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 -
-2-
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 ~
~.
-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 ~ ~ ~
-4-
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~
-5-
. ,
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
--6--
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
R2
~CHz~
H3C ~ H C
W
CH30 OCH3
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
2093129
-7-
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 "
--8--
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~
--11--
HO--P{~",3~34
CH30~ 2~ CH3
10 ~=
27 ~
CH30 OCH3
and C-32 phosphorylated ~-520,
2s
:,
- ~ . .,.... . . . . . . - . , .
WO 92/06992 9 PCI/US91/068l6 ~
.
~9
--12--
HO 7 o",3.~3~ H
CH3~ 2B CH3
30 ,~H
~ ~ .
~D ~20
CH3~ C~1B -
Z~ ~ :
~ 14 .
CH3O CH3
a nd C_32 PhO8PhOrY1ated Fg 523;
: .
. "'.
- .
:
.
~092/06992 PCT/US91/~16
2~93'~
--13--
HO-P~"3h34~
CH3~ 2~ CH3
CH3
~
CH3 21~ H3
~D \~20
CH3~00 C~1B
~J 1 6
~ 14 .
CH3O CH3
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 -
~ .,;.;
-14- -
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
-15-
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-
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 -
'
-18-
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~ -
-19-
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~ .
-20-
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 -
K2EP4 5-0
AdjuBt pX to 7.0
W092/069s2 ~ .~ PCT/US91/~16-~
'~'
-22-
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~
-23-
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_
-24-
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
-26-
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~
--27--
,
,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
.)
-28-
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
-30-
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~ .
-32-
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~
-33-
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
-34-
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~
--35--
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~
-36-
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).
10 NMR DATA
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
-37-
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
-38-
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
-39-
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~
~,,'
-40-
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~
-41-
.
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/~
-42-
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
-43-
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
-44-
(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~
-45-
Minimum Fungicidal Concentration
Fungus ~lml)
St~ain No. Compound IIIA
Candida albicans -
S MY 1~ 64
MY 102B 64
Candida tropicalis
MY 1012 - 32 -
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/~
-46-
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~
-47-
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-
.~ .,
-48-
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~
-49-
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
-50-
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 ~
- 51 -
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~
- 52 -
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
- 53 -
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_
~;
- 54 -
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
- 58 -
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~ .
- 60 -
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;~
- 63 -
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
- 6 4 -
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
~'
.,~ .
- 66 -
Zearalen~e
Also included by the process of thi~
invention is the biotransformation product: alpha
zearalenol 6'-phosphate: ¦
OH CH
Ho ~
OP3H2
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~
- 67 -
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
- 69 -
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 ~
- 71 -
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-~
- 72 -
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~
- 73 -
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,--
- 74 -
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~
- 75 -
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.
EXAMPLE 4
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--.
~' .
- 78 -
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~- ?,~
- 79 -
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 ~
- 81 -
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.
EXAMPLE 7
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~
- 82 -
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'
- 83 -
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-`
.
- 84 -
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 ~
- 85 -
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/~
- 86 -
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.
3D
.: . , ........ : . , .
. . .: -,,, ' , , ,, .' . ' '
~92/06992 PCT/US91/~
~9~d~
-- 87 --
~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
- 89 -
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
- 90 -
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
- 91 -
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
~'
. , .
- 92 -
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~ .
- 93 -
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.
EXAMPLE 12
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_
-- 94 --
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 ~
- 95 -
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 ~
- 97 -
Results are as follow~:
C~n~. L-706.579(~M~ %Inh ~ tion
100 100
89
1 20
.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~
-- 100 --
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~
-- 101 --
~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~
- 104 -
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-
,.
- , ., : ~ ,
.
.
. ~ . ,
., . ,. - ,: ., . :
', ' ' -
W092/06g92 PCT/US91/~16
2 O93~
- 105 -
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.
2S
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~ ,
- 106 -
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
. .
. - . . . .
, .. - . . . :
:
. . .. . ... . .. .
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 _
108 -
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.
', " ',~ , ' :
WOs~069s2 PCT/US9l/~16 ~
-- 110 --
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.
. ...... , .. : , . - ~ - :
.~ , . , , ~ .................................... .
,, , . . -, . .: . .,
W092/06gg2 PCT/US91/~16
20~3~ 9
111
EXAMPLE 19
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.
. ... ~ . . . . .
: . . . .. .
.. . . .
. . .
W092/06992 PCT~US9l/~16 -
112 -
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:
--,
- : . .... , :,... .. ~: :
- . ,:
~ .
: . .
W092/06992 PCT/US91~16
2~3r~9
- 113 -
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.
. . .: . ~ , . . .
: , . . . - , .~.
.. . . ..
- . . .. , ,. ,, ., . , ." . . , ~ . , , ,,, . .; . . ..
:~ - , .
, : . : '
.. :~ . :,. ` . `. ` ' . . , :
' ., ': , , . `.. : ' : ' .
W092/06992 PCT~US91/~16 _
- 114 -
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.
.. . , . . . - . - ,
- ~ . . . .: . . . .......................... . . : : .
. . . . ......................... . ~ ..
- -
~092~06gg2 PCT/US9l/~16
2 o 9 ~3 Ll, 2 ~
-- 115 --
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 _
- 116 -
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)
EXAMPLE 23
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 ~
- 117 -
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.
., .. , ~ . ., - ..
. .
- . . .
. ., ~ . - ,
,
,,
. ,, .,, - .
W092/06992 PCT~US91/~16 -
.
- 118 -
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~ '
-- 119 --
;
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.
~ ~ ... . .. .
.. ., " ,
- - . .. , . ~ . . ' ~ -,
. , : ; ~ . . : -
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~; ~
- 121 -
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~
- 122 -
(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~
- 123 -
(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 _
- 124 -
~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~
- 125 -
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 _
~.~
- 126 -
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~ ~ ~
- 127 -
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~
- 129 -
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
130 -
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 ~
- 131 -
The results of enzymatic hydrolysis are
summarized bel~w:
ENZYME ~ UNITs ACTIVITY
P-4653
Alk. Phosphatase 7.0 1.0 100% a-OH @ lhr
P-5521
Al~. Phosphatase 7.0 100.0 100% ~-O~ @ lhr
S-8629
Sulfatase 5.010.00% a-OH @ lhr
S-1629
Sulfatase 7.01.0100% -O~ @ lhs
P-8639
Alk. Phosphatase 7.0 10.0 6% ~-O~ @ lhr
P-1391
Alk. Phosphatase 7.0 10.0 100% a-O~ @ lh
S-9751
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.
