]dihydro-furan-~-(3H~,,-Qnç
To a stirred solution of (5S,l'S)-3-carbo-ethoxy-5-[1-((1~,1'-dimethylethoxycarbonyl)amino)-7-phenylethyl)-dihydrofuran-2-~3H)-one (product o~ Step E), 2 g ~5.3 mmol) in 25 mL of absolute ethanol was added a ~olution of 0.13 g of sodium in 2.2 mL of absolute ethanol followed by 1.30 g (5.5 mmol) of 4-benzylo y benzyl chlo~ide. The solution was heated 3~ to 50C under nitrogen for 1 hour, then cooled in an ice bath and acidified with 20 mL of 10% citric acid and diluted with 200 mL of wates. The mixture was . - - ~- . . . . . - . .. , .. . - .,.-.. .. . . . .......... ., . ,.. ; .

, ~ ; ~ ' . ' ` :' ' ' . ;. . ,~, , . - , W092/06992 PCT/US91/~16 _ ~'}~

extracted with 3 X 100 mL of ether and the combined ether extracts washed with 50 mL of water, 200 mL
of 6at'd Na~C03 and dried over MgS04. Remo~al of solvents under reduced pressure and purification by low pressure chromatography on silica gel, eluting with 40% ether in hexanes gave 1.~6 g (51% yield) of a clear colorless glass essentially homogeneous by TLC (SOZ ether/hexanes).

lo Step G: Preparation of (3R,5S,l'S)-3-(4-benzyloxy-phenylmethyl)-5-(1((1,1-dimethylethoxy-carbonyl)amin~)-2-phenylethyl)-dihydrofuran-2-(3H~-one.
The product of Step F, 13.6 g, was dissolved in 250 mL of 1,2-dimethoxyethane, and to it was added 117 mL of 1 M lithium hydroxide at room temperature.
After stirring for 12 hours, the solvents were removed under reduced pressure, the residue suspènded in 200 mL of 10% citric acid and extracted 3 ~ 500 mL of diethyl ether. The combined ether egtracts were washed with 500 mL of brine, dried tMgS~4) and the concentrated to dryness. The residue was dis601ved in 250 mL of toluene, heated to reflux for t2 hours, then concentrated to dryness under reduced pressure.
Purification by medium pressure chromatography over silica gel eluting with 15% ethyl acetate/heæanes gave 3.2 g of the 3R-lactone as a clear foam.
~urther elution with the same solvents gave 6.15 g of the 3S-lactone as a white solid.

, . . ' ~ ' ' ' ' .
.

~VO 92/06992 PCI~/US91/06816 - 103 _ 2~3~2~

Step H: Preparation of N'-(1,l-dimethylethoxy-carbonyl)-5(S)-amino-4(5)-(1l,1~-dimethyl-ethyl-l,l-dimethylsilyloxy)-6-phenyl-2(R)-(4-benzyloxv~henylmetbyi~h~anoic ~cid.
The product of Step G, 0.6 g, was dissolved in 30 mL of a 2:1 mixture of ethylene glycol dimethyl ether/water, and to it was added ~ mL of l M lithium hydroxide at room temperature. After stirring for 1 hour, the mixture was partitioned between 200 mL
lo chloroform and 20 mL 10% citric acid. The layers were separated and the aqueous phase extracted with 3 X 20 mL chloroform. The combined organic layers were dried (Na2S04) and the solvent removed to yield O.56 g of the crude hydroxy acid. This residue waæ
di~solved in 5 mL of dry DMF and 0.845 g tert-butyl dimethylsilyl chloride and 0.725 g of imidazole were added. After stirring for 18 hours, the reaction was poured into 50 mL of water and extracted with 3 X 20 mL of ethyl acetate. The combined organic extracts were washed with 3 X 20 mL of lOZ citric acid, 1 ~ 20 mL of water, 3 X 10 mL of saturated aqueous solution of Na2C03, and 20 mL of brine. After drying (Na2S04), the solvent was removed and the resulting residue dissolvet in a mixture of 5 mL of l~, 5 mL of glacial acetic acid, and 2 mL of water. The mixture was stirred for 4 hours, then poured into 50 mL of water and extracted with 3 g 20 mL of ether. The combined ether e~tracts were washed with 2 2 20 mL of water, brine, dried (Na2S04), and the solvent removed.
Purification by medium pressure chromatography over silica gel, eluting with MeO~/C~C13 gave 0.60 g of the product as a white glassy solid.

- . .. . . . .~ . , . .. .. - . . , . . . . -W092/06g92 PCT/US91/~16 _ 1.~3'~Q~

Ste~ esolution.of l-Amino-2-hy~lo~yind~n From the known racemic l-amino-2-hydroxyindan, the resolution was carried out as described for the 3-amino-1,2-dihydroxyindan in E~ample 7 ~elow (Steps D and E). The (lS,2R)-l-amino-2-hydro2yindan resulting from saponification of the higher Rf diastereomer was shown to have an aD of -58 (c = 1.0, CHC13). The (lR, 2S)-l-amino-2-hydroxyindan resulting from saponification of the lower Rf diastereomer was found to have an a~ of l62~ (c - 1.0, C~C13).

Step J: Preparation of N-(2(R)-hydroy -l(S)-indanyl)-5(S)-(l,l-dimethyletho~ycarbonylamino)-4(S)-hydroxy-6-phenyl-2(R)-(4-benzylo2yphenyl-methyl~ hexa~mide The product from Step H, 0.12 E~ was dissolved in 2 ml dry DMF and to it was added 40 mg of l(S)-amino-2(R)-hydroxyindane, (Step I) 25 mg of l-hydroxybenzotriazole hydrate and 70 mg of dimethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride.
Triethylamine was added to the stirred solution until the p~ was 8.5 (32 mL~. After stirring for concen-trated to dryness under reduced pressure, the residue waæ dissol~ed in lOO mL of chloroform and wor~ed with 1 X 50 mL of 10% citric acid, 1 ~ 50 mL H20~ 1 X 50 mL sat'd NaHC03, driet over MgS04 and concentrated to dryness. The residue was di~solved in 1 mL of tetrahydrofuran and added to 2 mL of 1 M tetrabutyl- - -ammonium fluoride in THF. After ætirring overnight at room temperature the reaction mi~ture was diluted with 10 mL of lOX citric acid and the white preci-,.
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', ' ' -W092/06g92 PCT/US91/~16 2 O93~

pitate collected by filtration. The product was purified by low pressure chromatography on silica ~el eluting with 2% methanol/C~2C12 to give 85 mg of product which was essentially homogeneous by TLC
(3% methanol/C~2C12).

Step K: Preparation of N-(2(R)-hydroxy-l(S)-indanyl)-5(S)-(l,l-dimethylethoxycarbonylamino)-4(S)-hydroxy-6-phenyl-2(R)-(4-hydroxyphenyl-methyl~hexana~
The product of Step J, 85 mg was dissolvedin 10 mL of methanol and lO mL of T~F, and to it was added 0.10 g of 10% palladium on carbon. The mixture was stirred under an atmosphere of hydrogen for 48 l~ hours at room temperature, then filtered and concen-trated to dryness. The residue was dissolved in lO mL
of hot ethanol and 20 mL water was added. On cooling the white solid precipitate was collected and dried under vacuum over P205. The yield was 72 mg (98%
yield~ of pure product: mp 218-219C (effervesces, sinters at 215) elemental analysis, Calc'd for C33H40N206: (560.696):
C, 70.69; ~, 7.19; N, 5.00;
Found: C, 70.62; ~, 7.39; N, 4.79.

Ste~ L: Preparation of N-(cis-2(R)-hydroxy-l(S)-indanyl)-5(S)-~l,l-dimethyletho y -carbonylamino)-4(S)-hydroxy-6-phenyl-2(R)-t(4-(2-(4-morpholinyl)ethoxy)phenyl]methyl3-hexa~a~ide A stirred mixture of Step ~ product, N-(2 (R)-hydroxy-l(S)-indanyl)-5(5)-[l,l-dimethylethoxy-WO92/06ss2 ~ ' PCT/US9~ 16 _ c~ , carbonyl)amino]-4(S)-hydroxy-6-phenyl-2(R)-(4-hydroxy-phenylmethyl) hexanamide (0.50 g, 0.9 mmol), anhydrous cesium carbonate (1.0 g, 3 mmol) and N-(2-chloroethyl) morpholine, free base (2.35 ~, 17 mmole) in lO0 mL
of anhydrous dioxane was heated to 80C (internal temperature) for 3 hrs. After cooling to room temperature the mixture was diluted with chloroform (50 mL) filtered, concentrated to dryness under reduced pressure, and the residue triturated with lo 50 ml of anhydrous ether and 10 mL of ethyl acetate.
The white solid product was collected and dried under vacuum over P205. The yield was 0.54 g ~89%) of pure product L-689,502: mp 195-7C. elemental analysis, Calc'd. for C3HslN30: (673.856):

C, 69.52 ~, 7.63; N, 6.23;
Found: C, 69.19 E, 7.45; N, 6.15.

maleate hydrate:

mp 112-113C dec. elemental analysis, Calc'd. for C3g~slN307.C4~404.H20: (807.946):
C, 63.92 ~, 7.11; N, 5.20;
Found: C, 64.23 ~, 6.94; N, 5.10.
EXAMeL~

A. F~rmen~atio~ o$ L-689.502 A frozen vial (2.0 ml) of Rhizo~ arrhi~U6 MT4974 was used to inoculate a 250 ml baffled shake fla6k containing 50 ml of seed medium A. The seed flas~ was incubated on a rotary ~ha~er (220 rpm) at . .

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W092/06992 PCT/USgl~16 - 107 ~ 2~93~

27OC for 24 hours. A 2.5 ml aliquot of the developed seed was used to inoculate a 250 ml non-baffled flask containing 50 ml of transformation medium B; L-689,502 in DMS0 was added to the fermentation at 0 hour to achieve a final concentration of 0.05 mg/ml. The sha~e flask contents were subsequently incubated at 27OC on a rotary shaker for 4 days. The resultant whole broth was e~tracted as described in Section B.

10 Media: Seed Medium A ~Ll Dextrose l.0 Dextrin 10.0 Beef Extract 3-Ardamine p~ 5.0 NZ Amine Type E 5.0 MgS4-7H2 0.05 K2~PD4 0-3 Adjust pH to 7.1 Add to CaC03 0.5 ~/1 Trans~ atio~ Medium~
Glucose 10 ~ycaæe SF 2 Beef Eatract Corn Steep Liquor 3 Adjust p~ to 7.0 B- IsQlaeis~ Ll~-LLLi~Ltion The whole broth (400 ml) was extlacted three 30 times with l-butaDol (3 x 400 ml). The e~tracts were combined and concentrated under vacuum to an oily residue. The residue was dissolved in methanol and - . . . .. . . : . ., . . ~ .

W092~06992 PCT/US91/~16 _ subjected to high performance liquid chromatography (HPLC). EPLC was carried out on Whatman Partisil 10 ODS-3,9.4 mm ~ 25 cm at room temperature and monitored at 215 mm. The column was developed at 3 ml/min with a linear gradient from 0.1% agueous ~3P04-CH3CN, 80:20, to 0.1% aQueous ~3P04-CH3CN, 20:80, in 60 minutes. The compound was collected during repeated injections of the above described e~tract.
The fractions at retention time, 19.3 minutes, were pooled, adjusted to pH 6.5 and evaporated to remove acetonitrile. The compound was further purified using a C18 Sep-Pak (Waters Associates) and methanol-water elution solvent to yield 11 mg of product.

Anal. Calc'd- for C39~5oolopN3Na2 C, 58.72 H, 6.27; N, 5.27; P, 3.89.
~ound: C, 59.78 ~, 6.58; N, 4.97; P, 4.17.

NMR, elemental analysis, and MS showed the structure to be 2s 0 O PO~H2 ~=/
~ -C- ~ ~H

3D ~ O

L-`696,432 : ' ;, '' ,, .
.- . . .. .

~092/06992 PCT/US91/~16 - 109 ~

Phosphorus was identi~ied and quantified by complexomatric and gra~imetric methods. Phosphoryl-ation at C4 was established by NMR from the~0.5 ppm downfield displacement of the C4H relati~e to its chemical shift in the parent L-689,502.

EXAM~L~ l7 Enzymatic Treatment o~_L~k2~ Z
The microbial transformation meta~olite L-696,432 was determined to be a phosphate ester of L-689,502, a highly potent ~IV protease inhibitor.
To determine if the phosphate ester bond can be enzymatically cleaved, treatment with alkaline phosphatase was performed.
To 0.5 ml of water containing 0.8 mg of L-696,432 was added 30 microliter of bacterial alkaline phosphatase (0.31 unit/microliter), and the reaction mixture was incubated at 37-C and monitored by HPLC. The reaction was complete a~ter four hours of'incubation. The reaction product was purified by ~PLC and subjected to FAB MS analysis. tM+~]+ion was obser~ed at m/z 674. The fragmentation ions at m/z 574,556,469 and 425 confirmed the product is L-689,502.
The results show that L-696,432 can be enzymatically hytrolyzed back to the parent compound L-689,502.

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WOs~069s2 PCT/US9l/~16 ~

EXAMpLE 18 Ass~y~for Inhibition of Rec~m~inan.t ~IV Prote~8 Inhibition studies of the reaction of ~IV
protease expressed in Escherichia coli were carried out with a tritiated peptide substrate, t3~]-acetyl-Val-Ser-Gln-Asn-(beta-napthyl Ala)-Pro-Ile-Val-Gln-Gly-Arg-Arg-N~2 (MW=1800). The two arginine residues at the carboxyl terminus give thi~ peptide an overall positive charge at acidic p~ and enable it to bind to the ~+ form of DOWEX AG-50W-~ resin and similar resin~. The ~IV protease cleaves between the ~-napthyl-Ala and proline residues to yield a pr.oduct ~3~]-acetyl-val-ser-asn-~n-napthyl-ala) that is either neutral or slightly negatively charged and does not bind to the cation exchange resin. It ie therefore possible to conveniently separate the labelled product from the substrate.
Aliqu~ts of 25 ~1 containing 6.0-8.0 nM ~IV
protease in assay buffer (100 mM 60dium acetate, p~
5.5 and 0.1% BSA) are placed in assay tubes. The reaction is initiated by addition of 25 ~1 aliquots of 4.2 ~M trîtated peptide substrate in 100 mM ~odium acetate, p~ 5.5. After incubation for 60 minutes at 37-C, the reaction is stopped with 100 ~1 of 5% E3P04, then analysed ~y application of column chro~atography. ~ :
L-696,432 demonstrated 32Z inhibition at a concentration of 1 ~g/ml in substantially purified form.

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,, , . . -, . .: . ., W092/06gg2 PCT/US91/~16 20~3~ 9 Organic Synthesis of L-~9~.432.
phosph~ ster o~ ~-689,~Q2 Or~anic S~nthesiQ of L-696~4~2 A. Ace~ lation of Ind~
~-(cis-2(R)-Acetoxy-l(S)indanyl)-5(S)-~l,l-dimethylethoxycarbonylamino)-4(S)-hydroxy-6-phenyl-2(R)-~(4-(2-(4-morpholinyl)ethoxy)phenyl~methyl~-hexanamide is prepared by the protocol of Example 1, except that l(S)-amino-2(R)-acetoxyindane substitutes for l(S)-amino-2~R)-hydroxyindane of step I.

. L-696.432 The product of step A is reacted with monophenyl phosphorodichloridate according to the principles and practice of Chambers, R.W. and ~
Khorana, J. Am. Chem. Soc. 80, 3749 (1958). Sub-sequent treatment with excesæ ammonia, followed by removal of the acetyl and phenyl groups under basic conditions, yields L-696,432.

2S C. Al~ernative $ynthetic Routes Another phosphorylating agent is dibenzyl phosphorochloridate. An exten~i~e tiscussion of phosphorylation and phosphorylating agents can be found in ~. Mizuno, Studies in Org. Chem, 24, 171-175 (1986) and reference6 cited therein.

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W092/06992 PCT~US9l/~16 -A culture of Rhizo~u~ arrhi~ MF 4974 from the Merck Culture Collection was inoculated in 50 milliliters of soy-glucose medium in a 250 milliliter flask and the flask incubated on a rotary shaker at 220 rpm at 27C for 24 hours to obtain the first stage seed culture. 2.5 milliliters of this seed culture was inoculated into each of 18 flasks containing 50 milliliters of soy-glucose medium which was also charged with Compound Z to a concentration Of 5 ~glml in 50 microliters of DMS0 and the resulting culture incubated on a rotary shaker at ~20 rpm at 27C for 18 hours.
Following incubation, each flask was harvested by centrifugation, the mycelium washed twice with sterile saline and resuspended in p~ 7.0 phosphate buffer containing 1 percent glycerol.
Compound Z was added to a final concentration of 58 ~g/ml using 100 ~1 of (DMS0). The flasks were incubated on a rotary shaker at 220 rpm at 27C for 48 hours.
At the end of this period, the contents of eighteen flasks (900 milliliters) were pooled and the whole broth centrifuged. The mycelial cake was slurried with 100 milliliters of water, the p~
adjusted to 3.5 and the slurry e2tracted twice with 100 milliliters of n-butanol. The 6upernatant was acidified to p~ 3.5 and extracted twice with one-half the volume of n-butanol. Each organic e2tract was assayed by ~PLC. The assay condition was as follows:

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~ .
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W092/06992 PCT/US91~16 2~3r~9 Column: "ZORBAX" (DuPont) C8 Rx 4.5 x 250 mm Mobile Phase: Acetonitrile + lOmM aq. KH2P04, gradient 20% to 80% o~er 2~ minutes Temperature: 45C
Flow rate: 1.5 ml/min.
Detection: 210 nm Sample Vol: 50 ~1 The extracts of the mycelium and the e2tracts of the supernatant were pooled and e~aporated to dryness at 30C at reduced pressure to obtain an oil as residue.
The oil was dissolved in the mobile phase of 40/60 acetonitrile/water and further purified using ZO~BAX C8 (9.6 mm ~ 25 cm) semi-preparative column.
The column was developed at 7.05 ml/min. using ~0 percent agueous acetonitrile containin~ 0.1 percent -trifluoroacetic acid (TFA) at 45C. Fractions having a retention time of 12.8 minutes were pooled, and the solvent evàporated to obtain 12 mg of Compound III in a yield which calculated to be 24Z.
The product had the mass spectral data previously set forth.
A portion of the product was converted to the monopotassium 6alt. This was carried out by dissolving the biophosphorylation product in 70 percent aqueous acetonitrile containing 10 mM ~2P04 (p~ 4.5). The mixture was subjected to retuced pres~ure to remove the acetonitrile and the aqueous residue loaded onto a water-equilibrated C-18 solid phase extraction column. ~he column was washed with water and then eluted with 70 percent aqueous acetonitrile and the eluate freeze-tried for retention of the salt.

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W092/06992 PCT~US91/~16 _ E~AMP~E 21 Spores of Rhizo~ arrhL~ were obtained from oatmeal agar slants of MF 4974 maintained in the Merck Culture Collection and employed to prepare a spore suspension in water of about 7 g 109 spores per milliliter for use in the preparation of seed culture.
Seed flasks each rontaining 500 milliliters of soy glucoæe bsoth of the composition previously given, were inoculated with 1 milliliter of spore suspension and incubated on a rotary shaker (220 rpm) at 27C for 24 hours.
Following incubation, the mycelia from each flask were harvested by filtering through a 10 micron nylon mesh and then resuspended in an equal volume of a 100 mM p~-6.3 phosphate buffer containing 3 percent glycerol and addet to the broth. Compound Z, was added to a concentration o$ about 50 ~g/ml in dimethylsulfoxide. The flasks were then incubated on a rotary ~haker at 220 rpm at 27C for 24 hours.
After completion of the incubation period, the contents of si~teen fla~ks totaling 8000 milliliters were pooled and filtered through a 10 micron nylon mesh. The mycelial cake was slurried with 1000 milliliters of 50 percent aqueous methanol and filtered. The mycelial cake was again extracted with aqueous met~anol and the two aqueous methanol filtrates combined and diluted with 2000 milliliteræ ~ -of water.
The resulting aq~eous solution was applied to a 15 mm x 300 mm column pac~ed to a 220 mm bed height with water equilibrated ~DIANION" HP20,resin.

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- -~092~06gg2 PCT/US9l/~16 2 o 9 ~3 Ll, 2 ~

The filtrate was pumped in a downflow mode at 15 mllmin. in 2000 ml aliquots. After loading, the column was washed with 500 milliliters or water and the desired phosphorylated product eluted with 500 5 milliliters of 20 percent aqueous acetonitrile. The -remaining substrate and metabolite wa~ eluted with 500 milliliters of 70 percent aqueous acetonitrile.
The diluted filtrates from the mycelial extracts were also pumped onto the column, the column washed and then eluted and the eluate assayed by HPLC. The assay condition was as described in Example 20.
HPLC indicated the following mas~ balance for microbial phosphorylation and "DIAION~ HP 20 15 isolation: :

Substrate Charged: 402.5 mg Metabolite & Substrate Recovered: 383.8 mg Total Recovery: 95.4%
20 Compound III 234.9.mg Bioconversion yield 61.2Z

The eluate was concentrated under reduced pressure and the residue dissolved in 40 percent aqueous acetonitrile containing 10mM RH2P04 to obtain a monopotassium salt. The aqueous ~olution was subjected to reduced pressure to remove the acetonitrile and the aqueous residue loaded onto a water-equilibrated C-18 solid phase extraction column. The column was thereafter washed with water and then the monopotassium salt of Compound III
eluted with 70 percent aqueous acetonitrile. The - . , -: . -. .
- , ~ . ...
: . -, .. ; , . , , ,, ~ , . . . .
. .. . . .. . .

~ ~ ' , , , W092/06g92 PCT/US91/~16 _ eluant wa~ freeze dried to obtain the salt. The NMR
spectrum of this salt is that previously detailed.
The salt is converted to the acid by careful acidification.

~L~

~ he following salts are prepared by reacting an appropriate phosphate salt and Compound III in the manner descri~ed in Example 21 and concentrating under vacuum.

Ex~le ~5~ R
IIIa PO(O~)(ONa) IIIb PO(O~)~OR>
IIIc PO(ONa)2 IIId PO(OLi)(O~) IIIe PO(O~)(OMg)U2 IIf PO(O~)(ON(CH3)4) 1000 compressed tablets each containin~ 500 mg of Compound IIIA are prepared from the following formulation: ;

Compound Grams 30 Compound IIIA 500 Starch 750 Dibasic calcium phosphate hydrous 5000 Calcium stearate 2.5 . . ' ' ' ' . ' ' ~ : ' . : ' . :

W092~06g92 PCT/US91/~6 2 ~

The finely powdered ingredients are mixed well and granulated with 10% starch paste. The granulation is dried and compressed into tablet~.

S EXAMPL~ 24 1000 hard gelatin capsules, each containing 500 mg of mono sodium salt of Compound III are preparet from the following formulation:

Compound Gram~

Compound III, mono ~odium salt 500 Starch 750 15 Dibasic calcium phosphate hydrous 5000 Calcium stearate 2.5 A uniform mixture of the ingredients is prepared by blending and used to fill two-piece hard gelatin capsuIes.
250 ml of an injectable solution are prepared by conventional procedures having the following for~ulation:

5 Dextrose 12.5 g Water 2~0 mL
Compound III, mono pota~sium ~alt 400 mg The ingredients are blended and thereafter stesilized for use.

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W092/06992 PCT~US91/~16 -.

EXAMPkE 25 An aero~ol composition may be prepared ha~ing the following fosmulation:

PeE_Can~ r Compound IIIA 24 mg Lecithin NF Liquid Concentrated 1.2 mg Trichlorofluoromethane, NF 4.026 g Dichlorodifluoromethane, NF 12.15 g Prepara~ion of Sta~tin~ Mate~ials Compound Z, the starting material, was prepared by inoculating 54 milliliters of P34-2 medium of the following composition per liter: corn steep liquor, 5 g; D-mannitol 25 g; glucose monohydrate, 10 g; ~PHARMAMEDIA," (nonhydrolyzed protein, Buckeye Oil6eed Products, Memphis, Tenn.) 20 g K~2P04, 9 g; FeS04-7~20, 10 mg MnS04^4H20 10 mg;
CuC12-2~20. 0.25 mg; CaC12-2~20~ 1 mg; ~3B03~ 0-5 mg (N~4)6 ~724-~2. 0-19 mg; Zns04-7~20~ 2 mg, with frozen vials of alerion arboricol~ MF5533 ATCC
74030 and the inoculated medium incubated with shaking at 220 rpm at 25C for four days. Twenty milliliters were used to inoculate four 2-liter flas~s containing SOO milliliters of P34-2 medium and the inoculated medium incubated at 25~C ~or four days at 220 rpm. The flask contents were pooled and used to inoculate into three fermenters each containing 180 liters of P34-2 medium and 2 ml/L of propylene W092/OCg92 PCT/US91/~16 2~J~f~ 2~ ' ;
glycol P-2000 (Dow Chemical) to reduce foaming and 3, the inoculated medium cultivated for si~ days at f 25~C, an air flow of 90 L/min. a pressure of 0.7 kg/cm2 gauge, and an agitator speed of 200 rpm. A
25 liter sample of the resulting broth was then u~ed to inoculate three fermenters each containing 475 liters of P34-2 medium containing 2 ml/L of P-2000 and cultivated for four days at 25C, air flow of 250 L/min, pressure 0.7 kg/cm2 gauge and 150 rpm.
425 liters of this ~eed broth was inoculated into each of three production f ermenters containing 13,700 liters of TG106 medium of the following composition per liter: D-mannitol, 100 g; NZ-Amine type E (casein hytrolysate, Sheffield Products, ~raft Inc.) 33 g; Eidco 8005 yeast e~tract (Difco), 10 g;
(N~4)2S04, 5 g; K~2P04, 9 g; P-2000, 2 ml, and the fermenters operated at a temperature of 27C, air flow of 2500 literstminute, a pressure of 0.7 kg/cm2 gauge, and an agitator speed of 50 rpm. The p~ was allowed to decrease from 6.0 to 5.5 and then maintained at 5.5 + 3. After about 2 1/2 weeks the broth was harvested for product i601ation.
The broth from the foregoing cultivation was first extracted with an equal ~olume of methanol.
The methanol-bIoth was clarified using a liquid-solid separator (centrifuge) to obtain clarified liquit as first extract and solid. The e~traction-clari~ication was repeated. The e~tracts were combined and the water content adjusted to about 50 percent. The resulting 601ution was passed through a "DIAION"
SP-207 adsorption column to adsorb Compound III and the column washed with aqueous methanol. Thereafter Compound III was recovered with 100 percent methanol.

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.. ., " , - - . .. , . ~ . . ' ~ -, . , : ; ~ . . : -W092/06~2 PCT/US91/~

` ~ - 120 -The water content of the methanol containing Compound III was adjusted to 50 percent and the aqueous methanol solution intimately mixed with an equal volume of 1:1 ethyl acetate/he~ane and the two liquid phases thereafter 6eparated. The aqueous methanol layer was passed through a column of "DIAION" SP-207, the column washed with aqueous methanol, and Compound III eluted with 100 percent methanol. The eluant was vacuum concentrated to a minimum volume and the sol~ent composition adjusted to about 75:20:5 ethyl acetate/methanol/water.
The feed thu~ prepared was passed through a silica gel column and Compound III eluted with 85:10:5 ethyl acetate/methanol/water. The fractions showing 85 percent or greater arèa purity by HPLC
were combined, vacuum concentrated to remove ethyl acetate and the concentrate adjusted to 50 percent aqueous methanol, the latter passed through "DIAION"
~P-20 in the manner pre~iou~ly described concentrated and Compound III precipitated with acetronitrile and recovered by vaccum filtration and then dried.
The starting material al~o may be prepared by method~ described in copending applications Serial Nos. 47/492,025 and 47/492,026.
~. ar~Li~Ql~ MF5533 ATCC 74030 is disclosed and claimed in copending application Serial No.
630,457, filed December 19, 1990. Briefly, it may be obtained by (a) i~oculating a frozen ~egatative mycelium of ~. arbo~icol~ ATCC 20957 (disclosed and claimed in copending application Serial No. 492,024>
into Æ seed medium of: corn steep liquor, 5 g/l;
tomato; tomato paste 40 g/l, oat flour 10 gll;

', ~ ' ' , ~ ' W092/06992 PCT/US91/~16 2~3~; ~

glucose lO gll; FeS04-7~20 10 mg/l; Mn504-4H20 10 mg/l; CuC12-2~20, 0.25 mg/l; CaC12-2~20, 1 mg/l;
B03, 0.56 mg/l; (N~4)6M724-~2~ 0-19 mg/ ;
ZNS04-7~20, 2 mg/l; adding to the medium N-methyl-N~-nitro-N-nitrosoguanidine (NTG) (b) cultivating, thereafter (c) plating a portion of the growth on potato dextrose agar and (d) incubating for 14 days at 25C to obtain spores then (e) harvesting the spores, (f) diluting the spores with sterile lo saline (g) plating on potato dextrose agar (h) incubatin~ for 7 days for colony formation, (i>
transferring separate colonies to slants of potato and (j) incubating for 14 days at 25C.
EXAM~LE_Z~

Biotransformation Enzymology-Isolation Of Cell Free Activity _ -Spores of Rhiz~o~us o~vzae ME 4974, cultivated on oatmeal agar, were inoculated in a Soy-Glucose medium consisting of 20.0 g dextrose, 5.O g Fidco yeast extract, 5.0 g NaCl, 5.O g ~2EP04 in a liter of distilled water. The p~ of the medium wa6 adjusted to 7.0 before autocla~ing. The cultures were incubated at 27-C for 24 hours on a rotary sha~er at 220 rpm. A fre~h Soy-Glucose medium wa~ inoculated with the 24-hour seed culture at a ratio of ~% (v/v) and the fermentations were continued in the ~anner described abo~e. The phosphorylating enzyme acti-vity, as a function of time, was determined.
Cell free extracts were obtained by suspend-in~ the cells in a buffer containing lO0 mM phosphate - , . , , , . . ~ ; . . , ,. ,, -", ; ' ' :

W092/OC~2 PCT/US91/~16.~

~993~

(p~ 7.5) and 2 mM EDTA. The cells to buffer rati~
was 2:1. Several procedures were then tried to break the cell membranes and release the cell contents, i.e., a high-pressure French press, sonication, grinding the frozen cells, and lysozyme treatment.
The enzyme reaction was carried out in 0.2 ml mixture containing 0.15 mM FK-520, 5 mM MgC12, 5 ~M ATP and ~arious amount of enzyme. The reac~ion mixture was incu~ated at different temperatures for 1 hour and terminated by the addition of 0.2 ml methanol. The resulting solution was æubjected to HPLC analysis on Whatman Partisil 10 ODS-3 at 55C.
The column was developed at 1 ml/min with a linear gradient from 45% acetonitrile in 0.1% pho~phoric l acid to 80% in O.lZ phosphoric acid in 30 minutes.
The retention time of phosphorylated ~K-520 and EK-520 was 16.5 and 20 minutes, respectively.
Protein concentration was determined by Pierce BCA protein a6say reagent using BSA aæ standard.
In a ~earch for phosphorylating enzyme in cell $ree extracts of Rhizogus sryz~ MF 4974 we ha~e found phosphorylating activity: 1) is highest in the extracts of cells isolated after 24 hours incubation in Soy-Glucose medium; 2) both sonication and macera-tion of the cells under liquid nitrogen can be usedto break the cell membraneæ for liberating the enzyme in soluble form. The pE optimum i8 7.5; 4) the temperature optimum is 37-C. 5) The phosphorylati~n of ~K-520 was linear with respect to time and enzyme concentration for one hour and from 0.2 to 2.0 mg protein. The e~fect of the FK-520 eoncentration on the rate of phosphorylation have been ~tudied using crude extract. The Km value for the substrate - - - - - - - - . . . .. . - . ..
.. , . . . ~
. . I ' ! . , , . ' . , ~ " ". " ,, . ' '' ' .
. .

W092/069g2 PCT/US91/~16 ~093~

(FK-520) was about 0.5 mM and the Vmax wa~ 2.1n mole/min/mg protein. To determine metal ion reguire-ment the crude enzyme was dialyzed thoroughly against 20 mM Tris-~Cl buffer and 2 mM EDTA. The results show that the phosphorylating acti~ity requires Mg~2 or Ca+2 as cofactor.

EX~L~ V
NMR analysis established that the biotrans-formation product obtained by incubating 24-deoxy FR-900520 with ME 4974 (Rhizopus arrhizus) is the 32 phosphorylated analog:

o 0~ 1 Me .

~
2s Mb ~ o J '~ b ~ .
OM~ 0~53 Contacting 24-deoxy FR 900520 made by the proce6s described in Exampleæ 28-29 with the R~i~o~us microorganism as pcr the analogous procedure described for 24-oxy FR 900520, yields the 32-phos-phorylated analog as determined by ~MR spectroscopy.

- . ' 1 .

W092/06~2 PCT/US91/~16 _ ~X~MPk~ 28 17-Ethyl-l-hydroxy-12-[2'-(3 " ,4 " -dihydroxycyclo-hexyl)-l'-methylvinyl~-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04~9]-octacos-l4 18-~iene-2 3 10~16-tetraon~
To a stirred solution of 31-de~methyl ~K-900,520, 17-ethyl-1,14-dihydroxy-12-~2l-(3'',4''-dihydroxycyclohexyl)-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 produced as described in EPO Publication. 0 349 061 ~77 mg in 3 ml benzene) was added 6 mg of --~-toluenesulfonic acid and the mixture warmed to 60C
in an oil bath. After 25 minutes, the reaction mixture was cooled to room temperature, neutralized by the addition of a saturated aqueous Na~C03 solution and extracted with ethyl acetate (3 times).
The combined organics were washed with saturated NaCl solution, dried over NazSO4 and purified by flash chromatography (20% hexanes in ethyl acetate and 1%
MeOH) to yield 40 mg of the title compound.
MASS: (FAB) 782 (m + Na).
Partial 1~ NMR (200 m~z): ~ 6.80 (dd, Jl = 16 ~Z. J2 _ 6 ~z), 6.16 (dd, Jl = 16 ~Z- J2 = 1-5 ~z~, 4-39 (broat d, J = 14 ~z~, 4.26 (broad d, J = 5 ~z), 3.91 (dt, Jl = 8.8 Rz, Jz = 3 ~z~

- - ,,; ~ . .............. ; ....... . . .

: - , - . . - . .~ .. . . ~ ; . .. . -W092/06992 PCT/US91/~16 ~0~3~

E~ LE 29 17-Ethyl-l-hydro~y-12-[2'-(3 " ,4 " -dihydroxycyclo-hexyl)-l'-methyl~inyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04~9]-oct~Q~ -ene-~l~.l0.1~tetr~Qae To a solution of 17-ethyl-1-hydro~y-12-~2'-(3 " ,4 " -dihydr~xycyclohexyl)-1'-methylvinyl3-23,25-dimethoxy-13,19,21,27-te~ramethyl-11,28-dioxa-4-azatricyclo~22.3.1.04~9]octacos-14,18-diene-2,3,10,16-tetraone (40 mg in 1.5 ml ethyl acetate) was added 3 mg of 5% Rh/Carbon catalyst. The reaction flask was fitted with a hydrogen balloon, evacuated and recharged with hydrogen gas (3 times). After 45 15 minutes, the mixture was filtered over Celite, concentrated and purified by fla~h chromatography (CH2Cl2: MeO~: ~exane (10:1:2)). to yield 33 mg of the title compound..
MASS: (~AB) 768 (m + Li).
20 Partial 1~ NMR (200 m~z): ~ 4.55 (broad d, J = 5 ~z), 4.39 (broad d, J = 14 ~z), 3.86 (dd, Jl = 8.8 J2 = 3 ~Z) EXAMPLE_~Q

P~OSPHORYLATED ANTI-C~OLESTEROLEMIC
I~QL~TES ~-706.52~ AND L-70~ 527 Contacting 8imvastatin, whose chemical name 30 is: 6(R)-~2-(8'(S)-2",2"-dimethylbutanoylogy-2'(S),6'(R)-dimethyl-1',2',6',7',8',8'a (R)-hexahydronaphthyl-l'(S)-ethyl]-4(R)-hydroxy-3,4,5,6-tetrahydro-2~-pyran-2-one with the Rhizopus, ~, . . . . . . ,~;

' ~ ' ' ,: -: ' ' -' . ' ' ' ~ : ' W092/06992 PCT/US91~16 _ ~.~

microorganism under the analogous conditions described in Example 3 produced two phosphorylated compounds, L-706,546 and L-706,527. The preparation and properties o~ simvastatin are described in EP
Publication No. 0033538 to Merck & Co., Inc.
NMR analyæis of the biotransformation products obtained by incubating the above compound simvistatin with R~izoPUs arrhi~ confirms their assigned structures as illustrated:

H0-P-0 HC~ H o ~4~H o ~C~}~ 3C/~ ~ - P- O- H ~ -Y~C H3C
L-706, 526 L-706, 527 - :

The compounds exhibit inhibition in the biosynthesis of cholesterol as al~o described for ~imvastatin in EP Publication No. 0 033 538.
Key features in the NMR of L-706,526 are the absence of a side chain methyl triplet and the psesence of a no~el C~3C~-0- moiety with signals at - l.lO ppm (C~3) and at 3.93 ppm (CE-0). Irradiating at l.lO ppm collapsed the methine quintet to a W092/06992 PCT/US91/~16 L~ ~ ~

doublet. Since the methine proton in the proposed structure has no vicinal neighbor the appearance aæ a toublet implies coupling with an unseen hetero atom.
i.e., the phosphorus.
Phosphorylation at the ~-6' sitea is sug-gested by the absence of a typical ~-6' signal at 3.69 ppm and the presence of a broad, fea~ureless signal at 4.26 ppm which is assi~ned to a displaced ~-6'. Both the magnitude of the displacement and the lo loss of detail are reasonable consequences of phos-phorylation.

~XAMPLE 31 Phosp~Q~v~tion of Zearal~none olH CH3 ~P03 H2 Chenic~l ~tructure oF
-zQaralQnol-6~-phosph~te Incubation of zearaleno~e available from Sigma with resting cells of R~L~Q~g~ arrh~ MF 4974 for 24 hours results in two biotransformation products. They have been identified as a-zearalenol and a-zearalenol-6'-phosphate. The fermentation, 3D iæolation, purification and enzymatic hydrolysis arè
described below.

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

W092/06~2 PCT/US91/~16 ~9 9~ - 128 -FERM~TATION

One Ml of a RhL~Q~us arrhi~us (MF 4974) spore suspension, harvested from oatmeal a~ar, wea incubated into 500 mL Soy-Glucose medium contained in a 2 liter Erlenmeyer flask and ~haken a 27C on a rotary shaker (220 rpm) for 24 hours.
loFollowing incubation, each flask was har~ested by filtration washed once with water, then resuspended in 500 mL or mM PO4 buffer (pH 7.0) containing 4% glycerol.
Zearalenone was added to a final concentration of .05 mg/mL, using 0.8 mL
DMSO. The charged flasks were incubated on a rotary shaker (220 rpm) at 27DC for 24 hours. Following incubation, the whole broth was worked up as described below.

Soy Glucose ~çdi~m ~/L

Dextrose 20.0 Soy Meal 5 0 ~ido yeast extract 5.0 NaCl 5.0 ~2~PO4 5.0 Adjust pH to 7.0 .. , .. .. , . - . . . . . . .

WO92/06ss2 PCT/US91/~16 2~93~

ISOLATION AND PuRIEIçATI~ j The whole broth (1000 mL) was diluted with an equal volume of methanol, than centrifuged. The mycelial cake was discarded. The clear filtrate was applied to a 15mm ~ 30 cm column packed with HP 20 (Mitsubishi Chemical, 220 mm bed height water equilibrated). The filtrate wa~
pumped onto the column in the downflow mode at 15 mL/min. After loading, the column was washed with 500 mL water. Metabolites or subætrates were not detected in the spent broth or wash. The column was eluted with a step gradient of methanol:water. The most polar metabolite eluted between 10% and 30%
methanol. The second metabolite and su~strate eluted in the column wash (100%
methanol). Appropriate fractions were combined and evaporated to dryness under reduced pres6ure at 30C to yield yellow oils that was subjected to further purification by preparative EPLC.
Preparative ~PLC was carried out on Whatman Magnum 9 Partisil 10 ODS-3 column (C18, 9.8 mm ID x 25cm) at room temperature and monitored at 237 nm. The column was developed at 4 mLImin with linear gradient from 20X acetonitrile in 0.1%phoRphoric acid 3 to 80% acetonitrile in O.lZ phosphoric acid in 40 minutes.

: . : . . . . ..... ... . . . . .

.. .. . ~ .

WOs2/06sg2 PCT/US91/~16 ., c~9 The compounds were collected during repeated injections of the above described extract.
Fractions of retenti~n times 17.~ and 20.1 minutes were collected, pooled, diluted with three volumns of water and desalted usin~ a Speed C18 column (Applied Separations) to yield 7 mg of 6-phosphate e3ter and 5 m~ of a-zearalenol, respecti~ely.
ENZYMATIC ~ o~

To prove that the 6'a-hydroxyl group is phosphorylated, enzymatic hydrolysis was examined using various sulfataæes and al~aline phosphatases obtained from Sigma.
In summary, a specific amount of enzyme was added to one mL of lOOmM phosphate ~uffer containing -50 ug/mL of zearalenol phosphate ester. The buffer solution was incubated at 37~C for 4 hours. After incubation, 300 uL was removed and analysed ~y ~PLC, using a Whatman Partisil 10 ODS-3 column (C18 10 um, 4.6 mm ID ~ 25 cm) at room temperat~re and monitored at 237 nm.
The column was de~eloped at 1 mL/min with a linear gradient gradient from 20%
acetonitrile in 0.1% phosphoric acid t~ 80Z
acetonitrile in 0.1% phosphoric acid in 20 minute~. ~nder these conditions, a-zearalenol-6'-phosphate ester and a-zearalenol have retention times of 13.93 and 18.17 minutes, respectively.

. . , . , ............ : . - , . . . ..
. , ...... .. . . . .: -,,- . , , . - , ' ~; , , " ,, wO92/06g92 PCT/US91/~16 20~3 ~

The results of enzymatic hydrolysis are summarized bel~w:

ENZYME ~ UNITs ACTIVITY

Alk. Phosphatase 7.0 1.0 100% a-OH @ lhr Al~. Phosphatase 7.0 100.0 100% ~-O~ @ lhr Sulfatase 5.010.00% a-OH @ lhr Sulfatase 7.01.0100% -O~ @ lhs Alk. Phosphatase 7.0 10.0 6% ~-O~ @ lhr Alk. Phosphatase 7.0 10.0 100% a-O~ @ lh Sulfatase 5.0100.052% a-O~ @ lhr C~ARACTERI~IO~

The compoundæ were identified by MS and NMR
and confirmed by enzymatic hydrolysis as a-zearalenol-6'-phosphate and a-zearalenol. Also, the presence of phosphoru~ was confirmed by an ICP elemental ~ analysis.

Claims (52)

WHAT IS CLAIMED IS:

1. A process for producing a biophosphory-lated hydroxyl containing organic compound, wherein said hydroxyl group is phosphate reactive, comprising the step of contacting a strain of Rhizopus oryzae microorganism together with the hydroxy containing organic compound in an aqueous medium containing a carbon nutrient at ambient temperature for a suffi-cient time to produce the biophosphorylated hydroxyl containing organic compound.

2. The process of Claim 1 wherein said microorganism comprises resting cells of Rhizopus oryzae in a phosphate buffer containing glycerol as a carbon nutrient.

3. The process of Claim 1 wherein said Rhizopus oryzae is cultured with said hydroxyl containing organic compound under submerged aerobic fermentation conditions in an aqueous carbohydrate medium containing a nitrogen nutrient at a pH below about 8.0 at ambient temperature.

4. The process of claim 1 wherein said Rhizopus oryzae strain is ATCC No. 11145.

5. A process for producing an immunos-suppressant, identified as a C-32 phosphorylated derivative of an FK-506 type macrolide comprising the step of contacting a strain of a Rhizopus oryzae microorganism together with a FK-506 type macrolide, containing a free C-32 hydroxy group, in an aqueous medium containing a carbon nutrient at ambient temperature for a sufficient time to produce the C-32 phosphorylated FK-506 type macrolide immunosup-pressant.

6. A compound being the C-32 phosphorylated derivative of an FK-506 type macrolide.

7. The compound of Claim 1 of the formula:

I

wherein:
R is H, C1-C4 alkyl, R1 is H2P04, R2 is hydrogen, hydroxy or lower alkanoyloxy, R3 is methyl, ethyl, propyl or allyl, n is an integer of 1 or 2, and the symbol of a line and dotted line is a single bond or a double bond, and a pharmaceutically acceptable salt thereof.

8. The compound of Claim 7 being the C-32 phsophorylated derivative of FK-506, FK-520, FK-523, FK-525, C-31 desmethyl FK-506, C-31 desmethyl FK-520.

9. The compound of Claim 7 being:

10. The compound of Claim 7 being:

11. The compound of Claim 7 being:

12. The compound of Claim 7 being:

13. The compound of Claim 7 being:

14. The compound of Claim 7 being:

15. The compound of Claim 7 being:

16. A pharmaceutical composition for the treatment of immunoregulatory disorders or diseases comprising a pharmaceutical carrier and a therapeutically effective amount of compound of Claim 7.

17. A pharmaceutical composition for the topical treatment of inflammatory and hyperproliferative skin diseases and or cutaneous manifestations of immunologically-mediated illnesses comprising a pharmaceutical carrier and a therapeutically effective amount of compound of Claim 7.

18. A method for the treatment of immunoregulatory disorders or diseases comprising the administration to a mammalian species in need of such treatment an effective of a compound of Claim 7.

19. A process for producing an immunosuppressant, identified as a phosphorylated rapamycin-type macrolide comprising the step of contacting a strain of a Rhizopus oryzae microorganism together with rapamycin macrolide in an aqueous medium containing a carbon nutrient at ambient temperature for a sufficient time to produce the C-43 phosphorylated rapamycin macrolide immunosuppressant.

20. The process of Claim 19 wherein said microorganism are resting cells of Rhizopus oryzae in a phosphate buffer containing glycerol as a carbon nutrient.

21. The process of Claim 19 wherein the resulting aqueous medium containing said phosphoryl-ated macrolide exhibits positive inhibition of T-Cell activation.

22. The process of Claim 19 wherein said Rhizopus oryzae is cultured with said rapamycin macrolide under submerged aerobic fermentation conditions in an aqueous carbohydrate medium con-taining a nitrogen nutrient at a pH below about 8.0 at ambient temperature.

23. A phosphorylated macrolide of the structural formula:
where

24. A phosphorylated macrolide exhibiting a characteristic proton NMR spectrum as illustrated in Figure 3 and exhibiting positive inhibition of T-cell proliferation, reversible by recombinant human IL-2, in a T-cell proliferation assay.

25. A phosphorylated macrolide having the structural formula as illustrated in Figure 4.

26. A method for producing a phosphorylated cyclic lipopeptide compound having the formula wherein R is which comprises cultivating Rhizopus arrhizus ATCC 11145 in a nutrient medium containing a compound having the formula:

and a phosphate salt, said medium maintained in a pH
range of from about 6.0 to 6.3.

27. A method according to Claim 26 in which the phosphate salt is in an amount of at least 10 percent by weight of the solid components.

28. A method according to Claim 26 in which the medium of the following composition is grams/liter: glucose 20.0; soya meal 5.0; Fidco yeast extract 5.0: sodium chloride 5.0; and potassium hydrogen phosphate 5.0; at pH 5.

29. A method according to Claim 26 in which the cultivation is carried out in the temperature range of from about 15°C to about 30°C with agitation in the range of 220 rpm to 400 rpm for a period of from 24 hours to about two days.

30. A method for selectively phosphorylating a compound having the formula at the 4-hydroxyproline hydroxyl which comprises cultivating Rhizopus arrhizus ATCC 11145 in a soy glucose medium containing said compound in an amount of 40-60 µg/ml, wherein said soy glucose medium is of the following composition in grams/liter: glucose 20.0; soya meal 5.0; Fidco yeast extract 5.0; sodium chloride 5.0; and potassium hydrogen phosphate 5.0;
at pH 5.

31. A phosphorylated cyclic lipopeptide compound obtained by biophosphorylating with Rhizopus arrhizus ATCC 11145, a cyclic lipopeptide related to echinocandins and having a peptide skeleton bearing several hydroxy groups wherein in said phosphorylated cyclic lipopeptide, the phosphate group is attached to the hydroxy group of the 4-hydroxyproline component of the lipopeptide,

32. A phosphorylated cyclic lipopeptide compound having the formula:

wherein R is or a cation salt thereof.

33. A compound according to Claim 32 wherein R is .

34. A compound according to Claim 32 wherein R is .

35. A compound according to Claim 32 wherein R is .

36. A composition comprising the compound of Claim 32 in admixture with a pharmaceutically acceptable carrier.

37. A method for controlling the growth of fungi comprising administering an antifungally effective amount of the compound of Claim 32.

38. A method for the treatment of or for the prevention of Pneumocystis carinii infections in mammals which comprises administering to mammals an anti-infective or therapeutic amount of the compound of Claim 32.

39. A compound of the Formula:

or pharmaceutically acceptable salt, hydrate, or ester thereof.

?O 92/06992 PCT/US91/06816

40. A method of preparing the compound of Claim 39, comprising the steps of (a) providing a quantity of N-(2(R)-hydroxy-1(S)-indanyl)-5(S)-(1,1-dimethylethoxy-carbonylamino)-4(S)-hydroxy-2(R)-[(4-(2-(4-morpholino)ethoxy)phenyl)prop-2-en-1-yl]-6-cyclohexyl-hexanamide;

(2) incubating the compound of step (a) in a microbial culture of Rhizopus arrhizus MF4974; and (3) isolating the compound of Claim 39.

41. A pharmaceutical composition useful for inhibiting HIV protease, comprising an effective amount of a compound of Claim 39, and a pharmaceutically acceptable carrier.

42. A pharmaceutical composition useful for preventing or treating infection of HIV or for treating AIDS or ARC, comprising an effective amount of a compound of Claim 39, and a pharmaceutically acceptable carrier.

43. A method of inhibiting HIV protease, comprising administering to a suitable mammal in need of such treatment an effective amount of a compound of Claim 39.

44. A method of preventing infection of HIV, or of treating infection by HIV or of treating AIDS
or ARC, comprising administering to a suitable mammal in need of such treatment an effective amount of a compound of Claim 39.

45. A compound of the Formula:

or pharmaceutically acceptable salt, hydrate, or ester thereof.

46. A method of preparing the compound of Claim 45, comprising the steps of (1) providing a quantity of N-(2(R)-hydroxy-1(S)-indanyl)-5(S)-(1,1-dimethylethoxy-carbonylamino)-4(S)-hydroxy-6-phenyl-2(R)-[(4-(2-(4-morpholinyl)ethoxy)-phenyl)methyl]-hexanamide;

(2) incubation the compound of step (1) in a microbial culture of Rhizopus arrhizus MF4974; and (3) isolating the compound of Claim 45.

47. A pharmaceutical composition useful for inhibiting HIV protease, comprising an effective amount of a compound of Claim 45, and a pharmaceutically acceptable carrier.

48. A pharamceutical composition useful for preventing or treating infection of HIV or for treating AIDS or ARC, comprising an effective amount of a compound of Claim 45, and a pharmaceutically acceptable carrier.

49. A method of inhibiting HIV protease, comprising administering to a mammal an effective amount of a compound of Claim 45.

50. A method of preventing infection of HIV, or of treating infection by XIV or of treating AIDS
or ARC, comprising administering to a mammal an effective amount of a compound of Claim 45.

51. A phosphorylated simvastatin derivative of the following structure:

L-706, 526 or L-706, 527

52. A phosphorylated zearalenol compound of the following structure:

.