Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
PHOSPHATE CONTAINING MACROCYCLIC IMMUNOMODULATORS
Reference to Related Application
This application is a conversion of United States Provisional Application
60/127,195, filed on March 31, 1999.
Field of the Invention
The present invention relates to novel chemical compounds having
immunomodulatory activity, and in particular to macrolide immunosuppressants.
More
particularly, the invention relates to semisynthetic analogs of ascomycin and
FK-506.
means for their preparation, pharmaceutical compositions containing such
compounds and
methods of treatment employing the same.
Background of the Invention
The compound cyclosporine (cyclosporin A) has found wide use since its
introduction in the fields of organ transplantation and immunomodulation, and
has brought
about a significant increase in the success rate for transplantation procedur
es.
Unsatisfactory side-effects associated with cyclosporine, however, such as
nephroto~icity,
have led to a continued search for immunosuppressant compounds having improved
efficacy and safety.
Recently, several classes of macrocyclic compounds having potent
immunomodulatory activity have been discovered. Okuhara et al., in European
Patent
Application No. 184162, published June 11, 1986, disclosed a number of
macrocyclic
compounds isolated from the genus Streptomyces. Immunosuppressant FK-506,
isolated
from a strain of S. tsukubaensis , is a 23-membered macrocyclic lactone ( 1 a,
Figure 1 ).
Other related natural products, such as FR-900520 ( 1 b, Figure 1 ) and FR-
900523 ( 1 c.
Figure 1) which differ from FK-506 in their alkyl substituent at C-17 (Figure
1), have been
isolated from S. hygroscopicus yaku.shimnaensis. Yet another analog, FR-900525
(ld,
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
2
Figure 1), produced by S. tsukubaensis, differs from FK-506 by the replacement
of the
piperdine moiety with a pyrrolidine moiety.
FR-900520, also known as ascomycin, has been previously disclosed by Arai et
al.
in U.S. Patent No. 3,244,592, issued April 5, 1966, where the compound is
described as an
antifungal agent. Monaghan, R.L., et al., on the other hand, described the use
of
ascomycin as an immunosuppressant in European Patent Application No. 323865,
published July 12, 1989.
Although the immunosuppressive activity of FK-506 has been clinically
confirmed, toxicity in mammals has limited its utility. 'The activity of FK-
506 has,
however, prompted efforts to discover novel analogs of FK-type compounds which
possess superior properties. These efforts include the isolation of new
fermentation
products, the microbial transformation of existing chemical entities, the
chemical
modification of these macrocycles, and the synthesis of hybrid species derived
from
smaller synthetic fragments.
HOn,./
I4 ~,6
12 14 ~ O
n~~..j2C) Q OH 17
o
O
0 21 .
H 0 0 '''%
1 ~;
.24 I~''OCH3
CH3
Figure 1
( 1 a): FK-506 R = CH2CH=CH2; n=2
(Ib): FR-900520 R = CH~CH3; n=2
( 1 c): FR-900523 R = CH3; n=2
(ld): FR-90052 R = CH2CH=CH2; n=1
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
-,
J
Fermentation products of FK-type compounds include C-17-epi derivatives of FK-
506; a 3'-demethylated derivative of FK-506; 3'-oxo-FK-506 ; compounds derived
from
FK-506, FR-900523 and FR-900525 which are characterized by the introduction of
hydroxy protecting groups; formation of a double bond by elimination of water
between
carbonsl4 and 15 ; oxidation of the hydroxy group at carbon 14 to the ketone,
and
reduction of the allyl side-chain at carbon 17 via hydrogenation (Figure 1 ).
Other
published derivatives include those derived from FK-506 and FR-900520 where
the
lactone ring is contracted to give a macrocyclic ring containing two fewer
carbons.
Several microbial transformations of FK-type compounds have been published,
such as the microbial demethylation of FR-900520 to form the bis-demethylated
3',25-
dihydroxy ring-rearranged derivative of FR-900520; the microbial
monodemethylation at
carbon 25 of FK-506 and FR-900520; and the microbial monodemethylation of FR-
900520 at C-3' (Figure 1 ), as well as a number of other macrocyclic microbial
transformation products.
Numerous chemical modifications of the FK-type compounds have been
attempted. These include the preparation of small synthetic fragments of FK-
type
derivatives; a thermal rearrangement of a variety of derivatives of FK-506
which expands
the macrocyclic ring by two carbons; and modifications which include methyl
ether
formation at C-4' and/or C-14, oxidation of C-4' alcohol to the ketone, and
epoxide
formation at C-2 (Figure 1 ).
Although some of these modified compounds exhibit immunosuppressive activity,
the need remains for macrocyclic immunosuppressants which do not have the
serious side
effects frequently associated with immunosuppressant therapy. Accordingly, one
object of
the present invention is to provide novel semisynthetic rnacrolides which
possess the
desired immunomodulatory activity but which may be found to minimize unwanted
side
effects. More particularly, the present invention provides novel semisynthetic
macrolides
which bear a phosphate moiety at C-4' (Figure 1 ).
Another object of the present invention is to provide synthetic processes for
the
preparation of such compounds from starting materials obtained by
fermentation, as well
as chemical intermediates useful in such synthetic processes.
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
4
A further object of the present invention is to provide pharmaceutical
compositions
containing, as an active ingredient, one of the above compounds. Yet another
object of the
invention is to provide a method of treating a variety of disease states,
including post-
transplant tissue rejection and autoimmune dysfunction.
Summary of the Invention
In one aspect of the present invention are disclosed compounds having formula
I:
Rs
R5 ~~,
o
n~H2C)
..,,R,
I R3 O
R2
HO
O,
~~~OC H 3
3
I,
or a pharmaceutically acceptable salt, ester, amide or prodrug thereof,
wherein,
n is an integer of 1-3;
R' is selected from the group consisting of
( 1 ) methyl,
(2) ethyl,
(3) propyl, and
(4) allyl;
R' and R3 are independently selected from the group consisting of
( 1 ) hydrogen and
(2) OR', wherein R' is selected from the group consisting of
a) hydrogen and
b) hydroxy protecting group, or
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
R' and R' taken together are selected from the group consisting of
( 1 ) oxo and
(2) thioxo;
R4 is selected from the group consisting of
5 ( 1 ) hydrogen and
(2) OR', wherein R' is previously defined;
R' and R6 are independently selected from the group consisting of
( 1 ) hydrogen and
(2)
Z
I I
P
-O~ I ~A
wherein Z is selected from the group consisting of O and S;
A is selected from the group consisting of
(a) NR8°Rg' wherein Rg° and R8' are independently selected from
the group
consisting of
(i) alkyl and
(ii) arylalkyl,
(b) morpholine,
(c) thiomorpholine, and
(d) OR8 wherein R8 is selected from the group consisting of
(i) alkenyl,
(ii) alkyl,
(iii) alkynyl,
(iv) aryl,
(v) arylalkyl,
(vi) cycloalkyl,
(vii) cycloalkylalkyl,
(viii) heterocycle,
(ix) heterocyclealkyl,
(x) NRg'R83alkylene, wherein Rg' and R8' are independently
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
6
selected from the group consisting of
hydrogen,
alkylcarbonyl, and
formyl,
provided that at least one of R8' and R8' is other than hydrogen;
B is ORS, wherein R9 is selected from the group consisting of
( 1 ) alkyl,
(2) aryl,
(3) cyanoalkyl, and
(4) haloalkyl;
provided that at least one of R' and R6 is other than hydrogen; and
a broken line represents the presence of an optional double bond, provided
that when R4 is OR', wherein R' is hydrogen, the double bond is absent.
In a further aspect of the present invention are disclosed pharmaceutical
compositions which comprise a compound of formula I in combination with a
pharmaceutically acceptable carrier alone or in combination with another
compound of
formula I.
In yet another aspect of the present invention is disclosed a method for
suppressing
the immune system of a mammal comprising administering to the mammal a
therapeutically effective amount of a compound of formula I.
In yet another aspect of the present invention is disclosed a method for
treating or
preventing post-transplant organ or tissue rejection in a mammal comprising
administering
to the mammal a therapeutically effective amount of a compound of formula I.
In yet another aspect of the present invention is disclosed a method for
treating or
preventing autoimmune diseases in a mammal comprising administering to the
mammal a
therapeutically effective amount of a compound of formula I.
Compounds of this invention include, but are not limited to,
( 1 R,2R,4R)-4-{ (E)-2-[( 1 R,9S,12S,13R,145,17R,21 S,23 S,24R,25 S,27R)-17-
ethyl-
1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3,10,16-tetraoxo-
11,28-
dioxa-4-azatricyclo[22.3.1.04°~]octacos-18-en-12-yl]-1-propenyl}-2-
methoxycyclohexyl
methyl 4-pyridinylmethyl phosphate,
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
( 1 R,2R,4R)-4-{(E)-2-[( 1 R,9S,12S,13R,14S,17R,21 S,23 S,24R,25S,27R)-17-
ethyl-
1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3,10,16-tetraoxo-
11,28-
dioxa-4-azatricyclo [22.3.1.04'9]octacos-18-en-12-yl]-1-propenyl } -2-
methoxycyclohexyl
methyl 3-pyridinylmethyl phosphate,
( 1 R,2R,4R)-4-{(E)-2-[( 1 R,9S,125,13R,14S,17R,21 S,23S,24R,25S,27R)-17-ethyl-
1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3,10,16-tetraoxo-
11,28-
dioxa-4-azatricyclo [22.3.1.04~~]octacos-18-en-12-yl]- I -propenyl } -2-
methoxycyclohexyl
methyl 2-pyridinylmethyl phosphate,
( I R,2R,4R)-4-{(E)-2-[(1 R,9S,12S,13R,145,17R,21 S,23S,24R,25S,27R)-17-ethyl-
1,14-dihydroxy-23,25-dimethoxy-13, I 9,21,27-tetramethyl-2,3,10,16-tetraoxo-
11,28-
dioxa-4-azatricyclo[22.3.1.O~v]octacos-18-en-12-yl]-1-propenyl}-2-
methoxycyclohexyl 2-
furylmethyl methyl phosphate,
benzyl (1R,2R,4R)-4-{(E)-2-[(1R,9S,12S,13R,14S,17R,21S,23S,24R,25S,27R)-
17-ethyl-I ,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3,10,16-
tetraoxo-
1~ 11,28-dioxa-4-azatricyclo[22.3.1.04'9]octacos-18-en-12-yl]-1-propenyl}-2-
methoxycyclohexyl methyl phosphate,
( 1 R,2R,4R)-4-{(E)-2-[( 1 R,9S,125,13R,145,17R,21 S,23S,24R,25S,27R)-17-ethyl-
1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3, I 0,16-tetraoxo-1
I ,28-
dioxa-4-azatricyclo [22.3.1.04v]octacos-18-en-12-yl]-1-propenyl } -2-
methoxycyclohexyl 3-
furylmethyl methyl phosphate,
( 1 R,2R,4R)-4-{(E)-2-[( I R,9S,12S, I 3R,14S,17R,21 S,23 S,24R,25 S,27R)-17-
ethyl-
1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3,10,16-tetraoxo-
11,28-
dioxa-4-azatricyclo [22.3.1.04°~]octacos-18-en- I 2-yl]-1-propenyl }-2-
methoxycyclohexy l
methyl 3-thienylmethyl phosphate,
( 1 R,2R,4R)-4-{(E)-2-[(1 R,9S,12S,13R,145,17R,21 S,23S,24R,25S,27R)-17-ethyl-
1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3,10,16-tetraoxo-
11,28-
dioxa-4-azatricyclo [22.3.1.0~~9]octacos-18-en- I 2-yl]- I -propenyl } -2-
methoxycyclohexyl 4-
fluorobenzyl methyl phosphate,
(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl (1R,2R,4R)-4-{(E)-2-
[(1R,9S,12S,13R,14S,17R,21S,23S,24R,25S,27R)-17-ethyl-1,14-dihydroxy-23,25-
dimethoxy-13,19,21,27-tetramethyl-2,3,10,16-tetraoxo-11,28-dioxa-4-
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
8
azatricyclo[22.3.1.04v]octacos-18-en-12-yl]-1-propenyl}-2-methoxycyclohexyl
methyl
phosphate,
( I R,2R,4R)-4-{ (E)-2-[( 1 R,9S,125,13R,14S,17R,215,23 S,24R,25 S,27R)-17-
ethyl-
1, I 4-dihydroxy-23,25-dimethoxy-I 3,19,21,27-tetramethyl-2,3,10,16-tetraoxo-
11,28-
dioxa-4-azatricyclo[22.3.1.049]octacos-18-en-12-yl]-1-propenyl}-2-
methoxycyclohexyl
methyl 4-nitrobenzyl phosphate,
( 1 R,2R,4R)-4-{ (E)-2-[( 1 R,9S,12S,13R,14S,17R,215,23 S,24R,25 S,27R)-17-
ethyl-
1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3, I 0,16-tetraoxo-
11,28-
dioxa-4-azatricyclo[22.3.1.04°9]octacos-18-en-12-yl]-1-propenyl}-2-
methoxycyclohexyl
methyl diisopropylphosphoramidoate,
2-cyanoethyl (1R,2R,4R)-4-{(E)-2-
[( 1 R,9S,125,13R,145,17R,215,23 S,24R,25S,27R)-17-ethyl-1,14-dihydroxy-23,25-
dimethoxy-13,19,21,27-tetramethyl-2,3, I 0,16-tetraoxo- I I ,28-dioxa-4-
azatricyclo[22.3.1.04v]octacos-18-en-12-yl]-1-propenyl}-2-methoxycyclohexyl
diisopropylphosphoramidoate,
( 1 R,2R,4R)-4- { (E)-2-[( 1 R,9S, I 2S,13 R,14S,17R,21 S,23 S,24R,25 S,27R)-
I 7-ethyl-
1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3,10, I 6-tetraoxo-
11,28-
dioxa-4-azatricyclo[22.3.1.04'9]octacos-18-en-12-yl]-1-propenyl}-2-
methoxycyclohexyl 3-
iodophenyl methyl phosphate,
2-(acetylamino)ethyl ( 1 R,2R,4R)-4-{ (E)-2-
[(1 R,9S,125,13R,145,17R,21 S,23S,24R,25S,27R)-17-ethyl-1,14-dihydroxy-23,25-
dimethoxy-13,19,21,27-tetramethyl-2,3,10,16-tetraoxo-11,28-dioxa-4-
azatricyclo[22.3.1.04°~]octacos-18-en-12-yl]-1-propenyl}-2-
methoxycyclohexyl methyl
phosphate,
( 1 R,2R,4R)-4-{(E)-2-[( 1 R,9S,12S,13R,145,17R,21 S,23S,24R,25S,27R)-17-ethyl-
1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3,10, I 6-tetraoxo-
11,28-
dioxa-4-azatricyclo[22.3.1.04v]octacos-18-en-12-yl]-I-propenyl}-2-
methoxycyclohexyl
methyl 2-(4-morpholinyl)ethyl phosphate,
( 1 R,2R,4R)-4-{ (E)-2-[( 1 R,9S,12S,13R,14S,17R,215,23 S,24R,25 S,27R)-17-
ethyl-
1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3,10,16-tetraoxo-
11,28-
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
9
dioxa-4-azatricyclo[22.3.1.04°9]octacos-18-en-12-yl]-1-propenyl}-2-
methoxycyclohexyl
methyl 2-(1-pyrrolidinyl)ethyl phosphate,
( 1 R,2R,4R)-4- { (E)-2-[( 1 R,9S,12S,13 R,14S,17R,21 S,23 S,24R,25 S,27R)-17-
ethyl-
1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3,10,16-tetraoxo- I
1,28-
dioxa-4-azatricyclo[22.3.1.04'9]octacos-18-en-12-yl]-1-propenyl}-2-
methoxycyclohexyl4-
fluorophenyl methyl phosphate,
( 1 R,2R,4R)-4-{(E)-2-[(1 R,9S,125,13R,145,17R,21 S,23 S,24R,25S,27R)-17-ethyl-
1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3,10, I 6-tetraoxo-
11,28-
dioxa-4-azatricyclo [22.3.1.04'9] octacos-18-en- I 2-yl]-1-propenyl } -2-
methoxycyclohexyl 4-
formylphenyl methyl phosphate,
4-acetylphenyl (1R,2R,4R)-4-{(E)-2-
[(I R,9S,12S,13R,145,17R,21 S,23S,24R,25S,27R)-17-ethyl-1,14-dihydroxy-23,25-
dimethoxy-13, I 9,21,27-tetramethyl-2,3,10,16-tetraoxo-11,28-dioxa-4-
azatricyclo[22.3.1.04-9]octacos-18-en-12-yl]-1-propenyl}-2-methoxycyclohexyl
methyl
phosphate,
diethyl ( 1 R,2R,4R)-4-{ (E)-2-[( 1 R,9S,12S,13R,14S,17R,215,23 S,24R,25
S,27R_)-
17-ethyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2,3,10,16-
tetraoxo-
11,28-dioxa-4-azatricyclo[22.3.1.04°~]octacos-18-en-12-yl]-1-propenyl}-
2-
methoxycyclohexyl phosphate, and
( 1 R,2R,4R)-4-{(E)-2-[( 1 R,9S,125,13R,145,17R,215,23 S,24R,25S,27R)-17-ethyl-
1,14-dihydroxy-23,25-dimethoxy- I 3,19,21,27-tetramethyl-2,3,10, I 6-tetraoxo-
I 1,28-
dioxa-4-azatricyclo[22.3.1.049]octacos-18-en-12-yl]-1-propenyl}-2-
methoxycyclohexyl
diphenyl phosphate.
Detailed Description of The Invention
It is understood that the foregoing detailed description and accompanying
examples are merely illustrative and are not to be taken as limitations upon
the scope of
the invention, which is defined solely by the appended claims and their
equivalents.
Various changes and modifications to the disclosed embodiments will be
apparent to those
skilled in the art. Such changes and modifications, including without
limitation those
relating to the chemical structures, substituents, derivatives, intermediates,
syntheses,
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
formulations and/or methods of use of the invention, may be made without
departing from
the spirit and scope thereof.
Compounds of the present invention can exist as stereoisomers, wherein
asymmetric or chiral centers are present. Stereoisomers are designated "R" or
" S,"
depending on the configuration of substituents around the chiral carbon atom.
The terms
"R" and "S" used herein are configurations as defined in IUPAC 1974
Recommendations
for Section E Fundamental Stereochemistry Pure Appl. Chem., (1976), 45: 13-30,
hereby
incorporated by reference. The present invention contemplates various
stereoisomers and
mixtures thereof and are specifically included within the scope of this
invention.
10 Stereoisomers include enantiomers, diastereomers, and mixtures of
enantiomers or
diastereomers. Individual stereoisomers of compounds of the present invention
may be
prepared synthetically from commercially available starting materials which
contain
asymmetric or chiral centers or by preparation of racemic mixtures followed by
resolution
well-known to those of ordinary skill in the art. These methods of resolution
are
exemplified by (1) attachment of a mixture of enantiomers to a chiral
auxiliary, separation
of the resulting mixture of diastereomers by recrystallization or
chromatography and
liberation of the optically pure product from the auxiliary or (2) direct
separation of the
mixture of optical enantiomers on chiral chromatographic columns.
Compounds of the present invention were named according to the rules described
in International Union of Pure and Applied Chemistry Organic Chemistry
Division,
Commission on Nomenclature of Organic Chemistry Nomenclature of Organic
Chemistry, Sections A, B, C, D, E, F, and H, 1979 edition, J. Rigaudy and S.P.
Klesney,
eds, Pergamon Press, Oxford, (1979) (Sections A, B, and C) and International
Union of
Pure and Applied Chemistry Organic Chemistry Division, Commission on
Nomenclature
of Organic Chemistry A Guide to IUPAC Nomenclature of Organic Compounds,
Recommendations 1993, Blackwell Science, 1993.
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
11
Definition of Terms
As used throughout this specification and in the appended claims, the
following
terms have the meanings specified:
The term "alkenyl," as used herein, refers to a straight or branched chain
hydrocarbon containing from 2 to 6 carbons and containing at least one carbon-
carbon
double bond formed by the removal of two hydrogens. Representative examples of
"alkenyl" include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-
propenyl, 3-
butenyl, 4-pentenyl, 5-hexenyl, and the like.
The term "alkoxy," as used herein, refers to an alkyl group, as defined
herein,
appended to the parent molecular moiety through an oxy group, as defined
herein.
Representative examples of alkoxy include, but are not limited to, methoxy,
ethoxy,
propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.
The term "alkoxycarbonyl," as used herein, refers to an alkoxy group, as
defined
herein, appended to the parent molecular moiety through a carbonyl group, as
defined
herein. Representative examples of alkoxycarbonyl include, but are not limited
to,
methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, and the like.
The term "alkyl," as used herein, refers to a straight or branched chain
hydrocarbon
containing from 1-to-6 carbon atoms. Representative examples of alkyl include,
but are
not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-
butyl, tert-butyl,
n-pentyl, n-hexyl, and the like.
The term "alkylcarbonyl," as used herein, refers to an alkyl group, as defined
herein, appended to the parent molecular moiety through a carbonyl group, as
defined
herein. Representative examples of alkylcarbonyl include, but are not limited
to, acetyl, 1-
oxopropyl, 2,2-dimethyl-1-oxopropyl, 1-oxobutyl, 1-oxopentyl. and the like.
The term "alkylcarbonyloxy," as used herein, refers to an alkylcarbonyl group,
as
defined herein, appended to the parent molecular moiety through an oxy group,
as defined
herein. Representative examples of alkylcarbonyloxy include, but are not
limited to,
acetyloxy, ethylcarbonyloxy, tert-butylcarbonyloxy, and the like.
The term "alkylene" denotes a divalent group derived from a straight or
branched
chain hydrocarbon of from 1 to 6 carbon atoms. Representative examples of
alkylene
include, but are not limited to, - _ _ - -CH,-, -CH,CH,-, -CH~CH,CH--. -
CH,CH,CH,CH,-,
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
12
-CH,CH(CH;)CH,-, and the like.
The term "alkylthio," as used herein, refers to an alkyl group, as defined
herein,
appended to the parent molecular moiety through a thio group, as defined
herein.
Representative examples of alkylthio include, but are not limited, methylthio,
ethylthio,
tert-butylthio, hexylthio, and the like.
The term "alkynyl," as used herein, refers to a straight or branched chain
hydrocarbon group containing from 2 to 6 carbon atoms and containing at least
one
carbon-carbon triple bond. Representative examples of alkynyl include, but are
not
limited, to acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, 1-
butynyl and the
like.
The term "allyl," _as used herein, refers to a -CH,CH=CH, group.
The term "amido" as used herein, refers to a -C(O)NR9°R~' group,
wherein R~° and
R9' are independently selected from the group consisting hydrogen, alkyl,
aryl, and
arylalkyl, as defined herein. Representative examples of -C(O)NR9°R~'
include, but are
not limited to, aminocarbonyl, dimethylaminocarbonyl, nnethylaminocarbonyl,
diethyaminocarbonyl, benzylaminocarbonyl, and the like.
The term "amino" as used herein, refers to a -NR9'R93 group, wherein R9' and
R93
are independently selected from the group consisting hydrogen, alkoxycarbonyl,
alkyl,
alkylcarbonyl, aryl, arylalkoxycarbonyl, arylalkyl, and formyl, as defined
herein.
Representative examples of -NR9zR~3 include, but are not limited to,
acetylamino,
benzyloxycarbonylamino, formylamino, ethoxycarbonylamino, acetylmethylamino,
and
the like.
The term "aryl," as used herein, refers to a phenyl group.
The aryl groups of this invention can be substituted with 1, 2, or 3
substituents
independently selected from alkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl,
alkylcarbonyloxy, alkylthio, amido, amino, carboxy, cyano, ethylenedioxy,
formyl,
halogen, haloalkyl, hydroxy, methylenedioxy, and nitro.
The term "arylalkoxy," as used herein, refers to an aryl group, as defined
herein,
appended to the parent molecular moiety through an alkoxy group, as defined
herein.
Representative examples of arylalkoxy include, but are not limited to, 2-
phenylethoxy, 3-
phenylpropoxy, 5-phenylpentyloxy, and the like.
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
13
The term "arylalkoxycarbonyl," as used herein, refers to an arylalkoxy group,
as
defined herein, appended to the parent molecular moiety through a carbonyl
group, as
defined herein. Representative examples of arylalkoxycarbonyl include, but are
not
limited to, benzyloxycarbonyl, 2-phenylethoxycarbonyl, and the like.
$ The term "arylalkyl," as used herein, refers to an aryl group, as defined
herein,
appended to the parent molecular moiety through an alkyl group, as defined
herein.
Representative examples of arylalkyl include, but are not limited to, benzyl,
2-phenylethyl,
3-phenylpropyl, and the like.
The term "carbonyl," as used herein, refers to a -C(O)- group.
The term "carboxy," _as used herein, refers to a -CO,H group.
The term "cyano," as used herein, refers to a -CN group.
The term "cyanoalkyl," as used herein, refers to a cyano group, as defined
herein,
appended to the parent molecular moiety through an alkyl group, as defined
herein.
Representative examples of cyanoalkyl include, but are not limited to, 2-
cyanoethyl,
3-cyanopropyl, and the like.
The term ''cycloalkyl", as used herein, refers to a saturated cyclic
hydrocarbon
group containing from 3 to 8 carbons. Representative examples of cycloalkyl
include, but
are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl,
cyclooctyl, and the like.
The cycloalkyl groups of this invention can be substituted with l, 2,or 3
substituents independently selected from alkoxy, alkyl, halogen, haloalkyl,
and -OR'.
The term "cycloalkylalkyl," as used herein, refers to cycloalkyl group, as
defined
herein, appended to the parent molecular moiety through an alkyl group, as
defined herein.
Representative examples of cycloalkylalkyl include, but are not limited to,
cyclopropylmethyl, 2-cyclobutylethyl, cyclopentylmethyl, cyclohexylmethyl,
4-cycloheptylbutyl, and the like.
The term "ethyl," as used herein, refers to a -CH~CH~ group.
The term "formyl," as used herein, refers to a -C(O)H group.
The term "halo" or "halogen," as used herein, refers to -C1, -Br, -I or -F.
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
14
The term "haloalkyl," as used herein, refers to at least one halogen, as
defined
herein, appended to the parent molecular moiety through an alkyl group, as
defined herein.
Representative examples of haloalkyl include, but are not limited to,
chloromethyl,
2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and
the like.
The term "heterocycle" or "heterocyclic," as used herein, refers to a
monocyclic,
bicyclic, or tricyclic ring system. Monocyclic ring systems are exemplified by
any 3- or 4-
membered ring containing a heteroatom independently selected from oxygen,
nitrogen and
sulfur; or a 5-, 6- or 7-membered ring containing one, two or three
heteroatoms wherein
the heteroatoms are independently selected from nitrogen, oxygen and sulfur.
The ~-
membered ring has from 0-2 double bonds and the 6- and 7-membered ring have
from 0-3
double bonds. Representative examples of monocyclic ring systems include, but
are not
limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane,
dithiane, furan,
imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline,
isothiazolidine,
isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline,
oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine,
pyran, pyrazine,
pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole,
pyrroline,
pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole,
thiadiazole,
thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene,
thiomorpholine,
thiomorpholine sulfone, thiopyran, triazine, triazole, trithiane, and the
like. Bicyclic ring
systems are exemplified by any of the above monocyclic ring systems fused to
an aryl
group as defined herein, a cycloalkyl group as defined herein, or another
monocyclic ring
system. Representative examples of bicyclic ring systems include but are not
limited to,
for example, benzimidazole, benzthiazole, benzothiophene, benzoxazole,
benzofuran,
benzopyran; benzothiopyran, benzodioxine, 1,3-benzodioxole, cinnoline,
indazole, indole,
indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene,
isoindole,
isoindoline, isoquinoline, phthalazine, pyranopyridine, quinoline,
quinolizine, quinoxaline,
quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine,
and the like.
Tricyclic rings systems are exemplified by any of the above bicyclic ring
systems fused to
an aryl group as defined herein, a cycloalkyl group as defined herein, or a
monocyclic ring
system. Representative examples of tricyclic ring systems include, but are not
limited to,
acridine, carbazole, carboline, dibenzofuran, dibenzothiophene, naphthofuran,
CA 02368463 2001-09-24
WO 00/58318 PCT/LTS00/07639
1~
naphthothiophene, oxanthrene, phenazine, phenoxathiin, phenoxazine,
phenothiazine,
thianthrene, thioxanthene, xanthene, and the like.
The heterocycles of this invention can be substituted with l, 2, or 3
substituents
independently selected from alkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl,
alkylcarbonyloxy, alkylthio, amido, amino, carboxy, cyano, formyl, halogen,
haloalkyl,
hydroxy, nitro, and oxo.
The term "heterocyclealkyl," as used herein, refers to a heterocycle, as
defined
herein, appended to the parent molecular moiety through an alkyl group, as
defined herein.
Representative examples of heterocyclealkyl include, but are not limited to,
pyrid-3-
ylmethyl, 2-pyrimidin-2-ylpropyl, and the like.
The term "hydroxy," as used herein, refers to an -OH group.
The term "hydroxy protecting group" or "O-protecting group" refers to a
substituent which protects hydroxyl groups against undesirable reactions
during synthetic
or semisynthetic procedures. Examples of hydroxy-protecting groups include,
but are not
limited to, substituted methyl ethers, for example, methoxymethyl,
benz~eloxymethyl, 2-
methoxyethoxymethyl, 2-(trimethylsiyll)-ethoxymethyl, benzyl, and
triphenylmethyl;
tetrahydropyranyl ethers; substituted ethyl ethers, for example, 2,2,2-
trichloroethyl and t-
butyl; silyl ethers, for example, trimethylsilyl, t-butyldimethylsily°l
and t-
butyldiphenylsilyl; cyclic acetals and ketals, for example, methylene acetal,
acetonide and
benzylidene acetal; cyclic ortho esters, for example, methoxymethylene; cyclic
carbonates;
and cyclic boronates. Commonly used hydroxy-protecting groups are disclosed in
T.H.
Greene and P.G.M: Wuts, Protective Groins in Or anic Synthesis, 2nd edition,
John
Wiley & Sons, New York ( 1991 ), which is hereby incorporated by reference.
The term "mammal," as used herein, has its ordinary meaning and includes human
beings.
The term "methyl," as used herein, refers to a -CHI group.
The term "-NRg'Rg3," as used herein, refers to two groups, R~' and RB;,
independently selected from the group consisting of hydrogen, alkylcarbonyl,
and formyl,
appended to the molecular moiety through a nitrogen atom. At least one of Rg'
and R8~
must be other than hydrogen. Representative examples of -NRg'Rg' include, but
are not
limited to, methylcarbonylamino, formylamino, ethylcarbonyl(formyl)amino, and
the like.
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
16
The term "NR8'Rg;alkylene," as used herein, refers to a -NRx'R8~ group, as
defined
herein, appended to the parent molecular moiety through an alkylene group, as
defined
herein. Representative examples of NR8'Rg;alkylene include, but are not
limited to,
2-(methylcarbonylamino)ethyl, 2-(formylamino)ethyl, 3-
(ethylcarbonylamino)propyl
2-[ethylcarbonyl(formyl)amino]ethyl, and the like.
The term "nitro," as used herein, refers to a -NO, group.
The term "oxo," as used herein, refers to (=O).
The term "oxy," as used herein, refers to (-O-).
The term "propyl," _as used herein, refers to a -CH,CH,CH; group.
The term "thio," as used herein, refers to (-S-)
The term "thioxo," as used herein, refers to (=S).
The term "pharmaceutically acceptable salts, esters, amides and prodrugs" as
used
herein refers to those carboxylate salts, amino acid addition salts, esters,
amides and
prodrugs of the compounds of the present invention which are, within the scope
of sound
medical judgement, suitable for use in contact with with the tissues of humans
and lower
animals with undue toxicity, irritation, allergic response, and the like,
commensurate with
a reasonable benefit/risk ratio, and effective for their intended use, as well
as the
zwitterionic forms, where possible, of the compounds of the invention. The
term "salts"
refers to the relatively non-toxic, inorganic and organic acid addition salts
of compounds
of the present invention. These salts can be prepared in sitar during the
final isolation and
purification of the compounds or by separately reacting the purified compound
in its free
base form with a suitable organic or inorganic acid and isolating the salt
thus formed.
Representative salts include the hydrobromide, hydrochloride, sulfate,
bisulfate,
phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate,
laurate, borate,
benzoate, lactate, phosphate, tosylate, citrate, maleate. fumarate, succinate,
tartrate,
naphthylate, mesylate, glucoheptonate, lactiobionate and laurylsulphonate
salts and the
like. These may include canons based on the alkali and alkaline earth metals,
such as
sodium, lithium, potassium, calcium, magnesium and the like, as well as
nontoxic
ammonium, quaternary ammonium and amine canons including, but not limited to,
ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,
trimethylamine, triethylamine, ethylamine and the like. (See, for example S.
M. Berge, et
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
17
al., "Pharmaceutical Salts," J. Pharm. Sci., 66: 1-19 (1977) which is
incorporated herein by
reference.)
Examples of pharmaceutically acceptable, non-toxic esters of the compounds of
this invention include C, to C6 alkyl esters wherein the alkyl group is a
straight or
branched chain. Acceptable esters also include C; to C, cycloalkyl esters as
well as
arylalkyl esters such as, but not limited to benzyl. Preferred alkyl esters
are C, to C4.
Esters of the compounds of the present invention may be prepared according to
conventional methods.
Examples of pharmaceutically acceptable, non-toxic amides of the compounds of
this invention include amides derived from ammonia, primary C, to C~ alkyl
amines and
secondary C, to C6 dialkyl amines wherein the alkyl groups are straight or
branched chain.
In the case of secondary amines the amine may also be in the form of a S or 6
membered
heterocycle containing one nitrogen atom. Amides derived from ammonia, C, to
C~ alkyl
primary amides and C, to C, dialkyl secondary amides are preferred. Amides of
the
compounds of the invention may be prepared according to conventional methods.
The term "prodrug" refers to compounds that are rapidly transformed in vivo to
yield the parent compound of the above formula, for example by hydrolysis in
blood. A
thorough discussion is provided in T. Higuchi and V. Stella, "Pro-drugs as
Novel Delivery
Systems", Vol 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers
in Drug
Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon
Press,
1987, both of which are incorporated herein by reference.
Where appropriate, prodrugs of derivatives of compounds of the present
invention
may be prepared by any suitable method. For those compounds in which the
prodrug
moiety is an amino acid or peptide functionality, the condensation of the
amino group with
amino acids and peptides may be effected in accordance with conventional
condensation
methods such as the azide method, the mixed acid anhydride method, the DCC
(dicyclohexylcarbodiimede) method, the active ester method (p-nitrophenyl
ester method,
N-hydroxysuccinic acid imide ester method, cyanomethyl ester method and the
like), the
Woodward reagent K method, the DCC-HOBT (1-hydroxy-benzotriazole) method and
the
like. Classical methods for amino acid condensation reactions are described in
"Peptide
Synthesis" Second Edition, M. Bodansky, Y.S. Klausner and M.A. Ondetti (1976).
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
18
Determination of Biological Activity
In Vitro Assay of Biological Activity
The immunosuppressant activity of the compounds of the present invention was
determined using the human mixed lymphocyte reaction (MLR) assay described by
Kino,
T. et al., in Transplantation Proceedings, XIX(5):36-39, Suppl. 6 (1987),
hereby
incorporated by reference. The results of the assay, shown below in Table 1,
demonstrate
that the compounds tested are effective immunomodulators.
Table 1
Example Number ICso (nM)
4.39
- 6 0.86
7 0.63
g 106
9 1.66
~ 20.2
11 43.6
12 0.36
13 14.6
14 1.94
1.98
16 27.1
17 1.49
18 53.1
19 0.87
0.44
21 1.08
22 0.75
23 72.4
24 15.6
1.3
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
19
In Vivo Assay of Biological Activity
Whole blood concetration in the Rat.
Lewis rats (obtained from Charles River, Wilmington, MA), weighing 225-250
grams, were acclimated for one week in a AAALAC approved facility. All animals
were
fasted overnight prior to dosing and throughout the study period but were
permitted free
access to water. Groups of animals were given FK-506 or Example 22 at a dose
of 5
mg/kg: Drugs were given in a 2 ml/kg volume of 10% ethanol, 40% propylene
glycol and
2% cremophore in 5% dextrose solution for both oral (p.o.) and intraperitoneal
(i.p.)
administration, and in a 1 ml/kg volume of the same vehicle without cremophor
for
intravenous (i.v.) dosing. Blood samples were collected from the tail vein in
heparinized
tubes at selected time points 0.25, 2.0, and 4.0 hours after dosing.
Drugs were separated from the hemolyzed whole blood contaminants utilizing
liquid-liquid extraction with ethyl acetate:hexane (1:1 by volume). Samples
were
centrifuged at 1200 x g for 10 minutes (4 °C) and a constant volume of
the organic layer
was transferred to a conical centrifuge tube and evaporated to dryness with a
gentle stream
of dry air over low heat (~35 °C). The samples were reconstituted with
40% (v/v)
acetonitrile in water with vortexing. The compounds of interest were separated
from the
co-extracted components by a 5 cm X 4.6 mm, 3 qm Spherisorb ODS~-2 column
(Regis,
Morton Grove, IL) with an acetonitrile:methanol: 0.1 % trifluoroacetic acid /
0.01 M
tetramethylammonium perchlorate mixture (45:5:50 by volume for FK506, 40:5:55
for
analogue Example 22) at a flow rate of 1.0 ml/min with UV detection at 205 nm.
The
temperature of the HPLC column was maintained at 70 °C.
The concentration of each sample in whole blood was calculated by a least
squares
linear regression analysis of the peak area compared to spiked rat blood
standards. The
data for whole blood concentration is shown in Table 2.
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
Table 2
Example number Whole Blood
Concentration
(pg/mL) at
0.25 hours
2 hours 4
hours
FK-506 1.14 0.44 0.43
Example 22 0.00 0.00 0.00
The data in Table 2 shows that Example 22 was eliminated from the blood at 15
minutes. This result suggests that phosphates of the present invention may
have reduced
5 adverse systemic effects and therefore may be an advantage when a topical
application is
considered.
The compounds of the invention, including but not limited to those specified
in the
examples, possess immunomodulatory activity in animals. As immunosuppressants,
the
compounds of the present invention may be useful for the treatment and
prevention of
10 immune-mediated diseases such as the resistance by transplantation of
organs or tissue
such as heart, kidney, liver, medulla ossium, skin, cornea, lung, pancreas,
intestinum
tenue; limb, muscle, nervus, duodenum; small-bowel, pancreatic-islet-cell,
etc.; graft-
versus-host diseases brought about by medulla ossium transplantation;
autoimmune
diseases such as rheumatoid arthritis, systemic lupus erythematosus,
Hashimoto's
15 thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes
uveitis, allergic
encephalomyelitis, glomerulonephritis, and the like; and further infectious
diseases caused
by pathogenic microorganisms. Further uses may include the treatment and
prophylaxis of
inflammatory and hyperproliferative skin diseases and cutaneous manifestations
of
immunologically-mediated illnesses, such as psoriasis, atopical dermatitis,
contact
20 dermatitis and further eczematous dermatitises, seborrhoeis dermatitis,
Lichen planus,
Pemphigus, bullous pemphigoid, Epidermolysis bullosa, urticaria, angioedemas,
vasculitides, erythemas, cutaneous eosinophilias, Lupus erythematosus, acne
and Alopecia
areata; various eye diseases (autoimmune and otherwise) such as
keratoconjunctivitis,
vernal conjunctivitis, uveitis associated with Behcet's disease, keratitis,
herpetic keratitis,
conical cornea, dystrophia epithelialis corneae, corneal leukoma, ocular
pemphigus,
Mooren's ulcer, Scleritis, Graves' opthalmopathy, Vogt-Koyanagi-Harada
syndrome,
sarcoidosis, etc.; reversible obstructive airway disease, which includes
condition such as
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
21
asthma (for example, bronchial asthma, allergic asthma, intrinsic asthma,
extrinsic asthma
and dust asthma), particularly chronic or inveterate asthma (for example, late
asthma and
airway hyper-responsiveness), bronchitis and the like; inflammation of mucosa
and blood
vessels such as gastric ulcers, vascular damage caused by ischemic diseases
and
thrombosis, ischemic bowel diseases, inflammatory bowel diseases, necrotizing
enterocolitis, intestinal lesions associated with thermal burns and
leukotriene B4-mediated
diseases; intestinal inflammations/allergies such as Coeliac diseases,
proctitis, eosinophilic
gastroenteritis, mastocytosis, Crohn's disease and ulcerative colitis; food-
related allergic
diseases which have symptomatic manifestation remote from the gastro-
intestinal tract
(e.g. migraine, rhinitis and eczema); renal diseases such as interstitial
nephritis,
Goodpasture's syndrome, hemolytic-uremic syndrome and diabetic nephropathy;
nervous
diseases such as multiple myositis, Guillain-Barre syndrome, Meniere's
disease,
polyneuritis, multiple neuritis, mononeuritis and radiculopathy; endocrine
diseases such as
hyperthyroidism and Basedow's disease; hematic diseases such as pure red cell
aplasia,
aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura,
autoimmune
hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia and
anerythroplasia; bone diseases such as osteoporosis; respiratory diseases such
as
sarcoidosis, fibroid lung and idiopathic interstitial pneumonia; skin disease
such as
dermatomyositis, leukoderma vulgaris, ichthyosis vulgaris, photoallergic
sensitivity and
cutaneous T cell lymphoma; circulatory diseases such as arteriosclerosis,
atherosclerosis,
aortitis syndrome, polyarteritis nodosa and myocardosis; collagen diseases
such as
scleroderma, Wegener's granuloma and Sjogren's syndrome; adiposis;
eosinophilic
fasciitis; periodontal disease such as lesions of gingiva, periodontium,
alveolar bone and
substantia ossea dentis; nephrotic syndrome such as glomerulonephritis; male
pattern
aleopecia or alopecia senilis by preventing epilation or providing hair
germination and/or
promoting hair generation and hair growth; muscular dystrophy; Pyoderma and
Sezary's
syndrome; Addison's disease; active oxygen-mediated diseases, as for example
organ
injury such as ischemia-reperfusion injury of organs (such as heart, liver,
kidney and
digestive tract) which occurs upon preservation, transplantation or ischemic
disease (for
example, thrombosis and cardiac infraction): intestinal diseases such as
endotoxin-shock,
pseudomembranous colitis and colitis caused by drug or radiation; renal
diseases such as
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
22
ischemic acute renal insufficiency and chronic renal insufficiency; pulmonary
diseases
such as toxinosis caused by lung-oxygen or drug (for example, paracort and
bleomycins),
lung cancer and pulmonary emphysema; ocular diseases such as cataracta,
siderosis,
retinitis, pigmentosa, senile macular degeneration, vitreal scarring and
corneal alkali burn;
dermatitis such as erythema multiforme, linear IgA ballous dermatitis and
cement
dermatitis; and others such as gingivitis, periodontitis, sepsis,
pancreatitis, diseases caused
by environmental pollution (for example, air pollution), aging, carcinogenis,
metastasis of
carcinoma and hypobaropathy; disease caused by histamine or leukotriene-C4
release;
Behcet's disease such as intestinal-, vasculo- or neuro-Behcet's disease, and
also Behcet's
which affects the oral cavity, skin, eye, vulva, articulation, epididymis,
lung, kidney and so
on. Furthermore, the compounds of the invention are useful for the treatment
and
prevention of hepatic disease such as immunogenic diseases (for example,
chronic
autoimmune liver diseases such as the group consisting of autoimmune
hepatitis, primary
biliary cirrhosis and sclerosing cholangitis), partial liver resection, acute
liver necrosis
I ~ (e.g. necrosis caused by toxin, viral hepatitis, shock or anoxia), B-virus
hepatitis, non-
Anon-B hepatitis, cirrhosis (such as alcoholic cirrhosis) and hepatic failure
such as
fulminant hepatic failure, late-onset hepatic failure and "acute-on-chronic"
liver failure
(acute liver failure on chronic liver diseases), and moreover are useful for
various diseases
because of their useful activity such as augmention of chemotherapeutic
effect, preventing
or treating activity of cytomegalovirus infection, particularly HCMV
infection, anti-
inflammatory activity, and so on.
Additionally, some compounds appear to possess FK-506 antagonistic properties.
The compounds of the present invention may thus be used in the treatment of
immunodepression or a disorder involving immunodepression. Examples of
disorders
involving immunodepression include AIDS, cancer, senile dementia, trauma
(including
wound healing, surgery and shock) chronic bacterial infection, and certain
central nervous
system disorders. The immunodepression to be treated may be caused by an
overdose of
an immunosuppressive macrocyclic compound, for example derivatives of 12-(2-
cyclohexyl-1-methylvinyl)-13, 19,21,27-tetramethyl-11,28-dioxa-4-
azatricyclo[22.3.1.0
4~9] octacos-18-ene such as FK-506, or rapamycin. Overdosing of such medicants
by
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
23
patients is quite common upon their realizing that they have forgotten to take
their
medication at the prescribed time and can lead to serious side effects.
A further situation in which the compounds of the present invention may be
used to
treat immunosuppression is in vaccination. It is sometimes found that the
antigen
introduced into the body for the acquisition of immunity from disease acts as
an
immunosuppressive agent, and so antibodies are not produced by the body and
immunity
is not acquired. By introducing a compound of the invention into the body (as
in a
vaccine), the undesired immunosuppression may be overcome and immunity
acquired.
Aqueous liquid compositions of the present invention may be particularly
useful
for the treatment and prevention of various diseases of the eye such as
autoimmune
diseases (including, for example, conical cornea, keratitis, dysophia
epithelialis corneae,
leukoma, Mooren's ulcer, sclevitis and Graves' ophthalmopathy) and rejectian
of corneal
transplantation.
When used in the above or other treatments, a therapeutically effective amount
of
I 5 one of the compounds of the present invention may be employed in pure form
or, where
such forms exist, in pharmaceutically acceptable salt, ester or prodrug form.
Alternatively,
the compound may be administered as pharmaceutical compositions containing the
compound of interest in combination with one or more pharmaceuticwlly
acceptable
excipients. By a "therapeutically effective amount" of the compound of the
invention is
meant a sufficient amount of the compound to treat gastrointestinal disorders,
at a
reasonable benefit/risk ratio applicable to any medical treatment. It will be
understood,
however, that the total daily usage of the compounds and compositions of the
present
invention will be decided by the attending physician within the scope of sound
medical
judgement. The specific therapeutically effective dose level for any
particular patient will
depend upon a variety of factors including the disorder being treated and the
severity of
the disorder; activity of the specific compound employed; the specific
composition
employed; the age, body weight, general health, sex and diet of the patient;
the time of
administration, route of administration, and rate of excretion of the specific
compound
employed; the duration of the treatment; drugs used in combination or
coincidental with
the specific compound employed; and like factors well known in the medical
arts. For
example, it is well within the skill of the art to start doses of the compound
at levels lower
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
24
than required for to achieve the desired therapeutic effect and to gradually
increase the
dosage until the desired effect is achieved.
The total daily dose of the compounds of this invention administered to a
human or
lower animal may range from about 0.001 to about 3 mg/kg/day. For purposes of
oral
administration, more preferable doses may be in the range of from about 0.005
to about
1.5 mg/kg/day. If desired, the effective daily dose may be divided into
multiple doses for
purposes of administration; consequently, single dose compositions may contain
such
amounts or submultiples thereof to make up the daily dose.
The pharmaceutical compositions of the present invention comprise a compound
of
the invention and a pharmaceutically acceptable carrier or excipient, which
may be
administered orally, rectally, parenterally, intracisternally, intravaginally,
intraperitoneally,
topically (as by powders, ointments, drops or transdermal patch), bucally, or
as an oral or
nasal spray. By "pharmaceutically acceptable carrier" is meant a non-toxic
solid, semi
solid or liquid filler, diluent, encapsulating material or formulation
auxiliary of any type.
The term "parenteral'" as used herein refers to modes of administration which
include
intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and
intraarticular
injection and infusion.
Pharmaceutical compositions of this invention for parenteral injection
comprise
pharmaceutically acceptable sterile aqueous or nonaqueous solutions,
dispersions,
suspensions or emulsions as well as sterile powders for reconstitution into
sterile injectable
solutions or dispersions just prior to use. Examples of suitable aqueous and
nonaqueous
carriers, diluents, solvents or vehicles include water, ethanol, polyols (such
as glycerol,
propylene glycol, polyethylene glycol; and the like). carboxymethylcellulose
and suitable
mixtures thereof, vegetable oils (such as olive oil), and injectable organic
esters such as
ethyl oleate. Proper fluidity can be maintained, for example, by the use of
coating
materials such as lecithin, by the maintenance of the required particle size
in the case of
dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservative, wetting
agents, emulsifying agents, and dispersing agents. Prevention of the action of
microorganisms may be ensured by the inclusion of various antibacterial and
antifungal
agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like.
It may also
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
be desirable to include isotonic agents such as sugars, sodium chloride, and
the like,
Prolonged absorption of the injectable pharmaceutical form may be brought
about by the
inclusion of agents which delay absorption such as aluminum monostearate and
gelatin.
In some cases, in order to prolong the effect of the drug, it is desirable to
slow the
absorption of the drug from subcutaneous or intramuscular injection. This may
be
accomplished by the use of a liquid suspension of crystalline or amorphous
material with
poor water solubility. The rate of absorption of the drug then depends upon
its rate of
dissolution which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally administered drug form is
10 accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of the drug
in
biodegradable polymers such as polylactide-polyglycolide. Depending upon the
ratio of
drug to polymer and the nature of the particular polymer employed, the rate of
drug release
can be controlled. Examples of other biodegradable polymers include
poly(orthoesters)
15 and poly(anhydrides) Depot injectable formulations are also prepared by
entrapping the
drug in liposomes or microemulsions which are compatible with body tissues.
The injectable formulations can be sterilized, for example, by filtration
through a
bacterial-retaining filter, or by incorporating sterilizing agents in the form
of sterile solid
compositions which can be dissolved or dispersed in sterile water or other
sterile injectable
20 medium just prior to use.
Solid dosage forms for oral administration include capsules, tablets, pills,
powders,
and granules. In such solid dosage forms, the active compound is mixed with at
least one
inert, pharmaceutically acceptable excipient or carrier such as sodium citrate
or dicalcium
phosphate and/or a) fillers or extenders such as starches, lactose, sucrose,
glucose,
25 mannitol; and silicic acid, b) binders such as, for example,
carboxymethylcellulose,
alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants
such as
glycerol, d) disintegrating agents such as agar-agar, calcium carbonate,
potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate, e) solution
retarding agents
such as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g)
wetting agents such as, for example, cetyl alcohol and glycerol monostearate,
h)
absorbents such as kaolin and bentonite clay, and i) lubricants such as talc,
calcium
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
26
stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and
mixtures thereof. In the case of capsules, tablets and pills, the dosage form
may also
comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft
and
hard-filled gelatin capsules using such excipients as lactose or milk sugar as
well as high
molecular weight polyethylene glycols and the like.
The solid dosage forms of tablets, dragees, capsules, pills, and granules can
be
prepared with coatings and shells such as enteric coatings and other coatings
well known
in the pharmaceutical formulating art. They may optionally contain opacifying
agents and
can also be of a composition that they release the active ingredients) only,
or
preferentially, in a certain part of the intestinal tract, optionally, in a
delayed manner.
Examples of embedding compositions which can be used include polymeric
substances
and waxes.
The active compounds can also be in micro-encapsulated form, if appropriate,
with
one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include pharmaceutically
acceptable
emulsions, solutions, suspensions, syrups and elixirs. In addition to the
active compounds,
the liquid dosage forms may contain inert diluents commonly used in the art
such as, for
example, water or other solvents, solubilizing agents and emulsifiers such as
ethyl alcohol,
isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl
benzoate,
propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in
particular, cottonseed,
groundnut, corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such
as
wetting agents, emulsifying and suspending agents, sweetening, flavoring, and
perfuming
agents.
Suspensions, in addition to the active compounds, may contain suspending
agents
as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and
sorbitan
esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-
agar, and
tragacanth, and mixtures thereof.
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
27
Topical administration includes administration to the skin or mucosa,
including
surfaces of the lung and eye. Compositions for topical administration,
including those for
inhalation, may be prepared as a dry powder which may be pressurized or non-
pressurized.
In non-pressurized powder compositions, the active ingredient in finely
divided form may
be used in admixture with a larger-sized pharmaceutically acceptable inert
carrier
comprising particles having a size, for example, of up to 100 micrometers in
diameter.
Suitable inert carriers include sugars such as lactose. Desirably, at least
95% by weight of
the particles of the active ingredient have an effective particle size in the
range of 0.01 to
micrometers.
10 Alternatively, the composition may be pressurized and contain a compressed
gas,
such as nitrogen or a liquified gas propellant. The liquified propellant
medium and indeed
the total composition is preferably such that the active ingredient does not
dissolve therein
to any substantial extent. The pressurized composition may also contain a
surface active
agent. The surface active agent may be a liquid or solid non-ionic surface
active agent or
may be a solid anionic surface active agent. It is preferred to use the solid
anionic surface
active agent in the form of a sodium salt.
A further form of topical administration is to the eye, as for the treatment
of
immune-mediated conditions of the eye such as automimmue diseases, allergic or
inflammatory conditions, and corneal transplants. The compound of the
invention is
delivered in a pharmaceutically acceptable ophthalmic vehicle, such that the
compound is
maintained in contact with the ocular surface for a sufficient time period to
allow the
compound to penetrate the corneal and internal regions of the eye, as for
example the
anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous
humor,
cornea, iris/cilary, lens, choroid/retina and sclera. The pharmaceutically
acceptable
ophthalmic vehicle may, for example, be an ointment, vegetable oil or an
encapsulating
material.
Compositions for rectal or vaginal administration are preferably suppositories
which can be prepared by mixing the compounds of this invention with suitable
non-
irritating excipients or carriers such as cocoa butter, polyethylene glycol or
a suppository
wax which are solid at room temperature but liquid at body temperature and
therefore melt
in the rectum or vaginal cavity and release the active compound.
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
28
Compounds of the present invention can also be administered in the form of
liposomes. As is known in the art, liposomes are generally derived from
phospholipids or
other lipid substances. Liposomes are formed by mono- or multi-lamellar
hydrated liquid
crystals that are dispersed in an aqueous medium. Any non-toxic,
physiologically
acceptable and metabolizable lipid capable of forming liposomes can be used.
The present
compositions in liposome form can contain, in addition to a compound of the
present
invention, stabilizers, preservatives, excipients, and the like. The preferred
lipids are the
phospholipids and the phosphatidyl cholines (lecithins), both natural and
synthetic.
Methods to form Iiposomes are known in the art. See. for example, Prescott,
Ed., Methods
in Cell Biolo~y, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et
seq.
Abbreviations
The following abbreviations are used: HCl for hydrochloric acid, THF for
tetrahydrofuran, TFA for trifluoroacetic acid, MgSOa for magnesium sulfate,
Na~SO~ for
sodium sulfate, NaCI for sodium chloride, NaHCO; for sodium bicarbonate, NH4C1
for
ammonium chloride, HPLC for high pressure liquid chromatography, PPTS for
pyridinium p-toluensulfonate.
Synthetic Methods
The compounds of the present invention may be prepared using one or more of
the
processes which follow. The starting materials for use in these processes are
preferably
one of the macrolides isolated from culture media obtained in accordance with
known
methods by fermentation of microorganisms of the genus Streptomyces, which are
disclosed in European Patent Application No. 0184162. Samples are available
from the
Fermentation Research Institute, Tsukuba, Ibaraki 30~, Japan under the
provisions of the
Budapest Treaty, under deposit No. FERM BP-927. This strain has been
redeposited on
April 27, 1989 with the Agricultural Research Culture Collection International
Depository,
Peoria, Illinois 61604, USA under the provisions of the Budapest Treaty, under
deposit
No. NRRL 18488. The macrolide FR-900520 (European Patent Application 0184162),
also known as ascomycin, may be prepared in accordance to the published
methods of (i)
H. Hatanaka, M. Iwami, T. Kino, T. Goto and M. Okuhara, FR-900520 and FR-
900523,
Novel immunosuppressants isolated from A streptomyces. I. Taxonomy of the
producing
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
29
strain. J. Antibiot., 1988. XLI(11), 1586-1591; (ii) H. Hafanaka, T. Kino, S.
Miyata, N.
Inamura, A. Kuroda, T. Goto, H. Tanaka and M. Okuhara, FR-90020 and FR-90023,
Novel immunosuppressants isolated from A streptomyces. 11. Fermentation,
isolation and
physico-chemical and biological characteristics. J. Antibiot., 1988. XLI(11),
1592-1601;
(iii) T. Arai, Y. Koyama, T. Suenaga and H. Honda. Ascomycin, An Antifungal
Antibiotic.
J. Antibiot., 1962. 15(231-2); and (iv) T. Arai in U.S. Patent No. 3,244,592.
One or more
of the processes discussed below may be then employed to produce the desired
compound
of the invention.
The compounds and processes of the present invention will be better understood
in
connection with the following synthetic Schemes 1-4, which illustrate the
methods by
which the compounds of the invention can be prepared.
The compounds of this invention can be prepared by a variety of synthetic
routes.
Representative procedures are shown in Schemes 1-4. For Schemes 1-4, a broken
line
represents an optional double bond, R', R', R3, and R~ are as defined in
formula I.
Scheme 1
RO
~P-Oi~,.4, \
HOi~,.
4, ." 6~ X
2%
n yv...
12 ' 14 O 12 14 O
7 O R4 17. 7 O R4 17
'~~,Rt
Rs o I RO(X)PCI N R3 0
(ii) o R2
O HO RZ ~., ~ HO
1 O; BaS2 1 O%
.24 .~~~OCH3 ,24 .~~~OCH3
'OCH3
3
(i) (Iii)
As shown in Scheme 1, the hydroxy group attached to carbon 4' of formula (i)
can
be phosphorylated with a phosphoramidochloridoite (ii), wherein X is a
secondary amine
and R is selected from alkyl, cyanoalkyl, haloalkyl, and phenyl, to give
phosphoramidoites
of formula (iii). Suitable phosphorylating reagents include, but are not
limited to, N,N-
diisopropylmethyl phosphonamidic chloride (Bruzik, K.S., Salamonczyk, G. and
Stec,
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
W.J. J. Org. Chem., (1986), 51, 2368-2370), N,N-diethylmethyl phosphonamidic
chloride,
N,N-dimethylmethyl phosphonamidic chloride, N,N-diisopropylphenyl
phosphonamidic
chloride (Hecker, S.J. et. al., J. Org. Chem., (1990), 55, 4904-4911), methyl
dichlorophosphite or phenyl dichlorophosphite (Martin, S.F. and Josey, J.A.,
Tetrahedron
5 Lett., (1988), 29, 3631-3634), 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite, 2-
cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite-diisopropylammonium
tetrazolide (Liu J. and Verdine, G. L. Tetrahedron Lett., (1992), 33, 4265-
4268),
morpholinomethyl phosphonamidic chloride (Seela, F., Kaiser, K. and Binding,
U. Helv.
Chim. Acta (1989), 72, 868-881), N,N-diisopropyl-bis-(2-
10 (trimethylsilyl)ethyl)phosphoramidite (Chao, H-G, Bernatowicz, M.S.,
Klimas, C.E. and
Matsueda; G.R. Tetrahedron Lett., (1993), 34, 3377-3380), and the reagents
described in
the following references (Reese; C.B. Tetrahedron, (1978), 34, 3143-3179),
such as 2,2,2-
trichloroethyl phosphodichloridate, 2-cyanoethyl phosphodichloridate, and the
like.
Further, the reaction is preferrably conducted in the presence of an organic
base such as
15 trialkylamines (e.g. triethylamine, etc.), pyridine compounds (e.g.
pyridine, lutidine,
picoline, 4-N,N-dimethylaminopyridine, etc.), quinoline, and the like,
preferred bases are
triethylamine, diisopropylethylamine, and pyridine.
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
31
Scheme 2
RO
I I
P-Oi~,. RO P-Oi~,.
4' 6' X 4' ' 6'
X .,
12 ' 14 , O ~.~__ I 12 '. 14 , O
7 ~ 17 7 p ~4' 17.,~ 1
.~~iR1 I ~ R
N 'O N 3 O
Rs R \
0 o R2 ,, oxidation o Ho~R2 ,,,,
o
o ~~ ~
1
/.24 ~~~~OCH3 ,24 .~~~OCH3
OCH3 OCH3
~III~ ~IV)
As shown in Scheme 2, phosphoramidoates or phosphoramidothioatesof formula
(iv), wherein Z is O or S respectively, can be prepared by oxidation of
phosphoramidoites
of formula (iii). The procedure described by (Hecker, S.J., Minich, M.L. and
Lackey, K.,
J. Org. Chem., (1990), 55, 4904-4911) can be used to give phosphoramidoates
and
elemental sulphur can give the phosphoramidothioates.
Scheme 3
RO RO
P-Oi~,. P-Oir,.
4
X 4~ R~O
12 r.~ 14 , O I 12 '. 14 , O
7 O ~ 17. 7 O 17.,, 1
'~iR1 I ~ R
N
N Ra O I Rs o
o Ho R2 ,, R'OH ° o R2 .,
o ,.
1 0= 1 H-tetrazole 1
/.24 I~~~OCH3 .24 .~~~OCH3
(III
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
32
As shown in Scheme 3, phosphates of formula (v) can be prepared by treating
phosphoramidoites of formula (iii) with an alcohol or phenol in the presence
of tetrazole.
Conversion of phosphoramidoites to phosphates utilizing 1 H-tetrazole is
described in
(Beaucage, S.L. and Iyer, R.P. Tetrahedron, (1993), 49, 6123-6194; Reese, C.B.
Tetrahedron, (1978), 34, 3143-3179; Liu, J. and Verdine, G.L. Tetrahedron
Lett., (1992),
33, 4265-4268; Zhao, K. and Landry, D.W., Tetrahedron Lett., (1993), 49, 363-
368. Other
activators that can be used for the conversion of phosphoramidoites to
phosphates include,
but are not limited to, additional members of the tetrazole class of
activators: 5-(p-
nitrophenyl) tetrazole (Froehler, B. C. and Mattcucci, M. D., Tetrahedron
Letters (1983),
24, 3171-3174); 5-(p-nitrophenyl) tetrazole and DMAP (Pon, R.T., Tetrahedron
Letters
(1987), 28, 3643-3646); and 5-(ethylthio)-1H-tetrazole (Wright, P. et al.,
Tetrahedron
Letters (1993), 34, 3373-3376). In addition to the tetrazole class of
activators, the
following activators have been employed: N-methylaniline trifluoroacetate
(Fourrey, J. L.
and Varenne, J., Tetrahedron Letters (1984), 25, 4511-4514); N-methyl
anilinium
trichloroacetate (Fourrey, J. L. et al., Tetrahedron Letters (1987), 28, 1769-
1772); I-
methylimidazoletrifluoromethane sulfonate (Arnold. L. et al., Collect. Czech.
Chem.
Commun. (1989), 54, 523-532); 1-methylimidazole HCI, 5-trifluoromethyl-1H-
tetrazole,
N,N-dimethylaniline HC1, and N,N-dimethylaminopyridine HCl (Hering, G. et al.,
Nucleosides and Nucleotides (1985), 4, 169-171).
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
33
Scheme 4
RO
I I
RO P-0~~~.4, 6'
P-Oi~,.
R'O
R,O
12 '~ 14 . O 12 '~ 14 , O
7 O 17 .~,
,iiR1 ~ i R
N
N Rs O ~ Rs o
0 o Rz ,~ oxidation o Ho R2
o; 1 0:
1 ,
.24 .~~~OCH3 ,24 .~~~OCH3
(VI)
As shown in Scheme 4, phosphites of formula (v) can be oxidized to phosphates
or
thiophosphates of formula (vi), wherein Z is O or S respectively. Oxidation of
phosphites
to phosphates can be accomplished using per acids such as tert-
butylhydroperoxide
(Hecker, S.J., Minich, M.L. and Lackey, K., J. Org. Chem., ( 1990), 55, 4904-
4911 ) and
hydrogen peroxide (Martin, S.F. and Josey, J.A., Tetrahedron Lett., (1988),
29, 3631-
3634). Other oxidizing agents that can be used include iodine in aquous
tetrahydrofuran at
low temperature (Lestinger, R.L. and Lunsford, W.B. J. Am. Chem. Soc., (1976),
98,
3655). Formation of a thiophosphate from a phosphite may be carried out using
elemental
sulfur (Bruzik, K.S., Salamonczyk, G. and Stec, W.J., J. Org. Chem., (1986),
51, 2368-
2370; Stec; W.J., Grajkowski, A., Koziolkiewicz, M. and Uznanski, B., Nucleic
Acid
Res., (1991), 19, 5883-5888; Martin, S.F. and Wagman, A.S., J. Org. Chem.,
(1993), 58,
5897-5899).
In the processes described in Schemes 1-4, the hydroxy group at the C-14
position
of formula (i) may or may not be necessary to protect. When it is protected,
suitable
protecting groups include. but are not limted to, dimethylthexylsilyl,
trisubstituted silyl
such as tri(lower)alkylsilyl (e.g. trimethylsilyl, triethylsilyl,
tributylsilyl, tri-i-propylsilyl,
tent-butyl-dimethylsilyl, tri-tent-butylsilyl, triphenylmethyl-dimethylsilyl,
etc.); lower
alkyldiarylsilyl (e.g. methyl-diphenylsilyl, ethyl-diphenylsilyl, propyl-
diphenylsilyl, tert-
butyl-diphenylsilyl; etc.), and the like; triarysilyl (e.g. triphenylsilyl,
tri-p-xylylsilyl, etc.);
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
34
triarylalkylsilyl (e.g. tribenzylsilyl, etc.), and the like, in which the
preferred one may be
tri(C,-C4)alkylsilyl and C,-C~ alkyldiphenylsilyl, and the most preferred one
may be tert-
butyldimethylsilyl. Suitable o-silylations may be carried out using a wide
variety of
organosilicon reagents including, but not limitted to, tert-butyldimethylsilyl
chloride, N-
(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide ( Mawhinney, T., and
Madison, M.
A. J. Org. Chem., (1982), 47. 3336), tert-butylchlorodiphenylsilane (
Hanessian, S. and
Lavallee, P Can. J. Chem., (1975), 63, 2975), tent-butyldimethylsilyl
trifluoromethanesulfonate ( Mander, L. N. and Sethi, S. P. Tetrahedron Lett.,
(1984), 25,
5953), dimethylthexylsilyl chloride or dimethylthexylsilyl
trifluoromethanesulfonate
(Wetter, H. and Oertle, K. Tetrahedron Lett., (1985), 26, 5515), 1-(tert-
butyldimethylsilyl)-imidazole, and the like. Carbonate hydroxy-protecting
groups may
also be used and introduced using a wide variety of a corresponding
helocarbonate
compound, such as methyl chloroformate, ethyl chloroformate, 2,2,2-
trichloroethyl
chloroformate, isobutyl chloroformate, vinyl chloroformate, allyl
chloroformate, benzyl or
substituted benzyl chloroformate such as p-methoxybenzyl, 3,4-dimethylbenzyl,
p-
nitrobenzyl etc., 2-(trimethylsilyl)ethyl chloroformate, 2-
(benzenesulfonyl)ethyl
chloroformate, 2-(trimethylsilyl)ethoxymethyl chloride and the like. Under
these
conditions, various tertiary base can be used such as pyridine, triethylamine,
imidazole,
diisopropylethylamine, and the like. (Tetrahedron Lett., (1980), 21, 3343;
ibid., 1981, 22,
3667; ibid. 1981, 22, 969; ibid. 1981, 22, 1933. ).
In the processes described in Schemes 1-4, when the C-14 hydroxy group of
formula (i) is protected by one of the above silyl groups, suitable reagents
for deprotection
include, but are not limited to aqueous hydrogen fluoride in acetonitrile
(Newton, R. F.,
Reynolds, D. P., Finch, M. A. W., Kelly, D. R. and Roberts, S. M. Tetrahedron
Lett.,
(1979), 3891), tetraalkyl ammonium fluoride in tetrahydrofuran (Corey, E. J.
and Snider,
B. B. J. Am. Chem. Soc., (1972), 94, 2549, Corey, E. J. and Venkateswarlu, A.
J. Am.
Chem. Soc., (1972), 94, 6190) or tetraalkyl ammonium chloride-potassium
fluoride in
acetonitrile (Carpino, L. A. and Sau, A. C. J. Chem. Soc., Chem. Commun.
(1979), 514;
Hurst, D. T. and MaInnes, A. G. Can. J. Chem., (1965), 43, 2004), citric acid
in methanol
(Bundy, G. L. and Peterson. D. C. Tetrahedron Lett., ( 1978), 41 ), acetic
acid:water (3:1 )
(Corey, E. J. and Varma, R. K. J. Am. Chem. Soc., (1971), 93, 7319), Dowex SOW-
X8 in
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
methanol (Corey, E. J., Ponder, J. W. and Ulrich, P. Tetrahedron Lett.,
(1980), 21, 137),
boron trifluoride etherate in chloroform (Kelly, D. R., Roberts, M. S. and
Newton, R. F.
Synth. Commun. (1979), 9, 295), methanolic hydrogen fluoride (Hanessian, S.
and
Lavallee; P. Can. J. Chem., (1975), 53, 297; ibid., 1977, 55, 562), and
pyridinuim
5 fluoride in tetrahydrofuran (Nicolaou, K. C., Seitz, S. P., Pavia, M. R. and
Petasis, N. A. J.
Org. Chem., (1979), 44, 4011); pyridinium p-toluenesulfonate in ethanol
(Prakash, C.,
Saleh, S. and Blair, I. A. Tetrahedron Lett., (1989), 30, 19) ; N-
bromosuccinimide in
dimethylsulfoxide (Batten, R. J., Dixon, A. J., Taylor, R. J. K. and Newton,
R. F.
Synthesis, (1980), 234 ); or Tetraethyldiboroxane in the presence of catalytic
amounts of
10 trimethylsilyl triflate (Dahlhoff, W. V. and Taba, K. M. Synthesis, (1986),
561).
The reactions described in Schemes 1-4 and the appended Examples may also be
carried out using a starting material having an opposite configuration at a
carbon center.
In this situation, the following two additional steps are required to yield a
starting material
having an epimeric hydroxyl moiety, i.e. ( 1 ) the alcohol is oxidized to its
corresponding
15 ketone, (2) the obtained ketone is reduced under selective conditions. Both
chiral centers
having either (R) or (S) configuration can be obtained selectively and
separately.
The compounds, processes and uses of the present invention will be better
understood in connection with the following examples, which are intended as an
illustration of and not a limitation upon the scope of the invention. Both
below and
20 throughout the specification, it is intended that citations to the
literature are expressly
incorporated by reference.
Example 1
(1R 9S 12S 13S 14S 17R 21S 23S 24R 25S,27R)-14-f ~tert-
25 butyl(dimeth~)sil~loxy~-12-'[(E)-2-((1R 3R 4R)-4-; [tert-
butyl(dimethyl)silylloxyl-3-
methoxyc~clohexyl)-1-meth ley thenyll-17-ethyl-1-hydroxy-23.2-dimethoxy-
13,19.21,27
tetramethyl-11 28-dioxa-4-azatricyclof22 3 1 04v]octacos-18-ene-2,3.10,16-
tetrone
A solution of ascomycin (25 g, 0.032 mol) and imidazole (43.03 g, 0.64 mol) in
dry N,N-dimethylformamide (500 mL) was treated with tert-
butyldimethylchlorosilane
30 (47.64 g, 0.32 mol) in portions and stirred at ambient temperature for 24
hours. N,N-
Dimethylformamide and excess tert-butyldimethylchlorosilane were removed by
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
36
evaporation (35 °C water bath ) under high vaccum. The solid residue
was dissolved in
350 mL of ethyl acetate, and the ethyl acetate layer was washed in succession
with
saturated NH4C1 solution (200 mL X3), 10% NaHSO~ solution(200 mL X3), brine.
saturated NaHCO~ solution (200 mL X3), brine (200 mL X3), and dried (MgS04).
The
solvent was removed in vacuo and the solid residue was purified by silica gel
chromatography, followed by HPLC eluting with 5% acetone in hexane to provide
the title
compound (27 g) in 84 % yield.
MS (FAB) m/z: M+K~=1058.
Example 2
~1R 9S 12S 13S 14S 17R 21 S 23S 24R 25S 27R)-14-; ~tert-
butyl(dimethyl)silylloxy)-17
ethyl 1-~droxy-12-~(E)-2-f(1R 3R 4R)-4-hydroxy-3-methoxycyclohexyll-1
methylethenyl~-23 25-dimethoxy-13 19 21 27-tetramethyl-11,28-dioxa-4-
azatricyclo[22 3 1 04°~loctacos-18-ene-2,3,10,16-tetrone
A solution of 48% hydrogen fluoride aqueous solution (5 nuL) was treated with
the
product from Example 1 (32 g, 0.031 mol) in acetonitrile (500 mL) followed by
stirring at
ambient temperature for 90 minutes. The reaction mixture was cooled to 0
°C in an ice
bath and treated with solid NaHCO; with stirring for 1 hour. The solids were
removed by
filtration and the acetonitrile was removed in vacuo. The residue was taken up
with ethyl
acetate (500 mL) and washed in succession with 10% NaHCO, solution (300 mL
X3),
brine (250 mL), 10% NaHS04 solution (300 mL X3), brine ( 350 mL X3), and dried
(NaS04). Evaporation of the solvent gave 35 g of crude title compound which
was
purified by silica gel column chromatography, followed by HPLC eluting with
25%
acetone in hexane to provide 24.28 g (85%) of the title compound.
MS (FAB) m/z: M+K+=844.
Example 3
(1R 2R 4R) 4 f(E) 2-((1R 9S 12S 13S 14S 17R ~ 1 S 23S 24R 25S 27R)-14-; ftert
butyl(dimeth ly_)silylloxyl-17-ethyl-1-hy_droxy-?3 ~5-dimethoxy-13 19,21,27-
tetramethyl
2 3 10 16 tetraoxo 11 28-dioxa-4-azatricyclo[22 3 1 0~~91octacos-18-en-12-yl)-
1-propenyll-
2 methoxycyclohexyl methyl diis~ropylphosphoramidoite
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
37
A solution of the product from Example 2 (1.81 g, 2 mmol) in methylene
chloride
(20 mL) at ambient temperature was treated in succession with
diisopropylethylamine
(0.42 mL, 2.4 mmol) and methyl diisopropylphosphoramidochloridoite (0.39 mL, 2
mmol). After stirring for 5 minutes at ambient, the solvent was removed and
the residue
dried under high vaccum for 2 hours. The dried residue was used without
further
purification.
Example 4
,(,1R 2R,4R)-4-[(E)-2-((1R,9S,12S,13S,14S.17R 21S.23S,24R.25S,27R)-14-~jtert-
butyl(dimeth~)silylloxy.)-17-ether-1-hydroxy-~3 25-dimethoxy-13 19 21,27-
tetramethyl-
2 3 10 16-tetraoxo-11 28-dioxa-4-azatricycloL22.3.1.04vloctacos-18-en-12-yl)-1-
propenyll-
2-methoxycyclohexyl 2-c~anoethyl diisopropylphosphoramidoite
A solution of the product from Example 2 ( 1.81 g, 2 mmol) in methylene
chloride
(20 mL) at ambient temperature was treated in succession with
diisopropylethylamine
(0.42 mL, 2.4 mmol), and 2-cyanoethyl diisopropylphosphoramidochloridoite (
0.446 mL,
2 mmol). After stirring for 5 minutes, the solvent was removed and the residue
dried under
high vaccum for 2 hours. The dried residue was used without further
purification.
Example 5
(1R 2R 4R)-4-1(E)-2-(~1R 9S 12S 13R 14S 17R 21S 23S.24R.25S,27R)-17-ethyl-1,14-
dih~droxy-23 ~5-dimethoxy-13 19 21 27-tetramethyl-2 3 10,16-tetraoxo-11,28-
dioxa-4-
azatricyclof~~ 3 1 04'9]octacos-18-en-12-yll-1-propenyll-2-methoxycyclohexyl
methyl 4-
~ r~ idinylmeth~l phosphate
A solution of the product from Example 3 (2.135 g, 2 mrnol) in
tetrahydrofuran:acetonitrile (1:1) (40 mL) under a nitrogen atmosphere was
treated with 4-
pyridinylmethanol (323 mg, 3 mmol) and 1H-tetrazole (70 mg, 1 mmol) with
stirring for
minutes. During this period, the starting material was consumed according to
TLC
(solvent used, 40 % acetone in hexane). Tert-butylhydroperoxide (0.09 mL, 3
mmol, in
hexane solution) was directly added to the reaction mixture and allowed to
stir over night.
30 The solvents were removed and the residue was dissolved in ethyl acetate
(50 mL),
quickly washed with 10% NaHCO; solution, brine, and dried (MgSO~). After
evaporated
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
38
to dryness, the crude residue (2.21 g) was purified by silica gel column
chromatography,
eluting with 40% acetone in hexane, followed by 5% methanol in methylene
chloride to
give (812 mg) of product, (MS (FAB) m/z: M+Kl=1129). A solution of the above
purified
product in acetonitrile (40 mL) was treated with 48% hydrofluoric acid
solution (4 mL)
with stirring for 5 hours. The reaction mixture was quenched with saturated
NaHS04
solution (20 mL) and extracted with ethyl acetate (100 mL). The ethyl acetate
layer was
washed in succession with saturated NaHSO~ solution (40 mL X3), brine (40 mL
X3), and
dried. The residue was purified by reverse phase (C-18) high performance
liquid
chromatography to provide the title compound (223 mg).
MS (FAB) m/z: M+K-=1 O1 ~. Mk=976;
'3C NMR (500 MHz in Pyridine-d;) 8 211.6 (q), 210.8 (q, minor), 199.7 (q,
minor), 198.9
(q), 170.2 (q, minor), 170.1 (q), 167.4 (q, minor), 167.1 (q), 162.4 and 162.1
(q,
CF3C00), 150.5 (t, pyridyl ring, major and minor), 146.1 (q, pyridyl ring),
139.4 (q,
minor), 138.5 (q), 133.5 (q, minor), 133.2 (q), 132.1 (t), 131.1 (t, minor),
124.7 (t, minor),
124.5 (t), 122.0 (t, pyridyl ring), 121.9 (t, py-ridyl, minor), 99.8 (q,
minor). 98.9 (q), 81.8
(t), 81.7 (t, minor), 81.3 (t), 81.1 (t, minor), 80.7 (t), 79.6 (t, minor),
77.9 (t, minor), 76.3
(t), 74.8 (t, minor), 74.4 (t), 74.0 (t, minor), 73.1 (t), 69.8 (t), 68.9 (t,
minor), 67.3 (s,
pyridyl methyl, minor), 67.1 (s, pyridyl), 57.8 (t, minor), 57.6 (t), X7.1
(t), 57.0 (t, major
and minor), 56.2 (t), 55.9 (t, minor), 55.5 (t), 55.3 (t, minor), 54.6, (p, P-
OCH3), 54.3, (p,
P-OCH3, minor), 53.1 (t), 49.4 (s), 48.0 (s, minor), 48.0 (s), 46.6 (s,
minor), 44.7 (s,
minor), 41.5 (t), 41.1 (t), 39.~ (s), 36.4 (s), 35.9 (t), 35.6 (t, minor),
35.2 (s, minor), 34.7
(t), 34.5 (s), 33.2 (s), 33.0 (s, minor), 31.9 (s), 31.8 (s, minor), 30.6 (s,
major and minor),
28.2 (s). 27.6 (s, minor), 27.6 (t, minor), 26.6 (t), 25.1 (s), 24.9 (s,
minor), 24.5 (s, minor),
21.9 (s, minor), 21.6 (s), 20.6 (p, minor), 20.0 (p), 16.9 (p), 16.9 (p,
minor), 16.3 (p,
minor), 16.0 (p), 13.7 (p, minor), 13.5 (p), 12.0 (p, minor), 11.9 (p), 10.8
(p), 10.6 (p,
minor);
'H NMR (500 MHz in Pyridine-d;) 8 0.82 (t, J=7.SHz, CH3), 0.87 (t, J=7.SHz,
CH;,
minor), 0.94 (s, CH3), 0.95 (s. CH3, minor), 1.0 (m, CH2), 1.10 (m, CH?), 1.17
(m, CHI
and CH, minor), 1.2 (d, J=7.~Hz, CH;), 1.27 (d, J=7.SHz, CH;), 1.42-1.58 (m,
CH2, CH2,
CH2, CH2, and CH2), 1.62 (s. CH;), 1.79 (s, CH;, minor), 1.80 (s, CH;), 1.86
(s, CH;,
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
39
minor), 1.98 (m; CH), 2.10 (m, CHI, CH2, and CH), 2.22 (m, CH2), 2.31, 2.35
(m, CH2
and CH), 2.92 (m, CH2, minor), 2.57 (m, CH), 2.67 (ddd, JI=J2=7.5Hz, J3=lSHz,
CH),
2.77 (m, CH, minor), 2.91 (m, CH, minor), 3.02 (m, CH, minor), 3.08 (m, CH),
3.23 (m,
CH2), 3.3 (m, CH), 3.34 (s, OCH3, minor), 3.38 (s, OCH;), 3.42 (s, OCH;, major
and
minor), 3.43 (s, OCH;), 3.44 (s, OCH;, minor), 3.55 (m, CH major and minor),
3.62 (m,
CHZ, minor), 3.68 (m, CH major and minor), 3.86 (s, P-OCH;), 3.90 (s, P-OCH3,
minor),
3.89 (m, CH major and minor), 4.19 (d, J=IOHz, CH), 4.28 (dd, J1=2.5Hz,
J2=IOHz, CH,
minor), 4.45 (m, CH), 4.50 (m, CH), 4.58 (m, CH), 4.64 (s. OH), 4.72 (d, CH2),
5.10 (m,
CH major and minor), 5.23 (m, CH), 5.31 (m, pyridylCH2), 5.41 (m, CH), 5.53
(d, J=SHz,
CH), 5.81 (m, J=SHz, CH), 5.87 (d, J=SHz, CH, minor), 7.43 (m, pyridyl ring 2
and 6-
CH), 8.74 (s, pyridyl ring 3 and 5-CH);
Elemental Analysis calculated for CSOH"N,O,;P 1 CF3COOH 2 HBO: C; 55.40;
H,7.33; N,
2.48. Found C, 55.76; H, 7.24; N, 2.03.
Example 6
~1R 2R 4R) 4 ~(E) 2-f(1R 9S 12S 13R 14S 17R 21 S 23S 24R 25S,27R~-17-ethyl-
1,14-
dihydroxy23 25 dimethoxy-13 19 21 27-tetramethyl-2 3 10 16-tetraoxo-11,28-
dioxa-4-
azatric~clo~2 3 I 04vloctacos-18-en-12- l~l-1-propenyl~-2-methoxycyclohexyl
methyl 3-
p~idinylmeth~phosphate
The product from Example 3, 3-pyridinylmethanol (323 mg, 3 mmol), and the
procedure described in Example 5 were used to provide the title compound.
MS (FAB) m/z M+K+=1015;
'3C NMR (500 MHz in Pyridine-ds) 8 211.7 (q), 210.9 (q, minor), 199.7 (q,
minor), 198.9
(q), 170.2 (q, minor), 170.1 (q), 167.4 (q, minor), 167.1 (q), 162.5 and 161.9
(q,
CF3C00), 149.7 (t, pyridyl ring CH), 139.4 (q, minor), 138.5 (q), 135.9 (t,
pyridyl ring
CH), 133.5 (q), minor), 133.2 (q), 132.8 (q, pyridyl ring), 132.2 (t), 131.2
(t, minor), 124.7
(t, minor), 124.5 (t), 99.8 (q, minor), 98.9 (q), 81.8 (t), 81.7 (t, minor),
81.2 (t), 81.1 (t,
minor), 80.7 (t), 79.6 (t, minor), 77.9 (t, minor), 76.3 (t), 74.8 (t, minor),
74.4 (t), 74.0 (t,
minor), 73.1 (t), 69.8 (t), 68.9 (t, minor), 67.0(s, pyridyl methyl, minor),
66.9 (s, pyridyl),
57.8 (t, minor), 57.6 (t), 57.1 (t), 57.0 (t, major and minor), 56.2 (t), 55.9
(t, minor), 55.5
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
(t), 55.3 (t, minor), 54.6, 54.5 (p, P-OCH3), 54.4, 54.2 (p, P-OCH3, minor),
53.1 (t), 49.4
(s), 48.0 (s, minor), 48.0 (s), 46.6 (s, minor), 44.7 (s, minor), 41.5 (t),
41.1 (t), 39.5 (s),
36.4 (s), 35.9 (t), 35.6 (t, minor), 35.2 (s, minor), 34.7 (t), 34.5 (s), 33.2
(s), 33.0 (s,
minor), 31.9 (s), 30.6 (s, major and minor), 28.2 (s), 27.6 (s, minor), 27.6
(t, minor), 26.6
5 (t), 25.1 (s), 24.9 (s, minor), 24.5 (s, minor), 21.9 (minor), 21.6 (s),
20.6 (p, minor), 20.1
(p), 16.9 (p), 16.9 (p, minor), 16.3 (p, minor), 16.1 (p), 13.7 (p, minor),
13.5 (p), 12.0 (p,
minor), 11.9 (p), 10.8 (p), 10.6 (p, minor);
'H NMR (500 MHz in Pyridine-d;) b 0.82 (t, J=7.SHz, CH;), 0.87 (t, J=7.SHz,
CH3,
minor), 0.94 (s, CH3), 0.95 (s, CH;, minor), 1.0 (rn, CH?l, 1.10 (m, CH2),
1.17 (m, 25 and
10 CH; minor), 1.2 (d, J=7.SHz, CH;), 1.27 (d, J=7.SHz, CH;), 1.42-1.58 (m,
CH2, CH2,
CH2, CHI, and CH2), 1.62 (s, CH;), 1.79 (s, CH;, minor), 1.80 (s, CH3), 1.86
(s, CH3,
minor), 1.98 (m, CH), 2.10 (m, CH2, CH2, and CH), 2.22 (m, CH2), 2.31, 2.35
(m, CH2
and CH), 2.92 (m, CH2, minor), 2.57 (m, CH), 2.67 (ddd, JI=J2=7.SHz, J3=lSHz,
CH),
2.77 (m, CH, minor), 2.91 (m, CH, minor), 3.02 (m, CH, minor), 3.08 (m, CH),
3.23 (m,
15 CH2)3.3 (m, CH), 3.35 (s, OCH;, minor), 3.38 (s, OCH;), 3.42 (s, OCH3),
3.44 (s,
OCH;), 3.44 (s, OCH;), 3.46 (s, OCH3, minor), 3.55 (m, CH major and minor),
3.62 (m,
CHI, minor), 3.68 (m, CH major and minor), 3.82 (s, P-OCH;), 3.84 (s, P-OCH;,
minor),
3.89 (m, CH major and minor), 4.19 (d, J=lOHz, CH), 4.28 (dd, J1=2.SHz,
J2=IOHz, Cfl,
minor), 4.45 (m, CH), 4.50 (m, CH), 4.58 (m, CH), 4.64 (s, OH), 4.72 (d, CH2),
5.10 (m,
20 CH major and minor), 5.13 (m, CH), 5.31 (m, pyridyl CH?), 5.41 (m, CH),
5.53 (d, J=SHz,
CH), 5.81 (d, J=SHz, CH), 5.87 (d, J=SHz, CH, minor), 7.27 (m, pyridyl ring
CH), 7.85 (d,
J=7.SHz, pyridyl ring CH), 8.92 (s, pyridyl ring CH);
Elemental Analysis calculated for C;oH"N,O,;P 2 CF~COOH: C; 53.81; H, 6.60; N,
2.32.
Found C, 53.65; H, 6.48; N, 2.33.
Example 7
(1R 2R 4R)-4-~~E)-2-j(1R 9S 12S 13R 14S 17R 21S 23S,24R,25S,27R)-17-ethyl-1,14-
dihydroxy-23 --25-dimethox~-13 19 21 27-tetramethvl-2 3 10 16-tetraoxo-11,28-
dioxa-4-
azatricyclo(22 3 I 04°9Loctacos-18-en-12-yll-1-propenyl)-2-
methoxycyclohexyl methyl 2-
pyridin l~methvl phosphate
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
41
The product from Example 3, 2-pyridinylmethanol (323 mg, 3 mmol), and the
procedure described in Example 5 were used to provide the title compound.
MS (FAB) m/z: M+H~=977; M+K+=1015;
'3C NMR (500 MHz in Pyridine-d;) S 211.7 (q), 210.9 (q, minor), 199.7 (q,
minor), 198.9
(q), 170.2 (q, minor), 170.1 (q), 167.4 (q, minor), 167.1 (q), 162.5 and 162.0
(q,
CF3C00), 157.1 (q, pyridyl), 149.7 (t, pyridyl), 139.5 (q, minor), 138.5 (q),
137.2 (t,
pyridyl), 133.5 (q), minor), 133.3 (q), 132.2 (t), 131.2 (t, minor), 124.7 (t,
minor), 124.5
(t), 123.6 (t, pyridyl), 121.8 (t, pyridyl), 121.7 (t, pyridyl, minor), 99.8
(q, minor), 98.9 (q),
81.8 (t); 81.7 (t, minor), 81.3 (t), 81.2 (t, minor), 80.7 (t), 79.6 (t,
minor), 78.0 (t, minor),
76.3 (t), 74.8 (t, minor), 74.4 (t), 74.0 (t, minor), 73.2 (t), 69.7 (s,
pyridyl methyl, 69.8 (t),
68.9 (t, minor), 57.8 (t, minor), 57.6 (t), 57.1 (t), 57.0 (t, major and
minor), 56.2 (t), 55.9
(t, minor), 55.5 (t), 55.3 (t, minor), 54.6, 54.5 (p, P-OCH3), 54.4, 54.2 (p,
P-OCH3,
minor), 53.1 (t): 49.4 (s), 48.0 (s, minor), 48.0 (s), 46.6 (s, minor), 44.7
(s, minor), 41.5 (t),
41.1 (t), 39.5 (s), 36.4 (s), 35.9 (t), 35.6 (t, minor), 35.2 (s, minor), 34.7
(t), 34.5 (s), 33.2
(s), 33.0)x, minor), 31.9 (s), 30.6 (s, major and minor), 28.3 (s), 27.6 (s,
minor), 27.6 (t,
minor), 26.7 (t), 25.1 (s), 24.9 (s, minor), 24.5 (s, minor), 21.9 (minor),
21.6 (s), 20.6 (p,
minor), 20.1 (p), 17.0 (p), 16.9 (p, minor), 16.4 (p, minor), 16.1 (p), 13.8
(p, minor), 13.5
(p), 12.0 (p, minor), 11.9 (p), 10.8 (p), 10.6 (p, minor);
'H NMR (500 MHz in Pyridine-d;) 8 0.82 (t, J=7.SHz, CH;), 0.87 (t, J=7.SHz,
CH;,
minor), 0.94 (s, CH3), 0.95 (s, CH;, minor), 0.99 (m, CH2), 1.09 (m, CH2),
1.16 (m, CH3,
minor), 1.18 (m; CH3, minor), 1.20 (m, CH3), 1.26 (d, J=SHz, CH3), 1.42 (m,
CH2), 1.47
(m, CH2), 1.51 (m, CH2), 1.55 (m, CHZ), 1.57 (m, CH2, CH2, CH2, minor), 1.60
(m, CHI,
CH2 minor, CH2 minor), 1.61 (s, CH3), 1.70 (m, CHI, minor), 1.75 (m, CHI),
1.76 (m,
CH2); 1.78 (m, CH2), 1.79 (s, CH3, minor), 1.80 (s, CH;), 1.85 (s, CH;,
minor), 1.87 (m,
CH; minor), 1.92 (m, CH?, minor), 1.95 (m, CH), 1.97 (m,CH2, minor), 2.00 (m.
CH?,
minor), 2.01 (m, CH, minor), 2.02 (m, CH2), 2.09 (m, CH), 2.10 (m, CH2), 2.11
(m, CH2,
minor), 2.13 (m, CH2), 2.19-2.24 (m, CHI major and minor, CH2 major and minor,
CH
minor), 2.33-2.43 (m, CH2 major and minor, CH minor and CH2 minor), 2.56 (m,
CH),
2.66 (ddd, Jl=J2=7.SHz, J3=lSHz, CH2), 2.76 (m, CH,minor), 2.90 (ddd,
Jl=J2=7.SHz,
J3=lSHz, CH2, minor), 3.03 (m, CH2, minor), 3.07 (m, CH2), 3.23 (m, CH2), 3.30
(CH
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
42
major and minor), 3.34 (s, OCH;, minor), 3.38 (s, OCH;), 3.42 (s, OCH;), 3.42
(s, OCH;,
minor), 3.44 (s, OCH3), 3.45 (s, OCH;, minor), 3.52 (m, CH), 3.55 (m, CH,
minor), 3.59-
3.61 (m; CHI, minor), 3.66 (m, CH), 3.68 (m, CH, minor), 3.86 (s, P-OCH3,
minor), 3.88
(s, P-OCH;), 4.18 (d, J=IOHz, CH), 4.27 (dd, Jl=2.SHz, J2=lOHz, CH, minor),
4.48 (m,
CH), 4.49 (m, CH), 4.57 (m, CH, minor), 4.71 (m, CH2), 5.11 (m, CH), 5.13 (m
CH major
and CH minor), 5.39 (CH minor), 5.41 (m, CH), 5.47 (m, pyridyl CH2), 5.52 (m,
CH,
minor), 5.80 (d, J=SHz, CH), 5.85 (d, J=SHz, CH, minor), 7.19 (m, pyridyl ring
CH), 7.63
(m, pyridyl ring CH), 7.69 (m, pyridyl ring CH), 8.65 (m, pyridyl ring CH);
Elemental Analysis calculated for CSOH"N,O,;P 1.5 CF3COOH: C; 55.44; H, 6.89;
N,
2.43. Found C, 55.57; H, 7.07; N, 2.09.
Example 8
(1R 2R 4R) 4 ~~El 2 [~1R 9S 12S 13R 14S 17R 21S 23S 24R 25S 2781-17-ethyl-1,14-
dih~droxy 23 25 dimethoxy-13 19 21 27-tetramethvl-2,3 10 16-tetraoxo-11,28-
dioxa-4-
azatricycloL2 s 1 04vloctacos-18-en-12-yl]-1-propenyl~-2-methoxycyclohexyl 2-
furYlmethyl meth~phosphate
The product from Example 3, 2-furylmethanol (259 qL, 3 mmol), and the
procedure described in Example 5 were used to provide the title compound.
MS (FAB) m/z: M+K+=1004;
'3C NMR (500 MHz in Pyridine-d;) 8 211.6 (q), 210.8 (q, minor), 199.6 (q,
minor), 198.8
(q), 170.1 (q, minor), 170.0 (q), 167.4 (q, minor), 167.1 (q), 150.6 (q, furan
ring), 144.2 (t,
furan ring), 144.1 (t, furan ring, minor), 139.4 (q, minor), 138.4 (q), 135.9
(t, pyridyl ring
CH), 133.4 (q), minor), 133.1 (q), 132.2 (t), 131.2 (t, minor), 124.6 (t,
minor), 124.5 (t),
111.3 (furan ring CH, minor), 111.3 (furan ring CH), 99.7 (q, minor), 98.9
(q), 81.8 (t),
81.7 (t; minor), 81.2 (t), 81.1 (t, minor), 80.7 (t), 79.6 (t, minor), 77.9
(t, minor), 76.3 (t),
74.8 (t, minor), 74.4 (t), 74.0 (t, minor), 73.1 (t), 69.7 (t), 68.8 (t,
minor), 61.6, 61.2 (s,
furan, minor), 61.0 (s, furan), 57.7 (t, minor), 57.5 (t), 57.0 (t), 57.1 (t,
major and minor),
56.1 (t), 55.9 (t, minor), 55.6 (t), 55.3 (t, minor), 54.3, (p, P-OCH3), 54.0
(p, P-OCH3,
minor), 53.0 (t), 49.4 (s), 48.0 (s, minor), 47.9(s), 46.6 (s, minor), 44.6
(s, minor), 41.4 (t),
41.0 (t), 39.5 (s), 36.4 (s), 35.9 (t), 35.6 (t, minor), 35.2 (s, minor), 34.7
(t), 34.4 (s), 33.1
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
43
(s), 32.9 (s, minor), 31.7 (s), 30.6 (s, minor), 30.5 (s) 28.2 (s), 27.6 (t,
minor), 27.5 (s,
minor), 26.6 (t); 25.0 (s), 24.9 (s, minor), 24.4 (s, minor), 21.8 (minor),
21.5 (s), 20.6 (p,
minor), 20.0 (p), 16.8 (p), 16.9 (p, minor), 16.3 (p, minor), 16.0 (p), 13.7
(p, minor), 13.4
(p) (p), 12.0 (p, minor), 11.9 (p), 10.8 (p), 10.6 (p, minor);
'H NMR (500 MHz in Pyridine-d;) 8 0.82 (t, J=7.SHz, CH;), 0.87 (t, J=7.SHz,
CH;,
minor), 0.94 (s, CH3), 0.9~ (s, CH;, minor), 1.0 (m, CH2), 1.10 (m, CH2), 1.17
(m, 25 and
CH3 minor), 1.2 (d, J=7.SHz, CH;), 1.27 (d, J=7.SHz, CH;), 1.42-1.58 (m, CH2,
CH2,
CH2, CH2, and CHI), 1.62 (s, CH;), 1.79 (s, CH;, minor), 1.80 (s, CH3), 1.86
(s, CH;,
minor), 1.98 (m, CH), 2.10 (m, CH2, CH2, and CH), 2.22 (m, CH2), 2.31, 2.35
(m, CHI
and CH), 2.92 (m, CH2, minor), 2.57 (m, CH), 2.67 (ddd, J1=J2=7.SHz, J3=lSHz,
CH),
2.77 (m, CH, minor), 2.91 (m, CH, minor), 3.02 (m, CH, minor), 3.08 (m, CH),
3.23 (m,
CH2), 3.3 (m, CH), 3.35 (s, OCH;, minor), 3.38 (s, OCH;), 3.42 (s, OCH;), 3.44
(s,
OCH;), 3.46 (s, OCH;, minor), 3.55 (m, CH major and minor), 3.62 (m, CHI,
minor),
3.68 (m, CH major and minor), 3.82 (s, P-OCH3), 3.84 (s, P-OCH;, minor). 3.89
(m, CH
major and minor), 4.19 (d, J=IOHz, CH), 4.28 (dd, Jl=2.SHz, J2=IOHz, CH,
minor), 4.45
(m, CH), 4.50 (m, CH), 4.58 (m, CH), 4.72 (d, CH2), 5.10 (m, CH major and
minor), 5.13
(m; CH), 5.19 (m, furan CH2), 5.41 (m, CH), 5.53 (d, J=SHz, CH), 5.81 (d,
J=SHz, CH),
5.87 (d, J=SHz, CH, minor), 6.68 (m, furan ring CH), 7.62 (m, furan ring CH),
7:78 (furan
ring CH);
Elemental Analysis calculated for C4~H,~,NO,6P 1.5 HBO: C; 59.26; H,8.10; N,
1.41.
Found C, 59.11; H, 7.96; N, 1.49.
Example 9
benzyl (1R 2R 4R)-4-~(E)-2-f(1R 9S 12S 13R 14S 17R 21S 23S,24R 25S.27R)-17-
ethyl-
1 14 dih,~xx-23 25-dimethoxy-13 19 21 27-tetramethyl-2.3 10.16-tetraoxo-11,28-
dioxa 4 azatricyclo[2~ 3 1 04~~]octacos-18-en-12-yll-1-propenyl~-2-
methoxycyclohexyl
methyl phosphate
The product from Example 3, phenylmethanol (0.310 mL, 3 mmol), and the
procedure described in Example 5 were used to provide the title compound.
MS (FAB) m/z: M+KT=1014;
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
44
'3C NMR (500 MHz in Pyridine-d;) 8 211.6 (q), 210.7 (q, minor), 199.6 (q,
minor), 198.8
(q), 170.1 (q, minor), 170.0 (q), 167.3 (q, minor), 167.0 (q), 149.7 (t,
pyridyl ring CH),
139.3 (q, minor), 138.4 (q), 137.2 (q, Ar-1C), 133.4 (q), minor), 133.1 (q),
132.1 (t), 131.2
(t, minor), 129.0, 128.9 (t, Ar), 128.7, 128.6 (t, Ar, minor), 128.3 (t, Ar),
128.2 (t, Ar,
minor), 124.6 (t, minor), 124.4 (t), 99.7 (q, minor), 98.9 (q), 81.8 (t), 81.7
(t, minor), 81.0
(t), 80.9 (t, minor), 80.6 (t), 79.6 (t, minor), 77.9 (t, minor), 76.2 (t),
74.8 (t, minor), 74.3
(t), 74.0 (t, minor), 73.1 (t), 69.7 (t), 68.9 (t, minor), 69.2 (s, benzyl,
minor), 69.0 (s,
benzyl), 57.7 (t, minor), 57.5 (t), 57.0 (t), 56.9 (t, major and minor), 56.1
(t), 55.8 (t,
minor), 55.5 (t), 55.2 (t, minor), 54.3, 54.2_ (p, P-OCH3), 54.0 (p, P-OCH3,
minor), 53.0
(t); 49.3 (s), 48.0 (s, minor), 47.9 (s), 46.6 (s, minor), 44.6 (s, minor),
41.4 (t), 41.0 (t),
39.5 (s), 36.4 (s), 35.9 (t), 35.6 (t, minor), 35.2 (s, minor), 34.6 (t), 34.4
(s), 33.1 (s), 32.9
(s; m.inor), 31.8 (s), 30.5 (s, major and minor), 28.2 (s), 27.5 (s, minor),
27.5 (t, minor),
26.5 (t); 25.0 (s), 24.8 (s, minor), 24.4 (s, minor), 21.8 (minor), 21.5 (s),
20.6 (p, minor),
20.0 (p), 16.9 (p), 16.8 (p, minor), 16.3 (p, minor), 16.0 (p), 13.7 (p,
minor), 13.4 (p) (p),
I5 12.0 (p, minor), 11.8 (p), 10.7 (p), 10.6 (p, minor);
'H NMR (500 MHz in pyridine-d;) 8 0.82 (t, J=7.5Hz, CH;), 0.87 (t, J=7.5Hz,
CH;,
minor), 0.94 (s, CH;), 0.95 (s, CH3, minor), 1.0 (m, CHI), 1.10 (m, CH2), 1.17
(m, 25 and
CH; minor), 1.2 (d, J=7.5Hz, CH;), 1.27 (d, J=7.5Hz, CH;j, 1.42-1.58 (m, CH2,
CHI,
CH2, CH2, and CH2), 1.62 (s, CH;), 1.79 (s, CH;, minor), 1.80 (s, CH;), 1.86
(s, CH;,
minor), 1.98 (m, CH), 2.10 (m, CH2, CH2, and CH), 2.22 (m, CH2), 2.31, 2.35
(m, CHI
and CH), 2.92 (m, CH2, minor), 2.57 (m, CH), 2.67 (ddd, JI=J2=7.5Hz, J3=lSHz,
CH),
2.77 (m, CH, minor), 2.91 (m, CH, minor), 3.02 (m, CH, minor), 3.08 (m, CH),
3.23 (m,
CHZ), 3.3 (m, CH), 3.31 (s, OCH3, minor), 3.38 (s, OCH3), 3.42 (s, OCH;), 3.44
(s,
OCH3), 3.46 (s, OCH;, minor), 3.55 (m, CH major and minor), 3.62 (m, CH2,
minor),
3.68 (m, CH major and minor), 3.86 (s, P-OCH;), 3.89 (s, P-OCH;, minor), 3.89
(m, CH
major and minor), 4.19 (d, J=IOHz, CH), 4.28 (dd, J1=2.SHz, J2=IOHz, CH,
minor), 4.50
(m, CH), 4.58 (m, CH), 4.64 (s, OH), 4.72 (d, CHI), 5.10 (m, CH amjor and
minor), 5.23
(m, CH), 5.41 (m, CH), 5.49 (m, benzylCH2), 5.53 (m, CH), 5.81 (d, J=SHz, CH),
5.87 (d,
J=SHz, CH, minor), 7.15 (m, ArCH), 7.6 (m, ArCH), 8.61 (m, ArCH);
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
Elemental Analysis calculated for C;,H,BNO,;P: C, 62.75; H, 8.05; N, 1.43.
Found C,
62.38; H, 7.92; N, 1.41.
Exa~le 10
5 (1R 2R 4R) 4 ~(E) 2 f(1R 9S 12S 13R 14S 17R 21S 23S 24R 25S 2781-17-ethyl-
1,14-
dihydrox 23 25 dimethoxy-13 19 21 27-tetramethyl-2.3 10.16-tetraoxo-11,28-
dioxa-4
azatricyclof22 3 1 O~vloctacos 18-en-12-yll-1-propenyl;-2-methoxycyclohexyl 3
furylmethyl methyl phosphate
The pxoduct from Example 3, 3-furylmethanol (258 mL, 3 mmol) and the
10 procedure described in Example 5 were used to provide the title compound.
MS (FAB) m/z: M+K'=1004;
'~C NMR (500 MHz in Pyridine-ds) ~ 211.6 (q), 210.8 (q, minor), 199.6 (q,
minor), 198.8
(q), 170.1 (q, minor), 170.0 (q), 167.4 (q, minor), 167.1 (q), I 44.2 (t,
furan ring), 144.1 (t,
furan ring, minor), 142.1 (t, furan ring), 139.4 (q, minor), 138.4 (q), 135.9
(t, pyridyl ring
15 CH), 133.4 (q), minor), 133.1 (q), 132.2 (t), 131.2 (t, minor), 124.6 (t,
minor), 124.5 (t),
122.2 (q, furan ring), 111.0 (furan ring, major and minor), 99.7 (q, minor),
98.9 (q), 81.8
(t), 81.7 (t, minor), 81.2 (t), 81.1 (t, minor), 80.7 (t), 79.6 (t, minor),
77.9 (t, minor), 76.3
(t), 74.8 (t, minor), 74.4 (t), 74.0 (t, minor), 73.1 (t), 69.7 (t), 68.8 (t,
minor), 61.3, 61.2 (s,
furan, minor), 61.0 (s, furan), 57.7 (t, minor), 57.5 (t), 57.0 (t), 57.1 (t,
major and minor),
20 56.1 (t), 55.9 (t, minor), 55.6 (t), 55.3 (t, minor), 54.3, (p, P-OCH3),
54.0 (p, P-OCH3,
minor), 53.0 (t); 49.3 (s), 48.0 (s, minor), 47.9(s), 46.6 (s, minor), 44.6
(s, minor), 41.4 (t),
41.0 (t); 39.5 (s), 36.4 (s), 35.9 (t), 35.6 (t, minor), 35.2 (s, minor), 34.7
(t), 34.4 (s), 33.1
(s), 32.9 (s, minor), 31.7 (s), 30.6 (s, minor), 30.5 (s) 28.2 (s), 27.6 (t,
minor), 27.5 (s,
minor), 26.6 (t), 25.0 (s), 24.9 (s, minor), 24.4 (s, minor), 21.8 (minor),
21.5 (s), 20.6 (p,
25 minor), 20.0 (p), 16.8 (p), 16.9 (p, minor), 16.3 (p, minor), 16.0 (p),
13.7 (p, minor), 13.4
(p), 12.0 (p, minor), 11.9 (p), 10.8 (p), 10.6 (p, minor).
Example 11
(1R 2R 4R) 4 ~(E) 2 [(1R 9S 12S 13R 14S 17R 21 S 23S 24R 25S 27R)-17-ethyl-
1,14-
30 dihydrox_y 23 25 dimethoxy-13 19 21 27-tetramethyl-2 3.10,16-tetraoxo-I
1,28-dioxa-4-
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
46
azatricyclo[2~ 3 1 O~v~octacos-18-en-12-~1-l~ropenyl~-2-methox~yclohexyl
methyl 3-
thien~lmethyl phosphate
The product from Example 3 (1.067 g, 1 mmol), 3-thienylmethanol (142 ~L, 1.5
mmol) and the procedure described in Example 5 were used to provide (61.6 mg)
the title
compound.
MS (FAB) m/z: M+KT=1020;
'3C NMR (500 MHz in Pyridine-d;) 8 211.5 (q), 210.7 (q, minor), 199.6 (q,
minor), 198.8
(q), 170.1 (q, minor), 170.0 (q), 167.4 (q, minor), 167.1 (q), 139.3 (q,
minor), 138.4 (q),
133.4 (q), minor), 13 3.1 (q), 1321 (t), 131.1 (t, minor), 127.9 (t, thiophene
ring), 3 26.9 (t,
thiophene ring), 124.7 (thiophene ring-), 124.6 (t, minor), 124.5 (t), 99.7
(q, minor), 98.9
(q), 81.8 (t), 81.7 (t, minor), 81.2 (t), 81.1 (t, minor), 80.7 (t), 79.6 (t,
minor), 77.9 (t,
minor), 76.3 (t), 74.8 (t, minor), 74.4 (t), 74.0 (t, minor), 73.0 (t), 69.6
(t), 68.8 (t, minor),
64.6, 64.5 (s, thiophene, minor), 64.3, 64.2 (s, furan), 57.6 (t, minor), 57.5
(t), 57.0 (t),
57.1 (t, major and minor), 56.1 (t), 55.9 (t, minor), 55.6 (t), 55.3 (t,
minor), 54.2, (p, P-
OCH3), 53.9 (p, P-OCH3, minor), 52.9 (t), 49.3 (s), 47.9 (s, minor), 47.9(x),
46.6 (s,
minor), 44.6 (s, minor), 41.4 (t), 41.0 (t), 39.4 (s), 36.3 (s), 35.8 (t),
35.6 (t, minor), 35.2 (s,
minor), 34.7 (t), 34.4 (s), 33.1 (s), 32.9 (s, minor), 31.7 (s), 30.6 (s,
minor), 30.5 (s) 28.1
(s), 27.6 (t, minor), 27.5 (s, minor), 26.6 (t), 25.0 (s), 24.9 (s, twinor),
24.4 (s, minor), 21.8
(minor), 21.5 (s), 20.6 (p, minor), 20.0 (p), 16.8 (p), 16.9 (p, minor), 16.3
(p, minor), 16.0
(p), 13.7 (p, minor), 13.4 (p), 12.0 (p, minor), 11.9 (p), 10.8 (p), 10.6 (p,
minor);
'H NMR (500 MHz in CDC1;) 8 0.84 (m, CH;), 0.87 (m, CH;), 0.92 (d, J=SHz, CH3,
minor), 0.96 (d, J=SHz, CH3), 0.98 (d, J=SHz, CH3, minor), 1.01 (d, J=SHz,
CH3), 1.05
(m, CH2), 1.6 (s, CH;), 1.63 (s, CH;, minor), 1.64 (s, CH;), 1.66 (s, CH;,
minor), 1.78 (m,
CH2, CH), 1.92 (m, CH2), 2.18 (m, CH2), 2.31 (m, CH, CH and CH), 2.75 (ddd,
J1=J2=2.5Hz, J3=lSHz, CH. minor), 2.8 (ddd, JI=J2=2.5Hz, J3=lSHz, CH), 3.3 (m,
CH2), 3.21 (m, 21 and CH), 3.31 (s, OCH;), 3.34 (s, OCH;, minor), 3.38 (s,
OCH;,
minor), 3.40 (s, OCH;), 3.43 (s, OCH; major and minor), 3.51 (m, CH), 3.58 (m,
CH),
3.67 (d, J=lOHz, CH), 3.73 (s. P-OCH3), 3.76 (s, P-OCH;, minor), 3.80 (m, CH
and CH
major and minor), 4.18 (m, CH), 4.23 (s, OH), 4.44 (m, CH2), 4.61 (d, J=SHz,
CH), 4.89
(s, OH), 5.04 (m, CH major and minor, CH), 5.50 (m, thiopheneCH2), 5.2 (d,
J=2.5Hz,
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
47
CH, minor), 5.35 (d, J=2.5Hz, CH), 7.14 (d, J=SHz, 1H, thiophene ring CH),
7.34 (m, 2H,
thiophene ring CH);
Elemental Analysis calculated for C4~H,6NO,;PS: C; 59.92; H, 7.79; N, 1.42.
Found C,
59.77; H, 7.94; N, 1.36.
Example 12
(1R 2R 4R)-4-~(E)-2-~(1R 9S 12S 13R 14S 17R 21 S 23S 24R 25S 27R)-17-ethyl-
1,14-
dihydroxy-23 25-dimethoxy-13 19 21 27-tetramethyl-2.3 10,16-tetraoxo-11,28-
dioxa-4-
azatricyclof22.3.1.04°9loctacos-18-en-12-yll-1-propenyl~ 2-
methoxvcyclohexyl 4-
fluorobenzyl methyl phosphate
The product from Example 3, (4-fluorophenyl)methanol, and the procedure
described in Example 5 were used to provide the title compound.
'3C NMR (500 MHz in Pyridine-d;) 8 211.7 (q), 210.8 (q, minor), 199.6 (q,
minor), 198.8
(q), 170.2 (q, minor), 170.1 (q), 167.4 (q, minor), 167.0 (q), 164.0 (q, Ar),
162.1 (q, Ar),
139.4 (q; minor), 138.5 (q), 133.5 (q), minor), 133.2 (q), 132.1 (t), 131.2
(t, minor), 130.5
(t, 2xAr), 124.7 (t, minor), 124.5 (t), 115.9 (t, 2xAr), 99.8 (q, minor), 98.9
(q), 81.8 (t),
81.7 (t, minor), 81.0 (t, major and minor), 80.7 (t), 79.5 (t, minor), 77.9
(t, minor), 76.3 (t),
74.8 (t, minor), 74.3 (t), 74.0 (t, minor), 73.1 (t), 69.7 (t), 68.8 (t,
minor), 68.5 (s, benzyl,
minor), 68.3 (s, benzyl), 57.7 (t, minor), 57.5 (t), 57.1 (t), 57.0 (t, major
and minor), 56.1
(t), 55.9 (t, minor), 55.5 (t), 55.2 (t, minor), 54.4, (p, P-OCH3), 54.0 (p, P-
OCH3, minor),
53.0 (t), 49.4 (s), 48.0 (s, minor), 47.9 (s), 46.6 (s, minor), 44.6 (s,
minor), 41.4 (t), 40.9(t,
minor), 39.5 (s), 36.2 (s), 35.9 (t), 35.6 (t, minor), 35.2 (s, minor), 34.7
(t), 34.4 (s), 33.1
(s), 32.9 (s, minor), 31.7 (s, major and minor), 30.5 (s, major and minor),
28.2 (s), 27.6 (t,
minor), 27.5 (s, minor), 26.6 (t), 25.0 (s), 24.9 (s, minor), 24.4 (s, minor),
21.8 (minor),
21.5 (s), 20.6 (p, minor), 20.0 (p), 16.9 (p), 16.8 (p, minor), 16.3 (p,
minor), 16.0 (p), 13.7
(p, minor), 13.4 (p), 12.0 (p, minor), 11.8 (p), 10.7 (p), 10.6 (p, minor);
'H NMR (500 MHz in Pyridine-d;) ~ 0.82 (t, J=7.5Hz, CH;), 0.87 (t, J=7.5Hz,
CH;,
minor), 0.94 (s, CH3), 0.95 (s, CH3, minor), 1.0 (m, CHI), 1.10 (m, CH2), 1.17
(m, 25 and
CH3 minor), 1.2 (d, J=7.5Hz, CH3), 1.27 (d, J=7.5Hz, CH;), 1.42-1.58 (m, CH2,
CH2,
CH2, CH2, and CH2), 1.62 (s, CH3), 1.79 (s, CH3, minor), 1.80 (s, CH3), 1.86
(s, CH;,
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
48
minor), 1.98 (m, CH), 2.10 (m, CH2, CH2, and CH), 2.22 (m, CH2), 2.31, 2.35
(m, CH2
and CH), 2.92 (m, CH2. minor), 2.57 (m, CH), 2.67 (ddd, J1=J2=7.SHz, J3=lSHz,
CH),
2.77 (m, CH, minor), 2.91 (m, CH, minor), 3.02 (m, CH, minor), 3.08 (m, CH),
3.23 (m,
CH2), 3.3 (m, CH), 3.36 (s, OCH;, minor), 3.38 (s, OCH3), 3.42 (s, OCH;, major
and
minor), 3.44 (s, OCH;), 3.46 (s, OCH3, minor), 3.55 (m, CH major and minor),
3.62 (m,
CH2; minor), 3.68 (m, CH major and minor), 3.82 (s, P-OCH;), 3.84 (s, P-OCH3,
minor),
3.89 (m, CH major and minor), 4.19 (d, J=IOHz, CH), 4.28 (dd, J1=2.SHz,
J2=lOHz, CH,
minor), 4.45 (m, CH), 4.50 (m, CH), 4.58 (m, CH), 4.72 (d, CH2), 5.10 (m, CH
major and
minor), 5.23 (m, CH), 5.25 (m, benzylCH2), 5.41 (m. CH), 5.53 (d, J=SHz, CH),
5.81 (d,
J=SHz, CH), 5.87 (d, J=SHz, CH, minor), 7.15 (m, 2xArCH), 7.52 (2xArCH);
Elemental Analysis calculated for C;,H"FNO,;P 0.5 HBO: C; 61.06; H, 7.83; N,
1.39.
Found C, 61.08; H, 8.05; N, 1.40.
Example 13
(1 3 dioxo-1 3-dihydro-2H-isoindol-2-yl)methyl (1R 2R 4R)-4-{(E)-2-
~(1R 9S 12S 13R 14S 17R 21 S 23S 24R 25S 27R)-17-ethyl-1.14-dihydroxy-23,25
dimethoxy-13 19 21 27-tetramethyl-2 3 10 16-tetraoxo-11,28-dioxa-4
azatri~cloL2 3 1 04-9~octacos-18-en-12-yll-1-propenyl~-2-methoxycyclchexyl
methyl
phosphate
The product from Example 3, 2-(hydroxymethyl)-1H-isoindole-1,3(2H)-dione, and
the procedure described in Example 5 were used to provide the title compound.
MS (FAB) m/z: M+Ky=1083;
'3C NMR (500 MHz in Pyridine-d;) ~ 211.6 (q), 210.8 (q, minor), 199.6 (q,
minor), 198.8
(q), 170.2 (q, minor), 170.1 (q), 167.4 (q, minor), 167.0 (q), 166.9 (q, from
phthalimide
group), 139.4 (q, minor), 138.4 (q), 135.0 (t, 2xAr), 133.4 (q), minor), 133.1
(q), 132.2 (t),
132.1 (q, Ar), 131.1 (t, minor), 124.7 (t, minor), 124. (t), 99.7 (q, minor),
98.9 (q), 81.8
(t), 81.7 (t, minor), 81.4 (t), 81.3 (t, minor), 80.7 (t). 79.5 (t, minor),
77.9 (t, minor), 76.2
(t); 74.8 (t, minor), 74.3 (t), 74.0 (t, minor), 73.1 (t). 69.7 (t), 68.9 (t,
minor), 63.7 (s, -
NCH2-OP, minor), 63.5 (s, -NCH2-OP), 57.7 (t, minor), 57.5 (t), 57.1 (t), 57.0
(t, major
and minor), 56.1 (t), 55.9 (t, minor), 55.5 (t), 5~.3 (t. minor), 54.6, (p, P-
OCH3), 54.3 (p,
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
49
P-OCH3, minor), 53.0 (t). 49.3 (s), 48.0 (s, minor), 47.9 (s), 46.5 (s,
minor), 44.6 (s,
minor), 41.4 (t), 41.1 (t, minor), 39.5 (s), 36.2 (s), 35.9 (t), 35.6 (t,
minor), 35.2 (s, minor),
34.6 (t), 34.5 (s), 33.1 (s), 32.9 (s, minor), 31.7 (s, major and minor), 30.5
(s, major and
minor), 28.2 (s), 27.5 (s, minor), 27.5 (t, minor), 26.6 (t), 25.0 (s), 24.9
(s, minor), 24.4 (s,
minor), 21.8 (minor), 21.5 (s), 20.6 (p, minor), 20.0 (p), 16.9 (p), 16.8 (p,
minor), 16.3 (p,
minor), 16.0 (p), 13.7 (p, minor), 13.4 (p), 12.0 (p, minor), 11.8 (p), 10.7
(p), 10.6 (p,
minor);
'H NMR (500 MHz in Pyridine-d; ) b 0.82 (t, J=7.5Hz, CH;), 0.87 (t, J=7.5Hz,
CH;,
minor). 0.94 (s, CH;), 0.95 (s, CH;, minor), 1.0 (m, CH2), 1.10 (m, CH2), 1.17
(m, 25 and
CH; minor), 1.2 (d, J=7.5Hz, CH3), 1.27 (d, J=7.5Hz, CH3), 1.42-1.58 (m, CH2,
CH2,
CH2, CH2, and CH2), 1.62 (s, CH3), 1.79 (s, CH;, minor), 1.80 (s, CH3), 1.86
(s, CH;,
minor), 1.98 (m, CH), 2.10 (m, CHI, CHI, and CH), 2.22 (m, CH2), 2.31, 2.35
(m, CH2
and CH), 2.92 (m, CH2, minor), 2.57 (m, CH), 2.67 (ddd, J1=J2=7.5Hz, J3=lSHz,
CH),
2.77 (m, CH, minor), 2.91 (m, CH, minor), 3.02 (m, CH, minor), 3.08 (m, CH),
3.23 (m,
CHI), 3.3 (m, CH), 3.35 (s, OCH;, minor), 3.37 (s, OCH;), 3.40 (s, OCH;), 3.42
(s,
OCH;), 3.45 (s, OCH;), 3.46 (s, OCH;, minor), 3.55 (m, CH major and minor),
3.62 (m,
CHI, minor), 3.68 (m, CH major and minor), 3.86 (s, P-OCH;), 3.88 (s, P-OCH;,
minor),
3.89 (m, CH major and minor), 4.19 (d, J=lOHz, CH), 4.28 (dd, Jl=2.5Hz,
J2=IOHz, CH,
minor), 4.50 (m, CH), 4.58 (m, CH), 4.72 (m, CH2), 5.10 (m, CH major and
minor), 5.23
(m; CH), 5.41 (m, CH), 5.53 (d, J=SHz, CH), 5.81 (d, J=SHz, CH), 5.87 (d,
J=SHz, CH,
minor), 5.96 (m, phthalimidoCH2), 7.6 (m, 2xArCH), 7.88 (m, 2xArCH).
Example 14
(1R 2R 4R)~,~E) 2-~(1R 9S 12S 13R 14S 17R 21 S 23S 24R 25S 27R)-17-ethyl-1 14-
dihydroxy-23 25-dimethoxy-13 19 21 27-tetramethyl-2 3 10 16-tetraoxo-11.28-
dioxa-4-
azatricyclof22 3 1 04'9]octacos-18-en-1~- ly_1-1-propenyl~-2-methoxycyclohexvl
methyl 4-
nitrobenz~phosphate
The product from Example 3, (4-nitrophenyl)methanol, and the procedure
described in Example 5 were used to provide the title compound.
MS (FAB) d M+K+=1059;
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
'3C NMR (500 MHz in Pyridine-d;) 8 211.6 (q), 210.8 (q, minor), 199.6 (q,
minor), 198.8
(q), 170.1 (q, minor), 170.0 (q), 167.3 (q, minor), 167.0 (q), 148.1 (q, Ar),
144.5 (q, Ar),
139.4 (q, minor), 138.5 (q), 137.2 (t, pyridyl), 135.9 (t, pyridyl ring CH),
133.5 (q),
minor), 133.2 (q); 132.1(t), 131.2 (t, minor), 128.4, 128.3 (t, Ar), 124.7 (t,
minor), 124.5
5 (t), 123.9, 123.8 (t, Ar), 99.8 (q, minor), 98.9 (q), 81.8 (t), 81.7 (t,
minor), 81.2 (t), 81.1 (t,
minor), 80.7 (t), 79.6 (t, minor), 77.9 (t, minor), 76.3 (t), 74.8 (t, minor),
74.4 (t), 74.0 (t,
minor), 73.1 (t), 69.8 (t), 68.9 (t, minor), 67.8(s, benzyl, minor), 67.5 (s,
benzyl), 57.8 (t,
minor), 57.6 (t), 57.1 (t), 57.0 (t, major and minor), 56.2 (t), 55.9 (t,
minor), 55.5 (t), 55.3
(t, minor), 54.6, 54.5 (p, P-OCH3), 54.4, 54.2 (p, P-OCH3, minor), 53.1 (t),
49.4 (s), 48.0
10 (s, minor), 47.9 (s), 46.6 (s, minor), 44.6 (s, minor), 41.5 (t), 41.0 (t),
39.5 (s), 36.4 (s),
35.9 (t); 35.6 (t, minor), 35.2 (s, minor), 34.7 (t), 34.5 (s), 33.2 (s), 33.0
(s, minor), 31.9
(s), 30.6 (s, major and minor), 28.2 (s), 27.6 (s, minor), 27.6 (t, minor),
26.6 (t), 25.1 (s),
24.9 (s, minor), 24.5 (s, minor), 21.9 (minor), 21.6 (s), 20.6 (p, minor),
20.1 (p), 16.9 (p),
16.9 (p, minor), 16.3 (p, minor), 16.1 (p), 13.7 (p, minor), 13.5 (p), 12.0
(p, minor), 11.9
15 (p), 10.8 (p), 10.6 (p, minor);
'H NMR (500 MHz in Pyridine-d5) 8 0.82 (t, 3=7.5Hz, CH3), 0.87 (t, J=7.5Hz,
CH;,
minor), 0.94 (s; CH;), 0.95 (s, CH3, minor), 1.0 (m, CH2), 1.10 (m, CH2), 1.17
(m, 25 and
CH3 minor), 1.2 (d, J=7.5Hz, CH3), 1.27 (d, J=7.5Hz, CH3), 1.42-1.58 (m, CH2,
CH2,
CHZ, CH2, and CHI), 1.62 (s, CH;), 1.79 (s, CH;, minor), 1.80 (s, CH;), 1.86
(s, CH;,
20 minor), 1.98 (m, CH), 2.10 (m, CHI, CH2, and CH), 2.22 (m, CHI), 2.31, 2.35
(m, CH2
and CH), 2.92 (m, CH2, minor), 2.57 (m, CH), 2.67 (ddd, J1=J2=7.5Hz, J3=lSHz,
CH),
2.77 (m; CH, minor), 2.91 (m, CH, minor), 3.02 (m, CH, minor), 3.08 (m, CH),
3.23 (m,
CH2), 3.3 (m, CH), 3.35 (s, OCH3, minor), 3.38 (s, OCH;), 3.42 (s. OCH3, major
and
minor), 3.44 (s; OCH;), 3.46 (s, OCH;, minor), 3.55 (m, CH major and minor),
3.62 (m,
25 CH2, minor), 3.68 (m, CH major and minor), 3.87 (s, P-OCH3, minor), 3.89
(s, P-OCH3),
3.89 (m, CH major and minor), 4.19 (d, J=IOHz, CH), 4.28 (dd, Jl=2.5Hz,
J2=IOHz, CH,
minor), 4.50 (m, CH), 4.58 (m, CH), 4.64 (s, OH), 4.72 (d, CHI). 5.10 (m, CH
major and
minor), 5.23 (m, CH), 5.40 (m, benzylCH2), 5.41 (m, CH), 5.53 (d, J=SHz, CH),
5.81 (d,
J=SHz, CH), 5.87 (d, J=SHz, CH, minor), 7.64 (m, 2xArCH), 8.22 (m, 2xArCH);
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
51
Elemental Analysis calculated for C;,H"N,O»P: C; 59.98; H, 7.60; N, 2.74.
Found C,
59.92; H, 7.56; N, 2.76.
Example 15
(1R 2R 4R) 4 ~(E) 2 f(1R 9S 12S 13R 14S 17R 21S 23S 24R 25S 27R)-17-ethyl-1,14-
dih drox -23 25-dimethox -13 19 21 27-tetramethvl-2,3,10 16-tetraoxo-11 28-
dioxa-4-
azatric~clof22 3 1 04~9Lctacos-18-en-12-yll-1-propenyl}-2-methoxycyclohexyl
methyl
diisopropylphosphoramidoate
The product from Example 3 was oxidized according to the method described in
(Hecker, S. J., Minich, M. L. and Lackey, K., J. Org. Chem., (1990), 55, 4904)
and
purified by reverse phase high performance liquid chromatography.
MS (FAB) m/z: M+K+=1007;
'3C NMR (500 MHz in Pyridine-d;) 8 211.6 (q), 210.7 (q, minor), 199.6 (q,
minor), 198.8
(q), 170.1 (q, minor), 170.0 (q), 167.3 (q, minor), 167.0 (q), 139.4 (q,
minor), 138.4 (q),
133.2 (q), minor), 132.9 (q), 132.4 (t), 131.4 (t, minor), 124.6 (t, minor),
124.4 (t), 99.7 (q,
minor), 98.9 (q), 82.2 (t), 82.1 (t, minor), 80.7 (t), 79.6 (t, minor),
79.3(t), 79.1 (t, minor),
77.9 (t; minor), 76.2 (t), 74.8 (t, minor), 74.3 (t), 74.0 (t, minor), 73.1
(t), 69.7 (t), 68.8 (t,
minor), 57.7 gyp, minor), 57.5 (p), 57.1 (p), 57.0 (t), 56.1 (t), 55.8 (t,
minor), 55.5 (t), 55:3
(t, minor), 53.0 (t), 52.4, 52.3 (p, P-OCH3), 49.3 (s), 48.0 (s, minor), 47.9
(s), 46.5 (s,
minor), 46.2, 46.1 (t,from isopropyl group), 44.6 (s, minor), 41.4 (t), 41.0
(t), 39.4 (s), 36.5
(s, major and minor), 35.9 (t), 35.5 (t, minor), 35.1 (s, minor), 34.8 (t),
34.4 (s), 33.1 (s),
32.9 (s, minor), 32.1 (s), 30.6 (s), 30.5 (s, minor), 28.2 (s), 27.5 (s,
minor), 27.5 (t, minor),
26.5 (t), 25.0 (s), 24.8 (s, minor), 24.4 (s, minor), 22.8 (p, isopropyl),
22.5 (p, isopropyl),
21.8 (minor), 21.5 (s), 20.6 (p, minor), 20.0 (p), 16.9 (p), 16.8 (p, minor),
16.3 (p, minor),
16.0 (p), 13.7 (p, minor), 13.4 (p), 12.0 (p, minor), 11.8 (p), 10.7 (p), 10.6
(p, minor);
'H NMR (500 MHz in Pyridine-d;) 8 0.82 (t, J=7.5Hz, CH;), 0.87 (t, J=7.SHz,
CH3,
minor), 0.94 (s, CH3), 0.95 (s, CH;, minor), 1.0 (m, CH2), 1.17 (m, CH3 and
CH; minor),
1.2 (d, J=7.5Hz, CH3), 1.27 (d, J=7.5Hz, CH;), 1.31 (s, isopropyl CH;), 1.34
(s, isopropyl
CH3), 1.42-1.58 (m, CH2, CH2, CH2, CH2, and CH2), 1.62 (s, CH3), 1.79 (s, CH3,
minor),
1.81 (s, CH;), 1.87 (s, CH;, minor), 1.98 (m, CH), 2.10 (m, CH2, CH2, and CH),
2.22 (m,
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
52
CH2); 2.31, 2.35 (m, CH2 and CH), 2.92 (m, CHI, minor), 2.57 (m, CH), 2.67
(ddd,
J1=J2=7.SHz, J3=lSHz, CH), 2.77 (m, CH, minor), 2.91 (m, CH, minor), 3.02 (m,
CH,
minor), 3.08 (m, CH), 3.23 (m, CH2), 3.40 (s, OCH;, minor), 3.42 (s, OCH;,
OCH;), 3.43
(s, OCH;), 3.55 (m, CH major and minor), 3.62 (m, CH2, minor), 3.68 (m, CH
major and
minor), 3.71 (s, P-OCH3), 3.74 (s, P-OCH;, minor), 3.89 (m, CH major and
minor), 4.19
(d; J=lOHz, CH), 4.28 (dd, J1=2.SHz, J2=lOHz, CH, minor), 4.40 (m, CH), 4.50
(m, CH),
4.58 (m; CH), 4.72 (d, CH2), 5.10 (m, CH major and minor), 5.13 (m, CH), 5.41
(m, CH),
5.53 (d, J=SHz, CH), 5.81 (d, J=SHz, CH), 5.87 (d, J=SHz, CH, minor);
Elemental Analysis calculated for C;oHB;N,O,~P 0.75 H2O: C; 61.11; H, 8.87; N,
2.85.
Found C, 61.29; H, 9.15; N, 2.56.
Example 16
2 c~anoethyl (1R 2R 4R)-4-~(E)-2-[(1R 9S 12S 13R 14S 17R 21 S 23S.24R,25S,27R)-
17-
ethyl 1 14-dihydrox~-23 25-dimethoxy-13 19 21 27-tetramethyl-2 3 10,16-
tetraoxo-11,28-
dioxa-4-azatricycloL,2 3 1 04°~~octacos-18- en-12-yll-1-propenyl~-2-
methox~yclohexyl
diisopropylphosphoramidoate
The product from Example 4 was oxidized according to the method described in
(Hecker, S. J., Minich, M. L. and Lackey, K., J. Org. Chem., ( 1990), 55,
4904) and
purified by reverse phase high performance liquid chromatography.
MS (FAB) m/z: M+K'=1046;
'3C NMR (500 MHz in Pyridine-d;) 8 211.6 (q), 210.7 (q, minor), 199.6 (q,
minor), 198.8
(q), 170.1 (q, minor), 170.0 (q), 167.3 (q, minor). 167.0 (q), 139.4 (q,
minor), 138.4 (q),
133.2 (q), minor), 132.9 (q), 132.4 (t), 131.4 (t, minor), 124.6 (t, minor),
124.4 (t), 118.5
(q, OCH~CH2CN), 99.7 (q, minor), 98.9 (q), 82.2 (t), 82.1 (t, minor), 80.7
(t), 79.6 (t,
minor), 79.3(t), 79.1 (t, minor), 77.9 (t, minor), 76.2 (t), 74.8 (t, minor),
74.3 (t), 74.0 (t,
minor), 73.1 (t), 69.7 (t), 68.8 (t, minor), 57.7 (p, minor), 57.5 (p), 57.2
(p), 57.1 (t), 56.1
(t), 55.8 (t, minor), 55.5 (t), 55.2 (t, minor), 53.0 (t), 49.3 (s), 48.0 (s,
minor), 47.9 (s), 46.5
(s, minor), 46.3 (t, from isopropyl group), 46.4 (t, from isopropyl group),
44.6 (s, minor),
414 (t); 41.0 (t), 39.4 (s), 36.6 (s, major and minor), 36.2 (s), 35.9 (t),
35.5 (t, minor), 35.0
(s, minor), 34.6 (t), 34.7 (s), 34.4 (s), 33.1 (s), 32.9 (s, minor), 32.0 (s),
31.8 (s), 30.6 (s),
CA 02368463 2001-09-24
WO 00/58318 PCT/LTS00/07639
53
30.5 (s, minor), 28.2 (s), 27.5 (s, minor), 27.5 (t, minor), 26.6 (t), 25.0
(s), 24.8 (s, minor),
24.4 (s, minor), 22.7 (p, isopropyl), 22.6 (p, isopropyl), 21.8 (minor), 21.5
(s), 20.6 (p,
minor), 20.0 (p), 16.9 (p), 16.8 (p, minor), 16.3 (p, minor), 16.0 (p), 13.7
(p, minor), 13.4
(p), 12.0 (p, minor), 11.8 (p), 10.7 (p), 10.6 (p, minor);
'H NMR (500 MHz in Pyridine-d;) 8 0.82 (t, J=7.5Hz, CH3), 0.87 (t, J=7.5Hz,
CH3,
minor), 0.94 (s, CH3), 0.95 (s, CH;, minor), 1.0 (m, CH2), 1.17 (m, CH3 and
CH3 minor),
1.2 (d, J=7.5Hz, CH;), 1.27 (d, J=7.5Hz, CH3), 1.31 (s, isopropyl CH;), 1.34
(s, isopropyl
CH;), 1.42-1.58 (m, CH2, CH2, CHI, CH2, and CH2), 1.62 (s, CH;), 1.79 (s, CH;,
minor),
1.8I (d, 3=SHz, CH3), 1.87 (d, J=SHz, CH;, minor), 1.98 (m, CH), 2.10 (m, CH2,
CH2,
and CH); 2.22 (m, CH2), 2.31, 2.35 (m, CH2 and CH), 2.92 (m, CH2, minor), 2.57
(m,
CH), 2.67 (m, CH), 2.77 (m, CH, minor), 2.91 (m, CH, minor), 3.02 (m, CH,
minor), 3.08
(m, CH), 3.23 (m, CH2), 3.39 (s, OCH;, minor), 3.41 (s, OCH;), 3.42 (s, OCH;),
3.44 (s,
OCH3), 3.45 (s, OCH3, minor), 3.55 (m, CH major and minor), 3.62 (m, CH2,
minor),
3.68 (m, CH major and minor), 3.89 (m, CH major and minor), 4.19 (d, J=IOHz,
CH), 4.28
(m, CH, minor), 4.40 (m, CH), 4.50 (m, CH), 4.58 (m, CH), 4.72 (m, CH2), 5.10
(m, CH
major and minor), 5.13 (m, CH), 5.41 (m, CH), 5.53 (d, J=SHz, CH), 5.81 (d,
J=SHz, CH),
5.87 (d, J=SHz, CH, minor);
Elemental Analysis calculated for CS,Hg~N3O,4P I.0 HBO: C; 60.85; H, 8.64; N,
4.09.
Found C, 60.90; H, 8.73; N, 3.88.
Example 17
~1R 2R 4R) 4-~(E)-2-~(1R 9S 12S 13R 14S 17R 21S 23S.24R.25S.27R)-17-ethyl-1,14-
dihydroxy 23 25-dimethoxy-13 19 21 27-tetramethvl-2 3 10 16-tetraoxo-11,28-
dioxa-4-
azatricyclof22 3 1 04~91octacos-18-en-12-~l-1-propenyl}-2-methoxycyclohexyl 3-
iodophen~l methyl phosphate
The product from Example 3 (1.067 g, 1 mmol), (3-iodophenyl)methanol (330 mg,
1.5 mmol), and the procedure described in Example 5 were used to provide
(280.5 mg) the
title compound.
MS (FAB) m/z: M+K+=I 126;
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
54
'3C NMR (500 MHz in Pyridine-d;) 8 211.6 (q), 210.7 (q; minor), 199.6 (q,
minor), 198.8
(q), 170.1 (q, minor), 170.0 (q), 167.3 (q, minor), 167.0 (q), 139.3 (q,
minor), 138.4 (q),
134.4 (t, Ar), 134.3 (t, Ar, minor), 133.5 (q), minor), 133.2 (q), 131.5 (t),
131.0 (t, minor),
131.5 (t, Ar), 130.7 (t, Ar), 130.5 (t, Ar,minor), 129.9 (t, Ar), 129.9 (t,
Ar, minor), 124.6 (t,
minor), 124.4 (t), 120.7 (t, Ar, minor), 120..2 (t, Ar), 99.7 (q, minor), 98.8
(q), 95.0 (q, Ar-
3C), 94.5 (q, Ar-3C, minor), 81.9 (t, minor), 81.8 (t), 81.6 (t), 81.5 (t,
minor), 80.6 (t), 79.5
(t, minor), 77.9 (t, minor), 76.2 (t), 74.7 (t, minor), 74.3 (t), 73.9 (t,
minor), 73.0 (t), 69.7
(t); 68.8 (t, minor), 57.6 (t, minor), 57.5 (t), 57.0 (t), 56.7 (t, major and
minor), 56.1 (t),
55.8 (t, minor), 55.5 (t), 55.2 (t, minor). 55.0 (p, P-OCH3), 53.0 (t), 49.3
(s), 47.9 (s,
minor), 47.9 (s), 46.5 (s, minor), 44.6 (s, minor), 41.4 (t), 40.9 (t), 39.4
(s), 36.1 (s, major
and minor), 35.8 (t), 35.5 (t. minor), 35.1 (s, minor), 34.6 (t), 34.4 (s),
33.1 (s), 32.9 (s,
minor), 31.7 (t, minor), 31.6 (s), 30.5 (s, minor), 30.4 (s), 28.1 (s), 27.5
(s, minor), 27.5 (t,
minor), 26.5 (t); 24.9(s), 24.8 (s, minor), 24.4 (s, minor), 21.8 (minor),
21.5 (s), 20.5 (p,
minor), 19.9 (p), 16.8 (p), 16.8 (p, minor), 16.2 (p, minor), 15.9(p), 13.6
(p, minor), 13.4
(p), 11.9 (p, minor), 11.8 (p), I0.7 (p), 10.5 (p, minor);
'H NMR (500 MHz in CDC13) 8 0.84 (m, CH3), 0.87 (m, CH;), 0.92 (d, J=SHz, CH;,
minor), 0.96 (d, J=SHz, CH;), 0.98 (d, J=SHz, CH;, minor), 1.01 (d, J=SHz,
CH;), 1.05
(m, CH2), 1.6 (s, CH3), 1.6 3 (s, CH3, minor), 1.64 (s, CH3), 1.66 (s, CH_;,
minor), 1.78 (m,
CH2, CH), 1.92 (m, CH2), 2.18 (m, CH2), 2.31 (m, CH, CH and CH), 2.75 (ddd,
J1=J2=2.SHz, J3=lSHz, CH, minor), 2.8 (ddd, J1=J2=2.SHz, J3=lSHz, CH), 3.3 (m,
CH2), 3.21 (m, 21 and CH). 3.31 (s, OCH3), 3.34 (s, OCH3, minor), 3.38 (s,
OCH;,
minor), 3.40 (s, OCH;), 3.43 (s, OCH; major and minor), 3.51 (m, CH), 3.58 (m,
CH),
3.67 (d, J=IOHz, CH), 3.87 (s, P-OCH;, minor), 3.9 (s, P-OCH;), 3.80 (m, CH
and CH
major and minor), 4.23 (s. OH), 4.28 (m, CH), 4.44 (m, CHI), 4.61 (d, J=SHz,
CH), 4.89
(s, OH), 5.04 (m, CH major and minor, CH), 5.2 (s, CH, minor), 5.35 (s, CH),
7.07 (t,
J=7.SHz, 1H, ArCH), 7.24 (m, 1H, ArCH), 7.52 (m, 1H, ArCH), 7.63 (m, 1H,
ArCH);
Elemental Analysis calculated for C;oH,;N0,5PI: C; 55.19; H, 6.94; N, 1.28.
Found C,
55.08; H,6.95; N, 1.24.
Example 18
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
2-(acetylamino)ethyl (1R,2R.4R)-4-~(E)-2-
j(1R 9S 12S 13R 14S 17R 21S 23S 24R 25S 27R)-17-ethyl-1 14-dihydroxy-23,25
dimethoxy-13 19 21 27-tetramethyl-2 3 10 16-tetraoxo-11,28-dioxa-4
azatricyclof22 3 1 04vloctacos-18-en-1~-yll-1-propenyl~-2-methoxycyclohexyl
methyl
5 phosphate
The product from Example 3, N-(2-hydroxyethyl)acetamide, and the procedure
described in Example 5 were used to provide the title compound.
MS (FAB) m/z: M+Ky=1009;
'3C NMR (500 MHz in Pyridine-d;) 8 211.7 (q), 210.9 (q, minor), 199.7 (q,
minor), 198.9
10 (q), 170.5 (q, acetoamido), 170.2 (q, minor), 170.1 (q), 167.4 (q, minor),
167.1 (q), 162.5
and 162.3, 162.0 (q, CF3C00), 139.5 (q, minor); 138.5 (q), 133.5 (q), minor),
133.2 (q),
132.2 (t), 131.2 (t, minor), 124.7 (t, minor), 124.5 (t), 99.8 (q, minor),
98.9 (q), 81.8 (t),
81.7 (t, minor), 81.0 (t), 80.7 (t), 79.6 (t, minor), 77.9 (t, minor), 76.3
(t), 74.8 (t, minor),
74.4 (t), 74.0 (t, minor), 73.1 (t), 69.8 (t), 68.9 (t, minor), 67.0 (s,
acetoamido-OCH2-,
15 minor), 66.7 (s, acetoamido-OCH2), 57.8 (t, minor), 57.6 (t), 57.1 (t),
57.0 (t, major and
minor), 56.2 (t), 55.9 (t, minor), 55.5 (t), 55.3 (t, minor), 54.6, 54.5 (p, P-
OCH3), 54.4,
54.2 (p, P-OCH3, minor), 53.1 (t), 49.4 (s), 48.0 (s, minor), 48.0 (s), 46.6
(s, minor), 44.7
(s, minor), 41.5 (t), 41.1 (t), 40.4 (s, acetoamido-NHCHZ), 40.3 (s,
acetoamido -NHCH2,
minor), 39.5 (s), 36.4 (s), 35.9 (t), 35.6 (t, minor), 35.2 (s, minor), 34.7
(t), 34.5 (s), 33.2
20 (s), 33.0 (s, minor), 31.9 (s, minor), 31.8 (s), 30.6 (s, major and minor),
28.2 (s), 27.6 (s,
minor), 27.6 (t, minor), 26.6 (t), 25.1 (s), 24.9 (s, minor), 24.5 (s, minor),
23.1 (p,
acetoamido-CH3), 21.9 (minor), 21.6 (s), 20.6 (p, minor), 20.1 (p), 16.9 (p),
16.9 (p,
minor), 16.3 (p, minor), 16.1 (p), 13.7 (p, minor), 13.5 (p), 12.0 (p, minor),
11.9 (p), 10.8
(p), 10.6 (p, minor);
25 'H NMR (500 MHz in Pyridine-d;) ~ 0.82 (t, J=7.5Hz. CH;), 0.87 (t, J=7.5Hz,
CH;,
minor), 0.94 (s, CH;), 0.95 (s, CH;, minor), 1.0 (m, CHI), 1.10 (m, CH2), 1.17
(m, 25 and
CH3 minor), 1.2 (m, CH3), 1.27 (d, J=7.5Hz, CH3), 1.42-1.58 (m, CH2, CH2, CH2,
CHI,
and CH2), 1.62 (s, CH3), 1.79 (s, CH;, minor), 1.80 (s, CH;), 1.86 (s, CH3,
minor), 1.98
(m, CH), 2.09 (s, acetoamidoCH3), 2.10 (m, CH2, CHI, and CH), 2.22 (m, CH2),
2.31,
30 2.35 (m, CH2 and CH), 2.92 (m, CH2, minor), 2.57 (m, CH), 2.67 (m, CH),
2.77 (m, CH,
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
56
minor), 2.91 (m, CH, minor), 3.02 (m, CH, minor), 3.08 ('m, CH), 3.23 (m,
CH2), 3.3 (m,
CH), 3.36 (s, OCH;, minor), 3.38 (s, OCH3), 3.42 (s, OCH;, major and minor),
3.44 (s,
OCH3), 3.45 (s, OCH;, minor), 3.55 (m, CH major and minor). 3.62 (m, CH2,
minor),
3.68 (m, CH major and minor), 3.77 (m, acetoamido-OCH2), 3.79 (s, P-OCH3),
3.83 (s, P-
OCH3; minor), 3.89 (m, CH major and minor), 4.19 (d, J=I OHz, CH), 4.28 (m,
CH,
minor), 4.41 (m, CH), 4.50 (m, CH), 4.58 (m, CH), 4.72 (d, CH2), 5.10 (m, CH
major and
minor), 5.23 (m, CH), 5.31 (m, pyridylCH~), 5.41 (m, CH), 5.53 (m, CH), 5.81
(m, CH),
5.87 (m, CH, minor).
Example 19
(1R ~R 4R) 4 ~(E)-2-[(,1R 9S 12S 13R 14S 17R 21 S 23S 24R 25S 27R)-17-ethyl-
1.14-
dihydroxy -23 25 dimethox~-13 19 21 27-tetramethyl-2 3 10 16-tetraoxo-11,28-
dioxa-4-
azatricyclo[22 3 1 04~91octacos-18-en-12-yll-I-propenyll-2-methoxycyclohexyl
methyl 2-
(4-mor~holinyl)ethyl phosphate
A solution of the product from Example 3 ( 1.067 g, I mmol) in tetrahydrofuran
acetonitril (1:1) (20 mL) under a nitrogen atmosphere was treated with 2-(4-
morpholinyl)-
1-ethanol (182 ml, 1.5 mmol) and 1H-tetrazole (35 mg, 0.5 mmol) and stirred
for 50
minutes. During this period, the starting material was consumed, according to
TLC
(solvent used: 40 % acetone in hexane). Tert-butylhydroperoxide (0.081 mL, 2
mmol, 3M
in hexanes) was directly added to the reaction mixture and stirred at ambient
temperature
for 2 hours. The solvents were removed and the residue was dissolved in ethyl
acetate (50
mL), quickly washed with 10% sodium bicarbonate, brine, and dried (MgS04). The
obtained product (886.6 mg) was dissolved in acetonitrile (SO mL) and treated
with 3 mL
of 48%hydrofluoric acid. After stirring at ambient temperature for 3 hours,
wet ice. ethyl
acetate ( I 00 mL), and 10% sodium hydrogen carbonate (20 mL) were carefully
added in
succession to the cooled reaction mixture. The phases were separated and the
aqueous
layer was extracted with ethyl acetate (50 mL). The combined ethyl acetate
layers were
washed in succession with 10% sodium hydrogen carbonate, brine, and dried
(MgSO;).
Evaporation of the solvent, followed by drying under high vaccum provided
650.8 mg of
crude product. The crude residue was purified by reverse phase high
performance liquid
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
57
chromatography, eluting with a gradient of 80%-water-20%-acetonitrile-0.1 %-
trifluoroacetic acid to acetonitrile-0.1%-trifluoroacetic acid to provide
(152.1 mg) the title
compound.
MS (FAB) m/z: M+H-=999; M+K-=1037;
'3C NMR (500 MHz in Pyridine-d;) ~ 211.5 (q), 210.7 (q, minor), 199.6 (q,
minor), 198.8
(q), 170.0 (q, major and minor), 167.2 (q, minor), 166.9 (q), 162.3 and 162.0
(q,
CF3C00), 139.3 (q, minor), 138.4 (q), 133.4 (q), minor), 133.1 (q), 132.0 (t),
131.0 (t,
minor), 124.6 (t, .minor), 124.4 (t), 99.7 (q, minor), 98.8 (q), 81.8 (t),
81.7 (t, minor), 80.8
(t, major and minor), 80.5 (t), 79.4 (t, minor), 77.8 (t, minor), 76.2 (t),
74.7 (t, minor), 74.3
(t), 73.9(t, minor), 73.0 (t), 69.7 (t), 68.8 (t, minor), 66.9(s, morpholine
ring-OCH2), 64.9
(s, P-OCH2-CH2-N, minor), 64.6 (s, P-OCH2-CH2-N), 58.5 (s, P-OCH2-CH2-N), 58.4
(s,
P-OCH2-CH2-N, minor), 57.6 (t, minor), 57.4 (t), 57.0 (t), 56.9 (t, major and
minor), 56.1,
(t), 55.8 (t, minor), 55.5 (t), 55.2 (t, minor), 54.3 (p, P-OCH3), 54.0 (s,
morpholine ring
NCH2), 52.9 (t), 49.3 (s), 47.8 (s, minor), 47.8 (s), 46.5 (s, minor), 44.5
(s, minor), 41.4
(t), 41.0 (t), 39.4 (s), 36.3 (s), 35.8 (t), 35.5 (t, minor), 35.0 (s, minor),
34.6 (t), 34.3 (s),
33.1 (s), 32.8 (s, minor), 31.8 (s), 30.5 (s, major and minor), 28.1 (s), 27.5
(s, minor and
minor), 26.~ (t), 25.0 (s), 24.8 (s, minor), 24.4 (s, minor), 21.8 (minor),
21.5 (s), 20.5 (p,
minor), 19.9 (p), 16.8 (p), 16.8 (p, minor), 16.2 (p, minor), 15.9 (p), 13. 6
(p, minor), 13.4
(p), 11.9 (p, minor), 11.8 (p), 10.7 (p), 10.5 (p, minor);
'H NMR (500 MHz in CDCl3) 8 0.84 (m, CH3), 0.87 (m, CH3), 0.92 (d, J=SHz, CH;,
minor), 0.96 (d, J=SHz, CH3), 0.98 (d, J=SHz, CH3, minor), 1.01 (d, J=SHz,
CH3), 1.05
(m, CH2), 1.6 (s, CH3), 1.63 (s, CH;, minor), 1.64 (s, CH;), 1.66 (s, CH;,
minor), 1.78 (m,
CH2, CH), 1.92 (m, CH2), 2.18 (m, CH2), 2.31 (m, CH, CH and CH), 2.75 (m, CH,
minor), 2.8 (m, CH), 3.3 (m, CH2), 3.21 (m, 21 and CH), 3.31 (s, OCH;), 3.34
(s, OCH3,
minor), 3.38 (s, OCH;, minor), 3.40 (s, OCH;), 3.43 (s, OCH3 major and minor),
3.51 (m,
CH), 3.58 (m, CH), 3.67 (d, J=lOHz, CH), 3.82 (s, P-OCH3, minor), 3.84 (s, P-
OCH3),
3.80 (m, CH and CH major and minor), 3.89 (m, P-OCH2 and morpholine ring-
OCH2),
4.18, (m, CH), 4.44 (m, CH2), 4.61 (d, J=SHz, CH), 5.04 (m, CH major and
minor, CH),
5.2 (br. s, CH, minor), 5.35 (br. s, CH);
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
58
Elemental Analysis calculated for C;oHB~N,O,6P 1.5 CF3COOH: C; 54.39; H, 7.27;
N,
2.39. Found C, 54.47; H, 7.29; N, 2.40.
Example 20
(IR 2R 4R)-4-~~E)-2-[(1R 9S 12S 13R 14S 17R 21 S 23S,24R,25S,27R)-17-ethyl-
1,14-
dih~droxy-23 25-dimethoxy-13 19 21 27-tetramethyl-2 3 10,16-tetraoxo-11,28-
dioxa-4-
azatricyclo[2~ 3 1 Oav]octacos-18-en-12- l~l-1-propenyl~-2-methoxycyclohexyl
methyl 2-
(I-py~'olidinyl)ethyl phosphate
The product from Example 3 (1.067 g, 1 mmol), 2-(I-pyrrolidinyl)-1-ethanol
(175.4 pL, 1.5 mmol), and the procedure described in Example 19 were used to
provide
( 122.3 mg) the title compound.
MS (FAB) m/z: M+H~=983; M+KT=1021;
"C NMR (500 MHz in Pyridine-d;) b 211.5 (q), 210.7 (q, minor), 199.6 (q,
minor), 198.8
(q), 170.0 (q, major and minor), 167.2 (q, minor), 166.9 (q), 162.3 and 162.0
(q,
CF3C00), 139.3 (q, minor), 138.4 (q), 133.4 (q), minor), 133.1 (q), 132.0 (t),
131.0 (t,
minor), 124.6 (t, .minor), 124.4 (t), 99.7 (q, minor), 98.8 (q), 81.8 (t),
81.7 (t, minor), 80.8
(t, major and minor), 80.5 (t), 79.4 (t, minor), 77.8 (t, minor), 76.2 (t),
74.7 (t, minor), 74 3
(t), 73.9(t, minor), 73.0 (t), 69.7 (t), 68.8 (t, minor), 63.9 (s, P-OCH2-C~I2-
N, minor), 63.6
(s, P-OCH2-CH2-N), 57.6 (t, minor), 57.4 (t), 57.0 (t), 56.9 (t, major and
minor), 56.1, (t),
55.8 (t, minor), 55.5 (t), 55.2 (t, minor), 54.6 (p, P-OCH3), 54.2 (s, P-OCH2-
CH2-N and
pyrrolidine ring 2-CH2 and 5-CH2), 52.9 (t), 49.3 (s), 47.8 (s, minor), 47.8
(s), 46.5 (s,
minor), 44.5 (s, minor), 41.4 (t), 41.0 (t), 39.4 (s), 36.1 (s), 35.8 (t),
35.4 (t, minor), 35.0 (s,
minor), 34.6 (t), 34.3 (s), 33.1 (s), 32.8 (s, minor), 31.7 (s), 30.5 (s,
major and minor), 28.1
(s), 27.5 (s, minor and minor), 26.5 (t), 25.0 (s), 24.8 (s, minor), 24.4 (s,
minor), 23.3 (s,
pyrrolidine ring 3-CH2 and 4-CH2), 21.8 (minor), 21.5 (s), 20.5 (p, minor),
19.9 (p), 16.8
(p), 16.8 (p, minor), 16.2 (p, minor), 15.9 (p), 13. 6 (p, minor), 13.4 (p),
11.9 (p, minor),
11.8 (p), 10.7 (p), 10.5 (p, minor);
'H NMR (500 MHz in CDCI~) 8 0.84 (m, CH3), 0.87 (m, CH;), 0.92 (d, J=SHz, CH;,
minor), 0.96 (d, J=SHz, CH;), 0.98 (d, J=SHz, CH;, minor), 1.01 (d, J=SHz,
CH3), 1.05
(m, CH2), 1.6 (s, CH3), 1.63 (s, CH3, minor), 1.64 (s, CH3), 1.66 (s, CH;,
minor), 1.78 (m,
CH2, CH), 1.92 (m, CH2), 2.18 (m, CH2), 2.31 (m, CH, CH and CH), 2.75 (m, CH,
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
59
minor), 2.8 (m, CH), 3.3 (m, CH2), 3.21 (m, 21 and CH); 3.31 (s, OCH;), 3.34
(s, OCH3,
minor), 3.38 (s, OCH;, minor), 3.40 (s, OCH;), 3.43 (s, OCH; major and minor),
3.51 (m,
CH), 3.58 (m, CH), 3.67 (d, J=IOHz, CH), 3.82 (s, P-OCH;, minor), 3.84 (s, P-
OCH;),
3.80 (m, CH and CH major and minor), 4.18, m, CH), 4.44 (m, CH2), 4.61 (d,
J=SHz,
CH), 5.04 (m, .CH major and minor, CH), 5.2 (br. s, CH, minor), 5.35 (br. s,
CH);
Elemental Analysis calculated for CSOHg3N~O,5P 1.5 CF3COOH: C, 55.15; H, 7.37;
N,
2.42. Found C, 54.98; H, 7.36; N, 2.45.
Example 21
(1R 2R 4R)-4-~(E)-2-f(1R 9S 12S 13R 14S 17R ~1S 23S 24R 25S 27R)-17-ethyl-1,14-
dihydroxy-23 25-dimethoxy-13 19 21 27-tetramethyl-2 3 10.16-tetraoxo-11,28-
dioxa-4
azatric~clo[22 3 1 O4°9loctacos-18-en-12-~l-l~ropenyl~-2-
methoxycyclohexyl 4
fluor~henyl methyl phosphate
The product from Example 3, 4-fluorophenol, and the procedure described in
Example 5 were used to provide the title compound.
MS (FAB) m/z: M+K+=1018;
'3C NMR (500 MHz in Pyridine-ds) 8 211.6 (q), 210.8 (q, minor), 199.6 (q,
minor), 198.8
(q), 170.2 (q, minor), 170.1 (q), 167.4 (q, minor), 167.0 (q), 161.0 (q, Ar),
159.1 (q, .Ar),
139.4 (q, minor), 138.5 (q), 133.5 (q), minor), 133.2 (q), 132.1 (t), 131.2
(t, minor), 124.7
(t, minor), 124.5 (t), 122.3 (t, 2x Ar), 116.7 (t, 2xAr), 99.8 (q, minor),
98.9 (q), 81.9 (t),
81.8 (t, minor), 81.7 (t), 81.6 (t, minor), 80.7 (t), 79.5 (t, minor), 77.9
(t, minor), 76.3 (t),
74.8 (t, minor), 74.3 (t), 74.0 (t, minor), 73.1 (t), 69.7 (t), 68.9 (t,
minor), 57.7 (t, minor),
57.5 (t), 57.1 (t), 57.0 (t, major and minor), 56.1 (t), 55.9 (t, minor), 55.5
(t), 55.3 (t,
minor), 55.1, (p, P-OCH3), 54.8 (p, P-OCH3, minor), 53.1 (t), 49.4 (s), 48.0
(s, minor),
47.9 (s), 46.6 (s, minor), 44.6 (s, minor), 41.4 (t), 40.9(t, minor), 39.5
(s), 36.2 (s), 35.9 (t),
35.6 (t, minor), 35.2 (s, minor), 34.6 (t), 34.5 (s), 33.1 (s), 32.9 (s,
minor), 31.7 (s, major
and minor), 30.5 (s, major and minor), 28.2 (s), 27.6 (t, minor), 27.5 (s,
minor), 26.6 (t),
25.0 (s), 24.9 (s, minor), 24.4 (s, minor), 21.8 (minor), 21.5 (s), 20.6 (p,
minor), 20.0 (p),
16.9 (p), 16.8 (p, minor), 16.3 (p, minor), 16.0 (p), 13.7 (p, minor), 13.4
(p), 12.0 (p,
minor), 11.8 (p), 10.7 (p), 10.6 (p, minor);
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
'H NMR (500 MHz in Pyridine-d;) 8 0.82 (t, J=7.SHz, CH3), 0.87 (t, J=7.SHz,
CH;,
minor), 0.94 (s, CH3), 0.95 (s, CH;, minor), 1.0 (m, CH2), 1.10 (m, CH2), 1.17
(m, 25 and
CH; minor), 1.2 (d, J=7.SHz, CH;), 1.27 (d, J=7.SHz, CH;), 1.42-1.58 (m, CH2,
CHI.
CH2, CH2, and CH2), 1.62 (s, CH3), 1.79 (s, CH;, minor), 1.80 (s, CH3), 1.86
(s, CH;,
5 minor), 1.98 (m, CH), 2.10 (m, CH2, CHI, and CH), 2.22 (m, CH2), 2.31, 2.35
(m, CH2
and CH)2.92 (m, CHI, minor), 2.57 (m, CH), 2.67 (ddd, Jl=J2=7.SHz, J3=lSHz,
CH),
2.77 (m, CH, minor), 2.91 (m, CH, minor), 3.02 (m, CH, minor), 3.08 (m, CH),
3.23 (m,
CH2), 3.3 (m, CH), 3.34 (s, OCH;, minor), 3.36 (s, OCH;), 3.42 (s, OCH;), 3.44
(s,
OCH;), 3.46 (s, OCH;, minor), 3.55 (m, CH major and minor), 3.62 (m, CH2,
minor),
10 3.68 (m, CH major and minor), 3.91 (s, P-OCH;), 3.94 (s, P-OCH3, minor),
3.89 (m, CH
major and minor), 4.19 (d, J=l OHz, CH), 4.28 (dd, J1=2.SHz, J2=l OHz, CH,
minor), 4.50
(m, CH), 4.58 (m, CH), 4.72 (m, CH2), 5.10 (m, CH major and minor), 5.23 (m,
CH),
5.41 (m, CH), 5.53 (d, J=SHz, CH), 5.81 (d, .1=SHz, CH), 5.87 (d, J=SHz, CH,
minorl,
7.16 (m, 2xArCH), 7.48 (m, 2xArCH);
15 Elemental Analysis calculated for C;oH,;FNO,;P 0.~ HpO: C, 60.71; H, 7.74;
N, 1.41.
Found C, 60.73; H, 7.44; N, 1.12.
Example 22
(1R 2R 4R) 4 ~~E) 2 ~(1R 9S 12S 13R 14S 17R 21S ?3S 24R 25S 27R)-17-ethyl-1,14-
20 dihydroxy 23 25 dimethoxy-13 19 21 27-tetramethyl-~ 3 10 16-tetraoxo-11,28-
dioxa-4-
azatricycloj22 3 1 04°~]octacos-18-en-1~-~l-I-propenyl~-2-
methoxycyclohexvl 4-
form~,phen~l methyl phosphate
A solution of the product from Example 3 (1.600 g, 1.5 mmol) in
tetrahydrofuran
acetonitrile ( 1:1 ) (30 mL) under a nitrogen atmosphere was treated with 4-
25 hydroxybenzaldehyde(274.8 mg, 2.25 mmol) and 1 H-tetrazole (52.5 mg, 0.7~
mmol).
After stirring for 30 minutes, tert-butylhydroperoxide (0.122 mL, 3 mmol, in
2,2,4-
trimethylpentane) was directly added to the reaction mixture and allowed to
stir at ambient
temperature overnight. The solvents were removed and the residue was dissolved
in ethyl
acetate (50 mL), quickly washed in succession with 10% sodium bicarbonate,
brine, and
30 dried (MgS04). The crude product (1.68 g) was dissolved in acetonitrile (45
mL) and
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
61
treated with 48% hydrofluoric acid (1.5 mL). After stirring for 30 minutes,
the mixture
was cooled and ethyl acetate and cold 10% sodium hydrogen sulfate were
carefully added
to the reaction mixture. The phases were separated and the aqueous layer
extracted twice
with ethyl acetate (50 mL). The combined ethyl acetate layers were washed in
succession
with 10% sodium hydrogen sulfate, brine, and dried (MgSO~). Solvent was
evaporated to
dryness to provide 1.26 g of crude product. The residue was purified by
reverse phase
high performance liquid chromatography, eluting with 30% acetone in hexane to
provide
(586 mg) the title compound.
MS (FAB) m/z: M+K-=1028;
'3C NMR (500 MHz in Pyridine-d5) b 211.7 (q), 210.8 (q, minor), 199.7 (q,
minor), 198.9
(q), 191.2 (t, CHO), 170.2 (q, minor), 170.1 (q), 167.4 (q, minor), 167.1 (q),
161.3 (q, Ar),
156.2, 156.2 (q; Ar-), 139.4 (q, minor), 138.5 (q), 133.5 (q), minor), 133.2
(q), 132.2 (t),
131.9 (t, Ar), 131.5 (t, Ar), 131.1 (t, minor), 124.7 (t, minor), 124.5 (t),
121.3 (t, Ar), 99.8
(q, minor), 98.9 (q), 82.3 (t), 82.2 (t, minor), 81.6 (t), 81.5 (t, minor),
80.7 (t), 79.8 (t,
minor), 77.9 (t, minor), 76.3 (t), 74.8 (t, minor), 74.4 (t), 74.0 (t, minor),
73.1 (t), 69.7 (t),
68.8 (t, minor), 57.7 (t, minor), 57.6 (t), 57.1 (t), 56.9 (t, minor), 56.7
(t), 56.2 (t), 55.9 (t,
minor), 55.6 (t), 55.3 (t, minor), 54.9, (p, P-OCH3), 53.1 (t), 49.4 (s), 48.0
(s, minor), 47.9
(s), 46.6 (s, minor), 44.7 (s, minor), 41.4 (t), 40.9 (t), 39.5 (s), 36.4 (s),
35.9 (t), 3~.6 (t,
minor), 35.2 (s, minor), 34.7 (t), 34.6 (s), 33.1 (s), 32.9 (s, minor), 31.7
(s), 30.6 (s, minor),
30.5 (s), 28.2 (s), 27.6 (s, minor), 27.6 (t, minor), 26.6 (t), 25.1 (s), 24.9
(s, minor), 24.5 (s,
minor), 21.9 (minor), 21.6 (s), 20.6 (p, minor), 20.0 (p), 16.9 (p), 16.9 (p,
minor), 16.3 (p,
minor). 16.0 (p), 13.7 (p, minor), 13.5 (p), 12.0 (p, minor), 11.9 (p), 10.8
(p), 10.6 (p,
minor);
'HNMR (500 MHz in Pyridine-d;) 8 0.82 (t, J=7.5Hz, 36-CH;), 0.87 (t, J=7.SHz,
CH;,
minor), 0.94 (s, CH3), 0.95 (s, CH3, minor), 1.0 (m, CH2), 1.10 (m, CH2), 1.17
(m, CHI
and CH; minor), 1.2 (d, J=7.5Hz, CH3), 1.27 (d, J=7.5Hz, CH3), 1.42-1.58 (m,
CH2, CH2,
CH2, CH2, and CH2), 1.62 (s, CH;), 1.79 (s, CH;, minor), 1.80 (s, CH;), 1.86
(s, CH;,
minor), 1.98 (m, CH), 2.10 (m, CH2, CH2, and CH), 2.22 (m, CH2), 2.31, 2.35
(m, CH2
and CH), 2.92 (m, CH2, minor), 2.57 (m, CH), 2.67 (m, CH), 2.77 (m, CH,
minor), 2.91
(m, CH, minor), 3.02 (m, CH, minor), 3.08 (m, CH), 3.23 (m, CH2), 3.3 (m, CH),
3.29 (s,
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
62
OCH;, minor), 3.34 (s, OCH;), 3.42 (s, OCH3), 3.44 (s, OCH;), 3.46 (s, OCH;,
minor),
3.55 (m, CH major and minor), 3.62 (m, CHI, minor), 3.68 (m, CH major and
minor), 3.89
(m, CH major and minor), 3.93 (s, P-OCH;), 3.95 (s, P-OCH;, minor), 4.19 (d,
J=IOHz,
CH), 4.28 (dd. Jl=2.5Hz, J2=IOHz, CH, minor), 4.50 (m, CH), 4.58 (m, CH minor
and
CH), 4.72 (d, CH2), 5.10 (m, CH major and minor), 5.13 (m, CH), 5.31 (m, CH2),
5.41 (m,
CH), 5.53 (d, J=SHz, CH), 5.81 (m, CH), 5.87 (m, CH, minor), 7.63 (m, Ar-CH),
7.97 (m,
Ar-CH), 10.04 (m, CHO);
Elemental Analysis calculated for C5,H,6NO,6P: C, 61.86; H, 7.73; N, 1.41.
Found C,
61.53; H, 8.03; N, 1.47.
Example 23
4-acet~phenyl (1R 2R 4R)-4-~(E~2-[(1R 9S 12S 13R 14S 17R.21S,23S,24R,25S,27R)-
17-ethyl-1 14-dihydroxy-~3 25-dimethoxy-13 19 21 27-tetramethyl-2,3,10,16-
tetraoxo-
11 28-dioxa-4-azatricyclo X22.3 .1.04°9]octacos-18-en-12-yl]-1-propenyl
) -2-
methox~cYclohexyl methyl phosphate
The product from Example 3, 1-(4-hydroxyphenyl)-1-ethanone, and the procedure
described in Example 5 were used to provide the title compound.
MS (FAB) m/z: M+K'=1042;
~3C NMR (500 MHz in Pyridine-d;) 8 211.7 (q), 210.8 (q, minor), 199.7 (q,
minor), 198.9
(q), 196.7 (q, COCH3, minor), 196.0 (q, COCH3), 170.2 (q, minor), 170.1 (q),
167.4 (q,
minor), 167.1 (q), 155.1, (q, Ar-1C), 139.4 (q, minor), 138.5 (q), 134.6 (q,
Ar-4C), 134.5
(Ar-4C, minor), 133.5 (q), minor), 133.3 (q), 132.1 (t), 131.1 (t, minor),
130.8 (t, Ar),
130.3 (t, Ar), 124.7 (t, minor), 124.5 (t), 120.7 (t, Ar), 99.8 (q, minor),
98.9 (q), 82.3 (t),
82.2 (t, minor), 81.6 (t), 81.5 (t, minor), 80.7 (t), 79.8 (t, minor), 77.9
(t, minor), 76.3 (t),
74.8 (t, minor), 74.4 (t), 74.0 (t, minor), 73.1 (t), 69.7 (t), 68.8 (t,
minor), 57.7 (t, minor),
57.6 (t), 57.1 (t), 56.9 (t, minor), 56.8 (t), 56.2 (t), 55.9 (t, minor), 55.6
(t), 55.3 (t, minor),
54.9, (p, P-OCH3), 53.1 (t), 49.4 (s), 48.0 (s, minor), 47.9 (s), 46.6 (s,
minor), 44.7 (s,
minor), 41.4 (t), 40.9 (t), 39.5 (s), 36.4 (s), 35.9 (t), 35.6 (t, minor),
35.2 (s, minor), 34.7
(t), 34.6 (s), 33.1 (s), 32.9 (s, minor), 31.7 (s), 30.6 (s, minor), 30.5 (s),
28.2 (s), 27.6 (s,
minor), 27.6 (t, minor), 26.6 (t), 25.1 (s), 24.9 (s, minor), 24.5 (s, minor),
21.9 (minor),
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
63
21.6 (s), 20.6 (p, minor), 20.0 (p), 16.9 (p), 16.9 (p, minor), 16.3 (p,
minor), 16.0 (p), 13.7
(p, minor),13.5 (p), 12.0 (p, minor), 11.9 (p), 10.8 (p), 10.6 (p, minor);
'H NMR (500 MHz in Pyridine-d;) ~ 0.82 (m, CH;), 0.87 (t, J=7.SHz, CH3,
minor), 0.94
(s, CH3), 0.95 (s, CH;, minor), 1.0 (m, CH2), 1.10 (m, CH2), 1.17 (m, 25 and
CH3 minor),
1.2 (m; CH;), 1.27 (m, CH;), 1.42-1.58 (m, CHI, CHI, CHI, CH2, and CH2), 1.62
(s,
CH3), 1.79 (s, CH;, minor), 1.80 (s, CH;), 1.86, (s, CH;, minor), 1.98 (m,
CH), 2.10 (m,
CH2, CH2, and CH), 2.22 (m, CH2), 2.31, 2.35 (m, CHI and CH), 2.44 (Phenyl-
COCH;,
diasteriomer); 2.5 (s, phenyl-COCH3, diasteriomer), 2.92 (m, CH2, minor), 2.57
(m, CH),
2.67 (m, CH), 2.77 Vim, CH, minor), 2.91 (m, CH, minor), 3.02 (m, CH, minor),
3.08 (m,
CH), 3.23 (m, CH2), 3.3 (m, CH), 3.29 (s, OCH;, minor), 3.34 (s, OCH;), 3.42
(s, OCH;),
3.44 (s, OCH;), 3.46 (s, OCH;, minor), 3.55 (m, CH major and minor), 3.62 (m,
CH2,
minor), 3.68 (m, CH major and minor), 3.89 (m, CH major and minor), 3.93 (s, P-
OCH3),
3.95 (s, P-OCH;, minor), 4.19 (m, CH), 4.28 (m, JCH, minor), 4.50 (m, CH),
4.58 (m, CH
minor and CH), 4.72 (m, CH2), 5.10 (m, CH major and minor), 5.13 (m, CH), 5.41
(m,
CH), 5.53 (m, CH), x.81 (m, CH), 5.87 (m, CH, minor), 6.33, 6.41 (m), 7.93 (m,
ArCH),
8.03 (m, ArCH), 8.09 (m, ArCH);
Elemental Analysis calculated for CSZH,8NO;6P 0.5 HBO: C, 61.84; H, 7.85; N,
1.38.
Found C, 61.54; H, 7.85; N, 1.41.
Example 24
_d_ieth 1 (1R 2R 4R) 4 ~(E) 2 f(1R 9S 12S 13R 14S 17R 21 S 23S 24R 2~S 27R1-17-
ethvl
1 14 dihydroxy 23 25 dimethoxy-13 19 21 27-tetramethvl-2,3,10,16-tetraoxo-
11,28
dioxa 4 azatricyclof22 s 1 04~91octacos 18-en-12-yl]-1-propenyll-2-
methoxvcvclohexvl
phosphate
A solution of ascomycin (474 mg, 0.6 mmol) and triethylamine (0.250 ml, 1.8
mmol) in benzene (8 ml) at 0 °C was treated with diethylchlorophosphate
(0.250 ml, 1.8
mmol) followed by 4-dimethylaminopyridine ( 15 mg, 0.12 mmol). The reaction
mixture
was stirred at 0 °C for 15 minutes, allowed to warm to ambient
temperature, and stir
overnight. The reaction mixture was quenched with 10% NaHS04 (20 ml) and
partitioned
with ethyl acetate (40 ml). The organic layer was successively washed with 10%
NaHSO~
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
64
(20 ml, X2), saturated NaHCO~ (20 ml, X2), brine (20 ml, X3), and dried
(Na,SO~).
Following evaporation to dryness, the crude residue (632 mg) was purified by
reverse
phase HPLC (Delta Prep) using a 1" phenyl column eluting with a gradient of
CH;CN:
10% CH;OH/H20 (program: 30% to 70% of 10% methanol/water over 35 minutes;
peaks
collected at 215 l; number of injections = 3, flow rate 35 ml/min). Peaks
collected were
lyophilized to provide the title compound (140 mg, 25%).
MS (FAB) m/z: M+K~=966
'3C NMR (500 MHz in CDC13) 8 213.64 (q, minor), 213.56 (q), 196.16(q), 192.38
(q,
minor), 168.96 (q); 168.62 (q, minor), 165.83 (q, minor), 164.69 (q), 139.67
(q, minor),
138.77 (q), 132.75 (q), 132.14 (q, minor), 128.88 (t, minor), 128..80 (t,
minor), 123.28 (t,
minor), 122.99 (t), 98.67 (q), 96.99 (q), 81.52 (t, minor), 81.48 (t), 80.4
(t, minor), 80.37
(t), 77.54 (t, minor), 77.67 (t), 76.61 (t, minor). 75.20 (t), 73..70 (t),
73.63 (t, minor), 72.91
(t), 72.16 (t, minor), 70.13 (t), 69.17 (t, minor), 63.62, 63.56 (s, P-
OCH2CH3), 63.38,
63.34 (s, P-OCH~CH;), 57.45 (p, minor), 56.94 (p), 56.61 (p), 56.31 (t), 54.98
(t, minor),
54.64 (t), 52.70 (t, minor), 48.60 (s), 48.47 (s, minor), 43.83 (s, minor),
43.04 (s, minor),
42.82 (s), 40.14 (t, minor), 39.6 (t), 39.23 (s), 36.27 (s), 36.15 (s, minor),
35.55 (s, minor),
34.56 (t), 34.43 (t), 33.59 (t, minor), 32.85 (s), 32.74 (s), 32.58 (s,
minor), 31.40 (s), 30.47
(s), 27.60 (s), 26.38 (t), 26.20 (s, minor), 25.98 (t, minor), 24.55 (s,
minor), 24.51 (s),
24.64 (s, minor), 24.14 (s), 21.18 (s), 20.84 (minor), 20.53 (p), 19.44 (p,
minor), 16.29 (p),
16.12 (p, P-OCH2CH3), 16.06 (p, P-OCH2CH;), 15.88 (p), 14.33 (p, minor), 14.28
(p),
11.68 (p), 9.74 (p, minor), 9.37 (p);
'H NMR (500 MHz in CDC13) 8 0.84 (m, CH;), 0.87 (m, CH;), 0.92 (d, J=SHz, CH;,
minor), 0.96 (d, J=SHz, CH;), 0.98 (d, J=SHz, CH;, minor), 1.01 (d, J=SHz,
CH3), 1.05
(m, CH2), 1.35 (t, J=7.SHz, P-OCH~CH3), 1.6 (s, CH;)> 1.63 (s, CH3, minor),
1.64 (s,
CH3), 1.66 (s, CH;, minor), 1.78 (m, CH2, CH), 1.92 (m, CHZ), 2.18 (m, CH2),
2.31 (m,
CH, CH and CH), 2.75 (m, CH, minor), 2.8 (ddd, J1=J2=2.SHz, J3=lSHz, CH), 3.3
(m,
CH2); 3.21 (m, 21 and CH), 3.31 (s, OCH3), 3.34 (s, OCH3, minor), 3.38 (s,
OCH;,
minor), 3.40 (s, OCH3), 3.43 (s, OCH3 major and minor), 3.51 (m, CH), 3.58 (m,
CH),
3.67 (d, J=IOHz, CH), 3.80 (m, CH and CH major and minor), 4.15 (m, CH and P-
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
OCH~CH3), 4.23 (s, OH), 4.44 (m, CH2), 4.61 (d, J=SHz, CH), 4.89 (s, OH), 5.04
(m, CH
major and minor, CH). ~.2 (br. s, CH, minor), 5.35 (br. s, CH);
Elemental Analysis calculated for C4,H,8NO,;P: C, 60.82; H, 8.47; N, 1.50.
Found C,
60.43; H, 8.59; N, 1.38.
5
Example 25
(1R 2R 4R) 4 ;(E)-2-[~1R 9S 12S 13R 14S 17R 21S ~3S 24R 25S 27R)-17-ethyl-1,14-
dihydrox~23 25-dimethoxy-13 19 21 27-tetramethvl-2 3.10.16-tetraoxo-11,28-
dioxa-4-
azatric~cloL22 3 1 OY~9loctacos-18-en-12-~l-1-propenyl r-2-methoxycyclohexyl
diphenvl
10 phosphate
A solution of ascomycin (500 mg, 0.63 mmol) and triethylamine (0.175 mL, 1.26
mmol) in benzene (10 ml) was treated with diphenylchlorophosphate (0.265 ml,
1.26
mmol) followed by 4-dimethylaminopyridine ( 10 mg, 0.082 mmol). The reaction
mixture
was stirred at 0 °C for 15 minutes, allowed to warm to ambient
temperature, and stir
15 overnight. The reaction mixture was quenched with 10% NaHS04 (20 ml) and
partitioned
with ethyl acetate (40 ml). The organic layer was successively washed with 10%
NaHSO4
(20 -ml, X2), saturated NaHCO~ (20 ml, X2), brine (20 ml, X3) and dried
(Na,S04).
Following evaporation to dryness, the crude residue (583 mg) was purified by
reverse
phase HPLC (Delta Prep) using a 2" phenyl column eluting with a gradient of
20 CH3CN:10% CH;OH/H20 (program: 60% to 0% of 10% methanol/water over 55
minutes; peaks collected at 215 1; number of injections = 7). Peaks collected
were
lyophilized to provide the title compound (105 mg, 16%).
MS (FAB) m/z: M+K~=1062;
'3C NMR (500 MHz in CDCI;) b 213.64 (q, minor), 213.56 (q), 196.16(q), 192.38
(q,
25 minor), 168.96 (q), 168.62 (q, minor), 165.83 (q, minor), 164.69 (q),
150.85 (q, Ar,
minor), 150.79 (q, Ar), 150.60 (q, Ar, minor), 150.54 (q, Ar), 139.67 (q,
minor), 138.77
(q), 132.75 (q), 132.14 (q, minor), 129.52 (t, Ar, minor). 129.49 (t, Ar),
128.59 (t), 125.08
(t, Ar, minor), 124.93 (t, Ar), 123.28 (t, minor), 122.99 (t), 120.19 (t, Ar,
minor), 120.15 (t,
Ar), 120.05 (t, Ar, minor), 120.01 (t, Ar), 98.67 (q), 96.99 (q), 82.29 (t),
82.17 (t, minor),
30 81.22 (t, minor), 81.17 (t), 77.54 (t, minor), 77.67 (t), 76.61 (t, minor).
75.20 (t), 73..70 (t),
73.63 (t; minor), 72.91 (t), 72.16 (t, minor), 70.13 (t), 69.17 (t, minor),
57.45 (p, minor),
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
66
56.94 (p), 56.61 (p), 56.31 (t), 54.98 (t, minor), 54.64 (t), 52.70 (t,
minor), 48.60 (s), 48.47
(s, minor), 43.83 (s, minor), 43.04 (s, minor). 42.82 (s), 40.14 (t, minor),
39.6 (t), 39.23
(s); 36.10 (s), 35.99 (s, minor), 35.45 (s, minor), 34.49 (t), 34.26 (t),
33.59 (t, minor),
32.85 (s), 32.74 (s), 32.58 (s, minor), 31.12 (s), 30.33 (s), 27.60 (s), 26.38
(t), 26.20 (s,
minor), 25.98 (t, minor), 24.55 (s, minor), 24.51 (s), 24.64 (s, minor), 24.14
(s), 21.18 (s),
20.84 (minor), 20.53 (p), 19.44 (p, minor), 16.29 (p), 15.88 (p), 14.33 (p,
minor), 14.28
(p), 11.68 (p), 9.74 (p, minor), 9.37 (p);
'H NMR (500 MHz in CDCI;) 8 0.84 (m, CH;), 0.87 (m, CH;), 0.92 (d, J=SHz, CH;,
minorl, 0.96 (d, J=SHz, CH;), 0.98 (d, J=SHz, CH;, minor), 1.01 (d, J=SHz,
CH3), 1.05
(m, CH2) 1.6 (s, CH;), 1.63 (s, CH3, minor), 1.64 (s, CH3), 1.66 (s, CH3,
minor), 1.78 (m,
CHz, CH), 1.92 (m, CH2), 2.18 (m, CH2), 2.31 (m, CH, CH and CH), 2.75 (m, CH,
minor), 2.8 (ddd, J1=J2=2.SHz, J3=lSHz, CH), 3.3 (m, CH2), 3.21 (m, CH and
CH), 3.31
(s, OCH3), 3.34 (s, OCH;, minor), 3.38 (s, OCH3, minor), 3.40 (s, OCH;), 3.4 3
(s, OCH3
major and minor), 3.51 (m, CH), 3.58 (m, CH), 3.67 (d, J=l OHz, CH), 3.80 (m,
CH and
CH major and minor), 4.44 (m, CH2), 4.61 (d, .1=SHz, CH), 5.04 (m, CH major
and minor,
CH), 5.2 (br. s, CH, minor), 5.35 (br. s, CH);
Elemental Analysis calculated for C;;H,8N0,;P 0.75 HBO: C, 63.66; H, 7.72;
N,1.34.
Found C, 63.86; H, 7.84; N, 1.05.
Example 26
(1R 9S 12S 13R 14S 17R 21S 23S 24R 25S 27R)-17-ethyl-1,14-dihydroxy-23,25-
dimethox, -~~~E)-2-[(1R 3R)-3-methoxy4-oxocyclohexyll-1-methylethenyl~-
13 19 21 27-tetramethyl-11 28-dioxa-4-azatricyclo[22.3.1.0;vloctacos-18-ene-
2,3,10,16-
tetrone
Methylsulfide-chlorine complex was prepared by adding oxalyl chloride
(0.32g) into a solution of dimethylsulfoxide (0.44g) in 4 mL of methylene
chloride and
stirred at -70 °C for 30 minutes. The solution of the complex was
slowly added to a stirred
solution of ascomycin (1.6g) in 5 mL of methylene chloride at -70 °C.
After stirring for 30
minutes, triethylamine (1.4g) was added and the mixture was allowed to stir
for an
additional 30 minutes at -70 °C. The reaction mixture was then allowed
to warm to room
CA 02368463 2001-09-24
WO 00/58318 PCT/US00/07639
67
temperature followed by additional stirring for 1 hour. The reaction mixture
was diluted
with 100 mL of ether, washed with 1N-HCl aq. solution (30 mL x 2), brine (30
mL), dried
over magnesium sulfate anhydrous, and the solvent removed in vacuo. The crude
product
was purified on 70 gram of silica gel, eluting with ether to obtain 0.95 g of
the title
compound.
MS (FAB) m/z: (M+H)°=790.
Example 27
( 1R 9S,12S,13R,14S,17R 21 S 23S 24R,25S,27R~-17-ethyl-I 14-dihydroxy-12-~(E~-
f(1R 3R 4S) 4 hydroxy 3 methoxycyclohexyl]-I-methylethenyl~-23 25-dimethoxy-
13 19 21 27 tetramethyl 1I 28-dioxa-4-azatricyclo(~~ 3 l.Owloctacos-18-ene-
2,3,10,16-
tetrone
Lithium tri-n-butoxyaluminum hydride (0.2 mL, iM in THF) was slowly added
to a stirred solution of the product from Example 15 (0.058g) in 1 mL of
anhydrous THF
at -70 °C under a nitrogen atmosphere. After stirring at -70 °C
for 3 hours, the mixture was
partitioned between 50 mL of ether and 10 mL of IN-HC1. The etheral solution
was
washed with brine, dried over magnesium sulfate anhydrous. The obtained crude
product
was purified by preparative TLC, using 35% acetone in hexane as a solvent to
afford 0.025
g of the title compound.
MS (FAB) m/z: (M+K)T=830.
Phosphate analogs wherein R6 is -OP(O)AB and R' is hydrogen can be prepared
using the product from Example 27 and the procedures described in Examples 1-
25.
