U.S. patent application number 09/956322 was filed with the patent office on 2002-05-23 for ethers of 7-desmethylrapamycin.
This patent application is currently assigned to American Home Products Corporation. Invention is credited to Enever, Robin, Zhu, Tianmin.
Application Number | 20020061905 09/956322 |
Document ID | / |
Family ID | 22893849 |
Filed Date | 2002-05-23 |
United States Patent
Application |
20020061905 |
Kind Code |
A1 |
Zhu, Tianmin ; et
al. |
May 23, 2002 |
Ethers of 7-desmethylrapamycin
Abstract
This invention provides ethers of 7-desmethylrapamycin 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) ; Enever, Robin; (New City, NY) |
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: |
22893849 |
Appl. No.: |
09/956322 |
Filed: |
September 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60237469 |
Oct 2, 2000 |
|
|
|
Current U.S.
Class: |
514/291 ;
540/456 |
Current CPC
Class: |
C07D 498/18 20130101;
A61P 11/00 20180101; A61P 29/00 20180101; A61P 9/10 20180101; A61P
35/00 20180101; A61P 37/06 20180101; A61P 25/00 20180101; A61P
31/10 20180101 |
Class at
Publication: |
514/291 ;
540/456 |
International
Class: |
A61K 031/4745; C07D
487/14 |
Claims
What is claimed is:
1. A compound having the structure 2wherein R.sup.1 and R.sup.2 are
each, independently, hydrogen, thioalkyl of 1-6 carbon atoms,
arylalkyl of 7-10 carbon atoms, hydroxyalkyl of 1-6 carbon atoms,
dihydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbon
atoms, hydroxyalkoxyalkyl of 2-12 carbon atoms, acyloxyalkyl of
3-12 carbon atoms, aminoalkyl of 1-6 carbon atoms, alkylaminoalkyl
of 1-6 carbon atoms per alkyl group, dialkylaminoalkyl of 1-6
carbon atoms per alkyl group, alkoxycarbonylaminoalkyl of 3-12
carbon atoms, acylaminoalkyl of 3-12 carbon atoms, alkenyl of 2-7
carbon atoms, arylsulfamidoalkyl having 1-6 carbon atoms in the
alkyl group, hydroxyalkylallyl of 4-9 carbon atoms,
dihydroxyalkylallyl of 4-9 carbon atoms, or dioxolanylallyl; with
the proviso that R.sup.1 and R.sup.2 are both not hydrogen, or a
pharmaceutically acceptable salt thereof.
2. The compound according to claim 1, wherein R.sup.2 is
hydrogen.
3. The compound according to claim 2, wherein R.sup.1 is
hydroxyalkyl of 1-6 carbon atoms.
4. The compound of claim 1, which is 42-O-(2-hydroxy)ethyl
7-desmethylrapamycin acid is particularly preferred..
5. 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 structure 3wherein R.sup.1 and R.sup.2 are each, independently,
hydrogen, thioalkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon
atoms, hydroxyalkyl of 1-6 carbon atoms, dihydroxyalkyl of 1-6
carbon atoms, alkoxyalkyl of 2-12 carbon atoms, hydroxyalkoxyalkyl
of 2-12 carbon atoms, acyloxyalkyl of 3-12 carbon atoms, aminoalkyl
of 1-6 carbon atoms, alkylaminoalkyl of 1-6 carbon atoms per alkyl
group, dialkylaminoalkyl of 1-6 carbon atoms per alkyl group,
alkoxycarbonylaminoalkyl of 3-12 carbon atoms, acylaminoalkyl of
3-12 carbon atoms, alkenyl of 2-7 carbon atoms, arylsulfamidoalkyl
having 1-6 carbon atoms in the alkyl group, hydroxyalkylallyl of
4-9 carbon atoms, dihydroxyalkylallyl of 4-9 carbon atoms, or
dioxolanylallyl; with the proviso that R.sup.1 and R.sup.2 are both
not hydrogen, or a pharmaceutically acceptable salt thereof.
6. 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 4wherein R.sup.1 and R.sup.2 are each,
independently, hydrogen, thioalkyl of 1-6 carbon atoms, arylalkyl
of 7-10 carbon atoms, hydroxyalkyl of 1-6 carbon atoms,
dihydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbon
atoms, hydroxyalkoxyalkyl of 2-12 carbon atoms, acyloxyalkyl of
3-12 carbon atoms, aminoalkyl of 1-6 carbon atoms, alkylaminoalkyl
of 1-6 carbon atoms per alkyl group, dialkylaminoalkyl of 1-6
carbon atoms per alkyl group, alkoxycarbonylaminoalkyl of 3-12
carbon atoms, acylaminoalkyl of 3-12 carbon atoms, alkenyl of 2-7
carbon atoms, arylsulfamidoalkyl having 1-6 carbon atoms in the
alkyl group, hydroxyalkylallyl of 4-9 carbon atoms,
dihydroxyalkylallyl of 4-9 carbon atoms, or dioxolanylallyl; with
the proviso that R.sup.1 and R.sup.2 are both not hydrogen, or a
pharmaceutically acceptable salt thereof.
7. 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 5wherein
R.sup.1 and R.sup.2 are each, independently, hydrogen, thioalkyl of
1-6 carbon atoms, arylalkyl of 7-10 carbon atoms, hydroxyalkyl of
1-6 carbon atoms, dihydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl
of 2-12 carbon atoms, hydroxyalkoxyalkyl of 2-12 carbon atoms,
acyloxyalkyl of 3-12 carbon atoms, aminoalkyl of 1-6 carbon atoms,
alkylaminoalkyl of 1-6 carbon atoms per alkyl group,
dialkylaminoalkyl of 1-6 carbon atoms per alkyl group,
alkoxycarbonylaminoalkyl of 3-12 carbon atoms, acylaminoalkyl of
3-12 carbon atoms, alkenyl of 2-7 carbon atoms, arylsulfamidoalkyl
having 1-6 carbon atoms in the alkyl group, hydroxyalkylallyl of
4-9 carbon atoms, dihydroxyalkylallyl of 4-9 carbon atoms, or
dioxolanylallyl; with the proviso that R.sup.1 and R.sup.2 are both
not hydrogen, or a pharmaceutically acceptable salt thereof.
8. 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 6wherein
R.sup.1 and R.sup.2 are each, independently, hydrogen, thioalkyl of
1-6 carbon atoms, arylalkyl of 7-10 carbon atoms, hydroxyalkyl of
1-6 carbon atoms, dihydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl
of 2-12 carbon atoms, hydroxyalkoxyalkyl of 2-12 carbon atoms,
acyloxyalkyl of 3-12 carbon atoms, aminoalkyl of 1-6 carbon atoms,
alkylaminoalkyl of 1-6 carbon atoms per alkyl group,
dialkylaminoalkyl of 1-6 carbon atoms per alkyl group,
alkoxycarbonylaminoalkyl of 3-12 carbon atoms, acylaminoalkyl of
3-12 carbon atoms, alkenyl of 2-7 carbon atoms, arylsulfamidoalkyl
having 1-6 carbon atoms in the alkyl group, hydroxyalkylallyl of
4-9 carbon atoms, dihydroxyalkylallyl of 4-9 carbon atoms, or
dioxolanylallyl; with the proviso that R.sup.1 and R.sup.2 are both
not hydrogen, or a pharmaceutically acceptable salt thereof.
9. 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 7wherein
R.sup.1 and R.sup.2 are each, independently, hydrogen, thioalkyl of
1-6 carbon atoms, arylalkyl of 7-10 carbon atoms, hydroxyalkyl of
1-6 carbon atoms, dihydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl
of 2-12 carbon atoms, hydroxyalkoxyalkyl of 2-12 carbon atoms,
acyloxyalkyl of 3-12 carbon atoms, aminoalkyl of 1-6 carbon atoms,
alkylaminoalkyl of 1-6 carbon atoms per alkyl group,
dialkylaminoalkyl of 1-6 carbon atoms per alkyl group,
alkoxycarbonylaminoalkyl of 3-12 carbon atoms, acylaminoalkyl of
3-12 carbon atoms, alkenyl of 2-7 carbon atoms, arylsulfamidoalkyl
having 1-6 carbon atoms in the alkyl group, hydroxyalkylallyl of
4-9 carbon atoms, dihydroxyalkylallyl of 4-9 carbon atoms, or
dioxolanylallyl; with the proviso that R.sup.1 and R.sup.2 are both
not hydrogen, or a pharmaceutically acceptable salt thereof.
10. 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 8wherein R.sup.1 and
R.sup.2 are each, independently, hydrogen, thioalkyl of 1-6 carbon
atoms, arylalkyl of 7-10 carbon atoms, hydroxyalkyl of 1-6 carbon
atoms, dihydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12
carbon atoms, hydroxyalkoxyalkyl of 2-12 carbon atoms, acyloxyalkyl
of 3-12 carbon atoms, aminoalkyl of 1-6 carbon atoms,
alkylaminoalkyl of 1-6 carbon atoms per alkyl group,
dialkylaminoalkyl of 1-6 carbon atoms per alkyl group,
alkoxycarbonylaminoalkyl of 3-12 carbon atoms, acylaminoalkyl of
3-12 carbon atoms, alkenyl of 2-7 carbon atoms, arylsulfamidoalkyl
having 1-6 carbon atoms in the alkyl group, hydroxyalkylallyl of
4-9 carbon atoms, dihydroxyalkylallyl of 4-9 carbon atoms, or
dioxolanylallyl; with the proviso that R.sup.1 and R.sup.2 are both
not hydrogen, or a pharmaceutically acceptable salt thereof.
11. 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 9wherein
R.sup.1 and R.sup.2 are each, independently, hydrogen, thioalkyl of
1-6 carbon atoms, arylalkyl of 7-10 carbon atoms, hydroxyalkyl of
1-6 carbon atoms, dihydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl
of 2-12 carbon atoms, hydroxyalkoxyalkyl of 2-12 carbon atoms,
acyloxyalkyl of 3-12 carbon atoms, aminoalkyl of 1-6 carbon atoms,
alkylaminoalkyl of 1-6 carbon atoms per alkyl group,
dialkylaminoalkyl of 1-6 carbon atoms per alkyl group,
alkoxycarbonylaminoalkyl of 3-12 carbon atoms, acylaminoalkyl of
3-12 carbon atoms, alkenyl of 2-7 carbon atoms, arylsulfamidoalkyl
having 1-6 carbon atoms in the alkyl group, hydroxyalkylallyl of
4-9 carbon atoms, dihydroxyalkylallyl of 4-9 carbon atoms, or
dioxolanylallyl; with the proviso that R.sup.1 and R.sup.2 are both
not hydrogen, or a pharmaceutically acceptable salt thereof.
12. A pharmaceutical composition which comprises a compound having
the structure 10wherein R.sup.1 and R.sup.2 are each,
independently, hydrogen, thioalkyl of 1-6 carbon atoms, arylalkyl
of 7-10 carbon atoms, hydroxyalkyl of 1-6 carbon atoms,
dihydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbon
atoms, hydroxyalkoxyalkyl of 2-12 carbon atoms, acyloxyalkyl of
3-12 carbon atoms, aminoalkyl of 1-6 carbon atoms, alkylaminoalkyl
of 1-6 carbon atoms per alkyl group, dialkylaminoalkyl of 1-6
carbon atoms per alkyl group, alkoxycarbonylaminoalkyl of 3-12
carbon atoms, acylaminoalkyl of 3-12 carbon atoms, alkenyl of 2-7
carbon atoms, arylsulfamidoalkyl having 1-6 carbon atoms in the
alkyl group, hydroxyalkylallyl of 4-9 carbon atoms,
dihydroxyalkylallyl of 4-9 carbon atoms, or dioxolanylallyl; with
the proviso that R.sup.1 and R.sup.2 are both not hydrogen, or a
pharmaceutically acceptable salt thereof, and a pharmaceutical
carrier.
Description
[0001] This application claims priority from copending provisional
application Serial No. 60/237,469, filed Oct. 2, 2000, the entire
disclosure of which is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates to ethers of 7-desmethylrapamycin,
which are useful in inducing immunosuppression and in the treatment
of transplantation rejection, autoimmune diseases, solid tumors,
fungal infections, and vascular disease or 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. Nos. 3,929,992; and 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] Ethers of rapamycin are disclosed in U.S. Pat. No.
5,665,772. In particular, 42-O-(2-hydroxy)ethyl rapamycin, also
known as SDZ-RAD, has been reported to be useful in treating or
inhibiting transplant rejection.
[0007] The preparation and use of 7-desmethylrapamycin and certain
derivatives thereof are disclosed in U.S. Pat. No. 5,728,710.
DESCRIPTION OF THE INVENTION
[0008] This invention provides ethers of 7-desmethylrapamycin
having the structure 1
[0009] wherein
[0010] R.sup.1 and R.sup.2 are each, independently, hydrogen,
thioalkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon atoms,
hydroxyalkyl of 1-6 carbon atoms, dihydroxyalkyl of 1-6 carbon
atoms, alkoxyalkyl of 2-12 carbon atoms, hydroxyalkoxyalkyl of 2-12
carbon atoms, acyloxyalkyl of 3-12 carbon atoms, aminoalkyl of 1-6
carbon atoms, alkylaminoalkyl of 1-6 carbon atoms per alkyl group,
dialkylaminoalkyl of 1-6 carbon atoms per alkyl group,
alkoxycarbonylaminoalkyl of 3-12 carbon atoms, acylaminoalkyl of
3-12 carbon atoms, alkenyl of 2-7 carbon atoms, arylsulfamidoalkyl
having 1-6 carbon atoms in the alkyl group, hydroxyalkylallyl of
4-9 carbon atoms, dihydroxyalkylallyl of 4-9 carbon atoms, or
dioxolanylallyl;
[0011] with the proviso that R.sup.1 and R.sup.2 are both not
hydrogen, 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.
[0012] 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.
[0013] The term alkyl includes both branched and straight chain
moieties. It is preferred that aryl groups are phenyl or naphthyl.
This invention covers compounds in which the stereochemistry of the
7-position is racemic (R, S) as well as the individual R and S
stereoisomers at the 7-position.
[0014] 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.
[0015] Of the compounds of this invention, it is preferred that
R.sup.2 is hydrogen, and more prefered that R.sup.2 is hydrogen and
R.sup.1 is hydroxyalkyl. 42-O-(2-Hydroxy)ethyl 7-desmethylrapamycin
acid is particularly preferred.
[0016] 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.
[0017] The preparation of the ethers of rapamycin, from which the
7-desmethylrapamycin ethers are made from, are described in U.S.
Pat. No. 5,665,772, which is hereby incorporated by reference. The
conversion of the 7-(S)-methoxy group of the rapamycin hydroxyester
to the 7-(R,S)-hydroxy group can be accomplished by nucleophilic
substitution in the mixture of water and aprotic organic solvent
such as acetonitrile in acidic condition. The ratio of aqueous to
organic solvent is preferred between 1:9 and 9:1, more preferred is
between 1:2 and 2:1. Most preferred ratio of aqueous to organic
solvent is 1:1. Resolution of the 7-isomers can be accomplished by
standard methodology, such as preparative HPLC.
[0018] The antifungal activity for the ethers of
7-desmethylrapamycin of this invention was confirmed in a standard
pharmacological test procedure which measured the ability of the
compound being evaluated to inhibit fungal growth.
42-O-(2-Hydroxy)ethyl 7-desmethylrapamycin acid (Compound I) was
evaluated as a representative compound of this invention. The
procedure used and results obtained are briefly described below. A
96 U-bottom microtiter plate was filled (50 .mu.l/well) with RPMI
1640. The compounds to be evaluated were placed in appropriate
wells, and serial diluted in successive wells to provide 11
dilutions. The concentrations ranged from 64 through 0.06 .mu.g/ml.
An adjusted inoculum of fungi (50 .mu.l) was added to each well and
the plates were incubated at 35.degree. C. for 24-48 hours. The MIC
is the lowest concentration of compound which completely inhibited
growth of organism in the wells. The following table shows the
results obtained in this standard pharmacological test procedure.
Where the same fungi is listed more than once, it indicates that
more than one strain was evaluated. Nystatin and amphotericin B
were used for the purpose of comparison.
1TABLE 1 ANTIFUNGAL ACTIVITY (MIC in .mu.g/mL) Com- Ampho-
Yeast/Fungi ID pound I Nystatin tericin .beta. Candida albicans
1063 1 1 .ltoreq.0.06 Candidu albicans 1117 2 1 0.12 Candida
albicans ATCC 90028 2 1 0.12 Candida parapsilosis 94 - 9 2 1 0.12
Candida parapsilosis 94 - 8 2 2 .ltoreq.0.06 Candida parapsilosis
ATCC 90018 2 2 .ltoreq.0.06 Candida pseudotropicalis ATCC 28838 2 1
.ltoreq.0.06 Candtda tropicalis 94 - 14 0.5 1 .ltoreq.0.06 Candida
tropicalis 94 - 13 0.5 1 .ltoreq.0.06 Candida krussii 94 - 2 0.5 1
0.12 Candida lusitaniae 94 - 3 1 1 .ltoreq.0.06 Candida rugosa 94 -
10 1 1 0.25 Aspergillus fumigatus ATCC 26933 64 2 0.25 Aspergillus
niger S430 64 1 0.25 Aspergillus niger S399 64 2 0.50
[0019] The results obtained in this standard pharmacological test
procedure demonstrate that the compounds of this invention are
useful as antifungal agents.
[0020] The antineoplastic 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 42-O-(2-hydroxy)ethyl 7-desmethylrapamycin (Compound I) as a
representative compound 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.
[0021] Growth Medium
[0022] BRL Minimum Essential Medium with Earle Salts (500 mL)
[0023] +5 mL BRL MEM Non-Essential Amino Acids (10 mM)
[0024] +5 mL BRL Penicillin-Streptomycin (10000 u/mL, 10000
.mu.g/mL)
[0025] +5 mL BRL Na Pyruvate Solution (100 mM)
[0026] +5 mL BRL L-Glutamine 200 mM
[0027] +50 mL BRL Fetal Bovine Serum (Qualified)
[0028] Test Procedure
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] Results
2 Compound IC.sub.50 Compound I 3.5 ng/mL
[0034] 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.
[0035] 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.
[0036] 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.
[0037] It is contemplated that when the compounds of this invention
are used as an immunosuppressive or antiinflammatory agent, they
can be administered in conjunction with one or more other
immunoregulatory agents. Such other immunoregulatory agents
include, but are not limited to azathioprine, corticosteroids, such
as prednisone and methylprednisolone, cyclophosphamide, rapamycin,
cyclosporin A, FK-506, OKT-3, mycophenolate, and ATG. By combining
the compounds of this invention with such other drugs or agents for
inducing immunosuppression or treating inflammatory conditions, the
lesser amounts of each of the agents are required to achieve the
desired effect. The basis for such combination therapy was
established by Stepkowski whose results showed that the use of a
combination of rapamycin and cyclosporin A at subtherapeutic doses
significantly prolonged heart allograft survival time.
[Transplantation Proc. 23: 507 (1991)].
[0038] When used in the treatment or inhibition of vascular
disease, it is contemplated that the compounds of this invention
may be used as the sole active ingredient to provide the
cardiovascular, cerebral, or peripheral vascular benefits covered
by this invention, or may be administered in combination with other
agents which provide beneficial cardiovascular, cerebral, or
peripheral vascular effects. Such agents are generally in the
classes of compounds known as ACE inhibitors, such as quinapril,
perindopril, ramipril, captoprii, trandolapril, fosinopril,
lisinopril, moexipril, and enalapril; angiotensin II receptor
antagonists, such as candesartan, irbesartan, losartan, valsartan,
and telmisartan; fibric acid derivatives, such as clofibrate, and
gemfibrozil; HMG Co-A reductase inhibitors, such as cerivastatin,
fluvastatin, atorvastatin, lovastatin, pravastatin, simvastatin;
beta adrenergic blocking agents, such as sotalol, timolol, esmolol,
carteolol, propranolol, betaxolol, penbutolol, nadolol, acebutolol,
atenolol, metoprolol, and bisoprolol; calcium channel blockers,
such as nifedipine, verapamil, nicardipine, diltiazem, nimodipine,
amlodipine, felodipine, nisoldipine, and bepridil; antioxidants;
anticoagulants such as, warfarin, dalteparin, heparin, enoxaparin,
and danaparoid; and agents useful in hormone replacement therapy
containing estrogens, such as conjugated estrogens, ethinyl
estradiol, 17-beta-estradiol, estradiol, and estropipate.
[0039] When administered for the treatment or inhibition of a
particular disease state or disorder, it is understood that the
effective dosage of the ether of 7-desmethylrapamycin 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 ether of
7-desmethylrapamycin 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.
[0040] 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.
[0041] 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.
[0042] In some cases it may be desirable to administer the
compounds directly to the airways in the form of an aerosol.
[0043] 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.
[0044] 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.
[0045] 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).
[0046] 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.
[0047] 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.
[0048] The preparation of representative examples of this invention
is described below.
EXAMPLE 1
Preparation of 42-O-(2-Hydroxy)ethyl 7-desmethylrapamycin (Compound
I)
[0049] 42-O -(2-Hydroxy)ethyl rapamycin (30 mg,
3.13.times.10.sup.-5 mole) was dissolved in 20 mL acetonitrile and
20 mL 0.1 N hydrochloric acid. The solution was kept at room
temperature overnight. Then the reaction mixture was extracted with
80 mL methylene chloride (CH.sub.2Cl.sub.2) in a separatory funnel.
The organic layer was washed 100 mL water, 50 mL 0.1 M sodium
phosphate buffer (pH 7) and then 100 mL water again. The methylene
chloride was removed by rotary evaporation. The pure compound I was
performed by preparative HPLC on a Prep Nova-pak HR C18
(300.times.19 mm) column from Waters. Compound I eluted at between
11.7-13.1 min and 42-O -(2-hydroxy)ethyl rapamycin eluted at 22.7
min using a gradient (0-5 min 40% A, 40% B, 5-25 min from 50% B to
70%, 25-30 min 70% B to 100%B). 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. Then most of
the solvent was removed by rotary evaporation and final product was
precipitated by hexane. Compound I, a white solid was obtained.
Positive ion mass spectrum shows the molecular ion specie
[M+NH.sub.4].sup.+ at m/z 961.6. The loss of 14 from the mass of
42-O-(2-hydroxy)ethyl rapamycin indicates the conversion of a
methoxy to a hydroxy. .sup.1H NMR (400 MHz) of compound I in
CDCl.sub.3 shows the loss of 7-position CH.sub.3O-resonance at 3.14
ppm comparing to the .sup.1H NMR of 42-O-(2-hydroxy)ethyl
rapamycin.
* * * * *