U.S. patent application number 09/954782 was filed with the patent office on 2002-05-09 for water soluble rapamycin esters.
This patent application is currently assigned to American Home Products Corporation. Invention is credited to Fawzi, Mahdi, Zhu, Tianmin.
Application Number | 20020055518 09/954782 |
Document ID | / |
Family ID | 22878646 |
Filed Date | 2002-05-09 |
United States Patent
Application |
20020055518 |
Kind Code |
A1 |
Zhu, Tianmin ; et
al. |
May 9, 2002 |
Water soluble rapamycin esters
Abstract
This invention provide pegylated hydroxyesters of rapamycin
which are useful in inducing immunosuppression and in the treatment
of transplantation rejection, autoimmune diseases, solid tumors,
fungal infections, and vascular disease.
Inventors: |
Zhu, Tianmin; (Monroe,
NY) ; Fawzi, Mahdi; (Morristown, NJ) |
Correspondence
Address: |
Arnold S. Milowsky
American Home Products Corporation
Patent Law Department - 2B
Five Giralda Farms
Madison
NJ
07940
US
|
Assignee: |
American Home Products
Corporation
Madison
NJ
|
Family ID: |
22878646 |
Appl. No.: |
09/954782 |
Filed: |
September 18, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60233776 |
Sep 19, 2000 |
|
|
|
Current U.S.
Class: |
514/291 ;
540/456 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 35/00 20180101; A61P 9/00 20180101; A61K 47/60 20170801; A61P
37/06 20180101; C07D 498/18 20130101; A61P 19/02 20180101; A61P
11/00 20180101; A61P 31/10 20180101; A61P 29/00 20180101 |
Class at
Publication: |
514/291 ;
540/456 |
International
Class: |
C07D 281/18; A61K
031/4745 |
Claims
What is claimed is:
1. A compound having the structure 5wherein R.sup.1 and R.sup.2 are
each, independently, hydrogen or
--CO(CR.sup.3R.sup.4).sub.b(CR.sup.5R.sup.6).s-
ub.dCR.sup.7R.sup.8R.sup.9; R.sup.3 and R.sup.4 are each,
independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7
carbon atoms, alkynyl of 2-7 carbon atoms, trifluoromethyl, or --F;
R.sup.5 and R.sup.6 are each, independently, hydrogen, alkyl of 1-6
carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon
atoms, --(CR.sup.3R.sup.4).sub.fOR.su- p.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.8 and R.sup.9 are each, independently,
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10,
--CF.sub.3, --F, or --CO.sub.2R.sup.11; R.sup.10 is hydrogen or
--COCH.sub.2--S--CH.sub.2C-
H.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub.3; R.sup.11 is
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, or phenylalkyl of 7-10 carbon atoms;
b=0-6; d=0-6; f=0-6; n=5-450; with the proviso that R.sup.1 and
R.sup.2 are both not hydrogen and further provided that either
R.sup.1 or R.sup.2 contains at least one
--(CR.sup.3R.sup.4).sub.fOR.sup.10 group in which R.sup.10 is
--COCH.sub.2--S--CH.sub.2CH.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub-
.3, or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1, wherein R.sup.2 is hydrogen.
3. The compound of claim 2, wherein n=5-200.
4. The compound of claim 3, wherein n=8-135.
5. The compound of claim 4, wherein n=8-20.
6. The compound of claim 4, wherein n=90-120.
7. The compound of claim 1, which is rapamycin 42-ester with
3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid
methoxypoly(ethylene glycol) thiol 5000 conjugate.
8. The compound of claim 1, which is rapamycin 42-ester with
3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid
bis(methoxypoly(ethylene glycol) thiol 5000) conjugate.
9. A method of treating or inhibiting transplant rejection or graft
vs. host disease in a mammal in need thereof, which comprises
providing to said mammal an effective amount of a compound having
the strucure 6wherein R.sup.1 and R.sup.2 are each, independently,
hydrogen or
--CO(CR.sup.3R.sup.4).sub.b(CR.sup.5R.sup.6).sub.dCR.sup.7R.sup.8R.sup.9;
R.sup.3 and R.sup.4 are each, independently, hydrogen, alkyl of 1-6
carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon
atoms, trifluoromethyl, or --F; R.sup.5 and R.sup.6 are each,
independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7
carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.8 and R.sup.9 are each, independently,
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10,
--CF.sub.3, --F, or --CO.sub.2R.sup.11; R.sup.10 is hydrogen or
--COCH.sub.2--S--CH.sub.2C-
H.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub.3; R.sup.11 is
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, or phenylalkyl of 7-10 carbon atoms;
b=0-6; d=0-6; f=0-6; n=5-450; with the proviso that R.sup.1 and
R.sup.2 are both not hydrogen and further provided that either
R.sup.1 or R.sup.2 contains at least one
--(CR.sup.3R.sup.4).sub.fOR.sup.10 group in which R.sup.10 is
--COCH.sub.2--S--CH.sub.2CH.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub-
.3, or a pharmaceutically acceptable salt thereof.
10. A method of treating or inhibiting a solid tumor in a mammal in
need thereof, which comprises providing to said mammal a compound
having the structure 7wherein R.sup.1 and R.sup.2 are each,
independently, hydrogen or
--CO(CR.sup.3R.sup.4).sub.b(CR.sup.5R.sup.6).sub.dCR.sup.7R.sup.8R.sup-
.9; R.sup.3 and R.sup.4 are each, independently, hydrogen, alkyl of
1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7
carbon atoms, trifluoromethyl, or --F; R.sup.5 and R.sup.6 are
each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl
of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.8 and R.sup.9 are each, independently,
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10,
--CF.sub.3, --F, or --CO.sub.2R.sup.11; R.sup.10 is hydrogen or
--COCH.sub.2--S--CH.sub.2C-
H.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub.3; R.sup.11 is
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, or phenylalkyl of 7-10 carbon atoms;
b=0-6; d=0-6; f=0-6; n=5-450; with the proviso that R.sup.1 and
R.sup.2 are both not hydrogen and further provided that either
R.sup.1 or R.sup.2 contains at least one
--(CR.sup.3R.sup.4).sub.fOR.sup.10 group in which R.sup.10 is
--COCH.sub.2--S--CH.sub.2CH.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub-
.3, or a pharmaceutically acceptable salt thereof.
11. A method of treating or inhibiting a fungal infection in a
mammal in need thereof, which comprises providing to said mammal an
effective amount of a compound having the structure 8wherein
R.sup.1 and R.sup.2 are each, independently, hydrogen or
--CO(CR.sup.3R.sup.4).sub.b(CR.sup.5-
R.sup.6).sub.dCR.sup.7R.sup.8R.sup.9; R.sup.3 and R.sup.4 are each,
independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7
carbon atoms, alkynyl of 2-7 carbon atoms, trifluoromethyl, or --F;
R.sup.5 and R.sup.6 are each, independently, hydrogen, alkyl of 1-6
carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon
atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.8 and R.sup.9 are each, independently,
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10,
--CF.sub.3, --F, or --CO.sub.2R.sup.11; R.sup.10 is hydrogen or
--COCH.sub.2--S--CH.sub.2C-
H.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub.3; R.sup.11 is
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, or phenylalkyl of 7-10 carbon atoms;
b=0-6; d=0-6; f=0-6; n=5-450; with the proviso that R.sup.1 and
R.sup.2 are both not hydrogen and further provided that either
R.sup.1 or R.sup.2 contains at least one
--(CR.sup.3R.sup.4).sub.fOR.sup.10 group in which R.sup.10 is
--COCH.sub.2--S--CH.sub.2CH.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub-
.3, or a pharmaceutically acceptable salt thereof.
12. A method of treating or inhibiting rheumatoid arthritis in a
mammal in need thereof, which comprises providing to said mammal an
effective amount of a compound having the structure 9wherein
R.sup.1 and R.sup.2 are each, independently, hydrogen or
--CO(CR.sup.3R.sup.4).sub.b(CR.sup.5-
R.sup.6).sub.dCR.sup.7R.sup.8R.sup.9; R.sup.3 and R.sup.4 are each,
independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7
carbon atoms, alkynyl of 2-7 carbon atoms, trifluoromethyl, or --F;
R.sup.5 and R.sup.6 are each, independently, hydrogen, alkyl of 1-6
carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon
atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.8 and R.sup.9 are each, independently,
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10,
--CF.sub.3, --F, or --CO.sub.2R.sup.11; R.sup.10 is hydrogen or
--COCH.sub.2--S--CH.sub.2C-
H.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub.3; R.sup.11 is
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, or phenylalkyl of 7-10 carbon atoms;
b=0-6; d=0-6; f=0-6; n=5-450; with the proviso that R.sup.1 and
R.sup.2 are both not hydrogen and further provided that either
R.sup.1 or R.sup.2 contains at least one
--(CR.sup.3R.sup.4).sub.fOR.sup.10 group in which R.sup.10 is
--COCH.sub.2--S--CH.sub.2CH.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub-
.3, or a pharmaceutically acceptable salt thereof.
13. A method of treating or inhibiting multiple sclerosis in a
mammal in need thereof, which comprises providing to said mammal an
effective amount of a compound having the structure 10wherein
R.sup.1 and R.sup.2 are each, independently, hydrogen or
--CO(CR.sup.3R.sup.4).sub.b(CR.sup.5-
R.sup.6).sub.dCR.sup.7R.sup.8R.sup.9; R.sup.3 and R.sup.4 are each,
independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7
carbon atoms, alkynyl of 2-7 carbon atoms, trifluoromethyl, or --F;
R.sup.5 and R.sup.6 are each, independently, hydrogen, alkyl of 1-6
carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon
atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.8 and R.sup.9 are each, independently,
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10,
--CF.sub.3, --F, or --CO.sub.2R.sup.11; R.sup.10 is hydrogen or
--COCH.sub.2--S--CH.sub.2C-
H.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub.3; R.sup.11 is
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, or phenylalkyl of 7-10 carbon atoms;
b=0-6; d=0-6; f=0-6; n=5-450; with the proviso that R.sup.1 and
R.sup.2 are both not hydrogen and further provided that either
R.sup.1 or R.sup.2 contains at least one
--(CR.sup.3R.sup.4).sub.fOR.sup.10 group in which R.sup.10 is
--COCH.sub.2--S--CH.sub.2CH.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub-
.3, or a pharmaceutically acceptable salt thereof.
14. A method of treating or inhibiting restenosis in a mammal in
need thereof, which comprises providing to said mammal an effective
amount of a compound having the structure 11wherein R.sup.1 and
R.sup.2 are each, independently, hydrogen or
--CO(CR.sup.3R.sup.4).sub.b(CR.sup.5R.sup.6).s-
ub.dCR.sup.7R.sup.8R.sup.9; R.sup.3 and R.sup.4 are each,
independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7
carbon atoms, alkynyl of 2-7 carbon atoms, trifluoromethyl, or --F;
R.sup.5 and R.sup.6 are each, independently, hydrogen, alkyl of 1-6
carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon
atoms, --(CR.sup.3R.sup.4).sub.fOR.su- p.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.8 and R.sup.9 are each, independently,
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10,
--CF.sub.3, --F, or --CO.sub.2R.sup.11; R.sup.10 is hydrogen or
--COCH.sub.2--S--CH.sub.2C-
H.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub.3; R.sup.11 is
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, or phenylalkyl of 7-10 carbon atoms;
b=0-6; d=0-6; f=0-6; n=5-450; with the proviso that R.sup.1 and
R.sup.2 are both not hydrogen and further provided that either
R.sup.1 or R.sup.2 contains at least one
--(CR.sup.3R.sup.4).sub.fOR.sup.10 group in which R.sup.10 is
--COCH.sub.2--S--CH.sub.2CH.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub-
.3, or a pharmaceutically acceptable salt thereof.
15. A method of treating or inhibiting pulmonary inflammation in a
mammal in need thereof, which comprises providing to said mammal an
effective amount of a compound having the structure 12wherein
R.sup.1 and R.sup.2 are each, independently, hydrogen or
--CO(CR.sup.3R.sup.4).sub.b(CR.sup.5-
R.sup.6).sub.dCR.sup.7R.sup.8R.sup.9; R.sup.3 and R.sup.4 are each,
independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7
carbon atoms, alkynyl of 2-7 carbon atoms, trifluoromethyl, or --F;
R.sup.5 and R.sup.6 are each, independently, hydrogen, alkyl of 1-6
carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon
atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.8 and R.sup.9 are each, independently,
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10,
--CF.sub.3, --F, or --CO.sub.2R.sup.11; R.sup.10 is hydrogen or
--COCH.sub.2--S--CH.sub.2C-
H.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub.3; R.sup.11 is
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, or phenylalkyl of 7-10 carbon atoms;
b=0-6; d=0-6; f=0-6; n=5-450; with the proviso that R.sup.1 and
R.sup.2 are both not hydrogen and further provided that either
R.sup.1 or R.sup.2 contains at least one
--(CR.sup.3R.sup.4).sub.fOR.sup.10 group in which R.sup.10 is
--COCH.sub.2--S--CH.sub.2CH.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub-
.3, or a pharmaceutically acceptable salt thereof.
16. A pharmaceutical composition which comprises a compound having
the structure 13wherein R.sup.1 and R.sup.2 are each,
independently, hydrogen or
--CO(CR.sup.3R.sup.4).sub.b(CR.sup.5R.sup.6).sub.dCR.sup.7R.s-
up.8R.sup.9; R.sup.3 and R.sup.4 are each, independently, hydrogen,
alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of
2-7 carbon atoms, trifluoromethyl, or --F; R.sup.5 and R.sup.6 are
each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl
of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11; R.sup.8 and R.sup.9 are each, independently,
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10,
--CF.sub.3, --F, or --CO.sub.2R.sup.11; R.sup.10 is hydrogen or
--COCH.sub.2--S--CH.sub.2C-
H.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub.3; R.sup.11 is
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, or phenylalkyl of 7-10 carbon atoms;
b=0-6; d=0-6; f=0-6; n=5-450; with the proviso that R.sup.1 and
R.sup.2 are both not hydrogen and further provided that either
R.sup.1 or R.sup.2 contains at least one
--(CR.sup.3R.sup.4).sub.fOR.sup.10 group in which R.sup.10 is
--COCH.sub.2--S--CH.sub.2CH.sub.2--(--CH.sub.2--CH.sub.2).sub.n--OCH.sub.-
3, or a pharmaceutically acceptable salt thereof, and a
pharmaceutical carrier.
17. A compound which is rapamycin 42-ester with
3-hydroxy-2-(2-iodo-acetox- ymethyl)-2-methyl-propionic acid.
18. A compound which is rapamycin 42-ester with
3-(2-lodo-acetoxy)-2-(2-io- do-acetoxymethyl)-2-methyl-propionic
acid.
Description
[0001] This application claims priority from copending provisional
application Ser. No. 60/233,776, filed Sep. 19, 2000, the entire
disclosure of which is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates to water soluble rapamycin esters
which are useful in inducing immunosuppression and in the treatment
of transplantation rejection, autoimmune diseases, solid tumors,
fungal infections, and vascular disease. More particularly, this
invention concerns methoxypoly(ethylene glycol) esters of
hydroxyesters of rapamycin and methods for using them for inducing
immunosuppression, and in the treatment of transplantation
rejection, graft vs. host disease, autoimmune diseases, diseases of
inflammation, adult T-cell leukemia/lymphoma, solid tumors, fungal
infections, cardiovascular disease, cerebral vascular disease,
peripheral vascular disease or hyperproliferative vascular
disorders.
[0003] Rapamycin is a macrocyclic triene antibiotic produced by
Streptomyces hygroscopicus, which was found to have antifungal
activity, particularly against Candida albicans, both in vitro and
in vivo [C. Vezina et al., J. Antibiot. 28, 721 (1975); S. N.
Sehgal et al., J. Antibiot. 28, 727 (1975); H. A. Baker et al., J.
Antibiot. 31, 539 (1978); U.S. Pat. No. 3,929,992; and U.S. Pat.
No. 3,993,749]. Additionally, rapamycin alone (U.S. Pat. No.
4,885,171) or in combination with picibanil (U.S. Pat. No.
4,401,653) has been shown to have antitumor activity.
[0004] The immunosuppressive effects of rapamycin have been
disclosed in FASEB 3, 3411 (1989). Cyclosporin A and FK-506, other
macrocyclic molecules, also have been shown to be effective as
immunosuppressive agents, therefore useful in preventing transplant
rejection [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); R. Y. Calne
et al., Lancet 1183 (1978); and U.S. Pat. No. 5,100,899]. R. Martel
et al. [Can. J. Physiol. Pharmacol. 55, 48 (1977)] disclosed that
rapamycin is effective in the experimental allergic
encephalomyelitis model, a model for multiple sclerosis; in the
adjuvant arthritis model, a model for rheumatoid arthritis; and
effectively inhibited the formation of IgE-like antibodies.
[0005] Rapamycin is also useful in preventing or treating systemic
lupus erythematosus [U.S. Pat. No. 5,078,999], pulmonary
inflammation [U.S. Pat. No. 5,080,899], insulin dependent diabetes
mellitus [U.S. Pat. No. 5,321,009], skin disorders, such as
psoriasis [U.S. Pat. No. 5,286,730], bowel disorders [U.S. Pat. No.
5,286,731], smooth muscle cell proliferation and intimal thickening
following vascular injury [U.S. Pat. Nos. 5,288,711 and 5,516,781],
adult T-cell leukemia/lymphoma [European Patent Application 525,960
A1], ocular inflammation [U.S. Pat. No. 5,387,589], malignant
carcinomas [U.S. Pat. No. 5,206,018], cardiac inflammatory disease
[U.S. Pat. No. 5,496,832], and anemia [U.S. Pat. No.
5,561,138].
[0006] A rapamycin ester, rapamycin 42-ester with
3-hydroxy-2-(hydroxymeth- yl)-2-methylpropionic acid [disclosed in
U.S. Pat. No. 5,362,718], also known as CCl-779, has been shown to
have antitumor activity against a variety of tumor cell lines, in
in vivo animal tumor models, and in Phase I clinical trials.
[Gibbons, J., Proc. Am. Assoc. Can. Res. 40: 301 (1999); Geoerger,
B., Proc. Am. Assoc. Can. Res. 40: 603 (1999); Alexandre, J., Proc.
Am. Assoc. Can. Res. 40: 613 (1999); and Alexandre, J., Clin.
Cancer. Res. 5 (November Supp.): Abstr. 7 (1999)].
[0007] Polyethylene glycol (PEG) is a linear or branched, neutral
polymer available in a variety of molecular weights and is soluble
in water and most organic solvents. At molecular weights less than
1000 are the viscous, colorless liquids; higher molecular weight
PEGs are waxy, white solids. The melting point of the solid is
proportional to the molecular weight, approaching a plateau at
67.degree. C. Molecular weight range from a few hundred to
approximately 20,000 are commonly used in biological and
biotechnological applications. Of much interest in the biomedical
areas is the fact that PEG is nontoxic and was approved by FDA for
internal consumption. Peglyated rapamycin is disclosed in U.S. Pat.
No. 5,780,462.
DESCRIPTION OF THE INVENTION
[0008] This invention provides methoxypoly(ethylene glycol) esters
of hydroxyesters of rapamycin having the structure 1
[0009] wherein
[0010] R.sup.1 and R.sup.2 are each, independently, hydrogen or
--CO(CR.sup.3R.sup.4).sub.b(CR.sup.5R.sup.6).sub.dCR.sup.7R.sup.8R.sup.9;
[0011] R.sup.3 and R.sup.4 are each, independently, hydrogen, alkyl
of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7
carbon atoms, trifluoromethyl, or --F;
[0012] R.sup.5 and R.sup.6 are each, independently, hydrogen, alkyl
of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7
carbon atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3,--F,
or --CO.sub.2R.sup.11;
[0013] R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of
2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.3R.sup.4).sub.fOR.su- p.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.11;
[0014] R.sup.8 and R.sup.9 are each, independently, hydrogen, alkyl
of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7
carbon atoms, --(CR.sup.3R.sup.4).sub.fOR.sup.10, --CF.sub.3, --F,
or --CO.sub.2R.sup.11;
[0015] R.sup.10 is hydrogen or
--COCH.sub.2--S--CH.sub.2CH.sub.2--(O--CH.s-
ub.2--CH.sub.2).sub.n--OCH.sub.3;
[0016] R.sup.11 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of
2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or phenylalkyl of
7-10 carbon atoms;
[0017] b=0-6;
[0018] d=0-6;
[0019] f=0-6;
[0020] n=5-450;
[0021] with the proviso that R.sup.1 and R.sup.2 are both not
hydrogen and further provided that either R.sup.1 or R.sup.2
contains at least one --(CR.sup.3R.sup.4).sub.fOR.sup.10 group in
which R.sup.10 is
--COCH.sub.2--S--CH.sub.2CH.sub.2--(O--CH.sub.2--CH.sub.2).sub.n--OCH.sub-
.3, or a pharmaceutically acceptable salt thereof which are useful
for inducing immunosuppression, and in the treatment of
transplantation rejection, graft vs. host disease, autoimmune
diseases, diseases of inflammation, adult T-cell leukemia/lymphoma,
solid tumors, fungal infections, cardiovascular disease, cerebral
vascular disease, peripheral vascular disease or hyperproliferative
vascular disorders. The compounds of this invention can also be
referred to as pegylated hydroxyesters of rapamycin.
[0022] When applicable, pharmaceutically acceptable salts can be
formed from organic and inorganic bases (i.e., when a compound
contains a free hydroxyl group), such as alkali metal salts (for
example, sodium, lithium, or potassium) alkaline earth metal salts,
ammonium salts, alkylammonium salts containing 1-6 carbon atoms or
dialkylammonium salts containing 1-6 carbon atoms in each alkyl
group, and trialkylammonium salts containing 1-6 carbon atoms in
each alkyl group, when the rapamycin or antiestrogen contains a
suitable acidic moiety.
[0023] As used in accordance with this invention, the term
"providing," with respect to providing a compound or substance
covered by this invention, means either directly administering such
a compound or substance, or administering a prodrug, derivative, or
analog which will form the equivalent amount of the compound or
substance within the body.
[0024] Of the pegylated hydroxyesters of rapamycin covered by this
invention, it is preferred that the hydroxyester of rapamycin is
CCl-779, in which one or both of the hydroxyl groups of the
3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid moiety are
pegylated. Of the compounds of this invention, it is preferred that
n=5-200; more preferred that n=8-135. Most preferred members are
those in which n=8-20 and those in which n=90-120. The values of n
refer to the range of repeating ethoxy units in the PEG side chain.
For example, when a compound is described as having n=5-200, it
means that such compound consists of a mixture of compounds having
a normal distribution between n=5 and n=200, with approximately
n=100 having the greatest frequency. With compounds III and IV, the
average n was 108, and 99% of n being between 65 and 155. The
compounds of this invention may also be described and understood
based upon the average molecular weight of the polyethylene glycol
chains used to produce their ester chains. For instance, an
CCl-779-PEG 5000 ester refers to a compound of the general formula
above in which one side chain PEG ester is formed utilizing a
polyethylene glycol derivative having an average molecular weight
range at or near 5,000; and CCl-779-(PEG 5000).sub.2 ester refers
to a compound of the general formula above in two side chain PEG
esters are formed utilizing a polyethylene glycol derivative having
an average molecular weight range at or near 5,000.
[0025] The reagents used in the preparation of the compounds of
this invention can be either commercially obtained or can be
prepared by standard procedures described in the literature.
[0026] The preparation of the hydroxyesters of rapamycin, from
which the pegylated hydroxyesters are made from, are described in
U.S. Pat. No. 5,362,718, which is hereby incorporated by reference.
One or more of the hydroxyesters may be acylated with a acylating
agent having the general structure X--CH.sub.2CO.sub.2H, where X is
a suitable leaving group, such as bromine or iodine, in the
presence of a coupling agent such as dicyclohexylcarbodiimide (DCC)
in the presence of a base catalyst, such as dimethylaminopyridine
(DMAP). Accordingly, rapamycin 42-ester with
3-hydroxy-2-(2-iodo-acetoxymethyl)-2-methyl-propionic acid
(Compound I) and rapamycin 42-ester with
3-(2-lodo-acetoxy)-2-(2-iodo-acetoxymethyl)-2- -methyl-propionic
acid (Compound II) are intermediates prepared using this
methodology, that are useful in the production of the corresponding
pegylated hydroxyesters of rapamycin (Compounds Ill and IV,
respectively).
[0027] The pegylated esters may be produced utilizing the
polyethylene glycols known in the art, such as those described on
pages 355 to 361 of the Handbook of Pharmaceutical Excipients,
Second Edition, 1994. The preferred compounds of this invention may
also be described as those of the formula esterified using
polyethylene glycols having an average molecular weight of from
about 200 to about 200,000. A preferred range of the PEG esters of
this invention includes those in which the molecular weight of the
polyethylene glycol portion of the ester chain has a molecular
weight in the range of from about 300 to about 20,000, more
preferably between about 350 and about 6,000. Reaction of the
hydroxyester of rapamycin which has been acylated as described
above, with a suitable polyethyleneglycol thiol in the presence of
a base, such as sodium bicarbonate provides the desired pegylated
hydroxyester of rapamycin.
[0028] The antitumor activity of the compounds of this invention
were confirmed in a standard pharmacological test procedure which
measures the inhibition of U87MG human glioblastoma cell growth (as
a function of .sup.3H-thymidine incorporation), using CCl-779-PEG
5000 ester (Compound III) and CCl-779-(PEG 5000).sub.2 ester
(Compound IV) as representative compounds of this invention. The
following briefly describes the procedure used and results
obtained. U87MG human glioblastoma cells (ATCC # HTB-14; available
from the American Type Culture Collection; 10801 University
Boulevard; Manassas, Va 20110;), were grown in the following
media.
[0029] Growth Medium:
[0030] BRL Minimum Essential Medium with Earle Salts (500 mL)
[0031] +5 mL BRL MEM Non-Essential Amino Acids (10 mM)
[0032] +5 mL BRL Penicillin-Streptomycin (10000 u/mL,10000
.mu.g/mL)
[0033] +5 mL BRL Na Pyruvate Solution (100 mM)
[0034] +5 mL BRL L-Glutamine 200 mM
[0035] +50 mL BRL Fetal Bovine Serum (Qualified)
[0036] Test Procedure:
[0037] 1. Cells were trypsinized and plated at a concentration of
10.sup.4 cells/well in a final volume of 200 .mu.L growth medium in
96-well flat bottom plates and allowed to adhere for 24 hours at
37.degree. C.
[0038] 2. The media was removed by aspiration with care to not
disturb the cell monolayer. 200 .mu.L of fresh growth media was
added per well, allowing enough wells for samples to be run in
triplicate. Test compounds were added in 10 .mu.L phosphate buffer
solution (PBS) and incubated for another 48 hours at 37.degree.
C.
[0039] 3. During the last 5 hours of incubation, plates were
labeled with 1 .mu.Ci .sup.3H thymidine per well. (New England
Nuclear thymidine, catalog # NET-027, 6.7 Ci/mmole). The 1 .mu.Ci
was added in 10 .mu.L of PBS (on the day of harvest). The plates
were returned to the incubator for the last 5 hours.
[0040] 4. The radioactive media was removed by aspiration, with
care not to disturb the cell monolayer. Then 50 .mu.L of BRL
10.times. Trypsin was added to each well, followed by incubation at
37.degree. C. for 10 minutes or until the monolayer was loosened
from the well bottom. Samples were harvested on a glass fiber
filter mat using a Skatron 96 well harvester. Mats were counted in
a Wallac Betaplate counter.
[0041] Results:
1 Compound IC.sub.50 CCI-779 0.6 ng/mL Compound III 1.0 ng/mL*
Compound IV 4.0 ng/mL* *ng/mL equivalent of CCI-779
[0042] The results obtained in this standard pharmacological test
procedure show that the compounds of this invention inhibit tumor
cell growth and are therefore useful as antineoplastic agents. In
particular, the compounds of this invention are useful in treating
or inhibiting the growth of solid tumors, including sarcomas and
carcinomas, such as astrocytomas, prostate cancer, breast cancer,
small cell lung cancer, and ovarian cancer.
[0043] The compounds of this invention are also useful treatment or
inhibition of transplantation rejection such as kidney, heart,
liver, lung, bone marrow, pancreas (islet cells), cornea, small
bowel, and skin allografts, and heart valve xenografts; in the
treatment or inhibition of graft vs. host disease; in the treatment
or inhibition of autoimmune diseases such as lupus, rheumatoid
arthritis, diabetes mellitus, myasthenia gravis, and multiple
sclerosis; and diseases of inflammation such as psoriasis,
dermatitis, eczema, seborrhea, inflammatory bowel disease,
pulmonary inflammation (including asthma, chronic obstructive
pulmonary disease, emphysema, acute respiratory distress syndrome,
bronchitis, and the like) and ocular uveitis; adult T-cell
leukemia/lymphoma; fungal infections; hyperproliferative vascular
diseases such as restenosis; graft vascular atherosclerosis; and
cardiovascular disease, cerebral vascular disease, and peripheral
vascular disease, such as coronary artery disease, cerebrovascular
disease, arteriosclerosis, atherosclerosis, nonatheromatous
arteriosclerosis, or vascular wall damage from cellular events
leading toward immune mediated vascular damage, and inhibiting
stroke or multiinfarct dementia.
[0044] When used for restenosis, it is preferred that the compounds
of this invention are used to treat restenosis that occurs
following an angioplasty procedure. When used for this treating
restenosis following an angioplasty, the compounds of this
invention can be administered prior to the procedure, during the
procedure, subsequent to the procedure, or any combination of the
above.
[0045] This invention also covers analogous pegylated hydroxyesters
of other rapamycins known in the art such as, but not limited to,
29-demethoxyrapamycin, [U.S. Pat. No. 4,375,464,
32-demethoxyrapamycin under C.A. nomenclature]; rapamycin
derivatives in which the double bonds in the 1-, 3-, and/or
5-positions have been reduced [U.S. Pat. No. 5,023,262];
29-desmethylrapamycin [U.S. Pat. No. 5,093,339,
32-desmethylrapamycin under C.A. nomenclature];
7,29-bisdesmethylrapamyci- n [U.S. Pat. No. 5,093,338,
7,32-desmethylrapamycin under C.A. nomenclature];
27-hydroxyrapamycin [U.S. Pat. No. 5,256,790] and
15-hydroxyrapamycin [U.S. Pat. No. 5,102,876]. This invention also
covers esters at the 31-position of 42-oxorapamycin [U.S. Pat. No.
5,023,263]. The disclosures in the above cited U.S. Patents are
hereby incorporated by reference.
[0046] When administered for the treatment or inhibition of a
particular disease state or disorder, it is understood that the
effective dosage of the pegylated hydroxyester of rapamycin may
vary depending upon the particular compound utilized, the mode of
administration, the condition, and severity thereof, of the
condition being treated, as well as the various physical factors
related to the individual being treated. As used in accordance with
invention, satisfactory results may be obtained when the pegylated
hydroxyester of rapamycin is administered in a daily oral dosage of
from about projected daily dosages of active compound would be 0.1
.mu.g/kg -100 mg/kg, preferably between 0.001-25 mg/kg, and more
preferably between 0.01-5 mg/kg. The projected daily dosages are
expected to vary with route of administration.
[0047] Such doses may be administered in any manner useful in
directing the active compounds herein to the recipient's
bloodstream, including orally, via implants, parenterally
(including intravenous, intraperitoneal and subcutaneous
injections), rectally, intranasally, vaginally, and transdermally.
The pegylated esters of this invention provide an advantage in ease
of formulation and administration over the non-pegylated esters of
rapamycin, in that they are significantly more water soluble than
corresponding non-pegylated esters. For example, both compounds 3
and 4 had a water solubility of >100 mg/mL, whereas CCl-779 had
a water solubility of <1.1 .mu.g/mL. This advantage is
particularly important for oral and parenteral administration.
[0048] Oral formulations containing the active compounds of this
invention may comprise any conventionally used oral forms,
including tablets, capsules, buccal forms, troches, lozenges and
oral liquids, suspensions or solutions. Capsules may contain
mixtures of the active compound(s) with inert fillers and/or
diluents such as the pharmaceutically acceptable starches (e.g.
corn, potato or tapioca starch), sugars, artificial sweetening
agents, powdered celluloses, such as crystalline and
microcrystalline celluloses, flours, gelatins, gums, etc. Useful
tablet formulations may be made by conventional compression, wet
granulation or dry granulation methods and utilize pharmaceutically
acceptable diluents, binding agents, lubricants, disintegrants,
surface modifying agents (including surfactants), suspending or
stabilizing agents, including, but not limited to, magnesium
stearate, stearic acid, talc, sodium lauryl sulfate,
microcrystalline cellulose, carboxymethylcellulose calcium,
polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan
gum, sodium citrate, complex silicates, calcium carbonate, glycine,
dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate,
lactose, kaolin, mannitol, sodium chloride, talc, dry starches and
powdered sugar. Preferred surface modifying agents include nonionic
and anionic surface modifying agents. Representative examples of
surface modifying agents include, but are not limited to, poloxamer
188, benzalkonium chloride, calcium stearate, cetostearl alcohol,
cetomacrogol emulsifying wax, sorbitan esters, colloidol silicon
dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum
silicate, and triethanolamine. Oral formulations herein may utilize
standard delay or time release formulations to alter the absorption
of the active compound(s). The oral formulation may also consist of
administering the active ingredient in water or a fruit juice,
containing appropriate solubilizers or emulsifiers as needed.
[0049] In some cases it may be desirable to administer the
compounds directly to the airways in the form of an aerosol.
[0050] The compounds of this invention may also be administered
parenterally or intraperitoneally. Solutions or suspensions of
these active compounds as a free base or pharmacologically
acceptable salt can be prepared in water suitably mixed with a
surfactant such as hydroxy-propylcellulose. Dispersions can also be
prepared in glycerol, liquid polyethylene glycols and mixtures
thereof in oils. Under ordinary conditions of storage and use,
these preparation contain a preservative to prevent the growth of
microorganisms.
[0051] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol and liquid polyethylene glycol),
suitable mixtures thereof, and vegetable oils.
[0052] For the purposes of this disclosure, transdermal
administrations are understood to include all administrations
across the surface of the body and the inner linings of bodily
passages including epithelial and mucosal tissues. Such
administrations may be carried out using the present compounds, or
pharmaceutically acceptable salts thereof, in lotions, creams,
foams, patches, suspensions, solutions, and suppositories (rectal
and vaginal).
[0053] Transdermal administration may be accomplished through the
use of a transdermal patch containing the active compound and a
carrier that is inert to the active compound, is non toxic to the
skin, and allows delivery of the agent for systemic absorption into
the blood stream via the skin. The carrier may take any number of
forms such as creams and ointments, pastes, gels, and occlusive
devices. The creams and ointments may be viscous liquid or
semisolid emulsions of either the oil-in-water or water-in-oil
type. Pastes comprised of absorptive powders dispersed in petroleum
or hydrophilic petroleum containing the active ingredient may also
be suitable. A variety of occlusive devices may be used to release
the active ingredient into the blood stream such as a
semi-permeable membrane covering a reservoir containing the active
ingredient with or without a carrier, or a matrix containing the
active ingredient. Other occlusive devices are known in the
literature.
[0054] Suppository formulations may be made from traditional
materials, including cocoa butter, with or without the addition of
waxes to alter the suppository's melting point, and glycerin. Water
soluble suppository bases, such as polyethylene glycols of various
molecular weights, may also be used.
[0055] The preparation of representative examples of this invention
is described below.
EXAMPLE 1
Preparation of Rapamycin 42-ester with
3-hydroxy-2-(2-iodo-acetoxymethyl)-- 2-methyl-propionic acid
(Compound I) and Rapamycin 42-ester with
3-(2-lodo-acetoxy)-2-(2-iodo-acetoxymethyl)-2-methyl-propionic acid
(Compound II)
[0056] 2
[0057] CCl-779 (1.03 g 1.0.times.10.sup.-3 mole),
4-Dimethylaminopyridine (3.0 mg) and 1,3-Dicyclohexylcarbodiimide
(0.136 g, 6.6.times.10.sup.-4 mole) were dissolved in 50 mL
anhydrous methylene chloride in a 250 mL round-bottom flask.
Iodoacetic acid (0.185 g, 1.0.times.10.sup.-3 mole) was dissolved
in 10 mL anhydrous methylene chloride. The iodoacetic acid solution
was added into reaction mixture over a period of 10 min with
stirring by a magnetic bar. Then the reaction mixture was stirred
at room temperature for another 2.5 h. The solution was then
filtered through a filter paper., The filtrate was transferred to a
separatory funnel, washed 50 mL of sodium bicarbonate solution (5.5
g/100 mL) and then washed with 2.times.50 mL of water. The
methylene chloride layer was dried with 10 g anhydrous sodium
sulfate for 2 h. Then sodium sulfate was filtered out and methylene
chloride was removed by rotary evaporation. A total of 0.93 g
yellow solid was obtained. Isolation of pure compound I and
compound 11 was performed by preparative HPLC on a Prep Nova-pak HR
C18 (300.times.19 mm) column from Waters. Compound I eluted at 18.4
min and compound 11 eluted at 24.4 min using a gradient ( 30% A,
70% B for 5 min. then to 100% B in 30 min.). A is 90% water, 10%
acetonitrile; B is 10% water, 90% acetonitrile. The fraction was
collected and extracted by 2.times.100 ml methylene chloride. The
organic layer was combined and dried with anhydrous sodium sulfate
for 4 h. The organic solvent was removed by rotary evaporation to
dryness. Compound I, a yellowish solid was obtained (0.14 g).
.sup.1H NMR (CDCL.sub.3, 400 MHz) .delta. 3.68 (s, 2 H,
I--CH.sub.2--CO.sub.2--), 4.28 (dd, 2 H, --CO.sub.2--CH.sub.2--).
MS m/z 1215.8 (M+NH.sub.4).sup.+. Compound II, a yellowish solid
was obtained (0.08 g). .sup.1H NMR (CDCL.sub.3, 400 MHz) .delta.
3.72 (s 4 H, 2 x I--CH.sub.2--CO.sub.2--), 4.28 (dd, 4 H, 2 x
--CO.sub.2--CH.sub.2--). MS m/z 1383.6 (M+NH.sub.4).sup.+.
EXAMPLE 2
Preparation of CCl-779-PEG 5000-Rapamycin 42-ester with
3-hydroxy-2-(hydroxymethyl)-2-methylpropionic Acid
Methoxypoly(Ethylene Glycol) Thiol 5000 Conjugate (Compound
III)
[0058] 3
[0059] Compound I (90 mg, 7.6.times.10.sup.-5 mole) was dissolved
in 40 mL of solution containing 50% acetonitrile and 50% aqueous
NaHCO.sub.3 (0.1 M) solution. The solution was flushed with N.sub.2
for 10 min. The original sample 10 .mu.L was taken for HPLC
analysis. Then mPEG-SH 5000 (450 mg, 9.1.times.10-5 mole) was added
to the reaction solution and the reaction mixture was stirred at
room temperature for another 45 min. The reaction was checked again
by taking 10 .mu.L sample for HPLC analysis. The chromatogram
showed that compound I was 100% converted to Compound Ill. The
reaction mixture was extracted with 2.times.100 mL methylene
chloride. The organic layer was dried with anhydrous sodium sulfate
then filtered. The filtrate was concentrated to a volume of 20 mL
by rotaty evaporation. The crude product was precipitated out after
adding 150 mL ether. A total of 404 mg white powder was obtained
after filtered out by a sintered glass funnel and dried under
vacuum. Isolation of pure compound III, which may also be referred
to as CCl-779-PEG 5000, was performed by preparative HPLC on a Prep
Nova-pak HR C18 (300.times.19 mm) column from Waters. Compound III
eluted at 18 min using a gradient (60% A, 40% B for 5 min then at
20% A, 80% B in 30 min). The fraction was collected and extracted
by 2.times.100 mL methylene chloride. The organic layer was
combined and dried with anhydrous sodium sulfate for 4 hr. The
organic solvent was removed by rotary evaporation to dryness. The
residue was dissolved in 5 mL methylene chloride and was
precipitated out after adding 150 mL ether. A white powder was
obtained after filtered out by a sintered glass funnel and dried
under vacuum. .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 2.83 (t, 2
H, --S--CH.sub.2--CH.sub.2--), 3.30 (s, 2 H, --CO--CH.sub.2--S),
3.38 (s, 3 H, --OCH.sub.3), 4.25 (dd, 2 H, --CO.sub.2--CH.sub.2--).
MS (MALD/TOF) m/z 5894.5 (ave. M. Wt.) which indicate the average
n=108 for compound III, with 99% of n being between 65 and 155.
EXAMPLE 4
Preparation of CCl-779-(PEG 5000).sub.2-Rapamycin 42-ester with
3-hydroxy-2-(Hydroxymethyl)-2-methylpropionic Acid
Bis(Methoxypoly(Ethylene Glycol) Thiol 5000) Conjugate (Compound
IV)
[0060] 4
[0061] Compound II (50 mg, 3.6.times.10.sup.-5 mole) was dissolved
in 30 mL of solution containing 50% acetonitrile and 50% aqueous
NaHCO.sub.3 (0.1 M) solution. The solution was flushed with N.sub.2
for 10 min. The original sample 10 .mu.L was taken for HPLC
analysis. Then mPEG-SH 5000 (500 mg, 1.0.times.10.sup.-4 mole) was
added to the reaction solution and the reaction mixture was stirred
at room temperature for about 70 min. The reaction was checked
again by taking 10 .mu.L sample for HPLC analysis. The chromatogram
showed that compound 11 was 100% converted to Compound IV. The
reaction mixture was extracted with 2.times.50 mL methylene
chloride. The organic layer was dried with anhydrous sodium sulfate
then filtered. The filtrate was concentrated to a volume of 10 mL
by rotaty evaporation. The crude product was precipitated out after
adding 150 mL ether. A total of 500 mg white powder was obtained
after filtered out by a sintered glass funnel and dried under
vacuum. Isolation of pure compound IV, which may also be referred
to as CCl-779-(PEG 5000)2, was performed by preparative HPLC on a
Prep Nova-pak HR C18 (300.times.19 mm) column from Waters. Compound
IV eluted at 22 min using a gradient (60% A, 40% B for 5 min then
at 20% A, 80% B in 30 min). The fraction was collected and
extracted by 2.times.100 mL methylene chloride. The organic layer
was combined and dried with anhydrous sodium sulfate for 4 hr. The
organic solvent was removed by rotary evaporation to dryness. The
residue was dissolved in 5 mL methylene chloride and was
precipitated out after adding 150 mL ether. A white powder was
obtained after filtered out by a sintered glass funnel and dried
under vacuum. .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta.2.81 (t, 4
H, 2 x S--CH.sub.2--CH.sub.2--), 3.29 (s, 4 H, 2 x
CO--CH.sub.2--S--), 3.38 (s, 6 H, 2 x --OCH.sub.3), 4.26 (dd, 4H, 2
x --CO.sub.2--CH.sub.2--). MS (MALD/TOF) m/z 10760.7 (ave. M. Wt.)
which indicate the average n=108 for compound IV, with 99% of n
being between 65 and 155.
* * * * *