U.S. patent application number 11/906665 was filed with the patent office on 2008-04-17 for nucleoside compounds and their use for treating cancer and diseases associated with somatic mutations.
This patent application is currently assigned to PTC Therapeutics, Inc.. Invention is credited to Neil G. Almstead, Westley J. Friesen, Paul D. Kennedy, James J. Takasugi, Ellen M. Welch, Richard G. Wilde.
Application Number | 20080090774 11/906665 |
Document ID | / |
Family ID | 30773080 |
Filed Date | 2008-04-17 |
United States Patent
Application |
20080090774 |
Kind Code |
A1 |
Wilde; Richard G. ; et
al. |
April 17, 2008 |
Nucleoside compounds and their use for treating cancer and diseases
associated with somatic mutations
Abstract
The invention encompasses nucleoside compounds, compositions
comprising the compounds and methods for treating or preventing
diseases associated with nonsense mutations of mRNA by
administering these compounds or compositions.
Inventors: |
Wilde; Richard G.;
(Somerville, NJ) ; Kennedy; Paul D.; (Kalamazoo,
MI) ; Almstead; Neil G.; (Holmdel, NJ) ;
Welch; Ellen M.; (Califon, NJ) ; Takasugi; James
J.; (Lawrenceville, NJ) ; Friesen; Westley J.;
(Huntingdon Valley, PA) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
PTC Therapeutics, Inc.
|
Family ID: |
30773080 |
Appl. No.: |
11/906665 |
Filed: |
October 2, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10625059 |
Jul 22, 2003 |
7291603 |
|
|
11906665 |
Oct 2, 2007 |
|
|
|
60398334 |
Jul 24, 2002 |
|
|
|
Current U.S.
Class: |
514/43 ;
514/49 |
Current CPC
Class: |
A61P 35/00 20180101;
C07H 19/00 20130101 |
Class at
Publication: |
514/043 ;
514/049 |
International
Class: |
A61K 31/706 20060101
A61K031/706; A61P 35/00 20060101 A61P035/00 |
Claims
1. A method of treating or preventing a disease resulting from a
somatic mutation in DNA or RNA comprising administering to a
patient in need thereof an effective amount of a compound having
the structure: ##STR80## or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate, racemate or stereoisomer thereof,
wherein: Z is substituted or unsubstituted alkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl; X is
CH.sub.2 or NH; R.sup.1 is hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocyclo, substituted or
unsubstituted arylalkyl, substituted or unsubstituted
heteroarylalkyl, substituted or unsubstituted cycloalkylalkyl,
substituted or unsubstituted heterocycloalkyl; R.sup.2 is
substituted or unsubstituted alkyl, carboxy, amido, acyl,
alkylcarbonyl, halogen, a biohydrolyzable group,
OP(O).sub.3.sup.2-, O[P(O).sub.3].sub.2.sup.3-,
O[P(O).sub.3].sub.3.sup.4-, N.sub.3, CH.sub.2--NR.sub.6R.sub.7 or
CH.sub.2--OR.sup.6; R.sup.3, R.sup.3', R.sup.4 and R.sup.4' are at
each occurrence independently OR.sup.7, OR.sup.8, hydrogen,
halogen, substituted or unsubstituted alkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl,
substituted or unsubstituted alkylcarbonyl, a biohydrolyzable
group, or R.sup.3 and R.sup.4 taken together form a bond, or
R.sup.3 and R.sup.4 taken together with the atoms to which they are
attached form a substituted or unsubstituted heterocyclo, or
R.sup.3 and R.sup.3' and/or R.sup.4 and R.sup.4' taken together
with the carbon to which they are attached form C(.dbd.O); and
R.sup.6, R.sup.7 and R.sup.8 are at each occurrence independently
hydrogen, substituted or unsubstituted alkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl,
substituted or unsubstituted alkylcarbonyl, a biohydrolyzable
group, or R.sup.3 and R.sup.4 taken together with the atoms to
which they are attached form a substituted or unsubstituted
heterocyclo.
2. The method of claim 1, wherein the compound, or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate or
stereoisomer thereof, is administered as a composition comprising
the compound and a pharmaceutically acceptable carrier or
diluent.
3. The method of claim 1, wherein the administration is
intravenous.
4. The method claim 1, wherein Z is substituted or unsubstituted
pyrimidinyl.
5. The method of claim 1, wherein each occurrence of R' is
hydrogen.
6. The method of claim 1, wherein the disease is cancer.
7. The method of claim 6, wherein the administration is
intravenous.
8. The method of claim 6, wherein the cancer is of the head and
neck, eye, skin, mouth, throat, esophagus, chest, bone, blood,
lung, colon, sigmoid, rectum, stomach, prostate, breast, ovaries,
kidney, liver, pancreas, brain, intestine, heart or adrenals.
9. The method of claim 6, wherein the compound, or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate or
stereoisomer thereof, comprises a pharmaceutically acceptable
carrier or diluent.
10. The method of claim 6, wherein the cancer is a solid tumor.
11. The method of claim 6, wherein the cancer is sarcoma,
carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,
retinoblastoma, a blood-born tumor or multiple myeloma.
12. The method of claim 6, wherein the cancer is acute
lymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acute
lymphoblastic T-cell leukemia, acute myeloblastic leukemia, acute
promyelocytic leukemia, acute monoblastic leukemia, acute
erythroleukemic leukemia, acute megakaryoblastic leukemia, acute
myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute
undifferentiated leukemia, chronic myelocytic leukemia, chronic
lymphocytic leukemia, hairy cell leukemia, or multiple myeloma.
13. A method of treating or preventing a disease associated with a
mutation of the p53 gene comprising administering to a patient in
need thereof an effective amount of a compound having the
structure: ##STR81## or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate, racemate or stereoisomer thereof,
wherein: Z is substituted or unsubstituted alkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl; X is
CH.sub.2 or NH; R.sup.1 is hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocyclo, substituted or
unsubstituted arylalkyl, substituted or unsubstituted
heteroarylalkyl, substituted or unsubstituted cycloalkylalkyl,
substituted or unsubstituted heterocycloalkyl; R.sup.2 is
substituted or unsubstituted alkyl, carboxy, amido, acyl,
alkylcarbonyl, halogen, a biohydrolyzable group,
OP(O).sub.3.sup.2-, O[P(O).sub.3].sub.2.sup.3-,
O[P(O).sub.3].sub.3.sup.4-, N.sub.3, CH.sub.2--NR.sub.6R.sub.7 or
CH.sub.2--OR.sup.6; R.sup.3, R.sup.3', R.sup.4 and R.sup.4' are at
each occurrence independently OR.sup.7, OR.sup.8, hydrogen,
halogen, substituted or unsubstituted alkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl,
substituted or unsubstituted alkylcarbonyl, a biohydrolyzable
group, or R.sup.3 and R.sup.4 taken together form a bond, or
R.sup.3 and R.sup.4 taken together with the atoms to which they are
attached form a substituted or unsubstituted heterocyclo, or
R.sup.3 and R.sup.3' and/or R.sup.4 and R.sup.4' taken together
with the carbon to which they are attached form C(.dbd.O); and
R.sup.6, R.sup.7 and R.sup.8 are at each occurrence independently
hydrogen, substituted or unsubstituted alkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl,
substituted or unsubstituted alkylcarbonyl, a biohydrolyzable
group, or R.sup.3 and R.sup.4 taken together with the atoms to
which they are attached form a substituted or unsubstituted
heterocyclo.
14. The method of claim 13, wherein the administration is
intravenous.
15. The method of claim 13, wherein the disease is sarcoma,
carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma or
retinoblastoma.
16. A method of inhibiting the growth of a cancer cell comprising
contacting the cancer cell with an effective amount of a compound
having the structure: ##STR82## or a pharmaceutically acceptable
salt, hydrate, solvate, clathrate, racemate or stereoisomer
thereof, wherein: Z is substituted or unsubstituted alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl; X is
CH.sub.2 or NH; R.sup.1 is hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocyclo, substituted or
unsubstituted arylalkyl, substituted or unsubstituted
heteroarylalkyl, substituted or unsubstituted cycloalkylalkyl,
substituted or unsubstituted heterocycloalkyl; R.sup.2 is
substituted or unsubstituted alkyl, carboxy, amido, acyl,
alkylcarbonyl, halogen, a biohydrolyzable group,
OP(O).sub.3.sup.2-, O[P(O).sub.3].sub.2.sup.3-,
O[P(O).sub.3].sub.3.sup.4-, N.sub.3, CH.sub.2--NR.sub.6R.sub.7 or
CH.sub.2--OR.sup.6; R.sup.3, R.sup.3', R.sup.4 and R.sup.4' are at
each occurrence independently OR.sup.7, OR.sup.8, hydrogen,
halogen, substituted or unsubstituted alkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl,
substituted or unsubstituted alkylcarbonyl, a biohydrolyzable
group, or R.sup.3 and R.sup.4 taken together form a bond, or
R.sup.3 and R.sup.4 taken together with the atoms to which they are
attached form a substituted or unsubstituted heterocyclo, or
R.sup.3 and R.sup.3' and/or R.sup.4 and R.sup.4' taken together
with the carbon to which they are attached form C(.dbd.O); and
R.sup.6, R.sup.7 and R.sup.8 are at each occurrence independently
hydrogen, substituted or unsubstituted alkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl,
substituted or unsubstituted alkylcarbonyl, a biohydrolyzable
group, or R.sup.3 and R.sup.4 taken together with the atoms to
which they are attached form a substituted or unsubstituted
heterocyclo, with the proviso that the cancer cell is not a
leukemia cancer cell.
17. A compound having the structure: ##STR83## or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
racemate or stereoisomer thereof, wherein: X is CH.sub.2, O, S or
NH; R.sup.1 is hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl; R.sup.2 is substituted or unsubstituted alkyl,
carboxy, amido, acyl, alkylcarbonyl, halogen, a biohydrolyzable
group, OP(O).sub.3.sup.2-, O[P(O).sub.3].sub.2.sup.3-,
O[P(O).sub.3].sub.3.sup.4-, N.sub.3, CH.sub.2--NR.sub.6R.sub.7 or
CH.sub.2--OR.sup.6; R.sup.3, R.sup.3', R.sup.4 and R.sup.4' are at
each occurrence independently OR.sup.7, OR.sup.8, hydrogen,
halogen, substituted or unsubstituted alkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl,
substituted or unsubstituted alkylcarbonyl, a biohydrolyzable
group, or R.sup.3 and R.sup.4 taken together form a bond, or
R.sup.3 and R.sup.4 taken together with the atoms to which they are
attached form a substituted or unsubstituted heterocyclo, or
R.sup.3 and R.sup.3' and/or R.sup.4 and R.sup.4' taken together
with the carbon to which they are attached form C(.dbd.O); R.sup.6,
R.sup.7 and R.sup.8 are at each occurrence independently hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
aryl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocyclo,
substituted or unsubstituted arylalkyl, substituted or
unsubstituted heteroarylalkyl, substituted or unsubstituted
cycloalkylalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or unsubstituted arylcarbonyl, substituted or
unsubstituted alkylcarbonyl, a biohydrolyzable group, or R.sup.7
and R.sup.8 taken together with the atoms to which they are
attached form a substituted or unsubstituted heterocyclo; A and B
are each independently C or N; Y.sup.1--Y.sup.5 are each
independently hydrogen, hydroxy, halogen, nitro, cyano, sulfate,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, alkoxy, alkylthioether, carboxyalkyl,
carbonylalkyl, amino, NR.sup.5R.sup.5', amido, or alkoxycarbonyl,
wherein if B is N, Y.sup.3 can also be O.sup.- and if A is N,
Y.sup.5 can also be O.sup.-, and wherein Y.sup.3 is not present if
B is N and Y.sup.5 is not present if A is N; and R.sup.5 and
R.sup.5' are each independently hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted alkenyl,
substituted or unsubstituted alkynyl, substituted or unsubstituted
aryl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocyclo,
substituted or unsubstituted arylalkyl, substituted or
unsubstituted heteroarylalkyl, substituted or unsubstituted
cycloalkylalkyl, substituted or unsubstituted heterocycloalkyl,
with the proviso that the compound is not
6-Amino-5-nitro-4-(.beta.-D-ribo-furanosylamino)pyrimidine.
18. The compound of claim 17, wherein the compound has the
structure: ##STR84## or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate or stereoisomer thereof.
19. The compound of claim 17, wherein the compound has the
structure: ##STR85## or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate or stereoisomer thereof.
20. The compound of claim 17, wherein the compound has the
structure: ##STR86## or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate or stereoisomer thereof.
21. The compound of claim 17, wherein the compound has the
structure: ##STR87## or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate or stereoisomer thereof.
22. The compound of claim 17, wherein the compound has the
structure: ##STR88## or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate or stereoisomer thereof.
23. The compound of claim 17, wherein the compound has the
structure: ##STR89## or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate or stereoisomer thereof.
24. The compound of claim 17, wherein the compound has the
structure: ##STR90## or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate or stereoisomer thereof, wherein each
occurrence of R'' is independently hydrogen, OP(O.sub.3).sup.-2,
C(.dbd.O)CH.sub.3 or a biohydrolyzable group.
25. A compound, wherein the compound is:
6-(N-methylamino)-5-nitro-4-(.beta.-D-ribo-furanosylamino)pyrimidine;
5,6-Diamino-4-(.beta.-D-ribofuranosyl-amino)pyrimidine;
3-Nitro-2-(.beta.-D-ribofuranosyl-amino)pyridine;
5-Nitro-2-(.beta.-D-ribofuranosyl-amino)pyridine;
(1R,2S,3R,5R)-3-(6-Amino-5-nitro-pyrimidin-4-ylamino)-5-hydroxymethyl-cyc-
lopentane-1,2-diol;
(1S,2R,3S,5S)-3-(6-Amino-5-nitro-pyrimidin-4-ylamino)-5-hydroxymethyl-cyc-
lopentane-1,2-diol;
6-methoxy-3-nitro-2-(.beta.-D-ribofuranosyl-amino)pyridine;
6-(dimethylamino)-5-nitro-4-(.beta.-D-ribo-furanosylamino)pyrimidine;
6-(thiomethyl)-5-nitro-4-(.beta.-D-ribo-furanosylamino)pyrimidine;
5-nitro-4-methyl-2-(.beta.-D-ribofuranosyl-amino)pyridine;
6-amino-5-nitro-4-(2,3-O-isopropylidene-.beta.-D-ribofuranosylamino)pyrim-
idine;
6-(2-hydroxy-ethylamino)-5-nitro-4-(.beta.-D-ribo-furanosylamino)p-
yrimidine;
6-(ethylamino)-5-nitro-4-(.beta.-D-ribo-furanosylamino)pyrimidine;
6-(4-methoxy-benzylamino)-5-cyano-4-(.beta.-D-ribo-furanosylamino)pyrimid-
ine; 3-cyano-2-(.beta.-D-ribofuranosyl-amino)pyridine;
6-hydroxy-5-nitro-4-(.beta.-D-ribofuranosylamino)pyrimidine;
6-amino-5-nitro-4-(.beta.-D-xylofuranosylamino)-pyrimidine;
6-amino-5-nitro-4-(.beta.-L-ribofuranosylamino)-pyrimidine;
6-amino-5-nitro-4-(5-deoxy-5-fluoro-.beta.-D-ribofuranosylamino)-pyrimidi-
ne;
6-amino-5-nitro-4-(5-deoxy-5-azido-.beta.-D-ribofuranosylamino)-pyrim-
idine; 6-amino-5-nitro-4-(.alpha.-D-ribofuranosylamino)-pyrimidine;
6-Amino-5-nitro-4-[(5-O-acetyl-.beta.-D-ribofuranosyl)amino]pyrimidine;
6-Amino-5-nitro-4-[(2,3,5-tri-O-benzoyl-.beta.-D-ribofuranosyl)amino]pyri-
midine; Methyl
6-amino-4-(.beta.-D-ribofuranosylamino)pyrimidine-5-carboxylate;
Methyl
6-chloro-4-(.beta.-D-ribofuranosylamino)pyrimidine-5-carboxylate;
Methyl
6-amino-4-(.alpha.-D-ribofuranosylamino)pyrimidine-5-carboxylate;
Methyl
6-chloro-4-(.alpha.-D-ribofuranosylamino)pyrimidine-5-carboxylate;
5-(6-Amino-5-nitro-pyrimidin-4-ylamino)-3,4-dihydroxy-tetrahydro-furan-2--
carboxylic acid;
5-(6-Amino-5-nitro-pyrimidin-4-ylamino)-3,4-dihydroxy-tetrahydro-furan-2--
carboxylic acid amide;
5-(6-Amino-5-nitro-pyrimidin-4-ylamino)-3,4-dihydroxy-tetrahydro-furan-2--
carbaldehyde;
2-(6-Amino-5-nitro-pyrimidin-4-ylamino)-5-(1-hydroxy-ethyl)-tetrahydro-fu-
ran-3,4-diol;
2-(6-Amino-5-nitro-pyrimidin-4-ylamino)-5-methyl-tetrahydro-furan-3,4-dio-
l;
2-(4-Amino-3-nitro-pyridin-2-ylamino)-5-hydroxymethyl-tetrahydro-furan-
-3,4-diol;
2-(5-Amino-4-nitro-pyridin-3-ylamino)-5-hydroxymethyl-tetrahydro-furan-3,-
4-diol;
5-(6-Amino-5-nitro-pyrimidin-4-ylamino)-2-hydroxymethyl-tetrahydr-
o-furan-3-ol;
5-(6-Amino-5-nitro-pyrimidin-4-ylamino)-4-fluoro-2-hydroxymethyl-tetrahyd-
ro-furan-3-ol;
5-(6-Amino-5-nitro-pyrimidin-4-ylamino)-4,4-difluoro-2-hydroxymethyl-tetr-
ahydro-furan-3-ol;
2-(6-Amino-5-nitro-pyrimidin-4-ylamino)-4-fluoro-5-hydroxymethyl-tetrahyd-
ro-furan-3-ol;
2-(6-Amino-5-nitro-pyrimidin-4-ylamino)-4,4-difluoro-5-hydroxymethyl-tetr-
ahydro-furan-3-ol;
[5-(6-Amino-5-nitro-pyrimidin-4-ylamino)-2,5-dihydro-furan-2-yl]-methanol-
;
5-(6-Amino-5-nitro-pyrimidin-4-ylamino)-2-hydroxymethyl-3-methyl-tetrah-
ydro-furan-3,4-diol;
2-(6-Amino-5-nitro-pyrimidin-4-ylamino)-4-hydroxy-5-hydroxymethyl-dihydro-
-furan-3-one;
5-(6-Amino-5-nitro-pyrimidin-4-ylamino)-4-hydroxy-2-hydroxymethyl-dihydro-
-furan-3-one;
2-Aminomethyl-5-(6-amino-5-nitro-pyrimidin-4-ylamino)-tetrahydro-furan-3,-
4-diol;
2-(6-Amino-5-nitro-pyrimidin-4-ylamino)-5-(2-hydroxy-ethyl)-tetra-
hydro-furan-3,4-diol;
2-(6-Amino-5-nitro-pyrimidin-4-ylamino)-5-hydroxymethyl-tetrahydro-thioph-
ene-3,4-diol;
2-(2-Amino-3-nitro-pyridin-4-ylamino)-5-hydroxymethyl-tetrahydro-furan-3,-
4-diol;
4-amino-6-(3,4-dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-ylami-
no)-pyrimidine-5-carboxylate;
4-Amino-6-(3,4-dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-ylamino)-pyri-
midine-5-carboxylic acid;
2-Hydroxymethyl-5-(5-nitro-pyrimidin-4-ylamino)-tetrahydro-furan-3,4-diol-
;
2-(6-Amino-5-nitro-pyridazin-4-ylamino)-5-hydroxymethyl-tetrahydro-fura-
n-3,4-diol;
2-(5-Amino-4-nitro-pyridazin-3-ylamino)-5-hydroxymethyl-tetrahydro-furan--
3,4-diol; or a pharmaceutically acceptable salt, hydrate, solvate,
clathrate or stereoisomer thereof.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 10/625,059, filed Jul. 22, 2003, allowed, which claims the
benefit of U.S. provisional application No. 60/398,334, filed on
Jul. 24, 2002, the disclosures of which are incorporated by
reference herein in their entireties.
1. FIELD OF INVENTION
[0002] The invention encompasses nucleoside compounds, compositions
comprising the compounds and methods for treating or preventing
diseases associated with nonsense mutations of mRNA by
administering these compounds or compositions.
2. BACKGROUND OF THE INVENTION
[0003] Gene expression in cells depends upon the sequential
processes of transcription and translation. Together, these
processes produce a protein from the nucleotide sequence of its
corresponding gene.
[0004] Transcription involves the synthesis of mRNA from DNA by RNA
polymerase. Transcription begins at a promoter region of the gene
and continues until termination is induced, such as by the
formation of a stem-loop structure in the nascent RNA or the
binding of the rho gene product.
[0005] Protein is then produced from mRNA by the process of
translation, occurring on the ribosome with the aid of tRNA, tRNA
synthetases and various other protein and RNA species. Translation
comprises the three phases of initiation, elongation and
termination. Translation is initiated by the formation of an
initiation complex consisting of protein factors, mRNA, tRNA,
cofactors and the ribosomal subunits that recognize signals on the
mRNA that direct the translation machinery to begin translation on
the mRNA. Once the initiation complex is formed, growth of the
polypeptide chain occurs by the repetitive addition of amino acids
by the peptidyl transferase activity of the ribosome as well as
tRNA and tRNA synthetases. The presence of one of the three
termination codons (UAA, UAG, UGA) in the A site of the ribosome
signals the polypeptide chain release factors (RFs) to bind and
recognize the termination signal. Subsequently, the ester bond
between the 3' nucleotide of the tRNA located in the ribosome's P
site and the nascent polypeptide chain is hydrolyzed, the completed
polypeptide chain is released, and the ribosome subunits are
recycled for another round of translation.
[0006] Mutations of the DNA sequence in which the number of bases
is altered are categorized as insertion or deletion mutations
(frameshift mutations) and can result in major disruptions of the
genome. Mutations of the DNA that change one base into another and
result in an amino acid substition are labeled missense mutations.
Base substitutions are subdivided into the classes of transitions
(one purine to another purine, or one pyrimidine to another
pyrimidine) and transversions (a purine to a pyrimidine, or a
pyrimidine to a purine).
[0007] Transition and transversion mutations can result in a
nonsense mutationchanging an amino acid codon into one of the three
stop codons. These premature stop codons can produce aberrant
proteins in cells as a result of premature translation termination.
A nonsense mutation in an essential gene can be lethal and can also
result in a number of human diseases, such as, cancers, lysosomal
storage disorders, the muscular dystrophies, cystic fibrosis and
hemophilia, to name a few.
[0008] The human p53 gene is the most commonly mutated gene in
human cancer (Zambetti, G. P. and Levine, A., FASEB 7:855-865
(1993)). Found in both genetic and spontaneous cancers, over 50
different types of human cancers contain p53 mutations and
mutations of this gene occur in 50-55% of all human cancers
(Hollstein, M., et al., Nucleic Acids Res. 22:3551-55 (1994);
International Agency for Research on Cancer (IARC) database).
Approximately 70% of colorectal cancer, 50% of lung cancer and 40%
of breast cancers contain mutant p53 (Koshland, D., Science
262:1953 (1993)). Aberrant forms of p53 are associated with poor
prognosis, more aggressive tumors, metastasis, and lower 5 year
survival rates (Id.). p53's role in the induction of cell growth
arrest and/or apoptosis upon DNA damage is believed to be essential
for the destruction of mutated cells that would have otherwise
gained a growth advantage. In addition, p53 sensitizes rapidly
dividing cells to apoptotic signals. Of greater than 15,000
reported mutations in the p53 gene, approximately 7% are nonsense
mutations. Accordingly, there is a need for a safe and effective
treatment directed to p53 nonsense mutations.
[0009] In bacterial and eukaryotic strains with nonsense mutations,
suppression of the nonsense mutation can arise as a result of a
mutation in one of the tRNA molecules so that the mutant tRNA can
recognize the nonsense codon, as a result of mutations in proteins
that are involved in the translation process, as a result of
mutations in the ribosome (either the ribosomal RNA or ribosomal
proteins), or by the addition of compounds known to alter the
translation process (for example, cycloheximide or the
aminoglycoside antibiotics). The result is that an amino acid will
be incorporate into the polypeptide chain, at the site of the
nonsense mutation and translation will not prematurely terminate at
the nonsense codon. The inserted amino acid will not necessarily be
identical to the original amino acid of the wild-type protein,
however, many amino acid substitutions do not have a gross effect
on protein structure or function. Thus, a protein produced by the
suppression of a nonsense mutation would be likely to possess
activity close to that of the wild-type protein. This scenario
provides an opportunity to treat diseases associated with nonsense
mutations by avoiding premature termination of translation through
suppression of the nonsense mutation.
[0010] The ability of aminoglycoside antibiotics to promote
readthrough of eukaryotic stop codons has attracted interest in
these drugs as potential therapeutic agents in human diseases
caused by nonsense mutations. One disease for which such a
therapeutic strategy may be viable is classical late infantile
neuronal ceroid lipofuscinosis (LINCL), a fatal childhood
neurodegenerative disease with currently no effective treatment.
Premature stop codon mutations in the gene CLN2 encoding the
lysosomal tripeptidyl-peptidase 1 (TPP-I) are associated with
disease in approximately half of children diagnosed with LINCL. The
ability of the aminoglycoside gentamicin to restore TPP-I activity
in LINCL cell lines has been examined. In one patient-derived cell
line that was compound heterozygous for a commonly seen nonsense
mutation (Arg208Stop) and a different rare nonsense mutation,
approximately 7% of normal levels of TPP-I were maximally restored
with gentamicin treatment. These results suggest that
pharmacological suppression of nonsense mutations by
aminoglycosides or functionally similar pharmaceuticals may have
therapeutic potential in LINCL (Sleat et. al., Eur. J. Ped. Neurol.
5:Suppl A 57-62 (2001)).
[0011] In cultured cells having premature stop codons in the Cystic
Fibrosis Transmembrane Conductance Regulator (CFTR) gene, treatment
with aminoglycosides led to the production of full length CFTR
(Bedwell et. al., Nat. Med. 3:1280-1284 (1997); Howard et. al. Nat.
Med. 2: 467-469 (1996)). In mouse models for Duchenne muscular
dystrophy, gentamicin sulfate was observed to suppress
translational termination at premature stop codons resulting in
full length dystrophin (Barton-Davis et. al., J. Clin. Invest.
104:375-381 (1999)). A small increase in the amount of full length
dystrophin provided protection against contraction-induced damage
in the mdx mice. The amino acid inserted at the site of the
nonsense codon was not determined in these studies.
[0012] Accordingly, small molecule therapeutics or prophylactics
that suppress premature translation termination by mediating the
misreading of the nonsense codon would be useful for the treatment
of a number of diseases. The discovery of small molecule drugs,
particularly orally bioavailable drugs, can lead to the
introduction of a broad spectrum of selective therapeutics or
prophylactics to the public which can be used against disease
caused by nonsense mutations is just beginning.
[0013] Clitocine
(6-Amino-5-nitro-4-(.beta.-D-ribo-furanosylamino)pyrimidine) is a
naturally occurring exocyclic amino nucleoside that was first
isolated from the mushroom Clitocybe inversa (Kubo et al., Tet.
Lett. 27: 4277 (1986)). The total synthesis of clitocine has also
been reported. (Moss et al., J. Med. Chem. 31:786-790 (1988) and
Kamikawa et al., J. Chem. Soc. Chem. Commun. 195 (1988)). Clitocine
has been reported to possess insecticidal activity and cytostatic
activity against leukemia cell lines (Kubo et al., Tet. Lett. 27:
4277 (1986) and Moss et al., J. Med. Chem. 31:786-790 (1988)).
However, the use of clitocine as a therapeutic for diseases
associated with a nonsense mutation has not been disclosed until
now. Nor has anyone reported the development of an analogue or
derivative of clitocine that has utility as a therapeutic for
cancer or a disease associated with a nonsense mutation.
[0014] Citation of any reference in Section 2 of this application
is not an admission that the reference is prior art to the
application.
3. SUMMARY OF THE INVENTION
[0015] The present invention is based in part on the discovery of
small molecules that modulate premature translation termination
and/or nonsense-mediated mRNA decay. The present invention
encompasses compounds of formula I, compositions comprising
compounds of formula I, and methods for the use of compounds of
formula I. Compounds of formula I have the structure: ##STR1## or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
racemate or stereoisomer thereof, wherein:
[0016] Z is substituted or unsubstituted alkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl;
[0017] X is CH.sub.2, O, S or NH;
[0018] R.sup.1 is hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl;
[0019] R.sup.2 is substituted or unsubstituted alkyl, carboxy,
amido, acyl, alkylcarbonyl, halogen, a biohydrolyzable group,
OP(O).sub.3.sup.2-, O[P(O).sub.3].sub.2.sup.3-,
O[P(O).sub.3].sub.3.sup.4-, N.sub.3, CH.sub.2--NR.sub.6R.sub.7, or
CH.sub.2--OR.sup.6;
[0020] R.sup.3, R.sup.3', R.sup.4 and R.sup.4' are at each
occurrence independently OR.sup.7, OR.sup.8, hydrogen, halogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
aryl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocyclo,
substituted or unsubstituted arylalkyl, substituted or
unsubstituted heteroarylalkyl, substituted or unsubstituted
cycloalkylalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or unsubstituted arylcarbonyl, substituted or
unsubstituted alkylcarbonyl, a biohydrolyzable group, or R.sup.3
and R.sup.4 taken together form a bond, or R.sup.3 and R.sup.4
taken together with the atoms to which they are attached form a
substituted or unsubstituted heterocyclo, or R.sup.3 and R.sup.3'
and/or R.sup.4 and R.sup.4' taken together with the carbon to which
they are attached form C(.dbd.O); and
[0021] R.sup.6, R.sup.7 and R.sup.8 are at each occurrence
independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl,
substituted or unsubstituted alkylcarbonyl, a biohydrolyzable
group, or R.sup.3 and R.sup.4 taken together with the atoms to
which they are attached form a substituted or unsubstituted
heterocyclo.
[0022] In another embodiment, compounds of formula I have the
structure: ##STR2##
[0023] or a pharmaceutically acceptable salt, hydrate, solvate,
clathrate, racemate or stereoisomer thereof, wherein:
[0024] A and B are each independently C or N;
[0025] Y.sup.1--Y.sup.5 are each independently hydrogen, hydroxy,
halogen, nitro, cyano, sulfate, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocyclo, substituted or
unsubstituted arylalkyl, substituted or unsubstituted
heteroarylalkyl, substituted or unsubstituted cycloalkylalkyl,
substituted or unsubstituted heterocycloalkyl, alkoxy,
alkylthioether, carboxyalkyl, carbonylalkyl, amino,
NR.sup.5R.sup.5', amido, alkylamino or alkoxycarbonyl, wherein if B
is N, Y.sup.3 can also be O.sup.- and if A is N, Y.sup.5 can also
be O.sup.-, and wherein Y.sup.3 is not present if B is N and
Y.sup.5 is not present if A is N; and
[0026] R.sup.5 and R.sup.5' are each independently hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl.
[0027] Without being limited by theory, the invention encompasses
methods for modulating premature translation termination and/or
nonsense-mediated mRNA decay in a cell, including mammalian cells
in a living host. The invention further encompasses a method for
suppressing premature translation termination and/or
nonsense-mediated mRNA decay in a cell comprising contacting a cell
exhibiting premature translation termination and/or
nonsense-mediated mRNA decay with an effective amount of a compound
of the invention. The methods of the invention are particularly
useful for treating or preventing diseases associated with a
nonsense mutation in the p53 gene due to p53's crucial role in
apoptosis. Without being limited by theory, it is thought that
apoptosis can be induced by contacting a cell with an effective
amount of a compound of the invention resulting in suppression of
the nonsense mutation which, in turn, allows the production of full
length p53 to occur. The invention further encompasses a method for
inducing nonsense suppression in a cell comprising contacting a
cell exhibiting a nonsense mutation with an effective amount of a
compound of the invention. A nonsense codon can be present in the
DNA or RNA of any type of cell and can arise naturally or result
from mutagenesis (e.g., arising from exposure to a chemical,
radiation, UV rays, X-rays or other environmental factors).
Accordingly, cells encompassed by the present methods include
animal cells, mammalian cells, bacterial cells, plant cells and
virally infected cells. In one embodiment, the nonsense mutation is
a genetic mutation (i.e., the nonsense codon was present in the
progenitor DNA). In another embodiment, the nonsense mutation is a
somatic mutation (i.e., the nonsense codon arose spontaneously or
from mutagenesis).
[0028] Without being limited by theory, the ability of the
compounds of the invention to promote readthrough of premature stop
codons makes them useful in the treatment or prevention of any
disease which is caused in whole or in part by a nonsense mutation.
Such diseases can occur due to the decreased amount of active
protein produced as a result of premature termination of
translation. Without being limited by theory, the compounds of the
invention allow the translation of mRNA to continue past the
nonsense mutation resulting in the production of full length
protein. A powerful aspect of the invention is that the therapeutic
activity of compounds of the invention are not necessarily limited
to a specific disease, instead are effective at treating or
preventing many diseases associated with a nonsense mutation.
Further, the methods of the invention include those that are
patient specific, that is, a patient can be screened to determine
if their disease is associated with a nonsense mutation. If so,
they can then be treated with a compound of the invention more
efficiently, specifically and effectively.
[0029] The compounds of the invention are useful for treating or
preventing diseases associated with a nonsense mutation. Diseases
that can be treated or prevented by compounds of the invention
include, but are not limited to, cancer, autoimmune diseases, blood
diseases, collagen diseases, diabetes, neurodegenerative diseases,
cardiovascular diseases, pulmonary diseases, inflammatory diseases,
lysosomal storage disease, tuberous sclerosis or central nervous
system diseases.
3.1 BRIEF DESCRIPTION OF THE FIGURES
[0030] FIGS. 1A and 1B demonstrate the ability of clitocine to
suppress nonsense mutations in the p53 gene of CAOV-3 and CALU-6
cells.
[0031] FIGS. 2A and 2B demonstrate that the p53 protein produced by
clitocine's nonsense suppression is functionally active.
[0032] FIG. 3 demonstrates that the p53 protein produced by
clitocine's nonsense suppression induces apoptosis.
[0033] FIG. 4 demonstrates the ability of clitocine to inhibit the
growth of CAOV-3 cells in vivo as compared to vehicle alone.
3.2 DEFINITIONS
[0034] As used herein, "premature translation termination" refers
to the result of a mutation that changes a codon corresponding to
an amino acid to a stop codon.
[0035] As used herein, "nonsense-mediated mRNA decay" refers to any
mechanism that mediates the decay of mRNAs containing a premature
translation termination codon.
[0036] As used herein, a "premature termination codon" or
"premature stop codon" refers to the occurrence of a stop codon
wherein a codon corresponding to an amino acid should be.
[0037] As used herein, a "nonsense mutation" is a point mutation
changing a codon corresponding to an amino acid to a stop codon.
The nonsense mutation can be either a genetic mutation or a somatic
mutation. A genetic mutation is one that occurs in a germ cell
(i.e., wherein the nonsense codon was present in the progenitor
DNA). A somatic mutation is one that occurs in the somatic tissue
(i.e., non germline tissue) through mutagenesis and will not be
heritable. The somatic mutation may be naturally occurring or the
result of exposure to a chemical, radiation, UV rays, X-rays or
other environmental factors.
[0038] As used herein, "nonsense suppression" refers to the
inhibition or suppression of premature translation and/or
nonsense-mediated mRNA decay.
[0039] As used herein, "modulation of premature translation
termination and/or nonsense-mediated mRNA decay" refers to the
regulation of gene expression by altering the level of nonsense
suppression. For example, if it is desirable to increase production
of a defective protein encoded by a gene with a premature stop
codon, i.e., to permit readthrough of the premature stop codon of
the disease gene so translation of the gene can occur, then
modulation of premature translation termination and/or
nonsense-mediated mRNA decay entails up-regulation of nonsense
suppression. Conversely, if it is desirable to promote the
degradation of an mRNA with a premature stop codon, then modulation
of premature translation termination and/or nonsense-mediated mRNA
decays entails down-regulation of nonsense suppression.
[0040] As used herein, the term "disease" means a condition in the
patient.
[0041] As used herein, the term "patient" means an animal (e.g.,
cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse,
rat, rabbit, guinea pig, etc.), preferably a mammal such as a
non-primate and a primate (e.g., monkey and human), most preferably
a human. In certain embodiments, the patient is an infant, child,
adolescent or adult. In one embodiment, it has been determined
through pre-screening that the patient possesses a non-sense
mutation. In another embodiment, it has been determined through
pre-screening which non-sense mutation the patient has (i.e., UAA,
UGA, or UAG). In another embodiment, the patient is infected with
bacterial cells (e.g., Pseudomonas aeruginosa). In another
embodiment, the cells of the patient are virally infected. In
another embodiment, the patient has a genetic mutation. In another
embodiment, the patient has a somatic mutation.
[0042] As used herein, unless otherwise specified, the phrase
"compound(s) of the invention" includes any compound of formula
I-IX, or any compound of table 1, including any pharmaceutically
acceptable salts, hydrates, polymorphs, solvates, clathrates,
racemates or stereoisomers thereof. The phrase "compound(s) of the
invention" also includes the a and .beta. anomer of any compound of
formula I-IX, or any compound of table 1.
[0043] As used herein, unless otherwise specified, the term
"substituted" means a group substituted by one to four or more
substituents, such as, alkyl, alkenyl, alkynyl, cycloalkyl, aroyl,
halo, haloalkyl (e.g., trifluoromethyl), haloalkoxy (e.g.,
trifluoromethoxy), hydroxy, alkoxy, alkylthioether, cycloalkyloxy,
heterocylooxy, oxo, alkanoyl, aryl, arylalkyl, alkylaryl,
heteroaryl, heteroarylalkyl, alkylheteroaryl, heterocyclo, aryloxy,
alkanoyloxy, amino, alkylamino, arylamino, arylalkylamino,
cycloalkylamino, heterocycloamino, mono- and di-substituted amino
(in which the two substituents on the amino group are selected from
alkyl, aryl or arylalkyl), alkanoylamino, aroylamino,
aralkanoylamino, substituted alkanoylamino, substituted arylamino,
substituted aralkanoylamino, thiol, alkylthio, arylthio,
arylalkylthio, cycloalkylthio, heterocyclothio, alkylthiono,
arylthiono, arylalkylthiono, alkylsulfonyl, arylsulfonyl,
arylalkylsulfonyl, sulfonamido (e.g., SO.sub.2NH.sub.2),
substituted sulfonamido, nitro, cyano, carboxy, carbamyl (e.g.,
CONH.sub.2), substituted carbamyl (e.g., CONH-alkyl, CONH-aryl,
CONH-arylalkyl or instances where there are two substituents on the
nitrogen selected from alkyl or arylalkyl), alkoxycarbonyl, aryl,
substituted aryl, guanidino, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted heteroaryl (such as,
indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl,
pyridyl, pyrimidyl and the like). In one embodiment, the
substituent is --O-alkyl-C(.dbd.O)-heterocyclo (substituted or
unsubstituted), wherein alkyl and heterocyclo are defined above.
Wherein, as noted above, the substituents themselves are further
substituted, such further substituents are selected from the group
consisting of halogen, alkyl, alkoxy, aryl, heteroaryl,
heterocyclo, cycloalkyl, and arylalkyl.
[0044] As used herein, unless otherwise specified, the term "alkyl"
means a saturated straight chain or branched non-cyclic hydrocarbon
having from 1 to 20 carbon atoms, preferably 1-10 carbon atoms and
most preferably 1-4 carbon atoms. Representative saturated straight
chain alkyls include -methyl, -ethyl, -n-propyl, -n-butyl,
-n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl and -n-decyl;
while saturated branched alkyls include -isopropyl, -sec-butyl,
-isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 3-methylbutyl,
2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl,
3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl,
2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl,
2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylpentyl,
2,2-dimethylhexyl, 3,3-dimtheylpentyl, 3,3-dimethylhexyl,
4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2-ethylhexyl,
3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl,
2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl,
2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl,
2-methyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3-diethylhexyl,
2,2-diethylhexyl, 3,3-diethylhexyl and the like. An alkyl group can
be unsubstituted or substituted. Unsaturated alkyl groups include
alkenyl groups and alkynyl groups, which are discussed below.
[0045] As used herein, unless otherwise specified the term "alkenyl
group" means a straight chain or branched non-cyclic hydrocarbon
having from 2 to 20 carbon atoms, more preferably 2-10 carbon
atoms, most preferably 2-6 carbon atoms, and including at least one
carbon-carbon double bond. Representative straight chain and
branched (C.sub.2-C.sub.10)alkenyls include -vinyl, -allyl,
-1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,
-3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl,
-1-hexenyl, -2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl,
-3-heptenyl, -1-octenyl, -2-octenyl, -3-octenyl, -1-nonenyl,
-2-nonenyl, -3-nonenyl, -1-decenyl, -2-decenyl, -3-decenyl and the
like. The double bond of an alkenyl group can be unconjugated or
conjugated to another unsaturated group. An alkenyl group can be
unsubstituted or substituted.
[0046] As used herein, unless otherwise specified the term "alkynyl
group" means a straight chain or branched non-cyclic hydrocarbon
having from 2 to 20 carbon atoms, more preferably 2-10 carbon
atoms, most preferably 2-6 carbon atoms, and including at lease one
carbon-carbon triple bond. Representative straight chain and
branched --(C.sub.2-C.sub.10)alkynyls include -acetylenyl,
-propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl,
-3-methyl-1-butynyl, -4-pentynyl, -1-hexynyl, -2-hexynyl,
-5-hexynyl, -1-heptynyl, -2-heptynyl, -6-heptynyl, -1-octynyl,
-2-octynyl, -7-octynyl, -1-nonynyl, -2-nonynyl, -8-nonynyl,
-1-decynyl, -2-decynyl, -9-decynyl, and the like. The triple bond
of an alkynyl group can be unconjugated or conjugated to another
unsaturated group. An alkynyl group can be unsubstituted or
substituted.
[0047] As used herein, unless otherwise specified the term
"halogen" or "halo" means fluorine, chlorine, bromine, or
iodine.
[0048] As used herein, unless otherwise specified the term
"haloalkyl" means -alkyl substituted with one or more halogens,
wherein alkyl and halogen are defined as above, including
--CF.sub.3, --CHF.sub.2, --CH.sub.2F, --CCl.sub.3, --CHCl.sub.2,
--CBr.sub.3, --CHBr.sub.2, --CH.sub.2CF.sub.3, --CH.sub.2CHF.sub.2,
--CH.sub.2CH.sub.2F, and the like.
[0049] As used herein, unless otherwise specified the term "alkyl
sulfonyl" means --SO.sub.2-alkyl, wherein alkyl is defined as
above, including --SO.sub.2--CH.sub.3,
--SO.sub.2--CH.sub.2CH.sub.3, --SO.sub.2--(CH.sub.2).sub.2CH.sub.3,
--SO.sub.2--(CH.sub.2).sub.3CH.sub.3,
--SO.sub.2--(CH.sub.2).sub.4CH.sub.3,
--SO.sub.2--(CH.sub.2).sub.5CH.sub.3, and the like.
[0050] As used herein, unless otherwise specified the term
"carboxyl" and "carboxy" mean --COOH or a salt thereof (e.g.,
--COO.sup.-Na.sup.+).
[0051] As used herein, unless otherwise specified the term "alkoxy"
means --O-(alkyl), wherein alkyl is defined above, including
--OCH.sub.3, --OCH.sub.2CH.sub.3, --O(CH.sub.2).sub.2CH.sub.3,
--O(CH.sub.2).sub.3CH.sub.3, --O(CH.sub.2).sub.4CH.sub.3,
--O(CH.sub.2).sub.5CH.sub.3, and the like.
[0052] As used herein, unless otherwise specified the term
"alkylthioether" means --S-(alkyl), wherein alkyl is defined above,
including --SCH.sub.3, --SCH.sub.2CH.sub.3,
--S(CH.sub.2).sub.2CH.sub.3, --S(CH.sub.2).sub.3CH.sub.3,
--S(CH.sub.2).sub.4CH.sub.3, --S(CH.sub.2).sub.5CH.sub.3, and the
like.
[0053] As used herein, unless otherwise specified the term
"haloalkoxy" means -alkoxy substituted with one or more halogens,
wherein alkoxy and halogen are defined as above, including
--OCF.sub.3, --OCHF.sub.2, --OCH.sub.2F, --OCCl.sub.3,
--OCHCl.sub.2, --OCBr.sub.3, --OCHBr.sub.2, --OCH.sub.2CF.sub.3,
--OCH.sub.2CHF.sub.2, --OCH.sub.2CH.sub.2F, and the like.
[0054] As used herein, unless otherwise specified the term
"alkoxycarbonyl" means --C(.dbd.O)O-(alkyl), wherein alkyl is
defined above, including --C(.dbd.O)O--CH.sub.3,
--C(.dbd.O)O--CH.sub.2CH.sub.3,
--C(.dbd.O)O--(CH.sub.2).sub.2CH.sub.3,
--C(.dbd.O)O--(CH.sub.2).sub.3CH.sub.3,
--C(.dbd.O)O--(CH.sub.2).sub.4CH.sub.3,
--C(.dbd.O)O--(CH.sub.2).sub.5CH.sub.3, and the like. In a
preferred embodiment, the esters are biohydrolyzable (i.e., the
ester is hydrolyzed to a carboxylic acid in vitro or in vivo).
[0055] As used herein, unless otherwise specified the term "acyl"
means --CH(.dbd.O).
[0056] As used herein, unless otherwise specified the term
"alkylcarbonyl" means --C(.dbd.O)-(alkyl), wherein alkyl is defined
above, including --C(.dbd.O)--CH.sub.3,
--C(.dbd.O)--CH.sub.2CH.sub.3,
--C(.dbd.O)--(CH.sub.2).sub.2CH.sub.3,
--C(.dbd.O)--(CH.sub.2).sub.3CH.sub.3,
--C(.dbd.O)--(CH.sub.2).sub.4CH.sub.3,
--C(.dbd.O)--(CH.sub.2).sub.5CH.sub.3, and the like.
[0057] As used herein, unless otherwise specified the term
"arylcarbonyl" means --C(.dbd.O)-(aryl), wherein aryl is defined
below, including --C(.dbd.O)-phenyl, --C(.dbd.O)-tolyl,
--C(.dbd.O)-anthracenyl, --C(.dbd.O)-fluorenyl,
--C(.dbd.O)-indenyl, --C(.dbd.O)-azulenyl,
--C(.dbd.O)-phenanthrenyl --C(.dbd.O)-naphthyl, and the like.
[0058] As used herein, unless otherwise specified the term
"carboxyalkyl" means -(alkyl)-carboxy, wherein alkyl and carboxy
are defined above, including --CH.sub.2--COOH,
--(CH.sub.2).sub.2--COOH, --(CH.sub.2).sub.3--COOH,
--(CH.sub.2).sub.4--COOH, and the like.
[0059] As used herein, unless otherwise specified the term
"alkoxyalkyl" means -(alkyl)-O-(alkyl), wherein each "alkyl" is
independently an alkyl group as defined above, including
--CH.sub.2OCH.sub.3, --CH.sub.2OCH.sub.2CH.sub.3,
--(CH.sub.2).sub.2OCH.sub.2CH.sub.3,
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2CH.sub.3, and the like.
[0060] As used herein, unless otherwise specified the term "aryl"
means a carbocyclic aromatic ring containing from 5 to 14 ring
atoms. The ring atoms of a carbocyclic aryl group are all carbon
atoms. Aryl ring structures include compounds having one or more
ring structures such as mono-, bi-, or tricylcic compounds as well
as benzo-fused carbocyclic moieties such as
5,6,7,8-tetrahydronaphthyl and the like. Preferably, the aryl group
is a monocyclic ring or bicyclic ring. Representative aryl groups
include phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl,
phenanthrenyl and naphthyl. A carbocyclic aryl group can be
unsubstituted or substituted.
[0061] As used herein, unless otherwise specified the term
"heteroaryl" means a carbocyclic aromatic ring containing from 5 to
14 ring atoms and the ring atoms contain at least one heteroatom,
preferably 1 to 3 heteroatoms, independently selected from
nitrogen, oxygen, or sulfur. Heteroaryl ring structures include
compounds having one or more ring structures such as mono-, bi-, or
tricyclic compounds as well as fused heterocyclic moities.
Representative heteroaryls are triazolyl, tetrazolyl, oxadiazolyl,
pyridyl, furanyl, benzofuranyl, thiophenyl, thiazolyl,
benzothiophenyl, benzoisoxazolyl, benzoisothiazolyl, quinolinyl,
pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl,
benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl,
isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,
cinnolinyl, phthalazinyl, quinazolinyl, benzoquinazolinyl,
acridinyl, pyrimidyl, oxazolyl, benzo[1,3]dioxole and
2,3-dihydro-benzo[1,4]dioxine. A group can be unsubstituted or
substituted.
[0062] As used herein, unless otherwise specified the term
"aryloxy" means --O-aryl group, wherein aryl is as defined above,
including, but not limited to --O-phenyl, --O-tolyl,
--O-anthracenyl, --O-fluorenyl, --O-indenyl, --O-azulenyl,
--O-phenanthrenyl and --O-naphthyl. An aryloxy group can be
unsubstituted or substituted.
[0063] As used herein, unless otherwise specified the term
"arylalkyl" means -(alkyl)-(aryl), wherein alkyl and aryl are
defined above, including, but not limited to --(CH.sub.2)phenyl,
--(CH.sub.2).sub.2phenyl, --(CH.sub.2).sub.3phenyl,
--CH(phenyl).sub.2, --CH(phenyl).sub.3, --(CH.sub.2)tolyl,
--(CH.sub.2)anthracenyl, --(CH.sub.2)fluorenyl,
--(CH.sub.2)indenyl, --(CH.sub.2)azulenyl, --(CH.sub.2)naphthyl,
and the like.
[0064] As used herein, unless otherwise specified the term
"alkylaryl" means -(aryl)-(alkyl), wherein aryl and aryl are
defined above, including, but not limited to
-phenyl-(CH.sub.3).sub.5, phenyl-(CH.sub.3).sub.4,
phenyl-(CH.sub.3).sub.3, phenyl-(CH.sub.3).sub.2,
phenyl-(CH.sub.3), -phenyl-(CH.sub.2CH.sub.3).sub.5,
phenyl-(CH.sub.2CH.sub.3).sub.4, phenyl-(CH.sub.2CH.sub.3).sub.3,
phenyl-(CH.sub.2CH.sub.3).sub.2, phenyl-(CH.sub.2CH.sub.3), and the
like wherein each alkyl group can be further substituted.
[0065] As used herein, unless otherwise specified the term
"heteroarylalkyl" means -(alkyl)-(heteroaryl), wherein alkyl and
heteroaryl are defined above, including, but not limited to,
--(CH.sub.2)pyridyl, --(CH.sub.2).sub.2 pyridyl, --(CH.sub.2).sub.3
pyridyl, --CH(pyridyl).sub.2, --C(pyridyl).sub.3,
--(CH.sub.2)triazolyl, --(CH.sub.2)thiazolyl,
--(CH.sub.2)tetrazolyl, --(CH.sub.2)oxadiazolyl, --(CH.sub.2)furyl,
--(CH.sub.2)benzofuranyl, --(CH.sub.2)thiophenyl,
--(CH.sub.2)benzothiophenyl, and the like.
[0066] As used herein, unless otherwise specified the term
"alkylheteroaryl" means -(heteroaryl)-(alkyl), wherein heteroaryl
and alkyl are defined above, including, but not limited to,
-pyridyl-(CH.sub.3), -triazolyl-(CH.sub.3), -thiazolyl-(CH.sub.3),
-tetrazolyl-(CH.sub.3), -oxadiazolyl-(CH.sub.3), -furyl-(CH.sub.3),
-benzofuranyl-(CH.sub.3), -thiophenyl-(CH.sub.3),
-benzothiophenyl-(CH.sub.3), and the like wherein each alkyl group
can be further substituted.
[0067] As used herein, unless otherwise specified the term
"arylalkyloxy" means --O-(alkyl)-(aryl), wherein alkyl and aryl are
defined above, including, but not limited to
--O--(CH.sub.2).sub.2phenyl, --O--(CH.sub.2).sub.3phenyl,
--O--CH(phenyl).sub.2, --O--CH(phenyl).sub.3, --O--(CH.sub.2)tolyl,
--O--(CH.sub.2)anthracenyl, --O--(CH.sub.2)fluorenyl,
--O--(CH.sub.2)indenyl, --O--(CH.sub.2)azulenyl,
--O--(CH.sub.2)naphthyl, and the like.
[0068] As used herein, unless otherwise specified the term
"cycloalkyl" means a monocyclic or polycyclic saturated ring
comprising carbon and hydrogen atoms and having no carbon-carbon
multiple bonds. A cycloalkyl group can be unsubstituted or
substituted. Examples of cycloalkyl groups include, but are not
limited to, (C.sub.3-C.sub.7)cycloalkyl groups, including
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl,
and saturated cyclic and bicyclic terpenes. A cycloalkyl group can
be unsubstituted or substituted. Preferably, the cycloalkyl group
is a monocyclic ring or bicyclic ring.
[0069] As used herein, unless otherwise specified the terms
"heterocyclyl" and "heterocyclo" mean a monocyclic or polycyclic
ring comprising carbon and hydrogen atoms, optionally having 1 or 2
multiple bonds, and the ring atoms contain at least one heteroatom,
preferably 1 to 3 heteroatoms, independently selected from
nitrogen, oxygen, and sulfur. Heterocyclyl ring structures include,
but are not limited to compounds having one or more ring structures
such as mono-, bi-, or trycylic compounds. Preferably, the
heterocyclyl group is a monocyclic ring or bicyclic ring.
Representative heterocycles include morpholinyl, pyrrolidinonyl,
pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl,
valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,
tetrahydrothiophenyl or tetrahydrothiopyranyl and the like. A
heterocyclyl ring can be unsubstituted or substituted.
[0070] As used herein, unless otherwise specified the term
"cycloalkyloxy" means --O-(cycloalkyl), wherein cycloalkyl is
defined above, including --O-cyclopropyl, --O-cyclobutyl,
--O-cyclopentyl, --O-cyclohexyl, --O-cycloheptyl and the like.
[0071] As used herein, unless otherwise specified the term
"cycloalkylalkyl" means -(alkyl)-(cycloalkyl), wherein cycloalkyl
and alkyl are defined above, including, but not limited to
--CH.sub.2-cyclopropyl, --CH.sub.2-cyclobutyl,
--CH.sub.2-cyclopentyl, --(CH.sub.2).sub.2-cyclohexyl,
--(CH.sub.2).sub.3-cyclohexyl, --(CH.sub.2).sub.4-cyclohexyl,
--CH.sub.2-cycloheptyl and the like.
[0072] As used herein, unless otherwise specified the term
"heterocycloalkyl" means -(alkyl)-(heterocyclo), wherein
heterocyclo and alkyl are defined above, including, but not limited
to --CH.sub.2-morpholinyl, --CH.sub.2-pyrrolidinonyl,
--CH.sub.2-pyrrolidinyl, --(CH.sub.2).sub.2-piperidinyl,
--(CH.sub.2).sub.3-piperidinyl, --(CH.sub.2).sub.4-piperidinyl,
--CH.sub.2-hydantoinyl and the like.
[0073] As used herein, unless otherwise specified the term
"cycloalkylalkyloxy" means --O-(alkyl)-(cycloalkyl), wherein
cycloalkyl and alkyl are defined above, including, but not limited
to --O--CH.sub.2-cyclopropyl, --O--CH.sub.2-cyclobutyl,
--O--CH.sub.2-cyclopentyl, --O--(CH.sub.2).sub.2-cyclohexyl,
--O--(CH.sub.2).sub.3-cyclohexyl, --O--(CH.sub.2).sub.4-cyclohexyl,
--O--CH.sub.2-cycloheptyl and the like.
[0074] As used herein, unless otherwise specified the term
"heterocycloalkyloxy" means --O-(alkyl)-(heterocyclo), wherein
heterocyclo and alkyl are defined above, including, but not limited
to --O--CH.sub.2-morpholinyl, --O--CH.sub.2-pyrrolidinonyl,
--O--CH.sub.2-pyrrolidinyl, --O--(CH.sub.2).sub.2-piperidinyl,
--O--(CH.sub.2).sub.3-piperidinyl,
--O--(CH.sub.2).sub.4-piperidinyl, --O--CH.sub.2-hydantoinyl and
the like.
[0075] As used herein, unless otherwise specified the term
"aminoalkoxy" means --O-(alkyl)-NH.sub.2, wherein alkyl is defined
above, including, but not limited to --O--CH.sub.2--NH.sub.2,
--O--(CH.sub.2).sub.2--NH.sub.2, --O--(CH.sub.2).sub.3--NH.sub.2,
--O--(CH.sub.2).sub.4--NH.sub.2, --O--(CH.sub.2).sub.5--NH.sub.2,
and the like.
[0076] As used herein, unless otherwise specified the term
"alkylamino" means --NH(alkyl) or --N(alkyl)(alkyl), wherein alkyl
is defined above, including, but not limited to NHCH.sub.3,
--NHCH.sub.2CH.sub.3, --NH(CH.sub.2).sub.2CH.sub.3,
--NH(CH.sub.2).sub.3CH.sub.3, --NH(CH.sub.2).sub.4CH.sub.3,
--NH(CH.sub.2).sub.5CH.sub.3, --N(CH.sub.3).sub.2,
--N(CH.sub.2CH.sub.3).sub.2, --N((CH.sub.2).sub.2CH.sub.3).sub.2,
--N(CH.sub.3)(CH.sub.2CH.sub.3), and the like.
[0077] As used herein, unless otherwise specified the term
"arylamino" means --NH(aryl), wherein aryl is defined above,
including, but not limited to --NH(phenyl), --NH(tolyl),
--NH(anthracenyl), --NH(fluorenyl), --NH(indenyl), --NH(azulenyl),
--NH(pyridinyl), --NH(naphthyl), and the like.
[0078] As used herein, unless otherwise specified the term
"arylalkylamino" means --NH-(alkyl)-(aryl), wherein alkyl and aryl
are defined above, including, but not limited to
--NH--CH.sub.2--(phenyl), --NH--CH.sub.2-(tolyl),
--NH--CH.sub.2-(anthracenyl), --NH--CH.sub.2-(fluorenyl),
--NH--CH.sub.2-(indenyl), --NH--CH.sub.2-(azulenyl),
--NH--CH.sub.2-(pyridinyl), --NH--CH.sub.2-(naphthyl),
--NH--(CH.sub.2).sub.2-(phenyl) and the like.
[0079] As used herein, unless otherwise specified the term
"cycloalkylamino" means --NH-(cycloalkyl), wherein cycloalkyl is
defined above, including, but not limited to --NH-cyclopropyl,
--NH-cyclobutyl, --NH-cyclopentyl, --NH-cyclohexyl,
--NH-cycloheptyl, and the like.
[0080] As used herein, unless otherwise specified the term
"aminoalkyl" means -(alkyl)-NH.sub.2, wherein alkyl is defined
above, including, but not limited to --CH.sub.2--NH.sub.2,
--(CH.sub.2).sub.2--NH.sub.2, --(CH.sub.2).sub.3--NH.sub.2,
--(CH.sub.2).sub.4--NH.sub.2, --(CH.sub.2).sub.5--NH.sub.2 and the
like.
[0081] As used herein, unless otherwise specified the term
"alkylaminoalkyl" means -(alkyl)-NH(alkyl) or
-(alkyl)-N(alkyl)(alkyl), wherein each "alkyl" is independently an
alkyl group defined above, including, but not limited to
--CH.sub.2--NH--CH.sub.3, --CH.sub.2--NHCH.sub.2CH.sub.3,
--CH.sub.2--NH(CH.sub.2).sub.2CH.sub.3,
--CH.sub.2--NH(CH.sub.2).sub.3CH.sub.3,
--CH.sub.2--NH(CH.sub.2).sub.4CH.sub.3,
--CH.sub.2--NH(CH.sub.2).sub.5CH.sub.3,
--(CH.sub.2).sub.2--NH--CH.sub.3, --CH.sub.2--N(CH.sub.3).sub.2,
--CH.sub.2--N(CH.sub.2CH.sub.3).sub.2,
--CH.sub.2--N((CH.sub.2).sub.2CH.sub.3).sub.2,
--CH.sub.2--N(CH.sub.3)(CH.sub.2CH.sub.3),
--(CH.sub.2).sub.2--N(CH.sub.3).sub.2, and the like.
[0082] As used herein, the term "liquid chromatography-mass
spectrometry (LC/MS)" means an analytical method in which a sample
is fractionated using liquid chromatography, and the column eluant
is characterized by mass spectrometry.
[0083] As used herein, a "therapeutically effective amount" refers
to that amount of the compound of the invention or other active
ingredient sufficient to provide a therapeutic benefit in the
treatment or management of the disease or to delay or minimize
symptoms associated with the disease. Further, a therapeutically
effective amount with respect to a compound of the invention means
that amount of therapeutic agent alone, or in combination with
other therapies, that provides a therapeutic benefit in the
treatment or management of the disease. Used in connection with an
amount of a compound of the invention, the term can encompass an
amount that improves overall therapy, reduces or avoids symptoms or
causes of disease, or enhances the therapeutic efficacy of or
synergies with another therapeutic agent.
[0084] As used herein, a "prophylactically effective amount" refers
to that amount of a compound of the invention or other active
ingredient sufficient to result in the prevention, recurrence or
spread of the disease. A prophylactically effective amount may
refer to the amount sufficient to prevent initial disease, the
recurrence or spread of the disease or the occurrence of the
disease in a patient, including but not limited to those
predisposed to the disease. A prophylactically effective amount may
also refer to the amount that provides a prophylactic benefit in
the prevention of the disease. Further, a prophylactically
effective amount with respect to a compound of the invention means
that amount alone, or in combination with other agents, that
provides a prophylactic benefit in the prevention of the disease.
Used in connection with an amount of a compound of the invention,
the term can encompass an amount that improves overall prophylaxis
or enhances the prophylactic efficacy of or synergies with another
prophylactic agent.
[0085] As used herein, a "therapeutic protocol" refers to a regimen
of timing and dosing of one or more therapeutic agents.
[0086] As used herein, a "prophylactic protocol" refers to a
regimen of timing and dosing of one or more prophylactic
agents.
[0087] A used herein, a "protocol" includes dosing schedules and
dosing regimens.
[0088] As used herein, "in combination" refers to the use of more
than one prophylactic and/or therapeutic agents.
[0089] As used herein, the terms "manage", "managing" and
"management" refer to the beneficial effects that a subject derives
from a prophylactic or therapeutic agent, which does not result in
a cure of the disease. In certain embodiments, a subject is
administered one or more prophylactic or therapeutic agents to
"manage" a disease so as to prevent the progression or worsening of
the disease.
[0090] As used herein, the terms "prevent", "preventing" and
"prevention" refer to the prevention of the onset, recurrence or
spread of the disease in a subject resulting from the
administration of a prophylactic or therapeutic agent.
[0091] As used herein, the terms "treat", "treating" and
"treatment" refer to the eradication or amelioration of the disease
or symptoms associated with the disease. In certain embodiments,
such terms refer to minimizing the spread or worsening of the
disease resulting from the administration of one or more
prophylactic or therapeutic agents to a subject with such a
disease.
[0092] As used herein, the term "pharmaceutically acceptable salts"
refer to salts prepared from pharmaceutically acceptable non-toxic
acids or bases including inorganic acids and bases and organic
acids and bases. Suitable pharmaceutically acceptable base addition
salts for the compound of the present invention include, but are
not limited to metallic salts made from aluminum, calcium, lithium,
magnesium, potassium, sodium and zinc or organic salts made from
lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and
procaine. Suitable non-toxic acids include, but are not limited to,
inorganic and organic acids such as acetic, alginic, anthranilic,
benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic,
formic, fumaric, furoic, galacturonic, gluconic, glucuronic,
glutamic, glycolic, hydrobromic, hydrochloric, isethionic, lactic,
maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic,
pantothenic, phenylacetic, phosphoric, propionic, salicylic,
stearic, succinic, sulfanilic, sulfuric, tartaric acid, and
p-toluenesulfonic acid. Specific non-toxic acids include
hydrochloric, hydrobromic, phosphoric, sulfuric, and
methanesulfonic acids. Examples of specific salts thus include
hydrochloride and mesylate salts. Others are well-known in the art,
see for example, Remington's Pharmaceutical Sciences, 18.sup.th
eds., Mack Publishing, Easton Pa. (1990) or Remington: The Science
and Practice of Pharmacy, 19.sup.th eds., Mack Publishing, Easton
Pa. (1995).
[0093] As used herein and unless otherwise indicated, the term
"polymorph" refers to solid crystalline forms of a compound of the
present invention or complex thereof. Different polymorphs of the
same compound can exhibit different physical, chemical and/or
spectroscopic properties. Different physical properties include,
but are not limited to stability (e.g., to heat or light),
compressibility and density (important in formulation and product
manufacturing), and dissolution rates (which can affect
bioavailability). Differences in stability can result from changes
in chemical reactivity (e.g., differential oxidation, such that a
dosage form discolors more rapidly when comprised of one polymorph
than when comprised of another polymorph) or mechanical
characteristics (e.g., tablets crumble on storage as a kinetically
favored polymorph converts to thermodynamically more stable
polymorph) or both (e.g., tablets of one polymorph are more
susceptible to breakdown at high humidity). Different physical
properties of polymorphs can affect their processing. For example,
one polymorph might be more likely to form solvates or might be
more difficult to filter or wash free of impurities than another
due to, for example, the shape or size distribution of particles of
it.
[0094] As used herein, the term "hydrate" means a compound of the
present invention or a salt thereof, that further includes a
stoichiometric or non-stoichiometric amount of water bound by
non-covalent intermolecular forces.
[0095] As used herein, the term "clathrate" means a compound of the
present invention or a salt thereof in the form of a crystal
lattice that contains spaces (e.g., channels) that have a guest
molecule (e.g., a solvent or water) trapped within.
[0096] As used herein and unless otherwise indicated, the term
"prodrug" means a derivative of a compound that can hydrolyze,
oxidize, or otherwise react under biological conditions (in vitro
or in vivo) to provide an active compound, particularly a compound
of the invention. Examples of prodrugs include, but are not limited
to, derivatives and metabolites of a compound of the invention that
include biohydrolyzable groups such as biohydrolyzable amides,
biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable
carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate
analogues (e.g., monophosphate, diphosphate or triphosphate).
Preferably, prodrugs of compounds with carboxyl functional groups
are the lower alkyl esters of the carboxylic acid. The carboxylate
esters are conveniently formed by esterifying any of the carboxylic
acid moieties present on the molecule. Prodrugs can typically be
prepared using well-known methods, such as those described by
Burger's Medicinal Chemistry and Drug Discovery 6.sup.th ed.
(Donald J. Abraham ed., 2001, Wiley) and Design and Application of
Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers
Gmfh).
[0097] As used herein and unless otherwise indicated, the terms
"biohydrolyzable amide," "biohydrolyzable ester," "biohydrolyzable
carbamate," "biohydrolyzable carbonate," "biohydrolyzable ureido,"
"biohydrolyzable phosphate" (collectively referred to herein as a
"biohydrolyzable group(s)") mean an amide, ester, carbamate,
carbonate, ureido, or phosphate, respectively, of a compound that
either: 1) does not interfere with the biological activity of the
compound but can confer upon that compound advantageous properties
in vivo, such as uptake, duration of action, or onset of action; or
2) is biologically inactive but is converted in vivo to the
biologically active compound. Examples of biohydrolyzable esters
include, but are not limited to, lower alkyl esters, alkoxyacyloxy
esters, alkyl acylamino alkyl esters, and choline esters. Examples
of biohydrolyzable amides include, but are not limited to, lower
alkyl amides, .alpha.-amino acid amides, alkoxyacyl amides, and
alkylaminoalkylcarbonyl amides. Examples of biohydrolyzable
carbamates include, but are not limited to, lower alkylamines,
substituted ethylenediamines, aminoacids, hydroxyalkylamines,
heterocyclic and heteroaromatic amines, and polyether amines.
[0098] As used herein and unless otherwise indicated, the term
"optically pure" or "stereomerically pure" means one stereoisomer
of a compound is substantially free of other stereoisomers of that
compound. For example, a stereomerically pure compound having one
chiral center will be substantially free of the opposite enantiomer
of the compound. A stereomerically pure a compound having two
chiral centers will be substantially free of other diastereomers of
the compound. A typical stereomerically pure compound comprises
greater than about 80% by weight of one stereoisomer of the
compound and less than about 20% by weight of other stereoisomers
of the compound, more preferably greater than about 90% by weight
of one stereoisomer of the compound and less than about 10% by
weight of the other stereoisomers of the compound, even more
preferably greater than about 95% by weight of one stereoisomer of
the compound and less than about 5% by weight of the other
stereoisomers of the compound, and most preferably greater than
about 97% by weight of one stereoisomer of the compound and less
than about 3% by weight of the other stereoisomers of the
compound.
[0099] As used herein and unless otherwise indicated, the term
"enantiomerically pure" means a stereomerically pure composition of
a compound having one chiral center.
[0100] It should be noted that if there is a discrepancy between a
depicted structure and a name given that structure, the depicted
structure controls. In addition, if the stereochemistry of a
structure or a portion of a structure is not indicated with, for
example, bold or dashed lines, the structure or portion of the
structure is to be interpreted as encompassing all stereoisomers of
it.
4. DETAILED DESCRIPTION OF THE INVENTION
4.1 Compounds of the Invention
[0101] As stated above, the present invention encompasses compounds
of formula I, compositions comprising compounds of formula I, and
methods of use thereof. Compounds of formula I have the structure:
##STR3## or a pharmaceutically acceptable salt, hydrate, solvate,
clathrate, racemate or stereoisomer thereof, wherein:
[0102] Z is substituted or unsubstituted alkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl;
[0103] X is CH.sub.2, O, S or NH;
[0104] R.sup.1 is hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl;
[0105] R.sup.2 is substituted or unsubstituted alkyl, carboxy,
amido, acyl, alkylcarbonyl, halogen, a biohydrolyzable group,
OP(O).sub.3.sup.2-, O[P(O).sub.3].sub.2.sup.3-,
O[P(O).sub.3].sub.3.sup.4-, N.sub.3, CH.sub.2--NR.sub.6R.sub.7 or
CH.sub.2--OR.sup.6;
[0106] R.sup.3, R.sup.3', R.sup.4 and R.sup.4' are at each
occurrence independently OR.sup.7, OR.sup.8, hydrogen, halogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
aryl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocyclo,
substituted or unsubstituted arylalkyl, substituted or
unsubstituted heteroarylalkyl, substituted or unsubstituted
cycloalkylalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or unsubstituted arylcarbonyl, substituted or
unsubstituted alkylcarbonyl, a biohydrolyzable group, or R.sup.3
and R.sup.4 taken together form a bond, or R.sup.3 and R.sup.4
taken together with the atoms to which they are attached form a
substituted or unsubstituted heterocyclo, or R.sup.3 and R.sup.3'
and/or R.sup.4 and R.sup.4' taken together with the carbon to which
they are attached form C(.dbd.O); and
[0107] R.sup.6, R.sup.7 and R.sup.8 are at each occurrence
independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl,
substituted or unsubstituted alkylcarbonyl, a biohydrolyzable
group, or R.sup.3 and R.sup.4 taken together with the atoms to
which they are attached form a substituted or unsubstituted
heterocyclo.
[0108] In one embodiment, Z is monocyclic. In another embodiment, Z
is bicyclic. In another embodiment, Z is tricyclic. In another
embodiment, Z is a 6-membered monocyclic ring.
[0109] In a preferred embodiment, Z is substituted or unsubstituted
pyridinyl.
[0110] In another preferred embodiment, Z is substituted or
unsubstituted pyrimidinyl.
[0111] In another preferred embodiment, Z is substituted or
unsubstituted pyrazinyl.
[0112] In another preferred embodiment, Z is substituted or
unsubstituted quinolinyl.
[0113] In another preferred embodiment, X is O.
[0114] In another preferred embodiment, R.sup.2 is
CH.sub.2--OR.sup.6.
[0115] In another preferred embodiment, R.sup.6 is H.
[0116] It should be recognized that the invention includes
embodiments which exclude clitocine and instead include other
compounds of the invention. In other words, in one embodiment, the
compound of formula I is not clitocine (i.e.,
6-amino-5-nitro-4-(.beta.-D-ribofuranosylamino)pyrimidine).
[0117] In another embodiment, compounds of formula I have the
structure: ##STR4##
[0118] or a pharmaceutically acceptable salt, hydrate, solvate,
clathrate, racemate or stereoisomer thereof, wherein:
[0119] X is CH.sub.2, O, S or NH;
[0120] R.sup.1 is hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl;
[0121] R.sup.2 is substituted or unsubstituted alkyl, carboxy,
amido, acyl, alkylcarbonyl, halogen, a biohydrolyzable group,
OP(O).sub.3.sup.2-, O[P(O).sub.3].sub.2.sup.3-,
O[P(O).sub.3].sub.3.sup.4-, N.sub.3, CH.sub.2--NR.sub.6R.sub.7 or
CH.sub.2--OR.sup.6;
[0122] R.sup.3, R.sup.3', R.sup.4 and R.sup.4' are at each
occurrence independently OR.sup.7, OR.sup.8, hydrogen, halogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
aryl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocyclo,
substituted or unsubstituted arylalkyl, substituted or
unsubstituted heteroarylalkyl, substituted or unsubstituted
cycloalkylalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or unsubstituted arylcarbonyl, substituted or
unsubstituted alkylcarbonyl, a biohydrolyzable group, or R.sup.3
and R.sup.4 taken together form a bond, or R.sup.3 and R.sup.4
taken together with the atoms to which they are attached form a
substituted or unsubstituted heterocyclo, or R.sup.3 and R.sup.3'
and/or R.sup.4 and R.sup.4' taken together with the carbon to which
they are attached form C(.dbd.O);
[0123] R.sup.6, R.sup.7 and R.sup.8 are at each occurrence
independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted arylcarbonyl,
substituted or unsubstituted alkylcarbonyl, a biohydrolyzable
group, or R.sup.7 and R.sup.8 taken together with the atoms to
which they are attached form a substituted or unsubstituted
heterocyclo;
[0124] A and B are each independently C or N;
[0125] Y.sup.1--Y.sup.5 are each independently hydrogen, hydroxy,
halogen, nitro, cyano, sulfate, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocyclo, substituted or
unsubstituted arylalkyl, substituted or unsubstituted
heteroarylalkyl, substituted or unsubstituted cycloalkylalkyl,
substituted or unsubstituted heterocycloalkyl, alkoxy,
alkylthioether, carboxyalkyl, carbonylalkyl, amino,
NR.sup.5R.sup.5', amido, or alkoxycarbonyl, wherein if B is N,
Y.sup.3 can also be O.sup.- and if A is N, Y.sup.5 can also be
O.sup.-, and wherein Y.sup.3 is not present if B is N and Y.sup.5
is not present if A is N; and
[0126] R.sup.5 and R.sup.5' are each independently hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl.
[0127] In one embodiment, the compounds of formula II are those
wherein:
[0128] R.sup.5 is substituted or unsubstituted alkyl, substituted
or unsubstituted alkenyl, substituted or unsubstituted alkynyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl.
[0129] In one embodiment, the compounds of formula II are those
wherein:
[0130] Y.sup.1 is hydrogen, hydroxy, halogen, cyano, sulfate,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocyclo, substituted or unsubstituted arylalkyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkyl, alkoxy, alkylthioether, carboxyalkyl,
carbonylalkyl, amino, NR.sup.5R.sup.5', amido or
alkoxycarbonyl.
[0131] In a preferred embodiment, Y.sup.1 is halogen, nitro,
sulfate, alkoxycarbonyl, cyano or carbonylalkyl.
[0132] In another preferred embodiment, Y.sup.2 is halogen, nitro,
sulfate, alkoxycarbonyl, cyano or carbonylalkyl.
[0133] In another preferred embodiment, R.sup.2 is
CH.sub.2--OR.sup.6.
[0134] In another preferred embodiment, R.sup.6 is H.
[0135] In another preferred embodiment, R.sup.1 is H.
[0136] It should be recognized that the invention includes
embodiments which exclude clitocine and instead include other
compounds of the invention. In other words, in one embodiment, the
compound of formula II is not clitocine (i.e.,
6-amino-5-nitro-4-(.beta.-D-ribofuranosylamino)pyrimidine).
[0137] In another embodiment, compounds of formula I have the
structure: ##STR5## wherein A, B, R.sup.1, R.sup.2, R.sup.3,
R.sup.3', R.sup.4, R.sup.4', Y.sup.1 and Y.sup.2 are as described
for formula I.
[0138] In a preferred embodiment, R.sup.2 is
CH.sub.2--OR.sup.6.
[0139] In another preferred embodiment, R.sup.6 is H.
[0140] In another embodiment, compounds of formula I have the
structure: ##STR6## wherein A, B, X, R.sup.2, R.sup.3, R.sup.3',
R.sup.4, R.sup.4', Y.sup.1 and Y.sup.2 are as described for formula
I.
[0141] In a preferred embodiment, R.sup.2 is
CH.sub.2--OR.sup.6.
[0142] In another preferred embodiment, R.sup.6 is H.
[0143] In another embodiment, compounds of formula I have the
structure: ##STR7## wherein A, B, X, R.sup.1, R.sup.2, R.sup.3,
R.sup.3', R.sup.4, R.sup.4' and Y.sup.2 are as described for
formula I.
[0144] In a preferred embodiment, R.sup.2 is
CH.sub.2--OR.sup.6.
[0145] In another preferred embodiment, R.sup.6 is H.
[0146] In another embodiment, compounds of formula I have the
structure: ##STR8## wherein A, B, X, R.sup.1, R.sup.2, R.sup.3,
R.sup.3', R.sup.4, R.sup.4' and Y.sup.1 are as described for
formula I.
[0147] In a preferred embodiment, R.sup.2 is
CH.sub.2--OR.sup.6.
[0148] In another preferred embodiment, R.sup.6 is H.
[0149] In another embodiment, compounds of formula I have the
structure: ##STR9## wherein A, B, X, R.sup.1, Y.sup.1 and Y.sup.2
are as described for formula I.
[0150] In another embodiment, compounds of formula I have the
structure: ##STR10## wherein X, R.sup.1, R.sup.2, R.sup.3,
R.sup.3', R.sup.4, R.sup.4', Y.sup.1 and Y.sup.2 are as described
for formula I.
[0151] In a preferred embodiment, R.sup.2 is
CH.sub.2--OR.sup.6.
[0152] In another preferred embodiment, R.sup.6 is H.
[0153] In another embodiment, the compound of formula I has the
structure: ##STR11##
[0154] Wherein each occurrence of R'' is independently hydrogen,
OP(O.sub.3).sup.-2, C(.dbd.O)CH.sub.3 or a biohydrolyzable
group.
[0155] Exemplary compounds of the present invention include those
listed below in Table 1 (data has been presented where it is
available): TABLE-US-00001 TABLE 1 Melting Point (.degree. C.) or
Compound Compound Name [M + H].sup.+ Activity 1 ##STR12##
6-amino-5-nitro-4-(.beta.-D- ribofuranosylamino)- pyrimidine
180.degree. C. (dec.) *** 2 ##STR13## 6-(N-methylamino)-5-
nitro-4-(.beta.-D-ribo- furanosylamino)pyrimidine 130- 135.degree.
C. ** 3 ##STR14## 5,6-Diamino-4-(.beta.-D- ribofuranosyl-
amino)pyrimidine [M + H].sup.+ =258 * 4 ##STR15##
3-Nitro-2-(.beta.-D- ribofuranosyl- amino)pyridine [M + H].sup.+
=271 *** 5 ##STR16## 5-Nitro-2-(.beta.-D- ribofuranosyl-
amino)pyridine [M + H].sup.+ =272 * 6 ##STR17##
(1R,2S,3R,5R)-3-(6-Amino-5- nitro-pyrimidin-4-ylamino)-
5-hydroxymethyl- cyclopentane-1,2-diol [M + H].sup.+ =286 0 7
##STR18## (1S,2R,3S,5S)-3-(6-Amino-5- nitro-pyrimidin-4-ylamino)-5-
hydroxymethyl-cyclopentane- 1,2-diol [M + H].sup.+ =286 0 8
##STR19## 6-methoxy-3-nitro-2-(.beta.-D- ribofuranosyl-
amino)pyridine 90.degree. C. (dec.) 0 9 ##STR20##
6-(dimethylamino)-5-nitro-4- (.beta.-D-ribo-furanosylamino)
pyrimidine [M + H].sup.+ =316 0 10 ##STR21##
6-(thiomethyl)-5-nitro-4-(.beta.-D- ribo-furanosylamino) pyrimidine
155- 158.degree. C. * 11 ##STR22## 5-nitro-4-methyl-2-(.beta.-D-
ribofuranosyl- amino)pyridine [M + H].sup.+ =286 * 12 ##STR23##
6-amino-5-nitro-4-(2,3-O- isopropylidene-.beta.-D-
ribofuranosylamino)pyrimidine 145- 147.degree. C. 0 13 ##STR24##
6-(2-hydroxy-ethylamino)-5- nitro-4-(.beta.-D-ribo-
furanosylamino)pyrimidine [M + H].sup.+ =332 0 14 ##STR25##
6-(ethylamino)-5-nitro-4- (.beta.-D-ribo- furanosylamino)pyrimidine
175.degree. C. (dec.) 0 15 ##STR26## 6-(4-methoxy-benzylamino)-
5-cyano-4-(.beta.-D-ribo- furanosylamino)pyrimidine [M + H].sup.+
=388 0 16 ##STR27## 3-cyano-2-(.beta.-D-ribofuranosyl-
amino)pyridine [M + H].sup.+ =252 * 17 ##STR28##
6-hydroxy-5-nitro-4-(.beta.-D- ribofuranosylamino)pyrimidine
197.degree. C. (dec.) * 18 ##STR29## 6-amino-5-nitro-4-(.beta.-D-
xylofuranosylamino)- pyrimidine 227- 228.degree. C. 0 19 ##STR30##
6-amino-5-nitro-4-(.beta.-L- ribofuranosylamino)- pyrimidine 226-
227.degree. C. ** 20 ##STR31## 6-amino-5-nitro-4-(5-deoxy-5-
fluoro-.beta.-D- ribofuranosylamino)- pyrimidine 129- 130.degree.
C. * 21 ##STR32## 6-amino-5-nitro-4-(5-deoxy-5-
azido-.beta.-D-ribofuranosylamino)- pyrimidine 204- 205.degree. C.
* 22 ##STR33## 6-amino-5-nitro-4-(.alpha.-D- ribofuranosylamino)-
pyrimidine 187- 214.degree. C. *** 23 ##STR34##
6-Amino-5-nitro-4-[(5-O- acetyl-.beta.-D-ribofuranosyl)
amino]pyrimidine 102- 125.degree. C. *** 24 ##STR35##
6-Amino-5-nitro-4-[(2,3,5-tri-O- benzoyl-.beta.-D-ribofuranosyl)
amino]pyrimidine [M + H].sup.+ =600 ** 25 ##STR36## Methyl
6-amino-4-(.beta.-D- ribofuranosylamino)pyrimidine- 5-carboxylate
154- 155.degree. C. * 26 ##STR37## Methyl 6-chloro-4-(.beta.-D-
ribofuranosylamino)pyrimidine- 5-carboxylate 161- 162.degree. C. 0
27 ##STR38## Methyl 6-amino-4-(.alpha.-D-
ribofuranosylamino)pyrimidine- 5-carboxylate 129- 130.degree. C. *
28 ##STR39## Methyl 6-chloro-4-(.alpha.-D-
ribofuranosylamino)pyrimidine- 5-carboxylate 135.degree. C. (dec.)
0 30 ##STR40## 5-(6-Amino-5-nitro-pyrimidin-
4-ylamino)-3,4-dihydroxy- tetrahydro-furan-2-carboxylic acid 31
##STR41## 5-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-3,4-dihydroxy-
tetrahydro-furan-2-carboxylic acid amide 32 ##STR42##
5-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-3,4-dihydroxy-
tetrahydro-furan-2-carbaldehyde 33 ##STR43##
2-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-5-(1-hydroxy-ethyl)-
tetrahydro-furan-3,4-diol 34 ##STR44##
2-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-5-methyl-
tetrahydro-furan-3,4-diol 35 ##STR45##
2-(4-Amino-3-nitro-pyridin-2- ylamino)-5-hydroxymethyl-
tetrahydro-furan-3,4-diol 36 ##STR46## 2-(5-Amino-4-nitro-pyridin-3
- ylamino)-5-hydroxymethyl- tetrahydro-furan-3,4-diol 37 ##STR47##
5-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-2-hydroxymethyl-
tetrahydro-furan-3-ol 38 ##STR48## 5-(6-Amino-5-nitro-pyrimidin-
4-ylamino)-4-fluoro-2- hydroxymethyl-tetrahydro- furan-3-ol 39
##STR49## 5-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-4-fluoro-2-
hydroxymethyl-tetrahydro- furan-3-ol 40 ##STR50##
5-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-4,4-difluoro-2-
hydroxymethyl-tetrahydro- furan-3-ol 41 ##STR51##
2-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-4-fluoro-5-
hydroxymethyl-tetrahydro- furan-3-ol 42 ##STR52##
2-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-4-fluoro-5-
hydroxymethyl-tetrahydro- furan-3-ol 43 ##STR53##
2-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-4,4-difluoro-5-
hydroxymethyl-tetrahydro- furan-3-ol 44 ##STR54##
[5-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-2,5-dihydro-furan-2-
yl]-methanol 45 ##STR55## 5-(6-Amino-5-nitro-pyrimidin-
4-ylamino)-2-hydroxymethyl-3- methyl-tetrahydro-furan-3,4- diol 46
##STR56## 2-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-4-hydroxy-5-
hydroxymethyl-dihydro-furan- 3-one 47 ##STR57##
5-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-4-hydroxy-2-
hydroxymethyl-dihydro-furan- 3-one 48 ##STR58##
2-Aminomethyl-5-(6-amino-5- nitro-pyrimidin-4-ylamino)-
tetrahydro-furan-3,4-diol 49 ##STR59##
2-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-5-(2-hydroxy-ethyl)-
tetrahydro-furan-3,4-diol 50 ##STR60##
2-(6-Amino-5-nitro-pyrimidin- 4-ylamino)-5-hydroxymethyl-
tetrahydro-thiophene-3,4-diol 51 ##STR61##
2-(2-Amino-3-nitro-pyridin-4- ylamino)-5-hydroxymethyl-
tetrahydro-furan-3,4-diol 52 ##STR62## 4-amino-6-(3,4-dihydroxy-5-
hydroxymethyl-tetrahydro- furan-2-ylamino)-pyrimidine-5-
carboxylate 53 ##STR63## 4-Amino-6-(3,4-dihydroxy-5-
hydroxymethyl-tetrahydro- furan-2-ylamino)-pyrimidine-5- carboxylic
acid 54 ##STR64## 2-Hydroxymethyl-5-(5-nitro- pyrimidin-4-ylamino)-
tetrahydro-furan-3,4-diol 55 ##STR65##
2-(6-Amino-5-nitro-pyridazin-4- ylamino)-5-hydroxymethyl-
tetrahydro-furan-3,4-diol 56 ##STR66##
2-(5-Amino-4-nitro-pyridazin-3- ylamino)-5-hydroxymethyl-
tetrahydro-furan-3,4-diol 57 ##STR67## 58 ##STR68## 59
##STR69##
[0156] Melting points (where available) were obtained on an
Electrothermal MeltTemp.TM. apparatus and are uncorrected. Mass
spec data (where available) was obtained on a Micro Mass (Beverly,
Mass.) ESI-MS (electrospray ionization-mass spectrometer).
[0157] Activity measurements in Table 1 were performed in a
cell-based luciferase reporter assay (as described in Section 4.2)
comprising a luciferase reporter construct containing a UGA
premature termination codon that was stably transfected in 293T
Human Embryonic Kidney cells. Gentamicin, a small molecule known to
allow readthrough of premature termination codons, was used as an
internal standard. Activity measurements are based on the
qualitative ratio between the minimum concentration of compound
required to produce a given protein in a cell versus the amount of
protein produced by the cell at that concentration. Compounds which
were found to have either or both very high potency and very high
efficacy of protein synthesis are classified as "***". Compounds
which were found to have significant potency and/or efficacy of
protein synthesis were classified as "**" Similarly, compounds
which were found to have potency and/or efficacy of protein
synthesis were classified as "*".
[0158] The present invention encompasses the in vitro or in vivo
use of a compound of the invention, and the incorporation of a
compound of the invention into pharmaceutical compositions and
single unit dosage forms useful in the treatment and prevention of
a variety of diseases and disorders. Specific diseases and
disorders include those ameliorated by the suppression of a
nonsense mutation in messenger RNA.
[0159] Pharmaceutical compositions including dosage forms of the
invention, which comprise a compound of the invention, can be used
in the methods of the invention.
[0160] Without being limited by theory, it is believed that a
compound of the invention can modulate premature translation
termination and/or nonsense-mediated mRNA decay. Consequently, a
first embodiment of the invention relates to a method of modulating
premature translation termination and/or nonsense-mediated mRNA
decay comprising contacting a cell exhibiting a nonsense mutation
with an effective amount of a compound of the invention. In a
particular embodiment, the invention relates to a method of
inducing nonsense suppression comprising contacting a cell
exhibiting a nonsense mutation with an effective amount of a
compound of the invention.
4.2 Biological Assays and Animal Studies
[0161] The test compounds identified in the nonsense suppression
assay (for convenience referred to herein as a compound of the
invention) can be tested for biological activity using host cells
containing or engineered to contain the target RNA element coupled
to a functional readout system. For example, the lead compound can
be tested in a host cell engineered to contain the RNA with the
premature translation termination codon controlling the expression
of a reporter gene. In this example, the lead compounds are assayed
in the presence or absence of the RNA with the premature
translation termination codon. Compounds that modulate premature
translation termination and/or nonsense-mediated mRNA decay in vivo
will result in increased expression of the full-length gene, i.e.,
past the premature termination codon. Alternatively, a phenotypic
or physiological readout can be used to assess activity of the
target RNA with the premature translation termination codon in the
presence and absence of the lead compound. Both the in vitro and in
vivo nonsense suppression assays used herein and as described in
International Patent Publication WO 01/44516, which is incorporated
by reference herein in its entirety, can be used to identify lead
compounds and can also be used to determine an EC.sub.50 for the
lead compounds.
[0162] Animal model systems can also be used to demonstrate the
safety and efficacy of a compound of the invention. The compounds
of the invention can be tested for biological activity using animal
models for a disease, condition, or syndrome of interest. These
include animals engineered to contain the target RNA element
coupled to a functional readout system, such as a transgenic
mouse.
[0163] Examples of animal models for cystic fibrosis include, but
are not limited to, cftr(-/-) mice (see, e.g., Freedman et al.,
2001, Gastroenterology 121(4):950-7), cftr(tm1 HGU/tm1 HGU) mice
(see, e.g., Bernhard et al., 2001, Exp Lung Res 27(4):349-66),
CFTR-deficient mice with defective cAMP-mediated Cl(-) conductance
(see, e.g., Stotland et al., 2000, Pediatr Pulmonol 30(5):413-24),
and C57BL/6-Cftr(m1 UNC)/Cftr(m1 UNC) knockout mice (see, e.g.,
Stotland et al., 2000, Pediatr Pulmonol 30(5):413-24).
[0164] Examples of animal models for muscular dystrophy include,
but are not limited to, mouse, hamster, cat, dog, and C. elegans.
Examples of mouse models for muscular dystrophy include, but are
not limited to, the dy-/- mouse (see, e.g., Connolly et al., 2002,
J Neuroimmunol 127(1-2):80-7), a muscular dystrophy with myositis
(mdm) mouse mutation (see, e.g., Garvey et al., 2002, Genomics
79(2):146-9), the mdx mouse (see, e.g., Nakamura et al., 2001,
Neuromuscul Disord 11(3):251-9), the utrophin-dystrophin knockout
(dko) mouse (see, e.g., Nakamura et al., 2001, Neuromuscul Disord
11(3):251-9), the dy/dy mouse (see, e.g., Dubowitz et al., 2000,
Neuromuscul Disord 10(4-5):292-8), the mdx(Cv3) mouse model (see,
e.g., Pillers et al., 1999, Laryngoscope 109(8):1310-2), and the
myotonic ADR-MDX mutant mice (see, e.g., Kramer et al., 1998,
Neuromuscul Disord 8(8):542-50). Examples of hamster models for
muscular dystrophy include, but are not limited to,
sarcoglycan-deficient hamsters (see, e.g., Nakamura et al., 2001,
Am J Physiol Cell Physiol 281(2):C690-9) and the BIO 14.6
dystrophic hamster (see, e.g., Schlenker & Burbach, 1991, J
Appl Physiol 71(5):1655-62). An example of a feline model for
muscular dystrophy includes, but is not limited to, the
hypertrophic feline muscular dystrophy model (see, e.g., Gaschen
& Burgunder, 2001, Acta Neuropathol (Berl) 101(6):591-600).
Canine models for muscular dystrophy include, but are not limited
to, golden retriever muscular dystrophy (see, e.g., Fletcher et
al., 2001, Neuromuscul Disord 11(3):239-43) and canine X-linked
muscular dystrophy (see, e.g., Valentine et al., 1992, Am J Med
Genet 42(3):352-6). Examples of C. elegans models for muscular
dystrophy are described in Chamberlain & Benian, 2000, Curr
Biol 10(21):R795-7 and Culette & Sattelle, 2000, Hum Mol Genet
9(6):869-77.
[0165] Examples of animal models for familial hypercholesterolemia
include, but are not limited to, mice lacking functional LDL
receptor genes (see, e.g., Aji et al., 1997, Circulation
95(2):430-7), Yoshida rats (see, e.g., Fantappie et al., 1992, Life
Sci 50(24):1913-24), the JCR:LA-cp rat (see, e.g., Richardson et
al., 1998, Atherosclerosis 138(1):135-46), swine (see, e.g.,
Hasler-Rapacz et al., 1998, Am J Med Genet 76(5):379-86), and the
Watanabe heritable hyperlipidaemic rabbit (see, e.g., Tsutsumi et
al., 2000, Arzneimittelforschung 50(2):118-21; Harsch et al., 1998,
Br J Pharmacol 124(2):227-82; and Tanaka et al., 1995,
Atherosclerosis 114(1):73-82).
[0166] An example of an animal model for human cancer in general
includes, but is not limited to, spontaneously occurring tumors of
companion animals (see, e.g., Vail & MacEwen, 2000, Cancer
Invest 18(8):781-92). Examples of animal models for lung cancer
include, but are not limited to, lung cancer animal models
described by Zhang & Roth (1994, In Vivo 8(5):755-69) and a
transgenic mouse model with disrupted p53 function (see, e.g.,
Morris et al., 1998, J La State Med Soc 150(4):179-85). An example
of an animal model for breast cancer includes, but is not limited
to, a transgenic mouse that overexpresses cyclin D1 (see, e.g.,
Hosokawa et al., 2001, Transgenic Res 10(5):471-8). An example of
an animal model for colon cancer includes, but is not limited to, a
TCRbeta and p53 double knockout mouse (see, e.g., Kado et al.,
2001, Cancer Res 61(6):2395-8). Examples of animal models for
pancreatic cancer include, but are not limited to, a metastatic
model of Panc02 murine pancreatic adenocarcinoma (see, e.g., Wang
et al., 2001, Int J Pancreatol 29(1):37-46) and nu-nu mice
generated in subcutaneous pancreatic tumours (see, e.g., Ghaneh et
al., 2001, Gene Ther 8(3):199-208). Examples of animal models for
non-Hodgkin's lymphoma include, but are not limited to, a severe
combined immunodeficiency ("SCID") mouse (see, e.g., Bryant et al.,
2000, Lab Invest 80(4):553-73) and an IgHmu-HOX11 transgenic mouse
(see, e.g., Hough et al., 1998, Proc Natl Acad Sci USA
95(23):13853-8). An example of an animal model for esophageal
cancer includes, but is not limited to, a mouse transgenic for the
human papillomavirus type 16 E7 oncogene (see, e.g., Herber et al.,
1996, J Virol 70(3):1873-81). Examples of animal models for
colorectal carcinomas include, but are not limited to, Apc mouse
models (see, e.g., Fodde & Smits, 2001, Trends Mol Med
7(8):369-73 and Kuraguchi et al., 2000, Oncogene 19(50):5755-63).
An example of an animal model for neurofibromatosis includes, but
is not limited to, mutant NF1 mice (see, e.g., Cichowski et al.,
1996, Semin Cancer Biol 7(5):291-8). Examples of animal models for
retinoblastoma include, but are not limited to, transgenic mice
that expression the simian virus 40 T antigen in the retina (see,
e.g., Howes et al., 1994, Invest Opthalmol Vis Sci 35(2):342-51 and
Windle et al, 1990, Nature 343(6259):665-9) and inbred rats (see,
e.g., Nishida et al., 1981, Curr Eye Res 1(1):53-5 and Kobayashi et
al., 1982, Acta Neuropathol (Berl) 57(2-3):203-8). Examples of
animal models for Wilm's tumor include, but are not limited to, a
WT1 knockout mice (see, e.g., Scharnhorst et al., 1997, Cell Growth
Differ 8(2): 133-43), a rat subline with a high incidence of
neuphroblastoma (see, e.g., Mesfin & Breech, 1996, Lab Anim Sci
46(3):321-6), and a Wistar/Furth rat with Wilms' tumor (see, e.g.,
Murphy et al., 1987, Anticancer Res 7(4B):717-9).
[0167] Examples of animal models for retinitis pigmentosa include,
but are not limited to, the Royal College of Surgeons ("RCS") rat
(see, e.g., Vollrath et al., 2001, Proc Natl Acad Sci USA 98(22);
12584-9 and Hanitzsch et al., 1998, Acta Anat (Basel)
162(2-3):119-26), a rhodopsin knockout mouse (see, e.g., Jaissle et
al., 2001, Invest Opthalmol Vis Sci 42(2):506-13), and Wag/Rij rats
(see, e.g., Lai et al., 1980, Am J Pathol 98(1):281-4).
[0168] Examples of animal models for cirrhosis include, but are not
limited to, CCl.sub.4-exposed rats (see, e.g., Kloehn et al., 2001,
Horm Metab Res 33(7):394-401) and rodent models instigated by
bacterial cell components or colitis (see, e.g., Vierling, 2001,
Best Pract Res Clin Gastroenterol 15(4):591-610).
[0169] Examples of animal models for hemophilia include, but are
not limited to, rodent models for hemophilia A (see, e.g., Reipert
et al., 2000, Thromb Haemost 84(5):826-32; Jarvis et al., 1996,
Thromb Haemost 75(2):318-25; and Bi et al., 1995, Nat Genet
10(1):119-21), canine models for hemophilia A (see, e.g.,
Gallo-Penn et al., 1999, Hum Gene Ther 10(11):1791-802 and Connelly
et al, 1998, Blood 91(9); 3273-81), murine models for hemophilia B
(see, e.g., Snyder et al., 1999, Nat Med 5(1):64-70; Wang et al.,
1997, Proc Natl Acad Sci USA 94(21):11563-6; and Fang et al., 1996,
Gene Ther 3(3):217-22), canine models for hemophilia B (see, e.g.,
Mount et al., 2002, Blood 99(8):2670-6; Snyder et al., 1999, Nat
Med 5(1):64-70; Fang et al., 1996, Gene Ther 3(3):217-22); and Kay
et al., 1994, Proc Natl Acad Sci USA 91(6):2353-7), and a rhesus
macaque model for hemophilia B (see, e.g., Lozier et al., 1999,
Blood 93(6):1875-81).
[0170] Examples of animal models for von Willebrand disease
include, but are not limited to, an inbred mouse strain RIIIS/J
(see, e.g., Nichols et al., 1994, 83(11):3225-31 and Sweeney et
al., 1990, 76(11):2258-65), rats injected with botrocetin (see,
e.g., Sanders et al., 1988, Lab Invest 59(4):443-52), and porcine
models for von Willebrand disease (see, e.g., Nichols et al., 1995,
Proc Natl Acad Sci USA 92(7):2455-9; Johnson & Bowie, 1992, J
Lab Clin Med 120(4):553-8); and Brinkhous et al., 1991, Mayo Clin
Proc 66(7):733-42).
[0171] Examples of animal models for b-thalassemia include, but are
not limited to, murine models with mutations in globin genes (see,
e.g., Lewis et al., 1998, Blood 91(6):2152-6; Raja et al., 1994, Br
J Haematol 86(1):156-62; Popp et al., 1985, 445:432-44; and Skow et
al., 1983, Cell 34(3):1043-52).
[0172] Examples of animal models for kidney stones include, but are
not limited to, genetic hypercalciuric rats (see, e.g., Bushinsky
et al., 1999, Kidney Int 55(1):234-43 and Bushinsky et al., 1995,
Kidney Int 48(6):1705-13), chemically treated rats (see, e.g.,
Grases et al., 1998, Scand J Urol Nephrol 32(4):261-5; Burgess et
al., 1995, Urol Res 23(4):239-42; Kumar et al., 1991, J Urol
146(5):1384-9; Okada et al., 1985, Hinyokika Kiyo 31 (4):565-77;
and Bluestone et al., 1975, Lab Invest 33(3):273-9), hyperoxaluric
rats (see, e.g., Jones et al., 1991, J Urol 145(4):868-74), pigs
with unilateral retrograde flexible nephroscopy (see, e.g., Seifmah
et al., 2001, 57(4):832-6), and rabbits with an obstructed upper
urinary tract (see, e.g., Itatani et al., 1979, Invest Urol
17(3):234-40).
[0173] Examples of animal models for ataxia-telangiectasia include,
but are not limited to, murine models of ataxia-telangiectasia
(see, e.g., Barlow et al., 1999, Proc Natl Acad Sci USA
96(17):9915-9 and Inoue et al., 1986, Cancer Res
46(8):3979-82).
[0174] Examples of animal models for lysosomal storage diseases
include, but are not limited to, mouse models for
mucopolysaccharidosis type VII (see, e.g., Brooks et al., 2002,
Proc Natl Acad Sci U S A. 99(9):6216-21; Monroy et al., 2002, Bone
30(2):352-9; Vogler et al., 2001, Pediatr Dev Pathol. 4(5):421-33;
Vogler et al., 2001, Pediatr Res. 49(3):342-8; and Wolfe et al.,
2000, Mol. Ther. 2(6):552-6), a mouse model for metachromatic
leukodystrophy (see, e.g., Matzner et al., 2002, Gene Ther.
9(1):53-63), a mouse model of Sandhoff disease (see, e.g., Sango et
al., 2002, Neuropathol Appl Neurobiol. 28(1):23-34), mouse models
for mucopolysaccharidosis type III A (see, e.g., Bhattacharyya et
al., 2001, Glycobiology 11(1):99-10 and Bhaumik et al., 1999,
Glycobiology 9(12):1389-96.), arylsulfatase A (ASA)-deficient mice
(see, e.g., D'Hooge et al., 1999, Brain Res. 847(2):352-6 and
D'Hooge et al, 1999, Neurosci Lett. 273(2):93-6); mice with an
aspartylglucosaminuria mutation (see, e.g., Jalanko et al., 1998,
Hum Mol Genet. 7(2):265-72); feline models of mucopolysaccharidosis
type VI (see, e.g., Crawley et al., 1998, J Clin Invest.
101(1):109-19 and Norrdin et al., 1995, Bone 17(5):485-9); a feline
model of Niemann-Pick disease type C (see, e.g., March et al.,
1997, Acta Neuropathol (Berl). 94(2):164-72); acid
sphingomyelinase-deficient mice (see, e.g., Otterbach &
Stoffel, 1995, Cell 81(7):1053-6), and bovine mannosidosis (see,
e.g., Jolly et al., 1975, Birth Defects Orig Arctic Ser.
11(6):273-8).
[0175] Examples of animal models for tuberous sclerosis ("TSC")
include, but are not limited to, a mouse model of TSC1 (see, e.g.,
Kwiatkowski et al., 2002, Hum Mol. Genet. 11(5):525-34), a Tsc1
(TSC1 homologue) knockout mouse (see, e.g., Kobayashi et al., 2001,
Proc Natl Acad Sci U S A. 2001 Jul. 17; 98(15):8762-7), a TSC2 gene
mutant(Eker) rat model (see, e.g., Hino 2000, Nippon Rinsho
58(6):1255-61; Mizuguchi et al., 2000, J Neuropathol Exp Neurol.
59(3):188-9; and Hino et al., 1999, Prog Exp Tumor Res. 35:95-108);
and Tsc2(+/-) mice (see, e.g., Onda et al., 1999, J Clin Invest.
104(6):687-95).
4.3 Synthesis and Preparation
[0176] The compounds of the invention can be obtained via standard,
well-known synthetic methodology, see e.g., March, J. Advanced
Organic Chemistry; Reactions Mechanisms, and Structure, 4th ed.,
1992. Starting materials useful for preparing the compounds of the
invention and intermediates therefore, are commercially available
or can be prepared from commercially available materials using
known synthetic methods and reagents.
[0177] Compounds of formula I can be prepared as shown in Scheme A.
An amine compound A1 is allowed to react with compound A2, wherein
G represents a leaving group such as chloro, bromo, iodo or
trifluoromethanesulfonyl. The reaction is usually performed in the
presence of a basic reagent, e.g., triethylamine or pyridine, in a
solvent such as methanol or dimethylformamide, and at temperatures
ranging from about ambient to the reflux temperature of the chosen
solvent. Alternatively, compounds of the invention, particularly
compounds of formula I may be prepared by using reagents having the
reversed substitution pattern. A compound A3, wherein L is an
appropriate leaving group such as bromo or acetoxy is reacted with
an amine A4, usually in the presence of an appropriate base and
solvent and at temperatures from about ambient to reflux. The
starting compounds A1-A4 for the preparation of compounds of the
invention including compounds of formula I can be obtained
commercially or can be prepared by methods familiar to one skilled
in the art of organic synthesis. ##STR70##
[0178] Compounds of formula II can be prepared as outlined in
Scheme B. In one embodiment, a compound of formula B1, which
possesses leaving groups G similar to that described in Scheme A,
is allowed to react with Y.sup.2 resulting in an intermediate of
formula B2. This intermediate can then react with a compound of
formula A1 as described in Scheme A to yield a compound of formula
II. Inversely, a compound of formula B1 can be treated first with
compound of formula A1 resulting in intermediate of formula B3
which is then allowed to react with Y.sup.2 to afford formula II.
The starting compounds of formula B1 are commercially available or
can be easily prepared by methods familiar to one skilled in the
art. ##STR71##
[0179] Compounds of formula VII can be prepared from compounds of
formula C1 (Scheme C), wherein P.sup.1, P.sup.2 and P.sup.2 can be
suitable hydroxyl protecting groups that can be removed using
protocols well established in the art of organic chemistry. A
source describing hydroxyl protecting groups and their removal can
be found in Greene and Wuts, Protective Groups in Organic
Synthesis, 3.sup.rd ed., pp17-245, 1999, John Wiley & Sons,
Inc., New York, N.Y. Representative hydroxyl groups include, but
are not limited to, acetyl, benzoyl, benzyl, dimethylacetal and
benzylidene. In the case where compound of formula A1 has
R.sup.6.dbd.P.sup.1, R.sup.7.dbd.P.sup.2 and R.sup.8.dbd.P.sup.3, a
compound of formula C1 can then be prepared as depicted in Scheme
B. ##STR72##
[0180] Compounds of formula D4 can be prepared as shown in Scheme
D. Selective removal of protecting group P1 from compounds of
formula C1 gives compounds of formula D1. Compounds of formula D1
can be converted to compounds of formula D2 by conversion of the
hydroxyl group to a leaving group G, in which G can be groups such
as, but not limited to, chloro, bromo, iodo,
trifluoromethanesulonate, methanesulfonate or toluenesulfonate.
Reaction of compounds of formula D2 with a nucleophile or metal
hydride gives compounds of formula D3. Alternatively compounds of
formula D1 can be converted to compounds of formula D3 (in which
R.dbd.F) directly by treatment with a fluorinating reagent such as
diethylamino sulfur trifluoride or related reagents as discussed in
Organic Reactions, vol. 35, (1988), John Wiley & Sons, Inc.,
New York, N.Y. or by utilizing the Mitsunobu reaction as discussed
in Organic Reactions, vol. 42, (1992), John Wiley & Sons, Inc.,
New York, N.Y. Removal of the protecting groups give compounds of
formula D4. ##STR73##
[0181] Compounds of formula E4 can be prepared as shown in Scheme
E. The hydroxyl group in compounds of formula D1 can be oxidized to
carboxylic acids of formula E1 which can be converted to activated
compounds of formula E2 in which G is a leaving group such as, but
not limited to, chloro, fluoro or bromo. Reaction of compounds of
formula E2 with nucleophiles gives compounds of formula E3.
Alternatively, compounds of formula E3 can be prepared directly
from compounds of formula E1 by methods familiar to one skilled in
the art. Removal of the protecting groups gives compounds of
formula E4. ##STR74##
[0182] Compounds of formula F2 and F4 can be prepared as shown in
Scheme F. Oxidation of the hydroxyl group in a compound of formula
D1 to the aldehyde gives compounds of formula F1. Removal of the
protecting groups from a compound of formula F1 gives compounds of
formula F2. In addition, reaction of compounds of formula F1 with a
nucleophilic compound such as RM, wherein M is a metal such as, but
not limited to, Zn, Mg or Li, and R is alkyl or an aromatic group,
gives compounds of formula F3. Compounds of formula F3 can be
converted to compounds of formula F4 by removal of the protecting
groups. ##STR75##
[0183] Compounds of formula G4 and G6 can be prepared as shown in
scheme G. The protecting group P2 can be removed selectively to
give the compounds of formula G1, which can be oxidized to give
compounds of formula G2. Compounds of formula G2 can be fluorinated
with a fluorinating reagent such as diethylaminosulfur trifluoride
to give the difluorinated compounds of formula G5, which upon
removal of the protecting groups can give compounds of formula G6.
Alternatively compounds of formula G1 can be fluorinated with a
fluorinating reagent such as diethylaminosulfur trifluoride to give
compounds of formula G3 which upon removal of the protecting groups
gives compounds of formula G4. ##STR76##
[0184] Compounds of formula H4 and H6 can be prepared as shown in
scheme H. The protecting group P3 can be removed selectively to
give the compounds of formula H1, which can be oxidized to give
compounds of formula H2. Compounds of formula H2 can be fluorinated
with a fluorinating reagent such as diethylaminosulfur trifluoride
to give the difluorinated compounds of formula H5, which upon
removal of the protecting groups can give compounds of formula H6.
Alternatively, compounds of formula H1 can be fluorinated with a
fluorinating reagent such as diethylaminosulfur trifluoride to give
compounds of formula H3, which upon removal of the protecting
groups gives compounds of formula H4. ##STR77##
[0185] Compounds of the formula J3 and J6 can be prepared as shown
in Scheme J. The hydroxyl group in H1 can be thioacylated to give
compounds of formula J2 in which T represents a thioacyl group.
Reaction of the thioacyl compounds of formula J2 following
literature procedure such as given in Barton et al., Tetrahedron
Lett. 3381 (1993), give compounds of formula J2 which upon removal
of protecting groups gives compounds of formula J3. Compounds of
formula J6 can be prepared from compounds of formula J2 by removal
of protecting group P2 to give compounds of formula J4. Conversion
of the hydroxyl group in compounds of formula J4 to a leaving group
G in which G can be groups such as, but not limited to, chloro,
bromo, iodo, trifluoromethanesulonate, methanesulfonate and
toluenesulfonate can give compounds of formula J5. Treatment of the
compounds of formula J5 with a base such as DBU followed by removal
of the protecting group can give compounds of formula J6.
##STR78##
[0186] Compounds of formula K3 can be prepared as shown in scheme
K. Compounds of formula K1 can be obtained as outlined in Iwakawa
et al., Carbohydrate Research 121:99 (1983). Reaction of compounds
of formula K1 with the amino substituted reactant can give
compounds of formula K2 as described in Paquette et al. ed.,
Organic Reactions, vol. 55, (2000). Removal of protecting groups in
compounds of formula K2 can give compounds of formula K3.
##STR79##
[0187] Compounds of the invention can also be synthesized using the
syntheses described in Moss et. al., J. Med. Chem. 31:786-790
(1988); Kamikawa et. al., J. Chem. Soc. Chem. Commun. 195 (1988);
Lee et. al., Bioorg. And Med. Chem. Lett. 11:293-301 (2001);
Nogueras et. al., Nucleosides and Nucleotides 13: 447-457 (1994);
Burgdorf et. al. Chem. Eur. J. 8:293-301 (2002); Mabry et. al.,
Nucleosides and Nucleotides 13:1125-1133 (1994); Palmer et. al.,
Tet. Lett. 31:279-282 (1990); Baxter et. al., Nucleosides and
Nucleotides 10:393-396 (1991); Franchetti et al. Nucleosides and
Nucleotides 10:543-545 (1991) and Organic Reactions, vol. 55,
(2000), Paquette et al. ed, each of which is incorporated by
reference herein in its entirety.
4.4 Methods of Use
[0188] The invention encompasses methods of treating and preventing
diseases or disorders ameliorated by the suppression of premature
translation termination and/or nonsense-mediated mRNA decay in a
patient which comprise administering to a patient in need of such
treatment or prevention a therapeutically effective amount of a
compound of the invention.
[0189] In one embodiment, the present invention encompasses the
treatment or prevention of any disease which is associated with a
gene exhibiting premature translation termination and/or
nonsense-mediated mRNA decay. In one embodiment, the disease is
due, in part, to the lack of or reduced expression of the gene
resulting from a premature stop codon. Specific examples of genes
which may exhibit premature translation termination and/or
nonsense-mediated mRNA decay and diseases associated with premature
translation termination and/or nonsense-mediated mRNA decay are
found in U.S. Provisional Patent Application No. 60/390,747,
titled: Methods For Identifying Small Molecules That Modulate
Premature Translation Termination And Nonsense Mediated mRNA Decay,
filed Jun. 21, 2002, and International Application PCT/US03/19760,
filed Jun. 23, 2003, both of which are incorporated herein by
reference in their entirety.
[0190] Diseases ameliorated by the suppression of premature
translation termination and/or nonsense-mediated mRNA decay
include, but are not limited to: genetic diseases, somatic
diseases, cancers, autoimmune diseases, blood diseases, collagen
diseases, diabetes, neurodegenerative diseases, proliferative
diseases, cardiovascular diseases, pulmonary diseases, inflammatory
diseases or central nervous system diseases.
[0191] In one embodiment, diseases to be treated or prevented by
administering to a patient in need thereof an effective amount of a
compound of the invention include, but are not limited to,
amyloidosis, hemophilia, Alzheimer's disease, Tay Sachs disease,
Niemann Pick disease, atherosclerosis, giantism, dwarfism,
hypothyroidism, hyperthyroidism, aging, obesity, Parkinson's
disease, cystic fibrosis, muscular dystrophy, heart disease, kidney
stones, ataxia-telangiectasia, familial hypercholesterolemia,
retinitis pigmentosa, Duchenne muscular dystrophy, and Marfan
syndrome. In one embodiment, the diseases are associated with a
nonsense mutation.
[0192] In one embodiment, the compounds of the invention are useful
for treating or preventing an autoimmune disease. In one
embodiment, the autoimmune disease is associated with a nonsense
mutation. In a preferred embodiment, the autoimmune disease is
rheumatoid arthritis or graft versus host disease.
[0193] In another embodiment, the compounds of the invention are
useful for treating or preventing a blood disease. In one
embodiment, the blood disease is associated with a nonsense
mutation. In a preferred embodiment, the blood disease is
hemophilia, Von Willebrand disease, ataxia-telangiectasia,
.beta.-thalassemia or kidney stones.
[0194] In another embodiment, the compounds of the invention are
useful for treating or preventing a collagen disease. In one
embodiment, the collagen disease is associated with a nonsense
mutation. In a preferred embodiment, the collagen disease is
osteogenesis imperfecta or cirrhosis.
[0195] In another embodiment, the compounds of the invention are
useful for treating or preventing diabetes. In one embodiment, the
diabetes is associated with a nonsense mutation.
[0196] In another embodiment, the compounds of the invention are
useful for treating or preventing an inflammatory disease. In one
embodiment, the inflammatory disease is associated with a nonsense
mutation. In a preferred embodiment, the inflammatory disease is
arthritis, rheumatoid arthritis or osteoarthritis.
[0197] In another embodiment, the compounds of the invention are
useful for treating or preventing a central nervous system disease.
In one embodiment, the central nervous system disease is associated
with a nonsense mutation. In one embodiment the central nervous
system disease is a neurodegenerative disease. In a preferred
embodiment, the central nervous system disease is multiple
sclerosis, muscular dystrophy, Duchenne muscular dystrophy,
Alzheimer's disease, Tay Sachs disease, Niemann Pick disease, late
infantile neuronal ceroid lipofuscinosis (LINCL) or Parkinson's
disease.
[0198] In another preferred embodiment, the compounds of the
invention are useful for treating or preventing cancer,
particularly in humans. In a preferred embodiment, the cancer is of
the head and neck, eye, skin, mouth, throat, esophagus, chest,
bone, blood, lung, colon, sigmoid, rectum, stomach, prostate,
breast, ovaries, kidney, liver, pancreas, brain, intestine, heart
or adrenals. In one embodiment, the cancer is a solid tumor. In one
embodiment, the cancer is associated with a nonsense mutation. In
another embodiment, the cancer is associated with a genetic
nonsense mutation. In another embodiment, the cancer is associated
with a somatic mutation. Without being limited by any theory, the
use of the compounds of the invention against cancer may relate to
its action against mutations of the p53 gene.
[0199] In one embodiment, the cancer is not a blood cancer. In
another embodiment, the cancer is not leukemia. In another
embodiment, the cancer is not multiple myeloma. In another
embodiment, the cancer is not prostate cancer.
[0200] In another preferred embodiment, the compounds of the
invention are useful for treating or preventing cancer associated
with a mutation of the p53 gene. In one embodiment, the mutation is
a genetic mutation. In another embodiment, the mutation is a
somatic mutation. The methods of the invention are particularly
useful for treating or preventing a cancer associated with a
nonsense mutation in the p53 gene due to p53's crucial role in
apoptosis. Without being limited by theory, it is thought that
apoptosis can be induced by contacting a cell with an effective
amount of a compound of the invention resulting in suppression of
the nonsense mutation which, in turn, allows the production of full
length p53 to occur. Nonsense mutations have been identified in the
p53 gene and have been implicated in cancer. Several nonsense
mutations in the p53 gene have been identified (see, e.g., Masuda
et al., 2000, Tokai J Exp Clin Med. 25(2):69-77; Oh et al., 2000,
Mol Cells 10(3):275-80; Li et al., 2000, Lab Invest. 80(4):493-9;
Yang et al., 1999, Zhonghua Zhong Liu Za Zhi 21(2):114-8;
Finkelstein et al., 1998, Mol. Diagn. 3(1):37-41; Kajiyama et al.,
1998, Dis Esophagus. 11(4):279-83; Kawamura et al., 1999, Leuk Res.
23(2):115-26; Radig et al., 1998, Hum Pathol. 29(11):1310-6;
Schuyer et al., 1998, Int J Cancer 76(3):299-303; Wang-Gohrke et
al., 1998, Oncol Rep. 5(1):65-8; Fulop et al., 1998, J Reprod Med.
43(2):119-27; Ninomiya et al., 1997, J Dermatol Sci. 14(3):173-8;
Hsieh et al., 1996, Cancer Lett. 100(1-2):107-13; Rall et al.,
1996, Pancreas. 12(1):10-7; Fukutomi et al., 1995, Nippon Rinsho.
53(11):2764-8; Frebourg et al., 1995, Am J Hum Genet. 56(3):608-15;
Dove et al., 1995, Cancer Surv. 25:335-55; Adamson et al., 1995, Br
J Haematol. 89(1):61-6; Grayson et al., 1994, Am J Pediatr Hematol
Oncol. 16(4):341-7; Lepelley et al., 1994, Leukemia. 8(8):1342-9;
McIntyre et al., 1994, J Clin Oncol. 12(5):925-30; Horio et al.,
1994, Oncogene. 9(4):1231-5; Nakamura et al., 1992, Jpn J Cancer
Res. 83(12):1293-8; Davidoff et al., 1992, Oncogene. 7(1):127-33;
and Ishioka et al., 1991, Biochem Biophys Res Commun. 177(3):901-6;
the disclosures of which are hereby incorporated by reference
herein in their entireties). Any disease associated with a p53 gene
encoding a premature translation codon including, but not limited
to, the nonsense mutations described in the references cited above,
can be treated or prevented by compounds of the invention.
[0201] In other embodiments, diseases to be treated or prevented by
administering to a patient in need thereof an effective amount of a
compound of the invention include, but are not limited to, solid
tumors such as sarcoma, carcinomas, fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and
retinoblastoma.
[0202] In another embodiment, diseases to be treated or prevented
by administering to a patient in need thereof an effective amount
of a compound of the invention include, but are not limited to, a
blood-born tumor such as acute lymphoblastic leukemia, acute
lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,
acute myeloblastic leukemia, acute promyelocytic leukemia, acute
monoblastic leukemia, acute erythroleukemic leukemia, acute
megakaryoblastic leukemia, acute myelomonocytic leukemia, acute
nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic
myelocytic leukemia, chronic lymphocytic leukemia, hairy cell
leukemia, or multiple myeloma. See e.g., Harrison's Principles of
Internal Medicine, Eugene Braunwald et al., eds., pp. 491-762 (15th
ed. 2001).
[0203] In yet another embodiment, the invention encompasses the
treatment of a human afflicted with a solid tumor or a blood
tumor.
[0204] In a preferred embodiment, the invention encompasses a
method of treating or preventing a disease ameliorated by
modulation of premature translation termination and/or
nonsense-mediated mRNA decay, or ameliorating one or more symptoms
associated therewith comprising contacting a cell with an effective
amount of a compound of the invention. Cells encompassed by the
present methods include animal cells, mammalian cells, bacterial
cells, plant cells and virally infected cells. In one embodiment,
the nonsense mutation is a genetic mutation (i.e., the nonsense
codon was present in the progenitor DNA). In another embodiment,
the nonsense mutation is a somatic mutation (i.e., the nonsense
codon arose spontaneously or from mutagenesis).
[0205] In certain embodiments, a compound of the invention is
administered to a patient, preferably a mammal, more preferably a
human, as a preventative measure against a disease associated with
premature translation termination and/or nonsense-mediated mRNA
decay.
[0206] In a preferred embodiment, it is first determined that the
patient is suffering from a disease associate with premature
translation termination and/or nonsense-mediated mRNA decay. In
another embodiment, the patient has undergone a screening process
to determine the presence of a nonsense mutation comprising the
steps of screening a subject, or cells extracted therefrom, by an
acceptable nonsense mutation screening assay. In a preferred
embodiment, the DNA of the patient can be sequenced or subjected to
Southern Blot, polymerase chain reaction (PCR), use of the Short
Tandem Repeat (STR), or polymorphic length restriction fragments
(RFLP) analysis to determine if a nonsense mutation is present in
the DNA of the patient. In one embodiment, it is determined whether
the nonsense mutation is a genetic mutation or a somatic mutation
by comparison of progenitor DNA. Alternatively, it can be
determined if altered levels of the protein with the nonsense
mutation are expressed in the patient by western blot or other
immunoassays. In another embodiment, the patient is an unborn child
who has undergone screening in utero for the presence of a nonsense
mutation. Administration of a compound of the invention can occur
either before or after birth. In a related embodiment, the therapy
is personalized in that the patient is screened for a nonsense
mutation screening assay and treated by the administration of one
or more compounds of the invention; particularly, the patient may
be treated with a compound particularly suited for the mutations in
question; e.g., depending upon the disease type, cell type, and the
gene in question. Such methods are well known to one of skill in
the art.
[0207] In another embodiment, the cells (e.g., animal cells,
mammalian cells, bacterial cells, plant cells and virally infected
cells) are screened for premature translation termination and/or
nonsense-mediated mRNA decay with a method such as that described
above (i.e., the DNA of the cell can be sequenced or subjected to
Southern Blot, polymerase chain reaction (PCR), use of the Short
Tandem Repeat (STR), or polymorphic length restriction fragments
(RFLP) analysis to determine if a nonsense mutation is present in
the DNA of the cell).
[0208] Specific methods of the invention further comprise the
administration of an additional therapeutic agent (i.e., a
therapeutic agent other than a compound of the invention). In
certain embodiments of the present invention, the compounds of the
invention can be used in combination with at least one other
therapeutic agent. Therapeutic agents include, but are not limited
to non-opioid analgesics; non-steroid anti-inflammatory agents;
antiemetics; .beta.-adrenergic blockers; anticonvulsants;
antidepressants; Ca.sup.2+-channel blockers; anticancer agent and
mixtures thereof.
[0209] In certain embodiments, the compounds of the invention can
be administered or formulated in combination with anticancer
agents. Suitable anticancer agents include, but are not limited to:
alkylating agents; nitrogen mustards; folate antagonists; purine
antagonists; pyrimidine antagoinists; spindle poisons;
topoisomerase inhibitors; apoptosis inducing agents; angiogenesis
inhibitors; podophyllotoxins; nitrosoureas; cisplatin; carboplatin;
interferon; asparginase; tamoxifen; leuprolide; flutamide;
megestrol; mitomycin; bleomycin; doxorubicin; irinotecan and
taxol.
[0210] In certain embodiments, the compounds of the invention can
be administered or formulated in combination with antibiotics. In
certain embodiments, the antibiotic is a macrolide (e.g.,
tobramycin), a cephalosporin (e.g., cephalexin, cephradine,
cefuroxime, cefprozil, cefaclor, cefixime or cefadroxil), a
clarithromycin (e.g., clarithromycin), an erythromycin (e.g.,
erythromycin), a penicillin (e.g., penicillin V) or a quinolone
(e.g., ofloxacin, ciprofloxacin or norfloxacin). In a preferred
embodiment, the antibiotic is active against Pseudomonas
aeruginosa.
[0211] The compounds of the invention and the other therapeutics
agent can act additively or, more preferably, synergistically. In a
preferred embodiment, a composition comprising a compound of the
invention is administered concurrently with the administration of
another therapeutic agent, which can be part of the same
composition or in a different composition from that comprising the
compounds of the invention. In another embodiment, a compound of
the invention is administered prior to or subsequent to
administration of another therapeutic agent.
[0212] The magnitude of a prophylactic or therapeutic dose of a
particular active ingredient of the invention in the acute or
chronic management of a disease or condition will vary, however,
with the nature and severity of the disease or condition, and the
route by which the active ingredient is administered. The dose, and
perhaps the dose frequency, will also vary according to the age,
body weight, and response of the individual patient. Suitable
dosing regimens can be readily selected by those skilled in the art
with due consideration of such factors. In general, the recommended
daily dose range for the conditions described herein lie within the
range of from about 0.1 mg to about 2000 mg per day. In one
embodiment, the compound of the invention is given as a single
once-a-day dose. In another embodiment, the compound of the
invention is given as divided doses throughout a day. More
specifically, the daily dose is administered in a single dose or in
equally divided doses. Preferably, a daily dose range should be
from about 5 mg to about 500 mg per day, more preferably, between
about 10 mg and about 200 mg per day. In managing the patient, the
therapy should be initiated at a lower dose, perhaps about 1 mg to
about 25 mg, and increased if necessary up to about 200 mg to about
2000 mg per day as either a single dose or divided doses, depending
on the patient's global response.
[0213] It may be necessary to use dosages of the active ingredient
outside the ranges disclosed herein in some cases, as will be
apparent to those of ordinary skill in the art. Furthermore, it is
noted that the clinician or treating physician will know how and
when to interrupt, adjust, or terminate therapy in conjunction with
individual patient response.
[0214] The phrases "therapeutically effective amount",
"prophylactically effective amount" and "therapeutically or
prophylactically effective amount," as used herein encompass the
above described dosage amounts and dose frequency schedules.
Different therapeutically effective amounts may be applicable for
different diseases and conditions, as will be readily known by
those of ordinary skill in the art. Similarly, amounts sufficient
to treat or prevent such diseases, but insufficient to cause, or
sufficient to reduce, adverse effects associated with conventional
therapies are also encompassed by the above described dosage
amounts and dose frequency schedules.
4.5 Pharmaceutical Compositions
[0215] Pharmaceutical compositions and single unit dosage forms
comprising a compound of the invention, or a pharmaceutically
acceptable polymorph, prodrug, salt, solvate, hydrate, or clathrate
thereof, are also encompassed by the invention. Individual dosage
forms of the invention may be suitable for oral, mucosal (including
sublingual, buccal, rectal, nasal, or vaginal), parenteral
(including subcutaneous, intramuscular, bolus injection,
intraarterial, or intravenous), transdermal, or topical
administration.
[0216] Pharmaceutical compositions and dosage forms of the
invention comprise a compound of the invention, or a
pharmaceutically acceptable prodrug, polymorph, salt, solvate,
hydrate, or clathrate thereof. Pharmaceutical compositions and
dosage forms of the invention typically also comprise one or more
pharmaceutically acceptable excipients.
[0217] A particular pharmaceutical composition encompassed by this
embodiment comprises a compound of the invention, or a
pharmaceutically acceptable polymorph, prodrug, salt, solvate,
hydrate, or clathrate thereof, and at least one additional
therapeutic agent. Examples of additional therapeutic agents
include, but are not limited to: anti-cancer drugs and
anti-inflammation therapies including, but not limited to, those
listed above in Section 4.4.
[0218] Single unit dosage forms of the invention are suitable for
oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or
rectal), parenteral (e.g., subcutaneous, intravenous, bolus
injection, intramuscular, or intraarterial), or transdermal
administration to a patient. Examples of dosage forms include, but
are not limited to: tablets; caplets; capsules, such as soft
elastic gelatin capsules; cachets; troches; lozenges; dispersions;
suppositories; ointments; cataplasms (poultices); pastes; powders;
dressings; creams; plasters; solutions; patches; aerosols (e.g.,
nasal sprays or inhalers); gels; liquid dosage forms suitable for
oral or mucosal administration to a patient, including suspensions
(e.g., aqueous or non-aqueous liquid suspensions, oil-in-water
emulsions, or a water-in-oil liquid emulsions), solutions, and
elixirs; liquid dosage forms suitable for parenteral administration
to a patient; and sterile solids (e.g., crystalline or amorphous
solids) that can be reconstituted to provide liquid dosage forms
suitable for parenteral administration to a patient.
[0219] The composition, shape, and type of dosage forms of the
invention will typically vary depending on their use. For example,
a dosage form used in the acute treatment of inflammation or a
related disease may contain larger amounts of one or more of the
active ingredients it comprises than a dosage form used in the
chronic treatment of the same disease. Similarly, a parenteral
dosage form may contain smaller amounts of one or more of the
active ingredients it comprises than an oral dosage form used to
treat the same disease or disorder. These and other ways in which
specific dosage forms encompassed by this invention will vary from
one another will be readily apparent to those skilled in the art.
See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack
Publishing, Easton Pa. (1990).
[0220] Typical pharmaceutical compositions and dosage forms
comprise one or more carriers, excipients or diluents. Suitable
excipients are well known to those skilled in the art of pharmacy,
and non-limiting examples of suitable excipients are provided
herein. Whether a particular excipient is suitable for
incorporation into a pharmaceutical composition or dosage form
depends on a variety of factors well known in the art including,
but not limited to, the way in which the dosage form will be
administered to a patient. For example, oral dosage forms such as
tablets may contain excipients not suited for use in parenteral
dosage forms. The suitability of a particular excipient may also
depend on the specific active ingredients in the dosage form.
[0221] This invention further encompasses anhydrous (e.g., <1%
water) pharmaceutical compositions and dosage forms comprising
active ingredients, since water can facilitate the degradation of
some compounds. For example, the addition of water (e.g., 5%) is
widely accepted in the pharmaceutical arts as a means of simulating
long-term storage in order to determine characteristics such as
shelf-life or the stability of formulations over time. See, e.g.,
Jens T. Carstensen, Drug Stability: Principles & Practice, 2d.
Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect, water
and heat accelerate the decomposition of some compounds. Thus, the
effect of water on a formulation can be of great significance since
moisture and/or humidity are commonly encountered during
manufacture, handling, packaging, storage, shipment, and use of
formulations.
[0222] Anhydrous pharmaceutical compositions and dosage forms of
the invention can be prepared using anhydrous or low moisture
containing ingredients and low moisture or low humidity conditions.
Pharmaceutical compositions and dosage forms that comprise lactose
and at least one active ingredient that comprises a primary or
secondary amine are preferably anhydrous if substantial contact
with moisture and/or humidity during manufacturing, packaging,
and/or storage is expected.
[0223] An anhydrous pharmaceutical composition should be prepared
and stored such that its anhydrous nature is maintained.
Accordingly, anhydrous compositions are preferably packaged using
materials known to prevent exposure to water such that they can be
included in suitable formulary kits. Examples of suitable packaging
include, but are not limited to, hermetically sealed foils,
plastics, unit dose containers (e.g., vials), blister packs, and
strip packs.
[0224] The invention further encompasses pharmaceutical
compositions and dosage forms that comprise one or more compounds
that reduce the rate by which an active ingredient will decompose.
Such compounds, which are referred to herein as "stabilizers,"
include, but are not limited to, antioxidants such as ascorbic
acid, pH buffers, or salt buffers.
[0225] Like the amounts and types of excipients, the amounts and
specific types of active ingredients in a dosage form may differ
depending on factors such as, but not limited to, the route by
which it is to be administered to patients. However, typical dosage
forms of the invention comprise a compound of the invention, or a
pharmaceutically acceptable salt, solvate, clathrate, hydrate,
polymorph or prodrug thereof lie within the range of from about 0.1
mg to about 2000 mg per day, given as a single once-a-day dose in
the morning but preferably as divided doses throughout the day
taken with food. More preferably, the daily dose is administered
twice daily in equally divided doses. Preferably, a daily dose
range should be from about 5 mg to about 500 mg per day, more
preferably, between about 10 mg and about 200 mg per day. In
managing the patient, the therapy should be initiated at a lower
dose, perhaps about 1 mg to about 25 mg, and increased if necessary
up to about 200 mg to about 2000 mg per day as either a single dose
or divided doses, depending on the patient's global response.
4.5.1. Oral Dosage Forms
[0226] Pharmaceutical compositions of the invention that are
suitable for oral administration can be presented as discrete
dosage forms, such as, but are not limited to, tablets (e.g.,
chewable tablets), caplets, capsules, and liquids (e.g., flavored
syrups). Such dosage forms contain predetermined amounts of active
ingredients, and may be prepared by methods of pharmacy well known
to those skilled in the art. See generally, Remington's
Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa.
(1990).
[0227] Typical oral dosage forms of the invention are prepared by
combining the active ingredient(s) in an intimate admixture with at
least one excipient according to conventional pharmaceutical
compounding techniques. Excipients can take a wide variety of forms
depending on the form of preparation desired for administration.
For example, excipients suitable for use in oral liquid or aerosol
dosage forms include, but are not limited to, water, glycols, oils,
alcohols, flavoring agents, preservatives, and coloring agents.
Examples of excipients suitable for use in solid oral dosage forms
(e.g., powders, tablets, capsules, and caplets) include, but are
not limited to, starches, sugars, micro-crystalline cellulose,
diluents, granulating agents, lubricants, binders, and
disintegrating agents.
[0228] Because of their ease of administration, tablets and
capsules represent the most advantageous oral dosage unit forms, in
which case solid excipients are employed. If desired, tablets can
be coated by standard aqueous or nonaqueous techniques. Such dosage
forms can be prepared by any of the methods of pharmacy. In
general, pharmaceutical compositions and dosage forms are prepared
by uniformly and intimately admixing the active ingredients with
liquid carriers, finely divided solid carriers, or both, and then
shaping the product into the desired presentation if necessary.
[0229] For example, a tablet can be prepared by compression or
molding. Compressed tablets can be prepared by compressing in a
suitable machine the active ingredients in a free-flowing form such
as powder or granules, optionally mixed with an excipient. Molded
tablets can be made by molding in a suitable machine a mixture of
the powdered compound moistened with an inert liquid diluent.
[0230] Examples of excipients that can be used in oral dosage forms
of the invention include, but are not limited to, binders, fillers,
disintegrants, and lubricants. Binders suitable for use in
pharmaceutical compositions and dosage forms include, but are not
limited to, corn starch, potato starch, or other starches, gelatin,
natural and synthetic gums such as acacia, sodium alginate, alginic
acid, other alginates, powdered tragacanth, guar gum, cellulose and
its derivatives (e.g., ethyl cellulose, cellulose acetate,
carboxymethyl cellulose calcium, sodium carboxymethyl cellulose),
polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch,
hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910),
microcrystalline cellulose, and mixtures thereof.
[0231] Examples of fillers suitable for use in the pharmaceutical
compositions and dosage forms disclosed herein include, but are not
limited to, talc, calcium carbonate (e.g., granules or powder),
microcrystalline cellulose, powdered cellulose, dextrates, kaolin,
mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch,
and mixtures thereof. The binder or filler in pharmaceutical
compositions of the invention is typically present in from about 50
to about 99 weight percent of the pharmaceutical composition or
dosage form.
[0232] Suitable forms of microcrystalline cellulose include, but
are not limited to, the materials sold as AVICEL-PH-101,
AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105 (available from FMC
Corporation, American Viscose Division, Avicel Sales, Marcus Hook,
Pa.), and mixtures thereof. An specific binder is a mixture of
microcrystalline cellulose and sodium carboxymethyl cellulose sold
as AVICEL RC-581. Suitable anhydrous or low moisture excipients or
additives include AVICEL-PH-103.TM. and Starch 1500 LM.
[0233] Disintegrants are used in the compositions of the invention
to provide tablets that disintegrate when exposed to an aqueous
environment. Tablets that contain too much disintegrant may
disintegrate in storage, while those that contain too little may
not disintegrate at a desired rate or under the desired conditions.
Thus, a sufficient amount of disintegrant that is neither too much
nor too little to detrimentally alter the release of the active
ingredients should be used to form solid oral dosage forms of the
invention. The amount of disintegrant used varies based upon the
type of formulation, and is readily discernible to those of
ordinary skill in the art. Typical pharmaceutical compositions
comprise from about 0.5 to about 15 weight percent of disintegrant,
specifically from about 1 to about 5 weight percent of
disintegrant.
[0234] Disintegrants that can be used in pharmaceutical
compositions and dosage forms of the invention include, but are not
limited to, agar-agar, alginic acid, calcium carbonate,
microcrystalline cellulose, croscarmellose sodium, crospovidone,
polacrilin potassium, sodium starch glycolate, potato or tapioca
starch, pre-gelatinized starch, other starches, clays, other
algins, other celluloses, gums, and mixtures thereof.
[0235] Lubricants that can be used in pharmaceutical compositions
and dosage forms of the invention include, but are not limited to,
calcium stearate, magnesium stearate, mineral oil, light mineral
oil, glycerin, sorbitol, mannitol, polyethylene glycol, other
glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated
vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil,
sesame oil, olive oil, corn oil, and soybean oil), zinc stearate,
ethyl oleate, ethyl laureate, agar, and mixtures thereof.
Additional lubricants include, for example, a syloid silica gel
(AEROSIL 200, manufactured by W. R. Grace Co. of Baltimore, Md.), a
coagulated aerosol of synthetic silica (marketed by Degussa Co. of
Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold
by Cabot Co. of Boston, Mass.), and mixtures thereof. If used at
all, lubricants are typically used in an amount of less than about
1 weight percent of the pharmaceutical compositions or dosage forms
into which they are incorporated.
4.5.2. Delayed Release Dosage Forms
[0236] Active ingredients of the invention can be administered by
controlled release means or by delivery devices that are well known
to those of ordinary skill in the art. Examples include, but are
not limited to, those described in U.S. Pat. Nos. 3,845,770;
3,916,899; 3,536,809; 3,598,123; and 4,008,719, 5,674,533,
5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556,
and 5,733,566, each of which is incorporated herein by reference.
Such dosage forms can be used to provide slow or controlled-release
of one or more active ingredients using, for example,
hydropropylmethyl cellulose, other polymer matrices, gels,
permeable membranes, osmotic systems, multilayer coatings,
microparticles, liposomes, microspheres, or a combination thereof
to provide the desired release profile in varying proportions.
Suitable controlled-release formulations known to those of ordinary
skill in the art, including those described herein, can be readily
selected for use with the active ingredients of the invention. The
invention thus encompasses single unit dosage forms suitable for
oral administration such as, but not limited to, tablets, capsules,
gelcaps, and caplets that are adapted for controlled-release.
[0237] Controlled-release pharmaceutical products can improve drug
therapy over that achieved by their non-controlled counterparts.
Ideally, the use of an optimally designed controlled-release
preparation in medical treatment is characterized by a minimum of
drug substance being employed to cure or control the condition in a
minimum amount of time. Advantages of controlled-release
formulations include extended activity of the drug, reduced dosage
frequency, and increased patient compliance. In addition,
controlled-release formulations can be used to affect the time of
onset of action or other characteristics, such as blood levels of
the drug, and can thus affect the occurrence of side (e.g.,
adverse) effects.
[0238] Most controlled-release formulations are designed to
initially release an amount of drug (active ingredient) that
promptly produces the desired therapeutic effect, and gradually and
continually release of other amounts of drug to maintain this level
of therapeutic or prophylactic effect over an extended period of
time. In order to maintain this constant level of drug in the body,
the drug must be released from the dosage form at a rate that will
replace the amount of drug being metabolized and excreted from the
body. Controlled-release of an active ingredient can be stimulated
by various conditions including, but not limited to, pH,
temperature, enzymes, water, or other physiological conditions or
compounds.
4.5.3. Parenteral Dosage Forms
[0239] Parenteral dosage forms can be administered to patients by
various routes including, but not limited to, subcutaneous,
intravenous (including bolus injection), intramuscular, and
intra-arterial. Because their administration typically bypasses
patients' natural defenses against contaminants, parenteral dosage
forms are preferably sterile or capable of being sterilized prior
to administration to a patient. Examples of parenteral dosage forms
include, but are not limited to, solutions ready for injection, dry
products ready to be dissolved or suspended in a pharmaceutically
acceptable vehicle for injection, suspensions ready for injection,
and emulsions. For example, lyophilized sterile compositions
suitable for reconstitution into particulate-free dosage forms
suitable for administration to humans.
[0240] Suitable vehicles that can be used to provide parenteral
dosage forms of the invention are well known to those skilled in
the art. Examples include, but are not limited to: Water for
Injection USP; aqueous vehicles such as, but not limited to, Sodium
Chloride Injection, Ringer's Injection, Dextrose Injection,
Dextrose and Sodium Chloride Injection, and Lactated Ringer's
Injection; water-miscible vehicles such as, but not limited to,
ethyl alcohol, polyethylene glycol, and polypropylene glycol; and
non-aqueous vehicles such as, but not limited to, corn oil,
cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl
myristate, and benzyl benzoate.
[0241] Compounds that increase the solubility of one or more of the
active ingredients disclosed herein can also be incorporated into
the parenteral dosage forms of the invention.
[0242] Parenteral dosage forms are preferred for the methods of
preventing, treating or managing disease in a cancer patient.
4.5.4. Transdermal and Topical Dosage Forms
[0243] Transdermal and topical dosage forms of the invention
include, but are not limited to, creams, lotions, ointments, gels,
solutions, emulsions, suspensions, or other forms known to one of
skill in the art. See, e.g., Remington's Pharmaceutical Sciences,
18th eds., Mack Publishing, Easton Pa. (1990); and Introduction to
Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger,
Philadelphia (1985). Transdermal dosage forms include "reservoir
type" or "matrix type" patches, which can be applied to the skin
and worn for a specific period of time to permit the penetration of
a desired amount of active ingredients.
[0244] Suitable excipients (e.g., carriers and diluents) and other
materials that can be used to provide transdermal and topical
dosage forms encompassed by this invention are well known to those
skilled in the pharmaceutical arts, and depend on the particular
tissue to which a given pharmaceutical composition or dosage form
will be applied. With that fact in mind, typical excipients
include, but are not limited to, water, acetone, ethanol, ethylene
glycol, propylene glycol, butane-1,3-diol, isopropyl myristate,
isopropyl palmitate, mineral oil, and mixtures thereof to form
lotions, tinctures, creams, emulsions, gels or ointments, which are
non-toxic and pharmaceutically acceptable. Moisturizers or
humectants can also be added to pharmaceutical compositions and
dosage forms if desired. Examples of such additional ingredients
are well known in the art. See, e.g., Remington's Pharmaceutical
Sciences, 18th eds., Mack Publishing, Easton Pa. (1990).
[0245] Depending on the specific tissue to be treated, additional
components may be used prior to, in conjunction with, or subsequent
to treatment with active ingredients of the invention. For example,
penetration enhancers can be used to assist in delivering the
active ingredients to the tissue. Suitable penetration enhancers
include, but are not limited to: acetone; various alcohols such as
ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as
dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide;
polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone;
Kollidon grades (Povidone, Polyvidone); urea; and various
water-soluble or insoluble sugar esters such as Tween 80
(polysorbate 80) and Span 60 (sorbitan monostearate).
[0246] The pH of a pharmaceutical composition or dosage form, or of
the tissue to which the pharmaceutical composition or dosage form
is applied, may also be adjusted to improve delivery of one or more
active ingredients. Similarly, the polarity of a solvent carrier,
its ionic strength, or tonicity can be adjusted to improve
delivery. Compounds such as stearates can also be added to
pharmaceutical compositions or dosage forms to advantageously alter
the hydrophilicity or lipophilicity of one or more active
ingredients so as to improve delivery. In this regard, stearates
can serve as a lipid vehicle for the formulation, as an emulsifying
agent or surfactant, and as a delivery-enhancing or
penetration-enhancing agent. Different salts, hydrates or solvates
of the active ingredients can be used to further adjust the
properties of the resulting composition.
4.5.5. Mucosal Dosage Forms and Lung Delivery
[0247] Mucosal dosage forms of the invention include, but are not
limited to, ophthalmic solutions, sprays and aerosols, or other
forms known to one of skill in the art. See, e.g., Remington's
Pharmaceutical Sciences, 18th eds., Mack Publishing, Easton Pa.
(1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed.,
Lea & Febiger, Philadelphia (1985). Dosage forms suitable for
treating mucosal tissues within the oral cavity can be formulated
as mouthwashes or as oral gels. In one embodiment, the aerosol
comprises a carrier. In another embodiment, the aerosol is carrier
free.
[0248] A compound of the invention can also be administered
directly to the lung by inhalation (see e.g., Tong et al., PCT
Application, WO 97/39745; Clark et al, PCT Application, WO
99/47196, which are herein incorporated by reference). For
administration by inhalation, a compound of the invention can be
conveniently delivered to the lung by a number of different
devices. For example, a Metered Dose Inhaler ("MDI") which utilizes
canisters that contain a suitable low boiling propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas can
be used to deliver a compound of the invention directly to the
lung. MDI devices are available from a number of suppliers such as
3M Corporation, Aventis, Boehringer Ingleheim, Forest Laboratories,
Glaxo-Wellcome, Schering Plough and Vectura.
[0249] Alternatively, a Dry Powder Inhaler (DPI) device can be used
to administer a compound of the invention to the lung (See, e.g.,
Raleigh et al., Proc. Amer. Assoc. Cancer Research Annual Meeting,
1999, 40, 397, which is herein incorporated by reference). DPI
devices typically use a mechanism such as a burst of gas to create
a cloud of dry powder inside a container, which can then be inhaled
by the patient. DPI devices are also well known in the art and can
be purchased from a number of vendors which include, for example,
Fisons, Glaxo-Wellcome, Inhale Therapeutic Systems, ML
Laboratories, Qdose and Vectura. A popular variation is the
multiple dose DPI ("MDDPI") system, which allows for the delivery
of more than one therapeutic dose. MDDPI devices are available from
companies such as AstraZeneca, GlaxoWellcome, IVAX, Schering
Plough, SkyePharma and Vectura. For example, capsules and
cartridges of gelatin for use in an inhaler or insufflator can be
formulated containing a powder mix of the compound and a suitable
powder base such as lactose or starch for these systems.
[0250] Another type of device that can be used to deliver a
compound of the invention to the lung is a liquid spray device
supplied, for example, by Aradigm Corporation. Liquid spray systems
use extremely small nozzle holes to aerosolize liquid drug
formulations that can then be directly inhaled into the lung.
[0251] In a preferred embodiment, a nebulizer device is used to
deliver a compound of the invention to the lung. Nebulizers create
aerosols from liquid drug formulations by using, for example,
ultrasonic energy to form fine particles that can be readily
inhaled (See e.g., Verschoyle et al., British J Cancer, 1999, 80,
Suppl 2, 96, which is herein incorporated by reference). Examples
of nebulizers include devices supplied by Sheffield/Systemic
Pulmonary Delivery Ltd. (See, Armer et al., U.S. Pat. No.
5,954,047; van der Linden et al., U.S. Pat. No. 5,950,619; van der
Linden et al., U.S. Pat. No. 5,970,974, which are herein
incorporated by reference), Aventis and Batelle Pulmonary
Therapeutics. Inhaled compound of the invention, delivered by
nebulizer devices, is currently under investigation as a treatment
for aerodigestive cancer (Engelke et al., Poster 342 at American
Association of Cancer Research, San Francisco, Calif., Apr. 1-5,
2000) and lung cancer (Dahl et al., Poster 524 at American
Association of Cancer Research, San Francisco, Calif., Apr. 1-5,
2000).
[0252] In a particularly preferred embodiment, an
electrohydrodynamic ("EHD") aerosol device is used to deliver a
compound of the invention to the lung. EHD aerosol devices use
electrical energy to aerosolize liquid drug solutions or
suspensions (see e.g., Noakes et al., U.S. Pat. No. 4,765,539;
Coffee, U.S. Pat. No. 4,962,885; Coffee, PCT Application, WO
94/12285; Coffee, PCT Application, WO 94/14543; Coffee, PCT
Application, WO 95/26234, Coffee, PCT Application, WO 95/26235,
Coffee, PCT Application, WO 95/32807, which are herein incorporated
by reference). The electrochemical properties of the compound of
the invention formulation may be important parameters to optimize
when delivering this drug to the lung with an EHD aerosol device
and such optimization is routinely performed by one of skill in the
art. EHD aerosol devices may more efficiently delivery drugs to the
lung than existing pulmonary delivery technologies. Other methods
of intra-pulmonary delivery of a compound of the invention will be
known to the skilled artisan and are within the scope of the
invention.
[0253] Liquid drug formulations suitable for use with nebulizers
and liquid spray devices and EHD aerosol devices will typically
include a compound of the invention with a pharmaceutically
acceptable carrier. Preferably, the pharmaceutically acceptable
carrier is a liquid such as alcohol, water, polyethylene glycol or
a perfluorocarbon. Optionally, another material may be added to
alter the aerosol properties of the solution or suspension of a
compound of the invention. Preferably, this material is liquid such
as an alcohol, glycol, polyglycol or a fatty acid. Other methods of
formulating liquid drug solutions or suspension suitable for use in
aerosol devices are known to those of skill in the art (See, e.g.,
Biesalski, U.S. Pat. Nos. 5,112,598; Biesalski, 5,556,611, which
are herein incorporated by reference). A compound of the invention
can also be formulated in rectal or vaginal compositions such as
suppositories or retention enemas, e.g., containing conventional
suppository bases such as cocoa butter or other glycerides.
[0254] In addition to the formulations described previously, a
compound of the invention can also be formulated as a depot
preparation. Such long acting formulations can be administered by
implantation (for example subcutaneously or intramuscularly) or by
intramuscular injection. Thus, for example, the compounds can be
formulated with suitable polymeric or hydrophobic materials (for
example, as an emulsion in an acceptable oil) or ion exchange
resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt.
[0255] Alternatively, other pharmaceutical delivery systems can be
employed. Liposomes and emulsions are well known examples of
delivery vehicles that can be used to deliver a compound of the
invention. Certain organic solvents such as dimethylsulfoxide can
also be employed, although usually at the cost of greater toxicity.
A compound of the invention can also be delivered in a controlled
release system. In one embodiment, a pump can be used (Sefton, CRC
Crit. Ref Biomed Eng., 1987, 14, 201; Buchwald et al., Surgery,
1980, 88, 507; Saudek et al., N. Engl. J Med, 1989, 321, 574). In
another embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley,
New York (1984); Ranger and Peppas, J Macromol. Sci. Rev. Macromol.
Chem., 1983, 23, 61; see also Levy et al., Science 1985, 228, 190;
During et al., Ann. Neurol., 1989, 25, 351; Howard et al., 1989, J.
Neurosurg. 71, 105). In yet another embodiment, a
controlled-release system can be placed in proximity of the target
of the compounds of the invention, e.g., the lung, thus requiring
only a fraction of the systemic dose (see, e.g., Goodson, in
Medical Applications of Controlled Release, supra, vol. 2, pp. 115
(1984)). Other controlled-release system can be used (see e.g.,
Langer, Science, 1990, 249, 1527).
[0256] Suitable excipients (e.g., carriers and diluents) and other
materials that can be used to provide mucosal dosage forms
encompassed by this invention are well known to those skilled in
the pharmaceutical arts, and depend on the particular site or
method which a given pharmaceutical composition or dosage form will
be administered. With that fact in mind, typical excipients
include, but are not limited to, water, ethanol, ethylene glycol,
propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl
palmitate, mineral oil, and mixtures thereof, which are non-toxic
and pharmaceutically acceptable. Examples of such additional
ingredients are well known in the art. See, e.g., Remington's
Pharmaceutical Sciences, 18th eds., Mack Publishing, Easton Pa.
(1990).
[0257] The pH of a pharmaceutical composition or dosage form, or of
the tissue to which the pharmaceutical composition or dosage form
is applied, can also be adjusted to improve delivery of one or more
active ingredients. Similarly, the polarity of a solvent carrier,
its ionic strength, or tonicity can be adjusted to improve
delivery. Compounds such as stearates can also be added to
pharmaceutical compositions or dosage forms to advantageously alter
the hydrophilicity or lipophilicity of one or more active
ingredients so as to improve delivery. In this regard, stearates
can serve as a lipid vehicle for the formulation, as an emulsifying
agent or surfactant, and as a delivery-enhancing or
penetration-enhancing agent. Different salts, hydrates or solvates
of the active ingredients can be used to further adjust the
properties of the resulting composition.
5. EXAMPLES
5.1 Synthesis of Illustrative Compounds
5.1.1. Synthesis of
6-Amino-5-Nitro-4-(.beta.-D-Ribofuranosylamino)-Pyrimidine (1)
[0258] A solution of
2',3'-O-Isopropylidene-.beta.-D-ribofuranosylamine
p-toluenesulfonate salt (2.65 g, 7.34 mmol) in 30 mL of
N,N-dimethylformamide at room temperature was treated first with
triethylamine (2.3 mL, 16.7 mmol) followed by the slow addition of
4,6-dichloro-5-nitropyrimidine (1.30 g, 6.68 mmol). After stirring
for 3 h, the yellow solution was diluted with ethyl acetate (50 mL)
and washed with water (50 mL). The aqueous phase was further
extracted with 2.times.20 mL of ethyl acetate and the combined
organic extracts were washed with 2.times.50 mL of brine, dried
over MgSO.sub.4, filtered and concentrated in vacuo. The resulting
crude product was purified by silica gel chromatography (3:1
hexane/ethyl acetate) to afford
6-chloro-5-nitro-4-(2,3-O-isopropylidene-.beta.-D-ribofuranosylamino)pyri-
midine (1.37 g, 59%) as a white powder (m.p. 117-119.degree. C.).
TLC Rf 0.3 (3:1 hexane/ethyl acetate). .sup.1H NMR (CDCl.sub.3, 300
MHz): .delta. 8.71 (1H, d, J=8.7 Hz), 8.43 (1H, s), 6.35 (1H, d,
J=8.7 Hz), 4.93 (1H, d, J=6.0 Hz), 4.69 (1H, d, J=6.0 Hz), 4.44
(1H, s), 3.88 (2H, dd, J=3.6 Hz, 1.8 Hz), 2.81 (1H, dd, J=2.4 Hz,
2.1 Hz), 1.57 (3H, s), 1.36 (3H, s). MS (ES+): 347.
[0259] To a solution of
6-chloro-5-nitro-4-(2',3'-O-isopropylidene-.beta.-D-ribofuranosylamino)py-
rimidine (760 mg, 2.19 mmol) in N,N-dimethylformamide (20 mL) at
room temperature was added a solution of 7N NH.sub.3 in MeOH (626
.mu.L, 4.38 mmol). The solution was stirred for 2 h and then
diluted with 20 mL of ethyl acetate and washed with water. The
aqueous layer was further extracted with 2.times.10 mL of ethyl
acetate and the combined organic layers were washed with 2.times.20
mL of brine, dried over MgSO4, filtered and concentrated in vacuo.
The resulting crude product was purified by chromatography on
silica gel (1:3 0:100 hexane/ethyl acetate) to yield
6-amino-5-nitro-4-(2,3-O-isopropylidene-.beta.-D-ribofuranosylam-
ino)pyrimidine (609 mg, 89%) as a slightly yellow solid. .sup.1H
NMR (d.sub.6-DMSO, 300 MHz): .delta. 9.95 (1H, d, J=8.4 Hz), 8.54
(2H, s), 7.98 (1H, s), 6.21 (1H, d, J=8.4 Hz), 5.62 (1H, t, J=3.0
Hz), 4.80 (1H, d, J=6.0 Hz), 4.64 (1H, d, J=6.3 Hz), 4.23 (1H, s),
3.47-3.63 (2H, m), 1.44 (3H, s), 1.26 (3H, s). MS (ES+): 329.
[0260] Deprotection of the acetonide was accomplished by adding 5
mL of 9:1 TFA/H.sub.2O to
6-amino-5-nitro-4-(2',3'-O-isopropylidene-.beta.-D-ribofuranosylamino)pyr-
imidine (538 mg, 1.64 mmol). The solution was stirred at room
temperature for 10 minutes and then the solvent was removed in
vacuo. The crude material was then triturated with diethyl ether
and the resulting off-white solid was filtered to yield 360 mg
(55%) of the desired
6-amino-5-nitro-4-(.beta.-D-ribofuranosylamino)pyrimidine as the
trifluoroacetate salt, as seen by .sup.19F NMR (d.sub.6-DMSO, 300
MHz): .delta. -75.5. .sup.1H NMR (d.sub.6-DMSO, 300 MHz): d 9.28
(1H, d, J=7.5 Hz), 8.57 (2H, s), 7.98 (1H, s), 5.76 (1H, dd, J=7.5
Hz, 3.3 Hz), 4.50 (6H, broad s), 4.06 (1H, dd, J=5.1 Hz), 3.91 (1H,
dd, J=3.6 Hz), 3.77 (1H, d, J=3.3 Hz, 2.1 Hz), 3.53 (1H, dd, J=11.7
Hz, 3.0 Hz), 3.43 (1H, dd, J=11.7 Hz, 2.4 Hz). MS (ES+): 288.
5.1.2. Synthesis of
6-(N-Methylamino)-5-Nitro-4-(.beta.-D-Ribofuranosylamino)-Pyrimidine
(2)
[0261] To a solution of
6-chloro-5-nitro-4-(2',3'-O-isopropylidene-.beta.-D-ribofuranosylamino)py-
rimidine (200 mg, 0.577 mmol) in N,N-dimethylformamide (2 mL) at
room temperature was added a 40% aqueous solution of N-methylamine
(124 .mu.L, 1.44 mmol). The solution was stirred for 4 h, diluted
with 15 mL water and extracted with ethyl acetate (2.times.15 mL)
and the combined organic layers were washed with brine (2.times.15
mL), dried over MgSO4, filtered and concentrated in vacuo. The
resulting crude product was deprotected following the procedure
listed above for Acetonide Deprotection Step. The product was
triturated with ether to give 131 mg of
6-(N-methylamino)-5-nitro-4-(.beta.-D-ribofuranosylamino)pyridmidine
as a yellow solid.
5.1.3. Synthesis of
5,6-Diamino-4-(.beta.-D-Ribofuranosylamino)-Pyrimidine (3)
[0262] To a solution of
6-amino-5-nitro-4-(2',3'-O-isopropylidene-.beta.-D-ribofuranosylamino)pyr-
imidine (50 mg, 0.153 mmol) in methanol (5 mL) was added 10% Pd/C
(20 mg) and the mixture was hydrogenated at 50 psi for 4 hours at
room temperature. The mixture was filtered through celite and the
celite washed with methanol. The filtrate was concentrated to give
45 mg of the product as a brown oil. The product was deprotected
following the procedure listed above for Acetonide Deprotection
Step to give 5,6-diamino-4-(.beta.-D-ribofuranosylamino)pyrimidine
as a brown oil.
5.1.4. Synthesis of
3-Nitro-2-(.beta.-D-Ribofuranosylamino)-Pyrimidine (4)
[0263] A solution of
2',3'-O-Isopropylidene-.beta.-D-ribofuranosylamine
p-toluenesulfonate salt (501 mg, 1.39 mmol) in 5 mL of
N,N-dimethylformamide at room temperature was treated first with
triethylamine (0.439 mL, 3.15 mmol) followed by the slow addition
of 2-chloro-3-nitropyridine (200 mg, 1.26 mmol). After stirring at
50.degree. C. for 48 h, the solution was diluted with ethyl acetate
(15 mL) and washed with water (15 mL). The aqueous phase was
further extracted with 15 mL of ethyl acetate and the combined
organic extracts were dried over MgSO.sub.4, filtered and
concentrated in vacuo. The resulting crude product was purified by
silica gel chromatography (4:1 to 1:1 hexane/ethyl acetate) to give
the product which was deprotected following the procedure listed
above for Acetonide Deprotection Step to give the crude product
which was triturated with ether to give
3-nitro-2-(.beta.-D-ribofuranosylamino)pyridine, 11 mg as a yellow
solid.
5.1.5. Synthesis of
5-Nitro-2-(.beta.-D-Ribofuranosylamino)-Pyrimidine (5)
[0264] A solution of
2',3'-O-isopropylidene-.beta.-D-ribofuranosylamine
p-toluenesulfonate salt (501 mg, 1.39 mmol) in 5 mL of
N,N-dimethylformamide at room temperature was treated first with
triethylamine (0.439 mL, 3.15 mmol) followed by the slow addition
of 2-chloro-5-nitropyridine (200 mg, 1.26 mmol). After stirring at
50.degree. C. for 48 h, the solution was diluted with ethyl acetate
(15 mL) and washed with water (15 mL). The aqueous phase was
further extracted with 15 mL of ethyl acetate and the combined
organic extracts were dried over MgSO.sub.4, filtered and
concentrated in vacuo. The resulting crude product was purified by
silica gel chromatography (4:1 to 1:1 hexane/ethyl acetate) to give
the product which was deprotected following the procedure listed
above for Acetonide Deprotection Step to give the crude product
which was triturated with ether to give
5-nitro-4-(.beta.-D-ribofuranosylamino)pyridine, 43 mg.
5.1.6. Synthesis of
(1R,2S,3R,5R)-3-(6-Amino-5-Nitro-Pyrmidin-4-Ylamino)-5-Hydroxymethyl-Cycl-
opentane-1,2-Diol (6)
[0265] To a solution of 4-amino-6-chloro-5-nitropyrimidine (320 mg,
1.83 mmol) in N,N-dimethyl formamide (5 mL) and triethylamine (512
.mu.L, 3.67 mmol) was added
(1R,2S,3R,5R)-3-Amino-5-hydroxymethyl-cyclopentane-1,2-diol (293
mg, 2.02 mmol). The mixture was stirred at room temperature and
monitored by thin layer chromatography until starting material was
no longer detected. The reaction mixture was diluted with water (20
mL) and extracted with diethyl ether (2.times.10 mL). The organic
extract was washed with saturated aqueous sodium chloride (20 mL),
dried over MgSO.sub.4, filtered and concentrated in vacuo to give
the crude product. The product was recrystallized from hot
methanol/water to give a 298 mg of
(1R,2S,3R,5R)-3-(6-amino-5-nitro-pyrimidin-4-ylamino)-5-hydroxymethyl-cyc-
lopentane-1,2-diol as a yellow solid.
5.1.7. Synthesis of
(1S,2R,3S,5S)-3-(6-Amino-5-Nitro-Pyridine-4-Ylamino)-5-Hydroxymethyl-Cycl-
opentane-1,2-DIOL (7)
[0266] To a solution of 4-amino-6-chloro-5-nitropyrimidine (320 mg,
1.83 mmol) in N,N-dimethyl formamide (5 mL) and triethylamine (512
.mu.L, 3.67 mmol) was added
(1S,2R,3S,5S)-3-Amino-5-hydroxymethyl-cyclopentane-1,2-diol (293
mg, 2.02 mmol). The mixture was stirred at room temperature and
monitored by thin layer chromatography until starting material was
no longer detected. The reaction mixture was diluted with water (20
mL) and extracted with diethyl ether (2.times.10 mL). The organic
extract was washed with saturated aqueous sodium chloride (20 mL),
dried over MgSO.sub.4, filtered and concentrated in vacuo to give
the crude product. The product was recrystallized from hot
methanol/water to give a 315 mg of
(1S,2R,3S,5S)-3-(6-amino-5-nitro-pyrimidin-4-ylamino)-5-hydroxymethyl-cyc-
lopentane-1,2-diol as a yellow solid.
5.1.8. Synthesis of
6-Methoxy-3-Nitro-2-(.beta.-D-Ribofuranosylamino)-Pyrimidine
(8)
[0267] A solution of
2',3'-O-Isopropylidene-.beta.-D-ribofuranosylamine
p-toluenesulfonate salt (422 mg, 1.17 mmol) in 5 mL of
N,N-dimethylformamide at room temperature was treated first with
triethylamine (0.369 mL, 2.65 mmol) followed by the slow addition
of 2-chloro-6-methoxy-3-nitropyridine (200 mg, 1.06 mmol). After
stirring at 50.degree. C. for 48 h, the solution was diluted with
ethyl acetate (15 mL) and washed with water (15 mL). The aqueous
phase was further extracted with 15 mL of ethyl acetate and the
combined organic extracts were dried over MgSO.sub.4, filtered and
concentrated in vacuo. The resulting crude product was purified by
silica gel chromatography (3:1 to 1:1 hexane/ethyl acetate) to give
the product which was deprotected following the procedure listed
above for Acetonide Deprotection Step to give the crude product
which was triturated with ether to give
6-methoxy-3-nitro-2-(.beta.-D-ribofuranosylamino)pyridine, 41 mg as
a brown oil.
5.1.9. Synthesis of
6-(Dimethylamino)-5-NITRO-4-(.beta.-D-Ribofuranosylamino)-Pyrimidine
(9)
[0268] To a solution of
6-chloro-5-nitro-4-(2',3'-O-isopropylidene-.beta.-D-ribofuranosylamino)py-
rimidine (121 mg, 0.349 mmol) in N,N-dimethylformamide (5 mL) at
room temperature was added a 2.0M solution of dimethylamine in
anhydrous tetrahydrofuran (698 .mu.L, 1.4 mmol). The solution was
stirred for 17 h, diluted with 15 mL ethyl acetate and washed with
water. The aqueous layer was re-extracted with 10 mL ethyl acetate.
The organic extracts were combined, washed with saturated aqueous
sodium chloride (2.times.15 mL), dried over MgSO4, filtered and
concentrated in vacuo. The resulting crude product was purified by
silica gel chromatography (3:1 hexane/ethyl acetate) to yield 88 mg
of the product which was deprotected following the procedure listed
above for Acetonide Deprotection Step to give
6-(dimethylamino)-5-nitro-4-(.beta.-D-ribofuranosylamino)pyrimidine.
5.1.10. Synthesis of
6-(Thiomethyl)-5-Nitro-4-(.beta.-D-Ribofuranosylamino)-Pyrimidine
(10)
[0269] To a solution of
6-chloro-5-nitro-4-(2',3'-O-isopropylidene-.beta.-D-ribofuranosylamino)py-
rimidine (200 mg, 0.577 mmol) in N,N-dimethylformamide (2 mL) at
room temperature was added sodium thiomethoxide (44 mg, 0.634
mmol). The solution was stirred for 5 h, diluted with 15 mL water
and extracted with ethyl acetate (2.times.15 mL) and the combined
organic layers were washed with brine (2.times.15 mL), dried over
MgSO4, filtered and concentrated in vacuo. The resulting 213 mg of
yellow solid product was deprotected following the procedure listed
above for Acetonide Deprotection Step to give crude product which
was triturated with ether to afford 100 mg of
6-(thiomethyl)-5-nitro-4-(.beta.-D-ribofuranosylamino)pyrimidine as
a yellow solid.
5.1.11. Synthesis of
5-Nitro-4-Methyl-2-(.beta.-D-Ribofuranosylamino)-Pyrimidine
(11)
[0270] A solution of
2',3'-O-Isopropylidene-.beta.-D-ribofuranosylamine
p-toluenesulfonate salt (461 mg, 1.27 mmol) in 5 mL of
N,N-dimethylformamide at room temperature was treated first with
triethylamine (0.404 mL, 2.90 mmol) followed by the slow addition
of 2-chloro-4-methyl-5-nitropyridine (200 mg, 1.16 mmol). After
stirring at 50.degree. C. for 48 h, the solution was diluted with
ethyl acetate (15 mL) and washed with water (15 mL). The aqueous
phase was further extracted with 15 mL of ethyl acetate and the
combined organic extracts were dried over MgSO.sub.4, filtered and
concentrated in vacuo. The resulting crude product was purified by
silica gel chromatography (3:1 to 1:1 hexane/ethyl acetate) to give
the product which was deprotected following the procedure listed
above for Acetonide Deprotection Step to give the crude product
which was triturated with ether to give
5-nitro-4-methyl-2-(.beta.-D-ribofuranosylamino)pyridine, 26 mg as
a brown oil. MS: M+1 286.
5.1.12. Synthesis of
6-Amino-5-Nitro-4-(2',3'-O-Isopropylidene-(.beta.-D-Ribofuranosy-lamino)--
Pyrimidine (12)
[0271] A solution of
2',3'-O-Isopropylidene-.beta.-D-ribofuranosylamine
p-toluenesulfonate salt (1.03 g, 2.85 mmol) in 10 mL of
N,N-dimethylformamide at room temperature was treated first with
triethylamine (0.662 mL, 4.75 mmol) followed by the slow addition
of 6-chloro-5-nitro-pyrimidin-4-ylamine (331 mg, 1.90 mmol). After
stirring at 50.degree. C. for 48 h, the solution was diluted with
ethyl acetate (30 mL) and washed with water (30 mL). The aqueous
phase was further extracted with 20 mL of ethyl acetate and the
combined organic extracts were washed with water (30 mL), brine (20
mL), dried over MgSO.sub.4, filtered and concentrated in vacuo. The
resulting crude product was purified by HPLC to give
6-amino-5-nitro-4-(2',3'-O-isopropylidene-.beta.-D-ribofuranosylamino)-py-
rimidine.
5.1.13. Synthesis of
6-(2-Hydroxy-Ethy)-5-Nitro-4-(.beta.-D-Ribofuranosy-lamino)-Pyrimidine
(13)
[0272] To a solution of
6-chloro-5-nitro-4-(2',3'-O-isopropylidene-.beta.-D-ribofuranosylamino)py-
rimidine (200 mg, 0.577 mmol) in N,N-dimethylformamide (10 mL) at
room temperature was added ethanolamine (70 .mu.L, 1.15 mmol). The
solution was stirred for 17 h, diluted with 15 mL water and
extracted with ethyl acetate (2.times.15 mL) and the combined
organic layers were washed with brine (2.times.15 mL), dried over
MgSO.sub.4, filtered and concentrated in vacuo. The crude product
was purified by preparative thin layer chromatography (3:1 ethyl
acetate/hexanes) to give the product as a yellow oil. The product
was deprotected following the procedure listed above for Acetonide
Deprotection Step to give crude product which was triturated with
ether to afford
6-(2-hydroxy-ethylamino)-5-nitro-4-(.beta.-D-ribo-furanosylamino)pyrimidi-
ne.
5.1.14. Synthesis of
6-(Ethylamino)-5-Nitro-4-(.beta.-D-Ribofuranosyl-amino)-Pyrimidine
(14)
[0273] To a solution of
6-chloro-5-nitro-4-(2',3'-O-isopropylidene-.beta.-D-ribofuranosylamino)py-
rimidine (200 mg, 0.577 mmol) in N,N-dimethylformamide (5 mL) and
triethylamine (201 .mu.L, 1.44 mmol) at room temperature was added
diethylamine hydrochloride (52 mg, 0.634 mmol). The solution was
stirred for 2 h, diluted with 20 mL water and extracted with ethyl
acetate (2.times.15 mL) and the combined organic layers were washed
with 20 mL of brine, dried over MgSO4, filtered and concentrated in
vacuo. The resulting crude product was purified by silica gel
chromatography (3:1 to 1:1 hexane/ethyl acetate) to afford 160 mg
of product. The product was deprotected following the procedure
listed above for Acetonide Deprotection Step to give 76 mg of
6-(ethylamino)-5-nitro-4-(.beta.-D-ribo-furanosylamino)pyrimidine
as a yellow solid.
5.1.15. Synthesis of
6-(4-Methoxy-Bemzy-lamino)-5-Cyano-4-(.beta.-D-Ribofuranosylamino)-Pyrimi-
dine (15)
[0274] A solution of
2',3'-O-Isopropylidene-.beta.-D-ribofuranosylamine
p-toluenesulfonate salt (158 mg, 0.437 mmol) in 5 mL of
N,N-dimethylformamide at room temperature was treated first with
triethylamine (0.127 mL, 0.910 mmol) followed by slow addition of
(6-Chloro-5-nitro-pyrimidin-4-yl)-(4-methoxy-benzyl)-amine (100 mg,
0.364 mmol). After stirring at 70.degree. C. for 48 h, the solution
was diluted with ethyl acetate (15 mL) and washed with water (15
mL). The aqueous phase was further extracted with ethyl acetate (10
mL) and the combined organic extracts were washed with water (15
mL), brine (10 mL), dried over MgSO.sub.4, filtered and
concentrated in vacuo. The resulting crude product was purified by
preparative thin layer chromatography to give the product. The
product was deprotected following the procedure listed above for
Acetonide Deprotection Step to give
6-(4-methoxy-benzylamino)-5-cyano-4-(.beta.-D-ribo-furanosylamino)pyrimid-
ine 8 mg.
5.1.16. Synthesis of
3-Cyano-2-(.beta.-D-Ribofuranosy-lamino)-Pyrimidine (16)
[0275] A solution of
2',3'-O-Isopropylidene-.beta.-D-ribofuranosylamine
p-toluenesulfonate salt (1.435 g, 3.97 mmol) in 20 mL of t-butyl
alcohol at room temperature was treated first with potassium
t-butoxide (810 mg, 7.22 mmol) followed by the slow addition of
2-chloro-3-cyano-pyridine (500 mg, 3.61 mmol). After stirring at
50.degree. C. for 17 h, the reaction mixture was concentrated in
vacuo and diluted with ethyl acetate (20 mL) and washed with water
(20 mL). The aqueous phase was further extracted with ethyl acetate
(20 mL) and the combined organic extracts were washed with brine
(20 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo.
The resulting crude product was purified by silica gel
chromatography (3:1 to 1:1 hexanes/ethyl acetate) to give 420 mg of
product. The product was deprotected following the procedure listed
above for Acetonide Deprotection Step to give
3-cyano-2-(.beta.-D-ribofuranosyl-amino)pyridine.
5.1.17. Synthesis of
6-Hydroxy-5-Nitro-4-(.beta.-D-Ribofuranosylamino-2,3,5-Tribenzoyl)
Pyrimidine (17)
[0276] To a suspension of 6-amino-4-hydroxy-5-nitropyrimidine (1.25
g, 8.01 mmol) in anhydrous hexamethyldisilazane (37.5 mL) was added
pyridine (6 mL), H.sub.2SO.sub.4 (0.25 mL), and
(NH.sub.4).sub.2SO.sub.4 (0.040 g). The reaction mixture was heated
at reflux for 17 h at which time the suspension gave way to a
homogeneous solution. The reaction mixture was cooled and
concentrated in vacuo to give a solid which was dissolved in
anhydrous acetonitrile (63 mL) and stirred at room temperature
while .beta.-D-ribofuranose 1-acetate 2,3,5-tribenzoate (4.85 g,
9.61 mmol) was added followed by trimethylsilyl
trifluoromethanesulfonate (2.50 mL, 12.5 mmol). The reaction
mixture was stirred at room temperature for 4.5 h, then
concentrated to give a viscous oil which was dissolved in methylene
chloride (40 mL), washed with saturated aqueous NaHCO.sub.3, dried
over MgSO.sub.4, filtered and concentrated. The crude product was
purified by silica gel chromatography (1-5% acetone/methylene
chloride) to give 4.03 g of
6-hydroxy-5-nitro-4-(.beta.-D-ribofuranosyl-amino-2,3,5-tribenzoyl)p-
yrimidine as a glassy solid.
[0277] To a solution of
6-hydroxy-5-nitro-4-(.beta.-D-ribofuranosyl-amino-2,3,5-tribenzoyl)pyrimi-
dine (2.10 g, 3.50 mmol) in absolute methanol (83 mL) and anhydrous
dioxane (16.6 mL) cooled in an ice bath was added 0.5 N sodium
methoxide in methanol (1.4 mL, 0.7 mmol). The reaction was stirred
in the ice bath for 1 hour, then sufficient Dowex 50WX4-50 resin
was added to give a pH of 6. The resin was filtered, washed with
methanol and discarded. The filtrate was concentrated and the crude
product triturated with ethyl ether (5.times.4 mL) to give 1.00 g
of 6-hydroxy-5-nitro-4-(.beta.-D-ribofuranosylamino)pyrimidine.
5.1.18. Synthesis of
6-Amino-5-Nitro-4-(.beta.-D-Xylofuranosyl-Amino-2,3,5-Tribenzoyl)
Pyrimidine (18)
[0278] To a suspension of 4,6-diamino-5-nitropyrimidine (203 mg,
1.31 mmol) in anhydrous hexamethyldisilazane (5.9 mL) was added
pyridine (0.95 mL), H.sub.2SO.sub.4 (40 .mu.L), and
(NH.sub.4).sub.2SO.sub.4 (6.7 mg). The reaction mixture was heated
at reflux for 5 h at which time the suspension gave way to a
homogeneous solution. The reaction mixture was cooled and
concentrated in vacuo to give a solid which was dissolved in
anhydrous acetonitrile (10 mL) and stirred at room temperature
while .beta.-D-xylofuranose 1,2,3,5-tetrabenzoate (0.60 g, 1.06
mmol) was added followed by trimethylsilyl
trifluoromethanesulfonate (0.40 mL, 2.2 mmol). The reaction mixture
was stirred at room temperature for 17 h and concentrated to give a
viscous oil which was dissolved in methylene chloride (30 mL),
washed with saturated aqueous NaHCO.sub.3, dried over MgSO.sub.4,
filtered and concentrated. The crude product was purified by silica
gel chromatography (1-10% acetonitrile/methylene chloride) to give
111 mg of
6-amino-5-nitro-4-(.beta.-D-xylofuranosylamino-2,3,5-tribenzoyl-
)pyrimidine as a light yellow glassy solid.
[0279] To a solution of
6-amino-5-nitro-4-(.beta.-D-xylofuranosylamino-2,3,5-tribenzoyl)pyrimidin-
e (111 mg, 0.185 mmol) in absolute methanol (2.36 mL) and anhydrous
dioxane (0.92 mL) cooled in an ice bath was added 0.5 N sodium
methoxide in methanol (76 .mu.L, 0.152 mmol). The reaction was
stirred in the ice bath for 17 hours, then sufficient Dowex
50WX4-50 resin was added to give a pH of 6.5. The resin was
filtered, washed with methanol and discarded. The filtrate was
concentrated and the crude product triturated with ethyl ether
(6.times.1.5 mL) to give 29 mg of crude product which was purified
by silica gel chromatography (35% tetrahydrofuran/hexanes to 100%
tetrahydrofuran) to give 15 mg of
6-amino-5-nitro-4-(.beta.-D-xylofuranosylamino)pyrimidine.
5.1.19. Synthesis of
6-Amino-5-Nitro-4-(.beta.-L-Ribofuranosyl-Amino)Pyrimidine (19)
[0280] To a suspension of 4,6-diamino-5-nitropyrimidine (260 mg,
1.68 mmol) in anhydrous hexamethyldisilazane (7.8 mL) was added
pyridine (1.3 mL), H.sub.2SO.sub.4 (0.52 .mu.L), and
(NH.sub.4).sub.2SO.sub.4 (12.3 mg). The reaction mixture was heated
at reflux for 15 h at which time the suspension gave way to a
homogeneous solution. The reaction mixture was cooled and
concentrated in vacuo to give a solid which was dissolved in
anhydrous acetonitrile (13 mL) and stirred at room temperature
while .beta.-L-ribofuranose 1-acetate 2,3,5-tribenzoate (1.00 g,
1.98 mmol) was added followed by trimethylsilyl
trifluoromethanesulfonate (0.6 mL, 3 mmol). The reaction mixture
was stirred at room temperature for 17 h, then concentrated to give
a crude product which was dissolved in methylene chloride (20 mL),
washed with saturated aqueous NaHCO.sub.3, dried over MgSO.sub.4,
filtered and concentrated. The crude product was purified by silica
gel chromatography (1-15% acetonitrile/methylene chloride) to give
0.77 g of
6-amino-5-nitro-4-(.beta.-L-ribofuranosyl-amino-2,3,5-tribenzoyl)pyrimidi-
ne as a light yellow glassy solid.
[0281] To a solution of
6-amino-5-nitro-4-(.beta.-L-ribofuranosyl-amino-2,3,5-tribenzoyl)pyrimidi-
ne (736 mg, 1.23 mmol) in absolute methanol (29 mL) and anhydrous
dioxane (5.8 mL) cooled in an ice bath was added 0.5 N sodium
methoxide in methanol (0.49 mL, 0.245 mmol). The reaction was
stirred at 4.degree. C. for 48 hours, then sufficient Dowex
50WX4-50 resin was added to give a pH of 6.5. The resin was
filtered, washed with methanol and discarded. The filtrate was
concentrated and the crude product triturated with ethyl ether
(6.times.1.5 mL) to give 0.39 g of crude product which was purified
by silica gel chromatography (35% tetrahydrofuran/hexanes to 100%
tetrahydrofuran) to give 237 mg of
6-amino-5-nitro-4-(.beta.-L-ribofuranosyl-amino)pyrimidine.
5.1.20. Synthesis of
6-Amino-5-Nitro-4-(5-Deoxy-5-Fluoro-.beta.-D-Ribofuranosylamino)-Pyrimidi-
ne (20)
[0282] To a suspension of 4,6-diamino-5-nitropyrimidine (1.32 g,
8.51 mmol) in anhydrous hexamethyldisilazane (40 mL) was added
pyridine (6.3 mL), H.sub.2SO.sub.4 (260 .mu.L), and
(NH.sub.4).sub.2SO.sub.4 (40 mg). The reaction mixture was heated
at reflux for 15 h at which time the suspension gave way to a
homogeneous solution. The reaction mixture was cooled and
concentrated in vacuo to give a solid which was dissolved in
anhydrous acetonitrile (63 mL) and stirred at room temperature
while .beta.-D-ribofuranose
5-t-butyldiphenylsilyloxy-1,2,3-tribenzoate (5.92 g, 8.45 mmol) was
added followed by trimethylsilyl trifluoromethanesulfonate (2.2 mL,
12.2 mmol). The reaction mixture was stirred at room temperature
for 30 min., then quenched by addition of saturated aqueous
NaHCO.sub.3 (15 mL) and stirred overnight. The mixture was filtered
through celite to remove suspended solids and the filtrate was
extracted with methylene chloride (3.times.15 mL). The extract was
washed with water, dried over MgSO.sub.4, filtered and
concentrated. The crude product was purified by silica gel
chromatography (1-6% acetonitrile/methylene chloride) to give 3.85
g of 6-amino-5-nitro-4-(.beta.-D-ribofuranose
5-t-butyldiphenylsilyloxy-1,2,-dibenzoyl)pyrimidine.
[0283] To a solution of
6-amino-5-nitro-4-(.beta.-D-ribofuranosyl-5-t-butyldiphenylsilyloxy-1,2-d-
ibenzoyl)pyrimidine (2.67 g, 3.64 mmol) in anhydrous
tetrahydrofuran (18 mL) was added a 1 M solution of
tetra-n-butylammonium fluoride in tetrahydrofuran (4.0 ml, 4.0
mmol). After 30 minutes water (10 mL) was added and the mixture was
concentrated to remove tetrahydrofuran. The aqueous mixture was
extracted with ethyl acetate (5.times.4 mL). The combined extract
was concentrated and purified by silica gel chromatography (5% to
40% acetonitrile/methylene chloride) to give 1.67 g of
6-amino-5-nitro-4-(.beta.-D-ribofuranose-1,2-dibenzoate)pyrimidine
as a glassy solid.
[0284] To a solution of
6-amino-5-nitro-4-(.beta.-D-ribofuranosylamino-2,3-dibenzoyl)pyrimidine
(205 mg, 0.414 mmol) in anhydrous methylene chloride (4 mL) was
added diethylaminosulfurtrifluoride (60 .mu.L, 0.46 mmol). The
mixture was stirred at room temperature for 48 hours then washed
with saturated aqueous sodium bicarbonate (10 mL), dried over
MgSO.sub.4 and concentrated to give 192 mg of crude product which
was purified by silica gel chromatography to give 85 mg of
6-amino-5-nitro-4-(.beta.-D-5-deoxy-5-fluoro-2,3-dibenzoylribofuranosylam-
ino)pyrimidine.
[0285] To a solution of
6-amino-5-nitro-4-(.beta.-D-5-deoxy-5-fluoro-2,3-dibenzoyl
ribofuranosylamino)pyrimidine (77 mg, 0.155 mmol) in absolute
methanol (3.7 mL) and anhydrous dioxane (0.73 mL) cooled in an ice
bath was added 0.5 N sodium methoxide in methanol (75 .mu.L, 0.0375
mmol). The reaction was stirred at 0.degree. C. for 6 hours, then
sufficient Dowex 50WX4-50 resin was added to give a pH of 6. The
resin was filtered, washed with methanol and discarded. The
filtrate was concentrated and the crude product was purified by
silica gel chromatography (1-10% methanol/methylene chloride) to
give 17 mg of
6-amino-5-nitro-4-(5-deoxy-5-fluoro-.beta.-D-ribofuranosylamino)-pyrimidi-
ne.
5.1.21. Synthesis of
6-Amino-5-Nitro-4-(5-Deoxy-5-Azido-.beta.-D-Ribofuranosylamino)-Pyrimidin-
e (21)
[0286] To a solution of
6-amino-5-nitro-4-(.beta.-D-ribofuranosylamino-2,3-dibenzoyl)pyrimidine
(248 mg) in anhydrous methylene chloride (2.50 mL) was added
triethylamine (140 .mu.L, 1.00 mmol) at 0.degree. C.
Methanesulfonyl chloride (43 .mu.L, 0.56 mmol) was added and the
mixture was stirred overnight at 0.degree. C. The reaction mixture
was washed with water (10 mL) dried over Na.sub.2SO.sub.4, filtered
and concentrated to give 289 mg of
6-amino-5-nitro-4-(.beta.-D-ribofuranosylamino-5-methanesulfonyloxy-2,-
3-dibenzoyl)pyrimidine.
[0287] To a solution of
6-amino-5-nitro-4-(.beta.-D-ribofuranosylamino-5-methanesulfonyloxy-2,3-d-
ibenzoyl)pyrimidine (271 mg, 0.473 mmol) in N,N-dimethylformamide
(1 mL) was added sodium azide (46.2 mg, 0.71 mmol). The mixture was
heated to 100.degree. C. for 7 hours, then diluted with water to
give a precipitate. The precipitate was filtered, washed with water
and dried to give 230 mg of
6-amino-5-nitro-4-(.beta.-D-ribofuranosylamino-5-deoxy-5-azido-2,3-dibenz-
oyl)pyrimidine.
[0288] To a solution of
6-amino-5-nitro-4-(.beta.-D-ribofuranosylamino-5-deoxy-5-azido-2,3-dibenz-
oyl)pyrimidine (199 mg, 0.382 mmol) in absolute methanol (9 mL) and
anhydrous dioxane (1.8 mL) cooled in an ice bath was added 0.5 N
sodium methoxide in methanol (152 .mu.L, 0.076 mmol). The reaction
was stirred at 0.degree. C. for 17 hours, then sufficient Dowex
50WX4-50 resin was added to give a pH of 6. The resin was filtered,
washed with methanol and discarded. The filtrate was concentrated
and the crude product was purified by silica gel chromatography
(5-25% dioxane/methylene chloride) to give 70 mg of
6-amino-5-nitro-4-(.beta.-D-5-deoxy-5-azidoribofuranosylamino)pyrimidine.
5.1.22. Synthesis of
6-Amino-5-Nitro-4-(.alpha.-D-Ribofuranosylamino)-Pyrimidine
(22)
[0289] A solution of
2',3'-O-Isopropylidene-.beta.-D-ribofuranosylamine
p-toluenesulfonate salt (10.83 g, 30 mmol) in 60 mL of acetonitrile
was stirred at room temperature with sodium sulfate (15 g) for 1
hour. 4,6-dichloro-5-nitropyrimidine (11.64 g, 60 mmol) was added
followed by diisopropylethylamine (5.97 g, 45 mmol). The reaction
mixture was stirred at room temperature for 30 h. The sodium
sulfate was removed by filtration and the filtrate was concentrated
to give an oil. The crude product was purified using silica gel
chromatography (1:3 to 1:1 ethyl acetate/hexanes) to give
6-chloro-5-nitro-4-(2',3'-O-isopropylidene-.alpha.-D-ribofuranosylamino)p-
yrimidine, 1.41 g.
[0290] A solution
6-chloro-5-nitro-4-(2',3'-O-isopropylidene-.alpha.-D-ribofuranosylamino)p-
yrimidine (1.41 g, 4 mmol) in anhydrous dioxane (25 mL) was stirred
at 0.degree. C. as a solution of 7 M ammonia in methanol (5 mL, 35
mmol) was added. The mixture was stirred overnight at 0.degree. C.
then filtered to remove a solid. The filtrate was concentrated and
purified by silica gel chromatography (5% methanol in methylene
chloride) to give 1.32 g of
6-amino-5-nitro-4-(2',3'-O-isopropylidene-.alpha.-D-ribofuranosylamino)py-
rimidine.
[0291] A solution of 98% aqueous trifluoroacetic acid was added to
6-amino-5-nitro-4-(2',3'-O-isopropylidene-.alpha.-D-ribofuranosylamino)py-
rimidine (200 mg, 0.611 mmol) at room temperature. The reaction
mixture was stirred for 1 hour at room temperature then
concentrated. The product was purified by HPLC to give
6-Amino-5-nitro-4-(.alpha.-D-ribofuranosylamino)pyrimidine 64
mg.
5.1.23. Synthesis of
6-Amino-5-Nitro-4-[(5-O-Acetyl-.beta.-D-Ribofuranosyl)Amino]Pyrimidine
(23)
[0292] To a solution of
6-amino-5-nitro-4-(2',3'-O-isopropylidene-.beta.-D-ribofuranosylamino)pyr-
imidine (200 mg, 0.611 mmol) in anhydrous tetrahydrofuran (3 mL)
stirring at room temperature was added triphenylphosphine (240 mg,
0.917 mmol) and acetic acid (0.05 mL, 0.92 mmol). The reaction
mixture was cooled in a -25.degree. C. bath and a solution of
diethyl azodicarboxylate (160 mg, 0.917 mmol) in anhydrous
tetrahydrofuran (1 mL) was added over 10 minutes. The mixture was
stirred at -25.degree. C. for 20 minutes after addition, then
allowed to warm to room temperature. The reaction mixture was
diluted with saturated aqueous sodium bicarbonate and extracted
with methylene chloride. The combined organic extracts were
concentrated and purified by silica gel chromatography (methylene
chloride then 1% methanol/methylene chloride) to give 103.5 mg of
6-amino-5-nitro-4-[(2,3-O-isopropylidene-5-O-acetyl-.beta.-D-ribofuranosy-
l)amino]pyrimidine.
[0293] To acetic acid
6-Amino-5-nitro-4-[(2,3-O-isopropylidene-5-O-acetyl-.beta.-D-ribofuranosy-
l)amino]pyrimidine (103.5 mg, 0.280 mmol) was added 98% aqueous
trifluoroacetic acid at -25.degree. C. The mixture was stirred for
20 minutes, then concentrated to give acetic acid
6-Amino-5-nitro-4-[(5-O-acetyl-.beta.-D-ribofuranosyl)amino]pyrimidine.
5.1.24. Synthesis of
6-Amino-5-Nitro-4-[(2,3,5-Tri-O-Benzoyl-.beta.-D-Ribofuranosyl)
Amino]Pyrimidine (24)
[0294] To a suspension of 4,6-diamino-5-nitropyrimidine (1.25 g,
8.05 mmol) in anhydrous hexamethyldisilazane (37.5 mL) was added
pyridine (6 mL), H.sub.2SO.sub.4 (0.25 mL) and
(NH.sub.4).sub.2SO.sub.4 (0.040 g). The reaction mixture was heated
at reflux for 1.5 h at which time the suspension gave way to a
homogeneous solution. The reaction mixture was cooled and
concentrated in vacuo to give a solid which was dissolved in
anhydrous acetonitrile (63 mL) and stirred at room temperature as
.beta.-D-ribofuranose 1-acetate 2,3,5-tribenzoate (4.85 g, 9.61
mmol) was added followed by trimethylsilyl
trifluoromethanesulfonate (2.50 mL, 12.5 mmol). The reaction
mixture was stirred at room temperature for 4.5 h, then
concentrated to give a viscous oil which was dissolved in methylene
chloride (40 mL), washed with saturated aqueous NaHCO.sub.3, dried
over MgSO.sub.4, filtered and concentrated. The crude product was
purified by silica gel chromatography (1-10% acetonitrile/methylene
chloride) to give 1.84 g of
6-amino-5-nitro-4-(.beta.-D-ribofuranosyl-amino-2,3,5-tribenzoy-
l)pyrimidine as a light yellow glassy solid.
5.1.25. Synthesis of Methyl
6-Chloro-4-(.beta.-D-Ribofuranosylamino)Pyrimidine-5-Carboxylate
(25) and Methyl
6-Chloro-4-(.alpha.-D-Ribofuranosylamino)Pyrimidine-5-Carboxylate
(26)
[0295] A solution of
2',3'-O-Isopropylidene-.beta.-D-ribofuranosylamine
p-toluenesulfonate salt (3.80 g, 11 mmol) in 50 mL of
N,N-dimethylformamide at room temperature was treated first with
methyl 4,6-dichloropyrimidyl-5-carboxylate (1.9 g, 9.2 mmol)
followed by the slow addition of diisopropylethylamine (5 mL, 27.5
mmol). The mixture was stirred for 18 hours at room temperature and
then concentrated. The crude product was purified by silica gel
chromatography (50% ethyl acetate/hexanes to 100% ethyl acetate) to
give 1.05 g of
6-chloro-4-[2',3'-O-isopropylidene-.beta.-D-ribofuranosyl)amino]pyrimidin-
e-5-carboxylate and 0.32 g of methyl
6-chloro-4-[2',3'-O-isopropylidene-.alpha.-D-ribofuranosyl)amino]pyrimidi-
ne-5-carboxylate.
[0296] Methyl
6-chloro-4-[2',3'-O-isopropylidene-.beta.-D-ribofuranosyl)amino]pyrimidin-
e-5-carboxylate was treated with 90% aqueous trifluoroacetic acid
(8 mL) at room temperature for 6 minutes then concentrated to give
the crude product which was co-evaporated with water (3.times.3 mL)
and then treated with dichloromethane (5 mL) and acetone (5 mL).
The resulting precipitate was filtered and dried under high vacuum
to give methyl
6-chloro-4-(.beta.-D-ribofuranosylamino)pyrimidine-5-carboxylate,
66 mg as a white solid.
[0297] Treatment of methyl
6-chloro-4-[2',3'-O-isopropylidene-.alpha.-D-ribofuranosyl)amino]pyrimidi-
ne-5-carboxylate (80 mg, 0.22 mmol) in the same manner gave methyl
6-chloro-4-(.alpha.-D-ribofuranosylamino)pyrimidine-5-carboxylate
30 mg as a solid.
5.1.26. Synthesis of Methyl
6-Amino-4-(.beta.-D-Ribofuranosylamino)Pyrimidine-5-Carboxylate
(27) and Methyl 6-Amino-4-(.alpha.-D-Ribofuranosylamino)
-Carboxylate)
[0298] A solution of methyl
6-chloro-4-[2',3'-O-isopropylidene-.beta.-D-ribofuranosyl)amino]pyrimidin-
e-5-carboxylate (0.4 g, 1.11 mmol) in 7 N ammonia in methanol (10
mL, 70 mmol) was stirred at room temperature for 18 hours and then
concentrated. The crude product was purified by silica gel
chromatography (90% acetonitrile/water to 100% acetonitrile) to
give methyl
6-amino-4-[2',3'-O-isopropylidene-.beta.-D-ribofuranosyl)amino]pyrimidine-
-5-carboxylate, 0.291 g.
[0299] A solution of methyl
6-amino-4-[2',3'-O-isopropylidene-.beta.-D-ribofuranosyl)amino]pyrimidine-
-5-carboxylate (60 mg, 0.176 mmol) in 90% aqueous trifloroacetic
acid (4 mL) was stirred for 6 minutes at room temperature and then
concentrated. The resulting gum was co-evaporated with water
(3.times.3 mL) and then treated with dichloromethane (5 mL),
acetone (5 mL) and hexanes (2 mL). The precipitate was filtered and
dried to give methyl
6-amino-4-(.beta.-D-ribofuranosylamino)pyrimidine-5-carboxylate 32
mg as an off-white solid.
[0300] The two step procedure used above was used to convert 168 mg
of methyl
6-chloro-4-(2',3'-O-isopropylidene-.alpha.-D-ribofuranosyl)amino)p-
yrimidine-5-carboxylate into methyl
6-amino-4-(.alpha.-D-ribofuranosylamino)pyrimidine-5-carboxylate,
50 mg.
5.2 EXAMPLE 2
Identification and Characterization of Compounds that Promote
Nonsense Suppression and/or Modulate Translation Termination
5.2.1. Increase in In Vitro Nonsense Suppression at UGA Codons
[0301] Compounds of the invention can be characterized further with
the in vitro luciferase nonsense suppression assay. To ensure that
the observed nonsense suppression activity of the selected
compounds is not limited to the rabbit reticulocyte assay system,
HeLa cell extract is prepared and optimized (Lie & Macdonald,
1999, Development 126(22):4989-4996 and Lie & Macdonald, 2000,
Biochem. Biophys. Res. Commun. 270(2):473-481). The nonsense
suppression activity of compounds of the invention, with respect to
the UGA codon, are compared to gentamicin in the HeLa cell
translation extracts.
5.2.2. Characterization of Compounds that Increase Nonsense
Suppression and Produce Functional Protein
[0302] A stable cell line harboring the UGA nonsense-containing
luciferase gene is treated with a test compound. Cells are grown in
standard medium supplemented with 1% penicillin-streptomycin (P/S)
and 10% fetal bovine serum (FBS) to 70% confluency and split 1:1
the day before treatment. The next day, cells are trypsinized and
40,000 cells are added to each well of a 96-well tissue culture
dish. Serial dilutions of each compound are prepared to generate a
six-point dose response curve spanning 2 logs (30 .mu.M to 0.3
.mu.M). The final concentration of the DMSO solvent remains
constant at 1% in each well. Cells treated with 1% DMSO serve as
the background standard, and cells treated with gentamicin serve as
a positive control.
5.2.3. Alteration of the Accessibility of Chemical Modifying Agents
to Specific Nucleotides in the 28S rRNA
[0303] Previous studies have demonstrated that gentamicin and other
members of the aminoglycoside family that decrease the fidelity of
translation bind to the A site of the 16S rRNA. By chemical
footprinting, UV cross-linking and NMR, gentamicin has been shown
to bind at the A site (comprised of nucleotides 1400-1410 and
1490-1500, E. coli numbering) of the rRNA at nucleotides 1406,
1407, 1494, and 1496 (Moazed & Noller, Nature 327(6121):389-394
(1978); Woodcock et al., EMBO J. 10(10):3099-3103 (1991); and
Schroeder et al., EMBO J. 19:1-9 (2000).
[0304] Ribosomes prepared from HeLa cells are incubated with the
small molecules (at a concentration of 100 mM), followed by
treatment with chemical modifying agents (dimethyl sulfate [DMS]
and kethoxal [KE]). Following chemical modification, rRNA is
phenol-chloroform extracted, ethanol precipitated, analyzed in
primer extension reactions using end-labeled oligonucleotides
hybridizing to different regions of the three rRNAs and resolved on
6% polyacrylamide gels. The probes used for primer extension cover
the entire 18S (7 oligonucleotide primers), 28S (24 oligonucleotide
primers), and 5S (one primer) rRNAs. Controls in these experiments
include DMSO (a control for changes in rRNA accessibility induced
by DMSO), paromomycin (a marker for 18S rRNA binding), and
anisomycin (a marker for 28S rRNA binding).
5.2.4. Readthrough of Premature Termination Codons in Cell-Based
Disease Models
[0305] To address the effects of the nonsense-suppressing compounds
on mRNAs altered in specific inherited diseases, a bronchial
epithelial cell line harboring a nonsense codon at amino acid 1282
(W1282X) is treated with a compound of the invention and CFTR
function is monitored as a cAMP-activated chloride channel using
the SPQ assay (Yang et al., Hum. Mol. Genet. 2(8):1253-1261 (1993)
and Howard et al., Nat. Med. 2(4):467-469 (1996)). The increase in
SPQ fluorescence in cells treated with a compound of the invention
is compared to those treated with cAMP and untreated cells. An
increase in SPQ fluorescence in cells is consistent with
stimulation of CFTR-mediated halide efflux and an increase in
readthrough of the nonsense codon. Full-length CFTR expression from
this nonsense-containing allele following treatment with a compound
of the invention demonstrates that cystic fibrosis cell lines
increase chloride channel activity when treated with a compound of
the invention.
5.2.5. Expression of Full Length Dystrophin Protein in the Nonsense
Mutation-Containing MDX Mouse Cell by Treatment
[0306] The mutation in the mdx mouse that premature termination of
the 427 kDa dystrophin polypeptide has been shown to be a C to T
transition at position 3185 in exon 23 (Sicinski et al., Science
244(4912):1578-1580 (1989)). Mouse primary skeletal muscle cultures
derived from 1-day old mdx mice are prepared as described
previously (Barton-Davis et al., J. Clin. Invest. 104(4):375-381
(1999)). Cells are cultured for 10 days in the presence of a
compound of the invention. Culture medium is replaced every four
days and the presence of dystrophin in myoblast cultures is
detected by immunostaining as described previously (Barton-Davis et
al., J. Clin. Invest. 104(4):375-381 (1999)). A primary monoclonal
antibody to the C-terminus of the dystrophin protein (F 19A 12) is
used undiluted and rhodamine conjugated anti-mouse IgG was used as
the secondary antibody. The F19A 12 antibody detects the
full-length protein produced by suppression of the nonsense codon.
Staining is viewed using a Leica DMR microscope, digital camera,
and associated imaging software at the University of
Pennsylvania.
5.2.6. Readthrough of Premature Termination Codons in the MDX
[0307] As previously described (Barton-Davis et al., J. Clin.
Invest. 104(4):375-381 (1999), compound is delivered by Alzet
osmotic pumps implanted under the skin of anesthetized mice. Two
doses of a compound of the invention are administered. Gentamicin
serves as a positive control and pumps filled with solvent only
serve as the negative control. Pumps are loaded with appropriate
compound such that the calculated doses to which tissue is exposed
are 10 mM and 20 mM. The gentamicin concentration is calculated to
achieve tissue exposure of approximately 200 mM. In the initial
experiment, mice are treated for 14 days, after which animals are
anesthetized with ketamine and exsanguinated. The tibialis anterior
(TA) muscle of the experimental animals is then excised, frozen,
and used for immunofluorescence analysis of dystrophin
incorporation into striated muscle. The presence of dystrophin in
TA muscles is detected by immunostaining, as described previously
(Barton-Davis et al., J. Clin. Invest. 104(4):375-381 (1999).
5.2.7. Suppression of nonsense mutations in p53 Tumor Suppressor
Genes
[0308] CAOV-3 have a nonsense mutation in the p53 gene at codon
136. CAOV-3 cells were plated in 3.5 mm wells (20,000 cells per
well). After 18 hours the media was changed and clitocine, a
representative compound of the invention, was added to each well
resulting in the following final concentrations: 0 nM, 32 nM, 80
nM, 200 nM and 500 nM. After incubating for 48 hours, cells were
scraped into media, centrifuged and resuspended in 200 .mu.L of
sodium dodecyl sulfate (SDS) sample buffer. 15 .mu.L of each sample
was separated by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE), transferred to nitrocellulose and a
western blot analysis was performed using an antibody specific for
p53 (DO-1). The membrane was stripped and re-probed with anti-actin
as a loading control. The results (FIG. 1A) demonstrate the ability
of clitocine to induce nonsense suppression in the p53 tumor
suppressor gene of CAOV-3 cells.
[0309] The above experiment was repeated using both CALU-6 and
CAOV-3 cells incubated with either clitocine (500 nM), G418 (330
.mu.M) or no treatment. The results (FIG. 1B) demonstrate the
ability of clitocine to induce nonsense suppression in the p53
tumor suppressor genes of both CALU-6 and CAOV-3 cells more
effectively than G418.
5.2.8. Suppression of Nonsense Mutation in Cancer Cell Lines with a
Known Nonsense Mutation
[0310] The procedure set forth above in Example 5.2.7 was carried
out with the following cell lines obtained from the American Type
Culture Collection (ATCC), which have a known nonsense mutation in
the p53 tumor suppressor gene: CAOV-3, NCI-H520, CALU-6, HCC1569,
NCI-H774 and HCI-H1926. Table 2, below, demonstrates the ability of
clitocine to suppress nonsense mutations in the p53 tumor
suppressor gene of these cells and, accordingly, its utility as a
nonsense suppressor. TABLE-US-00002 TABLE 2 Summary of anti-p53
antibody western blot results. Cell Line Mutation.sup.a clitocine
G418 Gentamicin CAOV-3 TAA-136 + + - NCI-H520 TGA-146 + + - CALU-6
TGA-196 + + - HCC1569 TAG-294 + nd nd NCI-H774 TGA-342 + + -
HCI-H1926 TGA-213 + + + .sup.a= nonsense codon-codon number; + =
full length p53 detected by western; - = full length p53 not
detected by western blot; nd = not determined.
5.2.9. Demonstration that p53 resulting from Nonsense Suppression
is Functionally Active
[0311] CALU-6 cells were incubated with clitocine (0 .mu.M, 0.25
.mu.M or 1 .mu.M) for 72 hours. Cells were harvested and protein
and RNA were extracted. Proteins were separated by SDS-PAGE and
western blotted with anti-p53 and anti-actin (loading control)
specific antibodies (FIG. 2A).
[0312] RNA was reverse transcribed and analyzed by real time
polymerase chain reaction (PCR) with probes specific for Bax mRNA
and p21.sup.WAFI mRNA (FIG. 2B). Bax and p21 genes are known to be
transcriptionally activated by p53. Accordingly, these results
demonstrate that the p53 produced by the suppression of the
nonsense mutation of the p53 gene by clitocine is functionally
active.
5.2.10. Demonstration that p53 resulting from Nonsense Suppression
Induces Apoptosis
[0313] According to the procedure set forth in Example 5.2.7,
CAOV-3 cells were treated with clitocine, a representative compound
of the invention, for 72 hours. The relative level of caspase 3/7
activity, a measure of relative apoptotic level, was measured.
Similarly, SKOV-3 cells which lack p53 (without a nonsense
mutation) were also treated with clitocine and relative caspase 3/7
activity level was measured. FIG. 3 shows that clitocine induced
apoptosis in CAOV-3 cells more potently an efficaciously than in
SKOV-3 cells. These data also show that p53 produced by the
suppression of the nonsense mutation of the p53 gene by clitocine
drives cells into apoptosis.
5.2.11. In Vivo Anti-Cancer Activity of Clitocine
[0314] CAOV-3 cells (1.times.10.sup.7) were injected into the
flanks of nude/nude mice. After 12 days, mice were randomized (10
mice per group) and treated subcutaneously (5 days per week) with 3
mg/kg of clitocine or intraperitonealy (1 day per week) with 30
mg/kg of clitocine. Tumor volumes were measured weekly (FIG. 4).
Error bars represent the standard error of the mean. These results
demonstrate that suppression of nonsense mutations in the p53 gene
by clitocine can inhibit cancer growth in vivo.
5.3 EXAMPLE 3
100 Mg Oral Dosage Form
[0315] Table 3 illustrates a batch formulation and a single dose
unit formulation containing 100 mg of clitocine. TABLE-US-00003
TABLE 3 Formulation for 100 mg tablet Percent Quantity Quantity
Material by Weight (mg/tablet) (kg/batch)
6-Amino-5-nitro-4-(.beta.-D-ribo- 40% 100.00 20.00
furanosylamino)pyrimidine (Clitocine) Microcrystalline Cellulose,
NF 53.5% 133.75 26.75 Pluronic F-68 Surfactant 4.0% 10.00 2.00
Croscarmellose Sodium Type A, 2.0% 5.00 1.00 NF Magnesium Stearate,
NF 0.5% 1.25 0.25 Total 100.0% 250.00 mg 50.00 kg
[0316] The microcrystalline cellulose, croscarmellose sodium, and
clitocine are passed through a #30 mesh screen (about 430.mu. to
about 655.mu.). The Pluronic F-68.RTM. (manufactured by JRH
Biosciences, Inc. of Lenexa, Kans.) surfactant is passed through a
#20 mesh screen (about 457.mu. to about 1041.mu.). The Pluronic
F-68.RTM. surfactant and 0.5 kgs of croscarmellose sodium are
loaded into a 16 qt. twin shell tumble blender and are mixed for
about 5 minutes. The mix is then transferred to a 3 cubic foot twin
shell tumble blender where the microcrystalline cellulose is added
and blended for about 5 minutes. The thalidomide is added and
blended for an additional 25 minutes. This pre-blend is passed
through a roller compactor with a hammer mill attached at the
discharge of the roller compactor and moved back to the tumble
blender. The remaining croscarmellose sodium and magnesium stearate
is added to the tumble blender and blended for about 3 minutes. The
final mixture is compressed on a rotary tablet press with 250 mg
per tablet (200,000 tablet batch size).
5.4 EXAMPLE 4
Aerosol Dosage Form
[0317] A concentrate is prepared by combining clitocine and a 12.6
kg portion of the trichloromonofluoromethane in a sealed stainless
steel vessel equipped with a high shear mixer. Mixing is carried
out for about 20 minutes. The bulk suspension is then prepared in
the sealed vessel by combining the concentrate with the balance of
the propellants in a bulk product tank that is temperature
controlled to 21.degree. to 27.degree. C. and pressure controlled
to 2.8 to 4.0 BAR. 17 ml aerosol containers which have a metered
valve which is designed to provide 100 inhalations of the
composition of the invention. Each container is provided with the
following: TABLE-US-00004 ipratropium bromide, 0.0021 g
6-Amino-5-nitro-4- (.beta.-D-ribofuranosylamino)pyrimidine
tetrahydro-furan-3,4-diol 0.0120 g trichloromonofluoromethane
1.6939 g dichlorodifluoromethane 3.7028 g dichlorotetrafluoroethane
1.5766 g total 7.0000 g
5.5 EXAMPLE 5
Intravenous Dosage Form
[0318] The intravenous formulation is prepared by reconstituting
clitocine with an appropriate liquid medium, such as water for
injection (WFI) or a 5% dextrose solution. A desired concentration
of the intravenous formulation can be obtained by reconstituting an
appropriate amount of clitocine with an appropriate volume of
liquid medium. A desired concentration of the intravenous
formulation provides a therapeutically effective amount of
clitocine to the patient, preferably a mammal, more preferably a
human, in need of the intravenous pharmaceutical formulation and
maintains a therapeutically effective level of clitocine in the
patient. The dose which is therapeutically effective will depend on
the rate at which the intravenous formulation is delivered to the
patient and the concentration of the intravenous formulation. For
example, two vials containing a composition (e.g., 500 mg of
clitocine per vial) are reconstituted with a 5% dextrose solution
(14 ml of 5% dextrose solution per vial) yielding a total of 28 mL
of solution. The reconstituted solution is incorporated into a
dextrose solution in an infusion bag and q.s. to 166 mL, resulting
in a solution containing 6 mg/ml of clitocine suitable for
intravenous infusion administration. The preferred concentration of
clitocine in the liquid medium, in the infusion bag, is about 3 to
about 10 mg/ml, preferably about 5 to about 6 mg/ml.
[0319] While the invention has been described with respect to the
particular embodiments, it will be apparent to those skilled in the
art that various changes and modifications may be made without
departing from the spirit and scope of the invention as defined in
the claims. Such modifications are also intended to fall within the
scope of the appended claims.
* * * * *