U.S. patent application number 12/005938 was filed with the patent office on 2008-10-23 for compounds and pharmaceutical compositions for the treatment of liver disorders.
This patent application is currently assigned to Idenix Pharmaceuticals, Inc.. Invention is credited to Gilles Gosselin, Christian Perigaud, Suzanne Peyrottes, Claire Pierra, Jean-Pierre Sommadossi.
Application Number | 20080261913 12/005938 |
Document ID | / |
Family ID | 39589128 |
Filed Date | 2008-10-23 |
United States Patent
Application |
20080261913 |
Kind Code |
A1 |
Sommadossi; Jean-Pierre ; et
al. |
October 23, 2008 |
Compounds and pharmaceutical compositions for the treatment of
liver disorders
Abstract
Provided herein are compounds, compositions and methods for the
treatment of liver disorders, including liver cancer and metabolic
diseases, such as diabetes, hyperlipidemia, atherosclerosis, and
obesity. Specifically, compounds and compositions of nucleoside
derivatives are disclosed, which can be administered either alone
or in combination with other anti-cancer agents.
Inventors: |
Sommadossi; Jean-Pierre;
(Cambridge, MA) ; Gosselin; Gilles; (Montpellier,
FR) ; Pierra; Claire; (Montarnaud, FR) ;
Perigaud; Christian; (Grabels, FR) ; Peyrottes;
Suzanne; (Grabels, FR) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
Idenix Pharmaceuticals,
Inc.
L'UNIVERSITE MONTPELLIER II;
|
Family ID: |
39589128 |
Appl. No.: |
12/005938 |
Filed: |
December 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60877944 |
Dec 28, 2006 |
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60936290 |
Jun 18, 2007 |
|
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60985891 |
Nov 6, 2007 |
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Current U.S.
Class: |
514/47 ; 514/120;
514/49; 514/52; 536/26.7; 536/26.8; 564/12 |
Current CPC
Class: |
A61P 1/16 20180101; A61P
9/04 20180101; C07F 9/65586 20130101; C07H 19/00 20130101; A61P
9/06 20180101; A61P 3/06 20180101; A61P 9/00 20180101; A61P 3/04
20180101; A61K 31/7056 20130101; A61P 5/14 20180101; A61K 31/7076
20130101; A61P 3/10 20180101; A61K 31/7072 20130101; A61P 9/10
20180101; A61P 19/10 20180101; A61P 31/20 20180101; A61P 31/12
20180101; A61K 31/708 20130101; C07F 9/65616 20130101; A61P 35/00
20180101; C07H 17/02 20130101; A61K 31/7068 20130101; A61P 31/14
20180101; A61P 13/12 20180101; A61P 5/50 20180101; A61K 31/675
20130101; A61P 25/24 20180101; A61P 27/06 20180101; A61K 31/7064
20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/47 ;
536/26.7; 536/26.8; 514/49; 564/12; 514/120; 514/52 |
International
Class: |
A61K 31/7076 20060101
A61K031/7076; C07H 19/20 20060101 C07H019/20; C07H 19/10 20060101
C07H019/10; A61K 31/7068 20060101 A61K031/7068; C07F 9/06 20060101
C07F009/06; A61K 31/661 20060101 A61K031/661; A61K 31/708 20060101
A61K031/708; A61P 35/00 20060101 A61P035/00; A61P 3/10 20060101
A61P003/10 |
Claims
1. A compound of formula ##STR00089## or a pharmaceutically
acceptable salt, solvate, a stereoisomeric, tautomeric or
polymorphic form thereof, wherein: R.sup.y is optionally
substituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl,
cycloalkenyl, amino, heterocyclyl or heteroaryl; R.sup.a and
R.sup.b are selected as follows: i) R.sup.a and R.sup.b are each
independently hydrogen or optionally substituted alkyl,
carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl,
aminocarbonylalkyl, alkoxycarbonylalkyl, aryl, arylalkyl,
cycloalkyl, heteroaryl or heterocyclyl; or ii) R.sup.a and R.sup.b
together with the nitrogen atom on which they are substituted form
a 3-7 membered heterocyclic or heteroaryl ring; and R.sup.1 is a
moiety derivable by removal of a hydrogen from a hydroxy group of
an anti-cancer drug.
2. The compound of claim 1, having the formula: ##STR00090## or a
pharmaceutically acceptable salt, solvate, a stereoisomeric,
tautomeric or polymorphic form thereof.
3. The compound of claim 1 having the formula: ##STR00091## or a
pharmaceutically acceptable salt, solvate, a stereoisomeric,
tautomeric or polymorphic form thereof.
4. The compound of claim 1 having the formula: ##STR00092## or a
pharmaceutically acceptable salt, solvate, a stereoisomeric,
tautomeric or polymorphic form thereof.
5. The compound of claim 1, wherein R.sup.y is optionally
substituted alkyl and R.sup.a and R.sup.b are each independently
hydrogen or optionally substituted benzyl.
6. The compound of claim 5, wherein R.sup.y is hydroxyalkyl or
aminoalkyl.
7. The compound of claim 1, wherein R.sup.y is --C(R.sup.c).sub.3
or --NHR.sup.c where each R.sup.c is independently optionally
substituted alkyl or optionally substituted aryl; and R.sup.a and
R.sup.b are independently hydrogen, optionally substituted alkyl or
optionally substituted arylalkyl.
8. The compound of claim 7, wherein R.sup.a and R.sup.b are each
independently hydrogen or substituted alkyl.
9. The compound of claim 5, wherein R.sup.y is selected from the
group consisting of alkyl and hydroxyalkyl.
10. The compound of claim 9, wherein R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH.
11. The compound of claim 9, wherein R.sup.a is hydrogen, R.sup.b
is benzyl and R.sup.y is --C(CH.sub.3).sub.2CH.sub.2OH.
12. The compound of any of claims 1, wherein R.sup.1 is
Aclarubicin, Decitabine, Daunorubicin, Dihydro-5-azacytidine,
Doxorubicin, Epirubicin, Estramustine, Etoposide, Fludarabine,
Neplanocin A, Tezacitabine, Troxacitabine, Vinblastin, Vincristin,
Vindesin, Teniposide, NK-611, Camptothecin, Irinotecan,
9-Aminocamptothecin, Topotecan, Paclitaxel, Azatoxin, Coformycin,
Pirarubicin, or Losoxantrone.
13. A compound of formula: ##STR00093## ##STR00094## wherein each
R, if present, is independently alkyl, halogen or hydroxyl; X, if
present, is CH.sub.2, O or S; R.sup.y is alkyl, alkenyl, alkynyl,
aryl, aryl alkyl, cycloalkyl, cycloalkenyl, amino, aminoalkyl,
heterocyclyl or heteroaryl, all optionally substituted; R.sup.a and
R.sup.b are selected as follows: i) R.sup.a and R.sup.b are each
independently hydrogen, alkyl, carboxyalkyl, hydroxyalkyl,
hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,
alkoxycarbonylalkyl, aryl, aryl alkyl, cycloalkyl, heteroaryl or
heterocyclyl, all optionally substituted; or ii) R.sup.a and
R.sup.b together with the nitrogen atom on which they are
substituted form a 3-7 membered heterocyclic or heteroaryl
ring.
14. The compound of claim 13, wherein each R, if present, is
independently alkyl, halogen or hydroxyl; X, if present, is
CH.sub.2, O or S; R.sup.y is optionally substituted alkyl, wherein
the substituents when present are hydroxy or amino; and R.sup.a and
R.sup.b are each independently hydrogen or alkyl, wherein the alkyl
group is optionally substituted with aryl, amino, amido, hydroxyl,
alkoxy or heteroaryl, each optionally substituted.
15. The compound of claim 13, wherein R.sup.a and R.sup.b are each
independently H or benzyl, wherein the benzyl group is optionally
substituted with hydroxy or amino.
16. The compound of claim 13, wherein R.sup.y is substituted alkyl
and R.sup.a and R.sup.b are each independently hydrogen or
optionally substituted benzyl.
17. The compound of claim 16, wherein R.sup.y is hydroxyalkyl or
aminoalkyl.
18. The compound of claim 13, wherein R.sup.y is --C(R.sup.c).sub.3
or --NHR.sup.c where each R.sup.c is independently optionally
substituted alkyl or optionally substituted aryl; and R.sup.a and
R.sup.b are independently hydrogen, optionally substituted alkyl or
optionally substituted arylalkyl.
19. The compound of claim 17, wherein R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH.
20. The compound of claim 19, wherein R.sup.a and R.sup.b are each
independently hydrogen or substituted alkyl.
21. The compound of claim 13, wherein R.sup.a is hydrogen, R.sup.b
is benzyl and R.sup.y is --C(CH.sub.3).sub.2CH.sub.2OH.
22. The compound of claim 13 having the structure: ##STR00095## or
a pharmaceutically acceptable salt, solvate, a stereoisomeric,
tautomeric or polymorphic form thereof.
23. A compound selected from formula: ##STR00096## wherein R.sup.x
and R.sup.z are each independently hydrogen or optionally
substituted alkyl; R.sup.w is optionally substituted alkyl; X.sup.1
is O or S; R.sup.y is alkyl, alkenyl, alkynyl, aryl, arylalkyl,
cycloalkyl, cycloalkenyl, amino, heterocyclyl or heteroaryl, all
optionally substituted; R.sup.a and R.sup.b are selected as
follows: i) R.sup.a and R.sup.b are each independently hydrogen,
alkyl, carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl,
aminocarbonylalkyl, alkoxycarbonylalkyl, aryl, arylalkyl,
cycloalkyl, heteroaryl or heterocyclyl, all optionally substituted;
or ii) R.sup.a and R.sup.b together with the nitrogen atom on which
they are substituted form a 3-7 membered heterocyclic or heteroaryl
ring.
24. The compound of claim 23, wherein R.sup.y is optionally
substituted alkyl, wherein the substituents when present are
selected from hydroxy and amino.
25. The compound of claim 24, wherein R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH.
26. The compound of claim 25, wherein R.sup.a is hydrogen and
R.sup.b is benzyl.
27. The compound of claim 26 having formula selected from:
##STR00097## ##STR00098##
28. The compound of claim 27 having formula: ##STR00099##
29. A method of treating cancer comprising administering a compound
of claim 1.
30. A method of lowering plasma lipid levels or lowering blood
glucose levels comprising administering a compound of claim 13.
31. A method of lowering blood glucose levels comprising
administering a compound of claim 23.
32. A pharmaceutical composition comprising a compound of any of
claims 1, 13 or 23 and a pharmaceutically acceptable carrier.
33. The composition of claim 32 that is suitable for oral
administration.
34. The composition of claim 33 wherein the composition is in the
form of a pill or tablet.
Description
1. CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent applications claims the benefit of priority
under 35 U.S.C. .sctn. 119 to 1) U.S. Provisional Appl. No.
60/877,944, filed Dec. 28, 2006; 2) U.S. Provisional Appl. No.
60/936,290, filed Jun. 18, 2007; and 3) U.S. Provisional
Application No. 60/985,891, filed Nov. 6, 2007. The disclosures of
the above referenced applications are incorporated by reference in
their entirety herein.
2. FIELD
[0002] The present invention relates to compounds, methods and
pharmaceutical compositions, for use in treatment and prevention of
disorders of the liver, including cancer.
3. BACKGROUND
[0003] Drug induced toxicities and pharmacological side effects are
often associated with interactions by the drug or drug metabolite
in tissues not associated with the pharmacological benefits of the
drug therapy. In other cases, the desired pharmacological effect is
poorly achieved either because of dose-limiting toxicities or
inadequate drug levels in the target tissues. Thus, there is a need
to deliver drugs to specific tissues or organs. High organ
specificity can be achieved by a variety of mechanisms including
local administration to the target organ and drug-protein
conjugates. Local administration to the target organ is an invasive
procedure. Drug-protein conjugates exhibit poor oral
bioavailability, limitations in carrier manufacturing and drug
loading, a potential for diminished liver uptake due to down
regulation of the receptor in diseased tissue, and a high incidence
of antibody induction. A third approach entails use of prodrugs
that are activated by enzymes highly enriched in the target
organ.
[0004] There is a particular need to deliver drugs to the liver to
treat diseases such as cancer and metabolic disorders. Many
therapies for these conditions have narrow therapeutic indices and
many therapeutic indications could be benefitted by selective
delivery of the therapeutic agent to the liver.
4. SUMMARY
[0005] Phosphoroamidate and phosphonoamidate compound forms of a
variety of therapeutic agents are provided, as well as methods for
their manufacture and use in the treatment of a variety of
disorders including liver cancer, inflammation, fibrosis and
metabolic disorders. In one embodiment, the compound is a
S-pivaloyl-2-thioethyl phosphoroamidate, S-pivaloyl-2-thioethyl
phosphonoamidate, S-hydroxypivaloyl-2-thioethyl phosphoroamidate or
S-hydroxypivaloyl-2-thioethyl phosphonoamidate. As used herein, a
"phosphoroamidate or phosphonoamidate compound of a therapeutic
agent" includes a therapeutic agent derivatized to include a
phosphoroamidate or phosphonoamidate group. The therapeutic agent
is, for example, an anti-cancer agent that includes, or has been
derivatized to include, a reactive group, such as a hydroxyl, for
attachment of the phosphoroamidate or phosphonoamidate moiety. Such
therapeutic agents include, but are not limited to nucleosides and
nucleoside analogs including acyclic nucleosides. In some
embodiments, phosphoroamidate or phosphonoamidate compounds of
nucleotides and nucleotide analogs, such as 2'-branched and
4'-branched nucleosides are provided. Such compounds can be
administered in an effective amount for the treatment of liver
disorders, including cancer.
[0006] In certain embodiments, while not being limited to any
theory, it is possible that the parent drug is obtained from
selective metabolism of the phosphoroamidate or phosphonoamidate
compound in the liver and thus the parent drug provided herein is
capable of accumulating in the liver. Accordingly, provided are
methods of directing phosphoroamidate or phosphonoamidate compounds
disclosed herein to the liver.
[0007] In certain embodiments, phosphoroamidate or phosphonoamidate
compounds of pharmaceutical agents for the treatment of a liver
disorder can be made and used therapeutically as described herein.
A variety of phosphoroamidate or phosphonoamidate compounds can be
used in the treatment of liver disorders. In particular,
therapeutic agents for the treatment of liver cancer can be
derivatized to form a phosphoroamidate or phosphonoamidate compound
as described herein, and used for the treatment of liver cancers.
Liver cancers that can be treated include benign tumors, malignant
tumors, hemangioma, hepatic adenomas, focal nodular hyperplasia,
hepatocellular carcinoma, fibrolamellar carcinoma,
cholangiocarcinomas, bile duct cancers, and other primary and
metastatic cancers of the liver.
[0008] Phosphoroamidate and phosphonoamidate compounds of a variety
of therapeutic agents are provided. The compounds can be formed
using methods available in the art and those disclosed herein. Such
compounds can be used in some embodiments to enhance delivery of
the drug to the liver. In one embodiment, the compound comprises an
S-acyl-2-thioethyl phosphoroamidate or an S-acyl-2-thioethyl
phosphonoamidate derivative, e.g., a S-pivaloyl-2-thioethyl
phosphoroamidate or a S-hydroxypivaloyl-2-thioethyl
phosphonoamidate derivative.
[0009] In some embodiments, the phosphoroamidate or
phosphonoamidate compounds, as well as salts thereof, and
compositions comprising the compounds, provided herein are useful
for treatment of disorders of the liver, including cancer. In other
embodiments, the phosphoroamidate or phosphonoamidate compounds, as
well as salts thereof, and compositions comprising the compounds,
provided herein are useful for treatment of metabolic diseases,
such as diabetes, hyperlipidemia, atherosclerosis, and obesity. In
other embodiments, the compounds, as well as salts thereof, and
compositions comprising the compounds, provided herein are useful
for treatment of liver fibrosis and inflammation.
[0010] In one embodiment, the compound provided herein is a
compound of Formula I:
##STR00001##
or a pharmaceutically acceptable salt, solvate, a stereoisomeric,
tautomeric or polymorphic form thereof, wherein
[0011] X.sup.a is
##STR00002##
[0012] Z is O or S;
[0013] each W is independently O or S;
[0014] R.sup.y and R.sup.u each independently represent alkyl,
alkenyl, alkynyl, aryl, aryl alkyl, cycloalkyl, cycloalkenyl,
amino, aminoalkyl, alkoxy, heterocyclyl, or heteroaryl, all
optionally substituted;
[0015] R.sup.a and R.sup.b are selected as follows:
[0016] i) R.sup.a and R.sup.b are each independently hydrogen,
alkyl, carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl,
aminocarbonylalkyl, alkoxycarbonylalkyl, aryl, aryl alkyl,
cycloalkyl, heteroaryl or heterocyclyl, all optionally substituted;
or
[0017] ii) R.sup.a and R.sup.b together with the nitrogen atom on
which they are substituted form a 3-7 membered heterocyclic or
heteroaryl ring;
[0018] n is 0-3; n2 is 1-4; and
[0019] R.sup.1 is a moiety derivable by removal of a hydrogen from
a group, such as a hydroxy group, of a therapeutic agent such as an
anti-cancer drug.
[0020] In another embodiment,
##STR00003##
[0021] Z is O, S, NH or NR.sup.w, where R.sup.w is, e.g., alkyl,
alkyl, alkenyl, alkynyl, aryl, aryl alkyl, cycloalkyl,
cycloalkenyl, amino, aminoalkyl, alkoxy, heterocyclyl, or
heteroaryl, all optionally substituted;
[0022] each W is O, S, NH or NR.sup.w, where R.sup.w is, e.g.,
alkyl, alkyl, alkenyl, alkynyl, aryl, aryl alkyl, cycloalkyl,
cycloalkenyl, amino, aminoalkyl, alkoxy, heterocyclyl, or
heteroaryl, all optionally substituted;
[0023] R.sup.y and R.sup.u each independently represent alkyl,
alkenyl, alkynyl, aryl, aryl alkyl, cycloalkyl, cycloalkenyl,
amino, aminoalkyl, alkoxy, heterocyclyl, or heteroaryl, all
optionally substituted;
[0024] R.sup.a and R.sup.b are selected as follows:
[0025] i) R.sup.a and R.sup.b are each independently hydrogen,
alkyl, carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl,
aminocarbonylalkyl, alkoxycarbonylalkyl, aryl, aryl alkyl,
cycloalkyl, heteroaryl or heterocyclyl, all optionally substituted;
or
[0026] ii) R.sup.a and R.sup.b together with the nitrogen atom on
which they are substituted form a 3-7 membered heterocyclic or
heteroaryl ring;
[0027] n is 0-3; n.sub.2 is 1-4; and
[0028] R.sup.1 is a moiety derivable by removal of a hydrogen from
a group, such as a hydroxy group, of a therapeutic agent such as an
anti-cancer drug.
[0029] Those of skill in the art will recognize that compounds of
Formula I can be designed or prepared by reaction, e.g., at a
hydroxy group of said drug, for example via condensation or
dehydration. For convenience, in the description herein when
exemplary substituents, such as R.sup.1 groups are identified as a
drug in a phosphoroamidate or phosphonoamidate compound, e.g. in a
formula, those of skill in the art will recognize that the compound
comprises a derivative, e.g. a radical of the anti-cancer drug.
Those derivatives can for example be prepared by elimination of a
hydrogen radical from a hydroxy group of the drug, for instance in
a dehydration reaction.
[0030] In certain embodiments of Formula I, R.sup.1 is a nucleoside
comprising a cyclic or acyclic sugar or an analog thereof.
[0031] In certain embodiments, R.sup.1 is an anti-cancer drug
selected from clarubicin, decitabine, daunorubicin,
dihydro-5-azacytidine, doxorubicin, epirubicin, estramustin,
etoposide, fludarabine, 7-hydroxychlorpromazin, neplanocin A,
podophyllotoxin, tezacitabine, troxacitabine, vinblastin,
vincristin, vindesin, etoposide, teniposide, NK-611, camptothecin,
irinotecan, 9-aminocamptothecin, GG-211, topotecan, paclitaxel,
Azatoxin, coformycin, pirarubicin, nelarabine and losoxantrone.
[0032] In one embodiment, R.sup.1 is an immunosuppressant, such as
pentostatin, combretastatin A-4, mycophenolic acid or
mitoxantrone.
[0033] In certain embodiments according to formula I, R.sup.y is
substituted alkyl, e.g. hydroxyalkyl or aminoalkyl; and R.sup.a and
R.sup.b are independently hydrogen, alkyl, substituted alkyl,
benzyl or substituted benzyl, for instance hydroxy- or
amino-substituted alkyl or benzyl.
[0034] In another embodiment, R.sup.y is --OR.sup.c,
--C(R.sup.c).sub.3 or --NHR.sup.c where each R.sup.c is
independently alkyl, substituted alkyl, aryl or substituted aryl,
for instance hydroxy- or amino-substituted alkyl or aryl; and
R.sup.a and R.sup.b are independently hydrogen, alkyl, substituted
alkyl, benzyl or substituted benzyl, for instance hydroxy- or
amino-substituted alkyl or benzyl.
[0035] In a further embodiment, R.sup.a and R.sup.b are
independently benzyl or substituted alkyl. In a further embodiment,
R.sup.y is selected from the group consisting of alkyl and
hydroxyalkyl. In certain embodiments, R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH.
[0036] In certain embodiments, the compounds provided herein are
selected such that R.sup.1 is not
3'-azido-2',3'-dideoxythymidine.
[0037] In another embodiment, the compound provided herein is a
compound of Formula IIa or IIb:
##STR00004##
or a pharmaceutically acceptable salt, solvate, a stereoisomeric,
tautomeric or polymorphic form thereof, wherein
[0038] R.sup.y is alkyl, alkenyl, alkynyl, aryl, aryl alkyl,
cycloalkyl, cycloalkenyl, amino, aminoalkyl, heterocyclyl or
heteroaryl, all optionally substituted;
[0039] R.sup.a and R.sup.b are selected as follows:
[0040] i) R.sup.a and R.sup.b are each independently hydrogen,
alkyl, carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl,
aminocarbonylalkyl, alkoxycarbonylalkyl, aryl, aryl alkyl
cycloalkyl, heteroaryl or heterocyclyl, all optionally substituted;
or
[0041] ii) R.sup.a and R.sup.b together with the nitrogen atom on
which they are substituted form a 3-7 membered heterocyclic or
heteroaryl ring; and
[0042] R.sup.1 is a drug such as an anti-cancer drug.
[0043] In certain embodiments according to Formula IIa or IIb,
R.sup.y is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl; and
R.sup.a and R.sup.b are each independently hydrogen, alkyl,
substituted alkyl, benzyl or substituted benzyl, for instance
hydroxy- or amino-substituted alkyl or benzyl. In another
embodiment, R.sup.y is --OR.sup.c, --C(R.sup.c).sub.3 or
--NHR.sup.c where each R.sup.c is independently alkyl, substituted
alkyl, aryl or substituted aryl, for instance hydroxy- or
amino-substituted alkyl or aryl; and R.sup.a and R.sup.b are
independently hydrogen, alkyl, substituted alkyl, benzyl or
substituted benzyl, for instance hydroxy- or amino-substituted
alkyl or benzyl. In a further embodiment, R.sup.a and R.sup.b are
each independently benzyl or substituted alkyl. In a further
embodiment, R.sup.y is selected from the group consisting of alkyl
and hydroxyalkyl. In certain embodiments, R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH.
[0044] In some embodiments, provided herein are: [0045] (a)
compounds as described herein, e.g. of Formula I, IIa or IIb, and
pharmaceutically acceptable salts and compositions thereof; [0046]
(b) compounds as described herein, e.g. of Formula I, IIa or IIb,
and pharmaceutically acceptable salts and compositions thereof for
use in the treatment and/or prophylaxis of a liver disorder; [0047]
(c) processes for the preparation of compounds as described herein,
e.g. of Formula I, IIa or IIb, as described in more detail below;
[0048] (d) pharmaceutical formulations comprising a compound as
described herein, e.g. of Formula I, IIa or IIb, or a
pharmaceutically acceptable salt thereof together with a
pharmaceutically acceptable carrier or diluent; and [0049] (e)
pharmaceutical formulations comprising a compound as described
herein, e.g. of Formula I, IIa or IIb, or a pharmaceutically
acceptable salt thereof together with one or more other effective
anti-cancer agents, optionally in a pharmaceutically acceptable
carrier or diluent.
[0050] In certain embodiments, the following phosphoroamidate and
phosphonoamidate formulas and compounds are provided, which
optionally act as thyroid hormone receptor effectors:
##STR00005## ##STR00006## ##STR00007##
wherein
[0051] each R, if present, is independently alkyl, halogen or
hydroxyl;
[0052] X, if present, is CH.sub.2, O or S;
[0053] R.sup.y, if present, is optionally substituted alkyl,
wherein the substituted alkyl is optionally hydroxyalkyl or
aminoalkyl, e.g., --C(CH.sub.3).sub.2CH.sub.2OH; and
[0054] R.sup.a and R.sup.b, if present, are independently hydrogen;
unsubstituted alkyl; or alkyl substituted with aryl, amino, amido,
hydroxyl, alkoxy, aminoalkyl, hydroxyalkyl, aryl, or heteroaryl,
each optionally substituted; wherein, in one embodiment, R.sup.a
and R.sup.b are independently H or a benzyl that is optionally
substituted, for example, with hydroxy or amino.
[0055] In certain embodiments according to formula IIIa or b, IVa
or b, Va or b, VIa or b, VIIa or b, VIII a or b, R.sup.a is
hydrogen, R.sup.b is --CH.sub.2--C.sub.6H.sub.5 and R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH.
[0056] In certain embodiments, the thyroid hormone receptor
effector compound provided herein has a formula selected from:
##STR00008##
wherein
[0057] R.sup.x and R.sup.z are each independently hydrogen or
alkyl;
[0058] R.sup.w is alkyl;
[0059] R.sup.y is alkyl, alkenyl, alkynyl, aryl, aryl alkyl,
cycloalkyl, cycloalkenyl, amino, aminoalkyl, heterocyclyl or
heteroaryl, all optionally substituted;
[0060] R.sup.a and R.sup.b are selected as follows:
[0061] i) R.sup.a and R.sup.b are each independently hydrogen,
alkyl, carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl,
aminocarbonylalkyl, alkoxycarbonylalkyl, aryl, aryl alkyl,
cycloalkyl, heteroaryl or heterocyclyl, all optionally substituted;
or
[0062] ii) R.sup.a and R.sup.b together with the nitrogen atom on
which they are substituted form a 3-7 membered heterocyclic or
heteroaryl ring.
[0063] In certain embodiments, the thyroid hormone receptor
effector provided herein is selected from:
##STR00009## ##STR00010##
[0064] In certain embodiments, R.sup.a is hydrogen, R.sup.b is
--CH.sub.2--C.sub.6H.sub.5 and R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH.
[0065] In certain embodiments, the compound or Formula selected
from IIIa or b, IVa or b, Va or b, VIa or b, VIIa or b, VIII a or b
is derived from a phosphonate compound useful for inhibiting
gluconeogenesis, optionally by inhibiting the enzyme fructose
1,6-bisphosphatase (FBPase).
[0066] In certain embodiments, the compound or Formula selected
from IXa or b, Xa or b, XIa or b, XIIa or b, XIIIa or b and XIVa or
b is derived from a compound useful for inhibiting gluconeogenesis,
optionally by inhibiting the enzyme fructose 1,6-bisphosphatase
(FBPase).
[0067] In certain embodiments, the compound or Formula selected
from IIIa or b, IVa or b, Va or b, VIa or b, VIIa or b, VIII a or b
is a phosphonic acid-containing compound that binds to a thyroid
receptor in the liver, and is optionally an agonist, antagonist,
partial agonist or partial antagonist of T3. Inhibition of
gluconeogenesis can result in blood glucose lowering in diabetic
subjects. Such compounds can exhibit enhanced pharmacokinetics
including oral bioavailability and liver drug levels.
[0068] In certain embodiments, provided is a method of treatment of
a subject in need thereof, the method comprising administering to
the subject a phosphoroamidate and phosphonoamidate compound or
Formula selected from IIIa or b, IVa or b, Va or b, VIa or b, VIIa
or b, VIII a or b, IXa or b, X a or b, XIa or b, XIIa or b, XIIIa
or b and XIVa or b or a pharmaceutically acceptable salt,
enantiomer, ester or prodrug thereof, in an amount effective for
one or more of the following:
[0069] reducing plasma lipid levels, lowering cholesterol levels,
reducing triglyceride levels, or increasing the ratio of HDL to
LDL;
[0070] lowering blood glucose levels;
[0071] treating hyperlipidemia or hypercholesterolemia;
[0072] treating obesity, reducing fat content, treating fatty
liver, reducing weight or preventing weight gain;
[0073] treating atherosclerosis, coronary heart disease, heart
failure, nephrotic syndrome, or chronic renal failure;
[0074] lowering blood glucose levels, treating diabetes, impaired
glucose tolerance, metabolic syndrome x, insulin resistance or
hyperinsulinemia;
[0075] increasing levels of genes associated with
gluconeogenesis;
[0076] decreasing hepatic glycogen levels or maintaining or
improving glycemic control;
[0077] amelioration of hyperinsulinemia and/or decrease of glucose
levels in diabetic subjects at doses that optionally do not affect
cardiac function, e.g., heart rate, force of systolic contraction,
duration of diastolic relaxation, vascular tone, or heart
weight;
[0078] treating thyroid disease, thyroid cancer, depression,
glaucoma, cardiac arrhythmias, heart failure, or osteoporosis;
[0079] increasing mitochondrial biogenesis, or increasing
expression of PGC-1, AMP activated protein kinase or nuclear
respiratory factor;
[0080] inhibiting hepatic gluconeogenesis; or
[0081] modulating expression of certain genes in the liver
resulting in effects on lipids (e.g., cholesterol), glucose,
lipoproteins, and triglycerides, or modulation of T3-responsive
genes.
[0082] In certain embodiments, the compounds do not affect thyroid
function, thyroid production of circulating iodinated thyronines
such as T3 and T4, and/or the ratio of T3 to T4.
[0083] Also provided are pharmaceutical compositions comprising the
compounds, e.g., in a dosage unit suitable for administration,
e.g., oral administration.
5. BRIEF DESCRIPTION OF DRAWINGS
[0084] FIG. 1 depicts depletion of B184 (NM108 hydroxySATE
phosphoroamidate) after incubation with and without NADPH in monkey
liver S9.
[0085] FIG. 2 depicts depletion of B102 (NM107 hydroxySATE
phosphoroamidate) after incubation with and without NADPH in monkey
liver S9.
6. DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0086] Provided herein are compounds, compositions and methods
useful for treating liver disorders, such as cancer, or metabolic
diseases, such as diabetes, hyperlipidemia, atherosclerosis, and
obesity. Further provided are dosage forms useful for such
methods.
6.1 Definitions
[0087] When referring to the compounds provided herein, the
following terms have the following meanings unless indicated
otherwise.
[0088] The term "alkyl", as used herein, unless otherwise
specified, includes a saturated straight, branched, or cyclic,
primary, secondary, or tertiary hydrocarbon of typically C.sub.1 to
C.sub.10, and specifically includes methyl, CF.sub.3, CCl.sub.3,
CFCl.sub.2, CF.sub.2Cl, ethyl, CH.sub.2CF.sub.3, CF.sub.2CF.sub.3,
propyl, isopropyl, cyclopropyl, butyl, isobutyl, secbutyl, t-butyl,
pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl,
cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl,
and 2,3-dimethylbutyl. The term includes both substituted and
unsubstituted alkyl groups, and particularly includes halogenated
alkyl groups, and even more particularly fluorinated alkyl groups.
Non-limiting examples of moieties with which the alkyl group can be
substituted are selected from the group consisting of halogen
(fluoro, chloro, bromo or iodo), hydroxyl, amino, alkylamino,
arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,
phosphonic acid, phosphate, or phosphonate, either unprotected, or
protected as necessary, as known to those skilled in the art, for
example, as taught in Greene, et al., Protective Groups in Organic
Synthesis, John Wiley and Sons, Second Edition, 1991, hereby
incorporated by reference.
[0089] The term "lower alkyl", as used herein, and unless otherwise
specified, includes a C to C.sub.4 saturated straight, branched, or
if appropriate, a cyclic (for example, cyclopropyl) alkyl group,
including both substituted and unsubstituted moieties.
[0090] "Alkylene" includes divalent saturated aliphatic hydrocarbon
groups particularly having up to about 11 carbon atoms and more
particularly 1 to 6 carbon atoms which can be straight-chained or
branched. This term is exemplified by groups such as methylene
(--CH.sub.2--), ethylene (--CH.sub.2CH.sub.2--), the propylene
isomers (e.g., --CH.sub.2CH.sub.2CH.sub.2-- and
--CH(CH.sub.3)CH.sub.2--) and the like.
[0091] "Alkenyl" includes monovalent olefinically unsaturated
hydrocarbon groups, in certain embodiment, having up to about 11
carbon atoms, from 2 to 8 carbon atoms, or from 2 to 6 carbon
atoms, which can be straight-chained or branched and having at
least 1 or from 1 to 2 sites of olefinic unsaturation. Exemplary
alkenyl groups include ethenyl (--CH.dbd.CH.sub.2), n-propenyl
(--CH.sub.2CH.dbd.CH.sub.2), isopropenyl
(--C(CH.sub.3).dbd.CH.sub.2), vinyl and substituted vinyl, and the
like.
[0092] "Alkenylene" includes divalent olefinically unsaturated
hydrocarbon groups, in certain embodiments, having up to about 11
carbon atoms or from 2 to 6 carbon atoms which can be
straight-chained or branched and having at least 1 or from 1 to 2
sites of olefinic unsaturation. This term is exemplified by groups
such as ethenylene (--CH.dbd.CH--), the propenylene isomers (e.g.,
--CH.dbd.CHCH.sub.2-- and --C(CH.sub.3).dbd.CH-- and
--CH.dbd.C(CH.sub.3)--) and the like.
[0093] "Alkynyl" includes acetylenically unsaturated hydrocarbon
groups, in certain embodiments, having up to about 11 carbon atoms
or from 2 to 6 carbon atoms which can be straight-chained or
branched and having at least 1 or from 1 to 2 sites of alkynyl
unsaturation. Non-limiting examples of alkynyl groups include
acetylenic, ethynyl (--C.ident.CH), propargyl
(--CH.sub.2C.ident.CH), and the like.
[0094] The term "aryl", as used herein, and unless otherwise
specified, includes phenyl, biphenyl, or naphthyl, and preferably
phenyl. The term includes both substituted and unsubstituted
moieties. The aryl group can be substituted with any described
moiety, including, but not limited to, one or more moieties
selected from the group consisting of halogen (fluoro, chloro,
bromo or iodo), alkyl, hydroxyl, amino, alkylamino, arylamino,
alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic
acid, phosphate, or phosphonate, either unprotected, or protected
as necessary, as known to those skilled in the art, for example, as
taught in Greene, et al., Protective Groups in Organic Synthesis,
John Wiley and Sons, Second Edition, 1991.
[0095] "Alkoxy" includes the group --OR where R is alkyl.
Particular alkoxy groups include, by way of example, methoxy,
ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy,
n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
[0096] "Alkoxycarbonyl" includes a radical --C(O)-alkoxy where
alkoxy is as defined herein.
[0097] "Amino" includes the radical --NH.sub.2.
[0098] "Carboxyl" includes the radical --C(O)OH.
[0099] The term "alkylamino" or "arylamino" includes an amino group
that has one or two alkyl or aryl substituents, respectively.
Unless otherwise specifically stated in this application, when
alkyl is a suitable moiety, lower alkyl is preferred. Similarly,
when alkyl or lower alkyl is a suitable moiety, unsubstituted alkyl
or lower alkyl is preferred.
[0100] "Halogen" or "halo" includes chloro, bromo, fluoro or
iodo.
[0101] "Monoalkylamino" includes the group alkyl-NR'--, wherein R'
is selected from hydrogen and alkyl.
[0102] "Thioalkoxy" includes the group --SR where R is alkyl.
[0103] The term "protected" as used herein and unless otherwise
defined refers to a group that is added to an oxygen, nitrogen, or
phosphorus atom to prevent its further reaction or for other
purposes. A wide variety of oxygen and nitrogen protecting groups
are known to those skilled in the art of organic synthesis.
[0104] "Pharmaceutically acceptable salt" includes any salt of a
compound provided herein which retains its biological properties
and which is not toxic or otherwise undesirable for pharmaceutical
use. Such salts may be derived from a variety of organic and
inorganic counter-ions well known in the art. Such salts include:
(1) acid addition salts formed with organic or inorganic acids such
as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric,
sulfamic, acetic, trifluoroacetic, trichloroacetic, propionic,
hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic,
lactic, malonic, succinic, sorbic, ascorbic, malic, maleic,
fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic,
picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic,
ethanesulfonic, 1,2-ethane-disulfonic, 2-hydroxyethanesulfonic,
benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic,
4-toluenesulfonic, camphoric, camphorsulfonic,
4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic,
3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl
sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic,
stearic, cyclohexylsulfamic, quinic, muconic acid and the like
acids; or (2) salts formed when an acidic proton present in the
parent compound either (a) is replaced by a metal ion, e.g., an
alkali metal ion, an alkaline earth ion or an aluminum ion, or
alkali metal or alkaline earth metal hydroxides, such as sodium,
potassium, calcium, magnesium, aluminum, lithium, zinc, and barium
hydroxide, ammonia or (b) coordinates with an organic base, such as
aliphatic, alicyclic, or aromatic organic amines, such as ammonia,
methylamine, dimethylamine, diethylamine, picoline, ethanolamine,
diethanolamine, triethanolamine, ethylenediamine, lysine, arginine,
ornithine, choline, N,N'-dibenzylethylene-diamine, chloroprocaine,
diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine
piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium
hydroxide, and the like.
[0105] Salts further include, by way of example only, sodium,
potassium, calcium, magnesium, ammonium, tetraalkylammonium and the
like, and when the compound contains a basic functionality, salts
of non-toxic organic or inorganic acids, such as hydrohalides, e.g.
hydrochloride and hydrobromide, sulfate, phosphate, sulfamate,
nitrate, acetate, trifluoroacetate, trichloroacetate, propionate,
hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate,
lactate, malonate, succinate, sorbate, ascorbate, malate, maleate,
fumarate, tartarate, citrate, benzoate,
3-(4-hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate,
phthalate, laurate, methanesulfonate (mesylate), ethanesulfonate,
1,2-ethane-disulfonate, 2-hydroxyethanesulfonate, benzenesulfonate
(besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate,
4-toluenesulfonate, camphorate, camphorsulfonate,
4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate,
3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl
sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate,
salicylate, stearate, cyclohexylsulfamate, quinate, muconate and
the like.
[0106] The term "alkaryl" or "alkylaryl" includes an aryl group
with an alkyl substituent. The term aralkyl or arylalkyl includes
an alkyl group with an aryl substituent.
[0107] The term "purine" or "pyrimidine" base includes, but is not
limited to, adenine, N.sup.6-alkylpurines, N.sup.6-acylpurines
(wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl),
N.sup.6-benzylpurine, N.sup.6-halopurine, N.sup.6-vinylpurine,
N.sup.6-acetylenic purine, N.sup.6-acyl purine,
N.sup.6-hydroxyalkyl purine, N.sup.6-alkylaminopurine,
N.sup.6-thioalkyl purine, N.sup.2-alkylpurines,
N.sup.2-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine,
5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2-
and/or 4-mercaptopyrmidine, uracil, 5-halouracil, including
5-fluorouracil, C.sup.5-alkylpyrimidines,
C.sup.5-benzylpyrimidines, C.sup.5-halopyrimidines,
C.sup.5-vinylpyrimidine, C.sup.5-acetylenic pyrimidine,
C.sup.5-acyl pyrimidine, C.sup.5-hydroxyalkyl purine,
C.sup.5-amidopyrimidine, C.sup.5-cyanopyrimidine,
C.sup.5-iodopyrimidine, C.sup.6-iodo-pyrimidine, C.sup.5--Br-vinyl
pyrimidine, C.sup.6--Br-vinyl pyrimidine, C.sup.5-nitropyrimidine,
C.sup.5-amino-pyrimidine, N.sup.2-alkylpurines,
N.sup.2-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,
triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and
pyrazolopyrimidinyl. Purine bases include, but are not limited to,
guanine, adenine, hypoxanthine, 7-deazaguanine, 7-deazaadenine,
2,6-diaminopurine, and 6-chloropurine. Functional oxygen and
nitrogen groups on the base can be protected as necessary or
desired. Suitable protecting groups are well known to those skilled
in the art, and include trimethylsilyl, dimethylhexylsilyl,
t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl
groups, and acyl groups such as acetyl and propionyl,
methanesulfonyl, and p-toluenesulfonyl.
[0108] The term "acyl" or "O-linked ester" includes a group of the
formula C(O)R', wherein R' is an straight, branched, or cyclic
alkyl (including lower alkyl), carboxylate reside of amino acid,
aryl including phenyl, alkaryl, arylalkyl including benzyl,
alkoxyalkyl including methoxymethyl, aryloxyalkyl such as
phenoxymethyl; or substituted alkyl (including lower alkyl), aryl
including phenyl optionally substituted with chloro, bromo, fluoro,
iodo, C.sub.1 to C.sub.4 alkyl or C.sub.1 to C.sub.4 alkoxy,
sulfonate esters such as alkyl or arylalkyl sulphonyl including
methanesulfonyl, the mono, di or triphosphate ester, trityl or
monomethoxy-trityl, substituted benzyl, alkaryl, arylalkyl
including benzyl, alkoxyalkyl including methoxymethyl, aryloxyalkyl
such as phenoxymethyl. Aryl groups in the esters optimally comprise
a phenyl group. In particular, acyl groups include acetyl,
trifluoroacetyl, methylacetyl, cyclpropylacetyl, propionyl,
butyryl, hexanoyl, heptanoyl, octanoyl, neo-heptanoyl,
phenylacetyl, 2-acetoxy-2-phenylacetyl, diphenylacetyl,
.alpha.-methoxy-.alpha.-trifluoromethyl-phenylacetyl, bromoacetyl,
2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl,
2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl,
trimethylacetyl, chlorodifluoroacetyl, perfluoroacetyl,
fluoroacetyl, bromodifluoroacetyl, methoxyacetyl,
2-thiopheneacetyl, chlorosulfonylacetyl, 3-methoxyphenylacetyl,
phenoxyacetyl, tert-butylacetyl, trichloroacetyl,
monochloro-acetyl, dichloroacetyl, 7H-dodecafluoro-heptanoyl,
perfluoro-heptanoyl, 7H-dodeca-fluoroheptanoyl,
7-chlorododecafluoro-heptanoyl, 7-chloro-dodecafluoro-heptanoyl,
7H-dodecafluoroheptanoyl, 7H-dodeca-fluoroheptanoyl,
nonafluoro-3,6-dioxa-heptanoyl, nonafluoro-3,6-dioxaheptanoyl,
perfluoroheptanoyl, methoxybenzoyl, methyl
3-amino-5-phenylthiophene-2-carboxyl,
3,6-dichloro-2-methoxy-benzoyl,
4-(1,1,2,2-tetrafluoro-ethoxy)-benzoyl, 2-bromo-propionyl,
omega-aminocapryl, decanoyl, n-pentadecanoyl, stearyl,
3-cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl,
pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl,
2,6-pyridinedicarboxyl, cyclopropane-carboxyl,
cyclobutane-carboxyl, perfluorocyclohexyl carboxyl,
4-methylbenzoyl, chloromethyl isoxazolyl carbonyl,
perfluorocyclohexyl carboxyl, crotonyl,
1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,
1-pyrrolidinecarbonyl, 4-phenylbenzoyl.
[0109] The term "amino acid" includes naturally occurring and
synthetic .alpha., .beta. .gamma. or .delta. amino acids, and
includes but is not limited to, amino acids found in proteins, i.e.
glycine, alanine, valine, leucine, isoleucine, methionine,
phenylalanine, tryptophan, proline, serine, threonine, cysteine,
tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,
arginine and histidine. In a preferred embodiment, the amino acid
is in the L-configuration. Alternatively, the amino acid can be a
derivative of alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl,
phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl,
threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl,
aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl,
.beta.-alanyl, .beta.-valinyl, .beta.-leucinyl,
.beta.-isoleuccinyl, .beta.-prolinyl, .beta.-phenylalaninyl,
.beta.-tryptophanyl, .beta.-methioninyl, .beta.-glycinyl,
.beta.-serinyl, .beta.-threoninyl, .beta.-cysteinyl,
.beta.-tyrosinyl, .beta.-asparaginyl, .beta.-glutaminyl,
.beta.-aspartoyl, .beta.-glutaroyl, .beta.-lysinyl,
.beta.-argininyl or .beta.-histidinyl.
[0110] As used herein, the term "substantially free of" or
"substantially in the absence of" with respect to a nucleoside
composition includes a nucleoside composition that includes at
least 85 or 90% by weight, preferably 95%, 98%, 99% or 100% by
weight, of the designated enantiomer of that nucleoside. In a
preferred embodiment, in the methods and compounds of this
invention, the compounds are substantially free of enantiomers.
[0111] Similarly, the term "isolated" with respect to a nucleoside
composition includes a nucleoside composition that includes at
least 85, 90%, 95%, 98%, 99% to 100% by weight, of the nucleoside,
the remainder comprising other chemical species or enantiomers.
[0112] "Solvate" includes a compound provided herein or a salt
thereof, that further includes a stoichiometric or
non-stoichiometric amount of solvent bound by non-covalent
intermolecular forces. Where the solvent is water, the solvate is a
hydrate.
[0113] As used herein, the terms "subject" and "patient" are used
interchangeably herein. The terms "subject" and "subjects" refer to
an animal, such as a mammal including a non-primate (e.g., a cow,
pig, horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey
such as a cynomolgous monkey, a chimpanzee and a human), and for
example, a human. In one embodiment, the subject is refractory or
non-responsive to current treatments for cancer. In another
embodiment, the subject is a farm animal (e.g., a horse, a cow, a
pig, etc.) or a pet (e.g., a dog or a cat). In one embodiment, the
subject is a human.
[0114] As used herein, the terms "therapeutic agent" and
"therapeutic agents" refer to any agent(s) which can be used in the
treatment or prevention of a disorder or one or more symptoms
thereof. In certain embodiments, the term "therapeutic agent"
includes a compound provided herein. In one embodiment, a
therapeutic agent is an agent which is known to be useful for, or
has been or is currently being used for the treatment or prevention
of a disorder or one or more symptoms thereof.
[0115] "Therapeutically effective amount" includes an amount of a
compound or composition that, when administered to a subject for
treating a disease, is sufficient to effect such treatment for the
disease. A "therapeutically effective amount" can vary depending
on, inter alia, the compound, the disease and its severity, and the
age, weight, etc., of the subject to be treated.
[0116] "Treating" or "treatment" of any disease or disorder refers,
in one embodiment, to ameliorating a disease or disorder that
exists in a subject. In another embodiment, "treating" or
"treatment" includes ameliorating at least one physical parameter,
which may be indiscernible by the subject. In yet another
embodiment, "treating" or "treatment" includes modulating the
disease or disorder, either physically (e.g., stabilization of a
discernible symptom) or physiologically (e.g., stabilization of a
physical parameter) or both. In yet another embodiment, "treating"
or "treatment" includes delaying the onset of the disease or
disorder.
[0117] As used herein, the terms "prophylactic agent" and
"prophylactic agents" as used refer to any agent(s) which can be
used in the prevention of a disorder or one or more symptoms
thereof. In certain embodiments, the term "prophylactic agent"
includes a compound provided herein. In certain other embodiments,
the term "prophylactic agent" does not refer a compound provided
herein. For example, a prophylactic agent is an agent which is
known to be useful for, or has been or is currently being used to
the prevent or impede the onset, development, progression and/or
severity of a disorder.
[0118] As used herein, the phrase "prophylactically effective
amount" includes the amount of a therapy (e.g., prophylactic agent)
which is sufficient to result in the prevention or reduction of the
development, recurrence or onset of one or more symptoms associated
with a disorder (, or to enhance or improve the prophylactic
effect(s) of another therapy (e.g., another prophylactic
agent).
6.2 Exemplary Embodiments
[0119] 6.2.1 Compounds
[0120] Phosphoroamidate and phosphonoamidate compounds of a variety
of therapeutic agents can be formed using methods available in the
art and those disclosed herein. Such compounds can be used in some
embodiments to enhance delivery of the drug to the liver. In one
embodiment, the compound is an S-acyl-2-thioethyl phosphoroamidate
or an S-acyl-2-thioethyl phosphonoamidate derivative, e.g., a
S-pivaloyl-2-thioethyl phosphoroamidate or a
S-hydroxypivaloyl-2-thioethyl phosphonoamidate. Therapeutic agents
that can be derivatized to compound form include an anti-cancer
agent that includes, or has been derivatized to include a reactive
group for attachment of the phosphoroamidate or phosphonoamidate
moiety, including but not limited to nucleosides and nucleoside
analogues including acyclic nucleosides.
[0121] Phosphoroamidate or phosphonoamidate compound forms of a
variety of nucleosides can be formed from nucleosides disclosed
herein and available in the art. In particular, anti-cancer
nucleosides can be derivatized to form a phosphoroamidate or
phosphonoamidate compound that can enhance delivery to the
liver.
[0122] In one embodiment, the phosphoroamidate or phosphonoamidate
compound provided herein is a compound of formula IIa or IIb:
##STR00011##
or a pharmaceutically acceptable salt, solvate, a stereoisomeric,
tautomeric or polymorphic form thereof, wherein;
[0123] R.sup.y is alkyl, alkenyl, alkynyl, aryl, arylalkyl,
cycloalkyl, cycloalkenyl, amino, heterocyclyl or heteroaryl, all
optionally substituted;
[0124] R.sup.a and R.sup.b are selected as follows:
[0125] i) R.sup.a and R.sup.b are each independently hydrogen,
alkyl, carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl,
aminocarbonylalkyl, alkoxycarbonylalkyl, aryl, arylalkyl,
cycloalkyl, heteroaryl or heterocyclyl, all optionally substituted;
or
[0126] ii) R.sup.a and R.sup.b together with the nitrogen atom on
which they are substituted form a 3-7 membered heterocyclic or
heteroaryl ring; and
[0127] R.sup.1 is a drug such as an anti-cancer drug.
[0128] In certain embodiments, the compound of formula IIa or IIb
is selected with a proviso that when R.sup.y is tert-butyl or
hydroxy-tert-butyl, then R.sup.1 is not
3'-azido-2',3'-dideoxythymidine.
[0129] In certain embodiments, R.sup.1, R.sup.a, R.sup.b and
R.sup.y are optionally substituted with one or more substituents as
defined herein, e.g., in the definitions.
[0130] In certain embodiments, the compounds are of Formula IIa or
IIb, wherein R.sup.y is alkyl, alkenyl, alkynyl, aryl, arylalkyl,
cycloalkyl, cycloalkenyl, amino, heterocyclyl or heteroaryl;
[0131] R.sup.a and R.sup.b are each independently hydrogen, alkyl,
carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl,
aminocarbonylalkyl, alkoxycarbonylalkyl, aryl, arylalkyl,
cycloalkyl, heteroaryl or heterocyclyl, all optionally substituted;
and
[0132] R.sup.1 is an anti-cancer drug.
[0133] In one embodiment, R.sup.1 or R.sup.1--CH.sub.2-- is a
nucleoside comprising a cyclic or acyclic sugar or analog thereof,
including any nucleoside or analogue thereof described herein or
known in the art.
[0134] In certain embodiments of Formula IIa or IIb, R.sup.y is
substituted alkyl, e.g. hydroxyalkyl or aminoalkyl; and R.sup.a and
R.sup.b are independently hydrogen, alkyl, substituted alkyl,
benzyl or substituted benzyl, for instance hydroxy- or
amino-substituted alkyl or benzyl. In another embodiment, R.sup.y
is --OR.sup.c, --C(R.sup.c).sub.3 or --NHR.sup.c where each R.sup.c
is independently alkyl, substituted alkyl, aryl or substituted
aryl, for instance hydroxy- or amino-substituted alkyl or aryl; and
R.sup.a and R.sup.b are independently hydrogen, alkyl, substituted
alkyl, benzyl or substituted benzyl, for instance hydroxy- or
amino-substituted alkyl or benzyl. In a further embodiment, R.sup.a
and R.sup.b are independently benzyl or substituted alkyl. In a
further embodiment, R.sup.y is selected from the group consisting
of alkyl and hydroxyalkyl. In certain embodiments, R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH. In certain embodiments according to
this paragraph, R.sup.2 and R.sup.3 are each hydrogen, R.sup.a is
hydrogen, R.sup.b is --CH.sub.2--C.sub.6H.sub.5 and R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH.
[0135] In one embodiment, R.sup.y is alkyl or hydroxyalkyl. In one
embodiment, R.sup.y is methyl, tert-butyl, hydroxy-tert-butyl or
hydroxyethyl. In certain embodiments, R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH.
[0136] In one embodiment, R.sup.a and R.sup.b are each
independently hydrogen, alkyl, carboxyalkyl, hydroxyalkyl,
hydroxyarylalkyl, acyloxyalkyl, aminocarbonylalkyl,
alkoxycarbonylalkyl, aryl or arylalkyl, wherein the alkyl groups
can be further substituted with one or more substitutents. In one
embodiment, at least one of R.sup.a or R.sup.b is other than
hydrogen. In one embodiment, R.sup.a and R.sup.b are each
independently hydrogen, methyl or benzyl.
[0137] In certain embodiments, R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH and R.sup.a and R.sup.b are each
independently hydrogen, methyl or benzyl. In certain embodiments,
R.sup.y is --C(CH.sub.3).sub.2CH.sub.2OH and R.sup.a is hydrogen
and R.sup.b is benzyl.
[0138] In another embodiment, the compound provided herein is a
compound of formula:
##STR00012##
wherein R.sup.1 and R.sup.y are as defined in formula IIa or IIb.
In one embodiment, R.sup.y is alkyl or hydroxyalkyl. In one
embodiment, R.sup.y is methyl, tert-butyl, hydroxy-tert-butyl or
hydroxyethyl. In one embodiment, R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH.
[0139] In certain embodiments according to formula XVa or XVb,
R.sup.y is substituted alkyl, e.g. hydroxyalkyl or aminoalkyl. In
another embodiment, R.sup.y is --OR.sup.c, --C(R.sup.c).sub.3 or
--NHR.sup.c where each R.sup.c is independently alkyl, substituted
alkyl, aryl or substituted aryl, for instance hydroxy- or
amino-substituted alkyl or aryl. In a further embodiment, R.sup.y
is selected from the group consisting of alkyl and hydroxyalkyl. In
certain embodiments, R.sup.y is --C(CH.sub.3).sub.2CH.sub.2OH.
[0140] In another embodiment, the compound provided herein is a
compound of formula:
##STR00013##
Wherein:
[0141] R.sup.1 is an anti-cancer drug, such as a nucleoside or
nucleoside derivative; and
[0142] R.sup.a and R.sup.b are each independently hydrogen, alkyl,
carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl,
aminocarbonylalkyl, alkoxycarbonylalkyl, aryl, arylalkyl,
cycloalkyl, heteroaryl or heterocyclyl, all optionally substituted;
and
[0143] wherein in one embodiment, one of R.sup.a and R.sup.b is H
and the other is alkyl optionally substituted with aryl, benzyl, or
heteroaryl, each optionally substituted.
[0144] In certain embodiments according to formula XVIa or XVIb,
R.sup.a and R.sup.b are independently hydrogen, alkyl, substituted
alkyl, benzyl or substituted benzyl, for instance hydroxy- or
amino-substituted alkyl or benzyl. In another embodiment, R.sup.a
and R.sup.b are independently hydrogen, alkyl, substituted alkyl,
benzyl or substituted benzyl, for instance hydroxy- or
amino-substituted alkyl or benzyl. In a further embodiment, R.sup.a
and R.sup.b are independently benzyl or substituted alkyl.
[0145] In another embodiment, the compound provided herein is a
compound of formula:
##STR00014##
wherein R.sup.1 is a drug such as an anti-cancer drug.
[0146] Exemplary anti-cancer drugs (R.sup.1's) that can be
derivatized as described herein, for example via a free hydroxyl
group, or after adding a hydroxylated linker, are:
TABLE-US-00001 Name Structure Aclarubicin ##STR00015## Decitabine
##STR00016## Daunorubicin ##STR00017## 5-azacytidine ##STR00018##
Doxorubicin ##STR00019## Epirubicin ##STR00020## Estramustine
##STR00021## Etoposide ##STR00022## Fludarabine ##STR00023##
Neplanocin A ##STR00024##
Tezacitabine([(E)-2'-deoxy-2'-(fluoromethylene)cytidine(FMdC)])
##STR00025## Troxacitabine((-)-2'-Deoxy-3'-oxacytidine)
##STR00026## Vinblastin ##STR00027## Vincristin ##STR00028##
Vindesin ##STR00029## Teniposide ##STR00030## NK-611 ##STR00031##
Camptothecin ##STR00032## Irinotecan ##STR00033##
9-Aminocamptothecin ##STR00034## Topotecan ##STR00035## Paclitaxel
##STR00036## Azatoxin ##STR00037## Coformycin ##STR00038##
Pirarubicin ##STR00039## Nelarabine ##STR00040## Losoxantrone
##STR00041## Fluxoridine ##STR00042## Mitomycin C ##STR00043##
Erlotinib (Tarceva .RTM.) ##STR00044## Thalidomide ##STR00045##
[0147] Exemplary immunosupressant drugs that can be derivatized as
described herein are:
TABLE-US-00002 Mitoxantrone ##STR00046## CombretastatinA-4
##STR00047## Mycophenolicacid ##STR00048## Pentostatin
##STR00049##
[0148] In certain embodiments, R.sup.1 is an anti-cancer drug such
as aclarubicin, decitabine, daunorubicin, dihydro-5-azacytidine,
doxorubicin, epirubicin, estramustin, etoposide, fludarabine,
7-hydroxychlorpromazin, neplanocin A, podophyllotoxin,
tezacitabine, troxacitabine, vinblastin, vincristin, vindesin,
etoposide, teniposide, NK-611, camptothecin, irinotecan,
9-aminocamptothecin, GG-211, topotecan, paclitaxel, azatoxin,
coformycin, pirarubicin and losoxantrone. In a particular
embodiment, the anti-cancer drug is camptothecin or azotoxin.
[0149] In another embodiment, R.sup.1 is a purine nucleoside analog
(see, e.g., Robak et al., Curr. Med. Chem. 2006, 13, 3165-3189).
R.sup.1 is, for example, a cytotoxic agent such as fludarabine
(9-.beta.-D-arabinofuranosyl-2-fluoradenine), cladribine
(2-chloro-2'-deoxyadenosine, CldA), pentostatin
(2'-deoxycoformycin, DCF), clofarabine (CAFdA), nelabarine,
immucillin H (BCX-1777, forodesine) or 8-chloroadenosine
(8-Cl-Ado).
[0150] The anti-cancer drug also can be
(2'S)-2'-deoxy-2'-C-methylcytidine (SMDC),
1-(2-deoxy-2-methylene-.beta.-D-erythro-pentofuranosyl)cytosine
(DMDC),
1-(2-C-cyano-2-deoxy-1-.beta.-D-arabino-pentofuranosyl)cytosine
(CNDAC) or 1-(3-C-ethynyl-.beta.-D-ribo-pentofuranosyl)cytosine
(ECyd). See, e.g., Matsuda et al., Cancer Sci, 2004,
95:105-111.
[0151] In one embodiment, R.sup.1 is an immunosuppressant, such as
combretastatin A-4, mycophenolic acid, pentostatin or
mitoxantrone.
[0152] The anti-cancer drug can be derivatized to include the
phosphoroamidate or phosphonoamidate at, e.g., a free OH or free
carboxy group.
[0153] In another embodiment, R.sup.1 is
(-)-2'-Deoxy-3'-oxacytidine (BCH-4556, Troxacitabine):
##STR00050##
[0154] In another embodiment, R.sup.1 is tezacitabine
(2'-fluoromethylene-2'-deoxycytidine). In another embodiment,
R.sup.1 is a 5'-aza-pyrimidine, such as 5'-aza-cytidine,
5'-azadeoxycytidine (decytabine), or fazarabine.
[0155] In certain embodiments, R.sup.1 is a
2'-deoxy-2'-methylidenepyrimidine nucleoside compound, disclosed,
e.g. in U.S. Pat. No. 5,401,726, such as
2'-deoxy-2'-methylidene-5-fluorocytidine,
2'-deoxy-2'-methylidene-5-chlorocytidine,
2'-deoxy-2'-methylidene-5-bromocytidine,
2'-deoxy-2'-methylidene-5-iodocytidine,
2'-deoxy-2'-methylidene-5-methylcytidine,
2'-deoxy-2'-methylidene-5-ethylcytidine,
2'-deoxy-2'-methylidene-5-ethyluridine,
2'-deoxy-2'-methylidene-5-ethynyluridine or
2'-deoxy-2'-methylidene-5-fluorocytidine-5'-phosphoric acid. In one
embodiment, R.sup.1 is 5-fluorouracil.
[0156] R.sup.1 also can be a pyrido[2,3-D]pyrimidine or
pyrimido[4,5-D]pyrimidine nucleoside as described in U.S. Pat. No.
7,081,449, such as
4-amino-5-oxo-8-(4-C-hydroxymethyl-.beta.-D-ribofuranosyl)pyrido-[2,3d]py-
rimidine;
4-amino-5-oxo-8-(5(R)--C-methyl-.beta.-D-ribofuranosyl)pyrido[2,-
3-d]pyrimidine;
4-amino-5-oxo-8-(5(R)--C-allyl-.beta.-D-ribofuranosyl)pyrido[2,3-d]pyrimi-
dine;
4-amino-5-oxo-8-(5(R,S)--C-ethynyl-.beta.-D-ribofuranosyl)pyrido[2,3-
-d]pyrimidine;
4-Amino-5-oxo-8-(5(R,S)--C-vinyl-.beta.-D-ribofuranosyl)pyrido[2,3-d]pyri-
midine;
4-Amino-5-oxo-8-(.beta.-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;
4-amino-5-oxo-8-(.beta.-L-ribofuranosyl)pyrido[2,3-d]pyrimidine;
4-amino-5-oxo-8-(4-C-methyl-.beta.-D-ribofuranosyl)pyrido[2,3-d]pyrimidin-
e;
4-amino-5-oxo-8-(4-C-ethyl-.beta.-D-ribofuranosyl)pyrido[2,3-d]pyrimidi-
ne;
4-amino-5-oxo-8-(5(R,S)--C-ethyl-.beta.-D-ribofuranosyl)pyrido-[2,3d]p-
yrimidine;
4-amino-5-oxo-8-(5(R)--C-propyl-.beta.-D-ribofuranosyl)pyrido[2-
,3-d]pyrimidine; or
4-amino-5-oxo-8-(2-deoxy-.beta.-D-ribofuranosyl)pyrido[2,3-d]pyrimidine.
In a particular embodiment, R.sup.1 is
4-amino-5-oxo-8-(.beta.-D-ribofuranosyl)pyrido[2,3-d]pyrimidine.
[0157] Other anti-cancer drugs include: dichloroacetal, Nexavar
(sorafenib), cimetidine, adriamycin, Cytoxan (cyclophosphamide),
methotrexate, vincristine, and 6-mercaptopurine.
[0158] In another embodiment, the anti-cancer drug is selected from
2',3-dideoxyinosine (ddl), or 2,3-didehydro-3-deoxythymidine (d4T).
See, e.g., WO/2006/125166.
[0159] In one embodiment, R.sup.1 is an anti-inflammatory drug,
such as a corticosteroid or a non-steroidal anti-inflammatory drugs
(NSAID) that can be derivatized to include the phosphoroamidate or
phosphonoamidate at, e.g., a free OH or free carboxy group.
[0160] Exemplary corticosteroid drugs suitable for use herein are
provided below:
##STR00051## ##STR00052##
[0161] Exemplary NSAID drugs suitable for use herein are provided
below:
Sodium salicylate Salicylsalicylic acid
##STR00053## ##STR00054##
[0162] In certain embodiments of the compounds of Formula IIa
below:
##STR00055##
[0163] the moiety:
##STR00056##
[0164] is derived from a drug that is an acyclic nucleoside
phosphonate, i.e.:
##STR00057##
[0165] Thus, compounds of Formula IIa, in one embodiment, are
phosphonoamidates of an acyclic nucleoside phosphonate that have
potential anti-cancer activity, such as
(S)-9-[3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine (HPMPC,
cidofovir), (S)-9-{3-hydroxy-2-(phosphonomethoxy)-propyl]adenine
((S)-HPMPA), phosphonomethoxyethylguanine (PMEG),
phosphonomethoxyethyl-adenine (PMEA, adefovir),
phosphonomethoxy-propyladenine (PMPA, tenofovir), acyclovir,
ganciclovir or penciclovir. See e.g., WO/2006/125166 and De Clercq
et al., Antiviral Research, Volume 75, Issue 1, July 2007, Pages
1-13.
Embodiments for Delivery of Thyroid Hormone Receptor Effectors
[0166] In certain embodiments, the following phosphoroamidate and
phosphonoamidate formulas and compounds are provided, which
optionally act as thyroid hormone receptor effectors:
##STR00058## ##STR00059## ##STR00060##
wherein
[0167] each R, if present, is independently alkyl, halogen or
hydroxyl;
[0168] X, if present, is CH.sub.2, O or S;
[0169] R.sup.y is alkyl, alkenyl, alkynyl, aryl, aryl alkyl,
cycloalkyl, cycloalkenyl, amino, aminoalkyl, heterocyclyl or
heteroaryl, all optionally substituted;
[0170] R.sup.a and R.sup.b are selected as follows:
[0171] i) R.sup.a and R.sup.b are each independently hydrogen,
alkyl, carboxyalkyl, hydroxyalkyl, hydroxyarylalkyl, acyloxyalkyl,
aminocarbonylalkyl, alkoxycarbonylalkyl, aryl, aryl alkyl,
cycloalkyl, heteroaryl or heterocyclyl, all optionally substituted;
or
[0172] ii) R.sup.a and R.sup.b together with the nitrogen atom on
which they are substituted form a 3-7 membered heterocyclic or
heteroaryl ring.
[0173] In certain embodiments, the compound provided herein has
formula selected from IIIa, IIIb, IVa, IVb, Va, Vb, VIa, VIb, VIIa,
VIIb, VIIIa or VIIIb, wherein
[0174] each R, if present, is independently alkyl, halogen or
hydroxyl;
[0175] X, if present, is CH.sub.2, O or S;
[0176] R.sup.y, if present, is optionally substituted alkyl,
wherein the substituted alkyl is optionally hydroxyalkyl or
aminoalkyl, e.g., --C(CH.sub.3).sub.2CH.sub.2OH; and
[0177] R.sup.a and R.sup.b, if present, are independently hydrogen;
unsubstituted alkyl; or alkyl substituted with aryl, amino, amido,
hydroxyl, alkoxy, aminoalkyl, hydroxyalkyl, aryl, or heteroaryl,
each optionally substituted; wherein, in one embodiment, R.sup.a
and R.sup.b are independently H or a benzyl that is optionally
substituted, for example, with hydroxy or amino; and [0178]
wherein, in another embodiment, if present, R.sup.a is hydrogen,
R.sup.b is --CH.sub.2--C.sub.6H.sub.5 and R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH.
[0179] In certain embodiments, the compound provided herein has a
formula selected from:
##STR00061##
wherein
[0180] R.sup.x and R.sup.z are each independently hydrogen or
alkyl;
[0181] R.sup.w is alkyl;
[0182] X.sup.1 is O or S;
[0183] R.sup.y is optionally substituted alkyl, wherein the
substituents when present are selected from hydroxy and amino;
[0184] R.sup.a and R.sup.b are each independently hydrogen or
optionally substituted alkyl; where the substituents when present
are selected from one or more, in one embodiment, one, two or three
groups selected from aryl, amino, amido, hydroxyl, alkoxy, aryl and
heteroaryl, each optionally substituted with hydroxy or amino.
[0185] In certain embodiments, the compound provided herein is
selected from:
##STR00062## ##STR00063##
[0186] In certain embodiments, R.sup.x and R.sup.z are each
hydrogen. In certain embodiments, R.sup.w is alkyl. In certain
embodiments, R.sup.w is isopropyl. In certain embodiments, R.sup.y
is optionally substituted alkyl, wherein the substituents when
present are selected from hydroxy and amino. In certain
embodiments, R.sup.y is --C(CH.sub.3).sub.2CH.sub.2OH. In certain
embodiments, R.sup.a and R.sup.b are each independently hydrogen or
optionally substituted alkyl; where the substituents when present
are selected from one or more, in one embodiment, one, two or three
groups selected from aryl, amino, amido, hydroxyl, alkoxy, aryl and
heteroaryl, each optionally substituted with hydroxy or amino. In
certain embodiments, R.sup.a is hydrogen and R.sup.b is benzyl.
[0187] In certain embodiments, R.sup.a is hydrogen, R.sup.b is
--CH.sub.2--C.sub.6H.sub.5 and R.sup.y is
--C(CH.sub.3).sub.2CH.sub.2OH.
[0188] In one embodiment, the thyroid receptor effector compound
has formula:
##STR00064##
[0189] In certain embodiments, the compound or Formula selected
from IIIa or b, IVa or b, Va or b, VIa or b, VIIa or b, VIII a or b
is derived from a phosphonate compound useful for inhibiting
gluconeogenesis, optionally by inhibiting the enzyme fructose
1,6-bisphosphatase (FBPase).
[0190] In certain embodiments, the compound or Formula selected
from IXa or b, X a or b, XIa or b, XIIa or b, XIIIa or b, XIVa or b
and XVIIIa or b is derived from a compound useful for inhibiting
gluconeogenesis, optionally by inhibiting the enzyme fructose
1,6-bisphosphatase (FBPase).
[0191] In certain embodiments, the compound or Formula selected
from IIIa or b, IVa or b, Va or b, VIa or b, VIIa or b, VIII a or b
is a phosphonic acid-containing compound that binds to a thyroid
receptor in the liver, and is optionally an agonist, antagonist,
partial agonist or partial antagonist of T3. Inhibition of
gluconeogenesis can result in blood glucose lowering in diabetic
subjects. Such compounds can exhibit enhanced pharmacokinetics
including oral bioavailability and liver drug levels.
[0192] In certain embodiments, provided is a method of treatment of
a subject in need thereof, the method comprising administering to
the subject a phosphoroamidate and phosphonoamidate compound or
formula selected from IIIa or b, IVa or b, Va or b, VIa or b, VIIa
or b, VIII a or b, IXa or b, X a or b, XIa or b, XIIa or b, XIIIa
or b, XIVa or b and XVIIIa or b or a pharmaceutically acceptable
salt, enantiomer, ester or prodrug thereof thereof, in an amount
effective for one or more of the following:
[0193] reducing plasma lipid levels, lowering cholesterol levels,
reducing triglyceride levels, or increasing the ratio of HDL to
LDL;
[0194] lowering blood glucose levels;
[0195] treating hyperlipidemia or hypercholesterolemia;
[0196] treating obesity, reducing fat content, treating fatty
liver, reducing weight or preventing weight gain;
[0197] treating atherosclerosis, coronary heart disease, heart
failure, nephrotic syndrome, or chronic renal failure;
[0198] lowering blood glucose levels, treating diabetes, impaired
glucose tolerance, metabolic syndrome x, insulin resistance or
hyperinsulinemia;
[0199] increasing levels of genes associated with
gluconeogenesis;
[0200] decreasing hepatic glycogen levels or maintaining or
improving glycemic control;
[0201] amelioration of hyperinsulinemia and/or decrease of glucose
levels in diabetic subjects at doses that optionally do not affect
cardiac function, e.g., heart rate, force of systolic contraction,
duration of diastolic relaxation, vascular tone, or heart
weight;
[0202] treating thyroid disease, thyroid cancer, depression,
glaucoma, cardiac arrhythmias, heart failure, or osteoporosis;
[0203] increasing mitochondrial biogenesis, or increasing
expression of PGC-1, AMP activated protein kinase or nuclear
respiratory factor;
[0204] inhibiting hepatic gluconeogenesis; or
[0205] modulating expression of certain genes in the liver
resulting in effects on lipids (e.g., cholesterol), glucose,
lipoproteins, and triglycerides, or modulation of T3-responsive
genes.
[0206] In certain embodiments, the compounds do not affect thyroid
function, thyroid production of circulating iodinated thyronines
such as T3 and T4, and/or the ratio of T3 to T4.
[0207] In certain embodiments, provided herein is a method for
treatment of liver fibrosis or inflammation by administering a
compound provided herein.
[0208] Also provided are pharmaceutical compositions comprising the
compounds, e.g., in a dosage unit suitable for administration,
e.g., oral administration.
[0209] Optically Active Compounds
[0210] It is appreciated that compounds provided herein have
several chiral centers and may exist in and be isolated in
optically active and racemic forms. Some compounds may exhibit
polymorphism. It is to be understood that any racemic,
optically-active, diastereomeric, polymorphic, or stereoisomeric
form, or mixtures thereof, of a compound provided herein, which
possess the useful properties described herein is within the scope
of the invention. Techniques known in the art can be used to
prepare optically active forms (for example, by resolution of the
racemic form by recrystallization techniques, by synthesis from
optically-active starting materials, by chiral synthesis, or by
chromatographic separation using a chiral stationary phase).
[0211] Examples of methods to obtain optically active materials are
known in the art, and include at least the following. [0212] i)
physical separation of crystals--a technique whereby macroscopic
crystals of the individual enantiomers are manually separated. This
technique can be used if crystals of the separate enantiomers
exist, i.e., the material is a conglomerate, and the crystals are
visually distinct; [0213] ii) simultaneous crystallization--a
technique whereby the individual enantiomers are separately
crystallized from a solution of the racemate, possible only if the
latter is a conglomerate in the solid state; [0214] iii) enzymatic
resolutions--a technique whereby partial or complete separation of
a racemate by virtue of differing rates of reaction for the
enantiomers with an enzyme; [0215] iv) enzymatic asymmetric
synthesis--a synthetic technique whereby at least one step of the
synthesis uses an enzymatic reaction to obtain an enantiomerically
pure or enriched synthetic precursor of the desired enantiomer;
[0216] v) chemical asymmetric synthesis--a synthetic technique
whereby the desired enantiomer is synthesized from an achiral
precursor under conditions that produce asymmetry (i.e., chirality)
in the product, which may be achieved using chiral catalysts or
chiral auxiliaries; [0217] vi) diastereomer separations--a
technique whereby a racemic compound is reacted with an
enantiomerically pure reagent (the chiral auxiliary) that converts
the individual enantiomers to diastereomers. The resulting
diastereomers are then separated by chromatography or
crystallization by virtue of their now more distinct structural
differences and the chiral auxiliary later removed to obtain the
desired enantiomer; [0218] vii) first- and second-order asymmetric
transformations--a technique whereby diastereomers from the
racemate equilibrate to yield a preponderance in solution of the
diastereomer from the desired enantiomer or where preferential
crystallization of the diastereomer from the desired enantiomer
perturbs the equilibrium such that eventually in principle all the
material is converted to the crystalline diastereomer from the
desired enantiomer. The desired enantiomer is then released from
the diastereomer; [0219] viii) kinetic resolutions--this technique
refers to the achievement of partial or complete resolution of a
racemate (or of a further resolution of a partially resolved
compound) by virtue of unequal reaction rates of the enantiomers
with a chiral, non-racemic reagent or catalyst under kinetic
conditions; [0220] ix) enantiospecific synthesis from non-racemic
precursors--a synthetic technique whereby the desired enantiomer is
obtained from non-chiral starting materials and where the
stereochemical integrity is not or is only minimally compromised
over the course of the synthesis; [0221] x) chiral liquid
chromatography--a technique whereby the enantiomers of a racemate
are separated in a liquid mobile phase by virtue of their differing
interactions with a stationary phase. The stationary phase can be
made of chiral material or the mobile phase can contain an
additional chiral material to provoke the differing interactions;
[0222] xi) chiral gas chromatography--a technique whereby the
racemate is volatilized and enantiomers are separated by virtue of
their differing interactions in the gaseous mobile phase with a
column containing a fixed non-racemic chiral adsorbent phase;
[0223] xii) extraction with chiral solvents--a technique whereby
the enantiomers are separated by virtue of preferential dissolution
of one enantiomer into a particular chiral solvent; [0224] xiii)
transport across chiral membranes--a technique whereby a racemate
is placed in contact with a thin membrane barrier. The barrier
typically separates two miscible fluids, one containing the
racemate, and a driving force such as concentration or pressure
differential causes preferential transport across the membrane
barrier. Separation occurs as a result of the non-racemic chiral
nature of the membrane which allows only one enantiomer of the
racemate to pass through.
Preparation of Compounds
[0225] The compounds provided herein can be prepared, isolated or
obtained by any method apparent to those of skill in the art.
Exemplary methods of preparation are described in detail in the
examples below.
[0226] In certain embodiments, compounds provided herein can be
prepared by coupling alcohols and H-phosphonate monoesters as
illustrated in the reaction scheme below:
##STR00065##
wherein R.sup.7, R.sup.8, R.sup.9, R.sup.10 are each independently
hydrogen, hydroxy, alkyl or alkoxy. Any reactive function on
R.sup.y, R.sup.7, R.sup.8, R.sup.9, R.sup.10 or on the base should
be protected during the coupling reaction. Any coupling agent known
to one of skill in the art can be used. Exemplary coupling agents
for use in the reaction include, but are not limited to HOBt
(N-Hydroxybenzotriazole), HBTU
(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate), DCC (N,N'-dicyclohexylcarbodiimide), BOP
(Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophospha-
te), PyBOP (1H-benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate) and others known to one of skill in the
art.
[0227] A general scheme for the synthesis of hydroxytBuSATE
N-benzylphosphoramidate nucleoside derivatives represented by B is
provided in Schemes B1-B3 below, in which modifications of
nucleosides are made by way of example, but the methodology may be
used for other active agents as well.
##STR00066##
where R=H, Tr, MMTr or DMTr in case of reactive amine; R.sup.1,
R.sup.2, R.sup.4, R.sup.6=H, alkyl or halo and R.sup.3/R.sup.5 are
both H or isopropylidene.
##STR00067##
##STR00068##
##STR00069##
[0228] In addition, certain nucleosides and analogs thereof and
prodrugs thereof can be prepared according to methods known to one
of skill in the art. Exemplary nucleosides and analogs are
described in International Publication No. WO 06/125166, contents
of which are hereby incorporated by reference in their
entireties.
[0229] The compounds of formula IIIa or b, IVa or b, Va or b, VIa
or b, VIIa or b and VIII a or b can be prepared by methods
described herein and methods known to one of skill in the art, for
example, see, Erion et al., Proc. Natl. Acad. Sci., 2007, 104,
15490-15495.
[0230] The compounds of formula IXa or b, X a or b, XIa or b, XIIa
or b, XIIIa or b, XIVa or b and XVIIIa or b can be prepared by
methods described herein and methods known to one of skill in the
art, for example, see, Dang et al., Discovery of Potent and
Specific Fructose-1,6-Bisphosphatase Inhibitors and a Series of
orally-Bioavailable Phosphoramidase-Sensitive Prodrugs for the
Treatment of Type 2 Diabetes, J. Am. Chem. Soc., 2007, Vol. 129,
No. 50, pp. 15491-502.
[0231] Assay Methods
[0232] Compounds can be assayed for accumulation in liver cells of
a subject according to any assay known to those of skill in the
art. In certain embodiments, a compound can be administered to the
subject, and a liver cell of the subject can be assayed for the
compound or a derivative thereof.
[0233] In one embodiment, a phosphoroamidate or phosphonoamidate
nucleoside compound is administered to cells, such as liver cells,
in vivo or in vitro, and the levels delivered intracellularly are
measured, to indicate delivery of the compound in the cell.
[0234] Assays for other activities, including anti-cancer activity
can be done as described in the art. Suitable in vitro assays can
be used to preliminarily evaluate the efficacy of a compound in
inhibiting growth of cancer cells. The compound can further be
examined for its efficacy in treating cancer by in vivo assays
known to those of skill in the art. For example, it can be
administered to an animal (e.g., a mouse model) having cancer and
its therapeutic effect can then assessed. Based on the results, an
appropriate dosage range and administration route can also be
determined. Exemplary assays are described in the paragraphs
below.
[0235] Anti-Cancer Activity
[0236] Compounds provided herein can be shown to inhibit tumor cell
proliferation, cell transformation and tumorigenesis in vitro and
in vivo using a variety of assays known in the art, or described
herein. Such assays can use cells of a cancer cell line, or cells
from a patient. Many assays well-known in the art can be used to
assess such survival and/or growth; for example, cell proliferation
can be assayed by measuring (.sup.3H)-thymidine incorporation, by
direct cell count, by detecting changes in transcription,
translation or activity of known genes such as proto-oncogenes
(e.g., fos, myc) or cell cycle markers (Rb, cdc2, cyclin A, D1, D2,
D3, E, etc.). The levels of such protein and mRNA and activity can
be determined by any method well known in the art. For example,
protein can be quantitated by known immunodiagnostic methods such
as Western blotting or immunoprecipitation using commercially
available antibodies (for example, many cell cycle marker
antibodies are available from Santa Cruz Biotechnology, Inc., Santa
Cruz, Calif.). mRNA can be quantitated by methods that are well
known and routine in the art, for example, by Northern analysis,
RNase protection, and the polymerase chain reaction in connection
with the reverse transcription. Cell viability can be assessed by
using trypan-blue staining or other cell death or viability markers
known in the art. Differentiation can be assessed, for example,
visually based on changes in morphology, etc.
[0237] Cell proliferation analysis can be performed using a variety
of techniques known in the art, including but not limited to the
following:
[0238] As one example, bromodeoxyuridine (BRDU) incorporation may
be used as an assay to identify proliferating cells. The BRDU assay
identifies a cell population undergoing DNA synthesis by
incorporation of BRDU into newly synthesized DNA. Newly synthesized
DNA can then be detected using an anti-BRDU antibody (see Hoshino
et al., 1986, Int. J. Cancer 38, 369; Campana et al., 1988, J.
Immunol. Meth. 107, 79).
[0239] Cell proliferation can also be examined using (3H)-thymidine
incorporation (see e.g., Chen, J., 1996, Oncogene 13:1395-403;
Jeoung, J., 1995, J. Biol. Chem. 270:18367-73). This assay allows
for quantitative characterization of S-phase DNA synthesis. In this
assay, cells synthesizing DNA incorporate (3H)-thymidine into newly
synthesized DNA. Incorporation can then be measured using standard
techniques in the art such as by counting of radioisotope in a
Scintillation counter (e.g. Beckman LS 3800 Liquid Scintillation
Counter).
[0240] Detection of proliferating cell nuclear antigen (PCNA) can
also be used to measure cell proliferation. PCNA is a 36 kilodalton
protein whose expression is elevated in proliferating cells,
particularly in early G1 and S phases of the cell cycle and
therefore can serve as a marker for proliferating cells. Positive
cells are identified by immunostaining using an anti-PCNA antibody
(see Li et al., 1996, Curr. Biol. 6:189-199; Vassilev et al., 1995,
J. Cell Sci. 108:1205-15).
[0241] Cell proliferation can be measured by counting samples of a
cell population over time (e.g. daily cell counts). Cells may be
counted using a hemacytometer and light microscopy (e.g. HyLite
hemacytometer, Hausser Scientific). Cell number may be plotted
against time in order to obtain a growth curve for the population
of interest. In a preferred embodiment, cells counted by this
method are first mixed with the dye Trypan-blue, such that living
cells exclude the dye, and are counted as viable members of the
population.
[0242] DNA content and/or mitotic index of the cells can be
measured, for example, based on the DNA ploidy value of the cell.
For example, cells in the GI phase of the cell cycle generally
contain a 2N DNA ploidy value. Cells in which DNA has been
replicated but have not progressed through mitosis (e.g. cells in
S-phase) exhibit a ploidy value higher than 2N and up to 4N DNA
content. Ploidy value and cell-cycle kinetics can be further
measured using propidum iodide assay (see e.g. Turner, T., et al.,
1998, Prostate 34:175-81). Alternatively, the DNA ploidy can be
determined by quantitation of DNA Feulgen staining (which binds to
DNA in a stoichiometric manner) on a computerized
microdensitometrystaining system (see e.g., Bacus, S., 1989, Am. J.
Pathol. 135:783-92). In an another embodiment, DNA content can be
analyzed by preparation of a chromosomal spread (Zabalou, S., 1994,
Hereditas. 120:127-40; Pardue, 1994, Meth. Cell Biol.
44:333-351).
[0243] The expression of cell-cycle proteins (e.g., CycA, CycB,
CycE, CycD, cdc2, Cdk4/6, Rb, p21, p27, etc.) provide information
relating to the proliferative state of a cell or population of
cells. For example, identification in an anti-proliferation
signaling pathway can be indicated by the induction of p21.
Increased levels of p21 expression in cells results in delayed
entry into G1 of the cell cycle (Harper et al., 1993, Cell
75:805-816; Li et al., 1996, Curr. Biol. 6:189-199). p21 induction
can be identified by immunostaining using a specific anti-p21
antibody available commercially (e.g. Santa Cruz Biotechnology,
Inc., Santa Cruz, Calif.). Similarly, cell-cycle proteins may be
examined by Western blot analysis using commercially available
antibodies. In another embodiment, cell populations are
synchronized prior to detection of a cell cycle protein. Cell cycle
proteins can also be detected by FACS (fluorescence-activated cell
sorter) analysis using antibodies against the protein of
interest.
[0244] Detection of changes in length of the cell cycle or speed of
cell cycle can also be used to measure inhibition of cell
proliferation by the compounds provided herein. In one embodiment
the length of the cell cycle is determined by the doubling time of
a population of cells (e.g., using cells contacted or not contacted
with one or more compounds identified using the pharmacophores of
the present invention). In another embodiment, FACS analysis is
used to analyze the phase of cell cycle progression, or purify G1,
S, and G2/M fractions (see e.g., Delia, D. et al., 1997, Oncogene
14:2137-47).
[0245] The compounds useful in the methods of the present invention
can also be demonstrated to inhibit cell transformation (or
progression to malignant phenotype) in vitro. In this embodiment,
cells with a transformed cell phenotype are contacted with one or
more compounds of the present invention, and examined for change in
characteristics associated with a transformed phenotype (a set of
in vitro characteristics associated with a tumorigenic ability in
vivo), for example, but not limited to, colony formation in soft
agar, a more rounded cell morphology, looser substratum attachment,
loss of contact inhibition, loss of anchorage dependence, release
of proteases such as plasminogen activator, increased sugar
transport, decreased serum requirement, or expression of fetal
antigens, etc. (see Luria et al., 1978, General Virology, 3d Ed.,
John Wiley & Sons, New York, pp. 436-446).
[0246] Loss of invasiveness or decreased adhesion may also be used
to demonstrate the anti-cancer effects of the compounds useful in
the methods of the present invention. For example, a critical
aspect of the formation of a metastatic cancer is the ability of a
precancerous or cancerous cell to detach from primary site of
disease and establish a novel colony of growth at a secondary site.
The ability of a cell to invade peripheral sites is reflective of a
potential for a cancerous state. Loss of invasiveness may be
measured by a variety of techniques known in the art including, for
example, induction of E-cadherin-mediated cell-cell adhesion. Such
E-cadherin-mediated adhesion can result in phenotypic reversion and
loss of invasiveness (Hordijk et al., 1997, Science
278:1464-66).
[0247] Loss of invasiveness may further be examined by inhibition
of cell migration. A variety of 2-dimensional and 3-dimensional
cellular matrices are commercially available
(Calbiochem-Novabiochem Corp. San Diego, Calif.). Cell migration
across or into a matrix may be examined by microscopy, time-lapsed
photography or videography, or by any method in the art allowing
measurement of cellular migration. In a related embodiment, loss of
invasiveness is examined by response to hepatocyte growth factor
(HGF). HGF-induced cell scattering is correlated with invasiveness
of cells such as Madin-Darby canine kidney (MDCK) cells. This assay
identifies a cell population that has lost cell scattering activity
in response to HGF (Hordijk et al., 1997, Science 278:1464-66).
[0248] Alternatively, loss of invasiveness may be measured by cell
migration through a chemotaxis chamber (Neuroprobe/Precision
Biochemicals Inc., Vancouver, BC). In such assay, a
chemo-attractant agent is incubated on one side of the chamber
(e.g., the bottom chamber) and cells are plated on a filter
separating the opposite side (e.g., the top chamber). In order for
cells to pass from the top chamber to the bottom chamber, the cells
must actively migrate through small pores in the filter.
Checkerboard analysis of the number of cells that have migrated may
then be correlated with invasiveness (see e.g., Ohnishi, T., 1993,
Biochem. Biophys. Res. Commun. 193:518-25).
[0249] The compounds provided herein can also be demonstrated to
inhibit tumor formation in vivo. A number of animal models of
hyperproliferative disorders, including tumorigenesis and
metastatic spread, are known in the art (see Table 317-1, Chapter
317, "Principals of Neoplasia," in Harrison's Principals of
Internal Medicine, 13th Edition, Isselbacher et al., eds.,
McGraw-Hill, New York, p. 1814, and Lovejoy et al., 1997, J.
Pathol. 181:130-135).
[0250] For example, a compound provided herein can be administered
to a test animal, preferably a test animal predisposed to develop a
type of tumor, and the test animal subsequently examined for
decreased incidence of tumor formation in comparison with controls
not administered the compound identified using the pharmacophores
of the present invention. Alternatively, a compound useful in the
methods of the present invention can be administered to test
animals having tumors (e.g., animals in which tumors have been
induced by introduction of malignant, neoplastic, or transformed
cells, or by administration of a carcinogen) and subsequently
examining the tumors in the test animals for tumor regression in
comparison to controls that were not administered the compound.
[0251] Thyroid Receptor Binding Activity
[0252] Thyroid receptor binding activity that can serve as a
mechanism for treatment of diseases sensitive thereto can be tested
using assays available in the art. Thyroid hormones (TH) are
synthesized in the thyroid in response to thyroid stimulating
hormone (TSH), which is secreted by the pituitary gland in response
to various stimulants. Thyroid hormones are iodinated O-aryl
tyrosine analogues excreted into the circulation primarily as
3,3',5,5'-tetraiodothyronine (T4). T4 is rapidly deiodinated in
local tissues by thyroxine 5'-deiodinase to
3,3',5'-triiodothyronine (T3), which is the most potent TH. Most of
the circulating T4 and T3 is eliminated through the liver.
[0253] THs have profound physiological effects in animals and
humans. Hyper-thyroidism is associated with increased body
temperature, general nervousness, weight loss despite increased
appetite, muscle weakness and fatigue, increased bone resorption
and enhanced calcification, and a variety of cardiovascular
changes, including increased heart rate, increased stroke volume,
increased cardiac index, cardiac hypertrophy, decreased peripheral
vascular resistance, and increased pulse pressure. Hypothyroidism
is generally associated with the opposite effects.
[0254] The biological activity of THs is mediated largely through
thyroid hormone receptors (TRs). TRs belong to the nuclear receptor
superfamily, which, along with its common partner, the retinoid X
receptor, form heterodimers that act as ligand-inducible
transcription factors.
[0255] The most widely recognized effects of THs are an increase in
metabolic rate, oxygen consumption and heat production. T3
treatment increases oxygen consumption in isolated perfused liver
and isolated hepatocytes. (Oh et al., J. Nutr. 125(1):112-24
(1995); Oh et al., Proc. Soc. Exp. Biol. Med. 207(3): 260-7
(1994))
[0256] THs also stimulate metabolism of cholesterol to bile acids.
Hyperthyroidism leads to decreased plasma cholesterol levels, which
is likely due to increased hepatic LDL receptor expression.
Hypothyroidism is a well-established cause of hypercholesterolemia
and elevated serum LDL. L-T3 is known to lower plasma cholesterol
levels. In addition, THs are known to affect levels of other
lipoproteins linked to atherosclerosis. THs stimulate apo AI and
the secretion of apo AI in HDL while reducing apo B100.
Accordingly, one would expect T3 and T3 mimetics to inhibit the
atherosclerotic process in the cholesterol fed animal.
[0257] THs simultaneously increase de novo fatty acid synthesis and
oxidation through effects on enzymes such as ACC, FAS, and spot-14.
THs increase circulating free fatty acids (FFA) levels in part by
increasing production of FFAs from adipose tissue via TH-induced
lipolysis. In addition, THs increase mitochondrial enzyme levels
involved in FFA oxidation, e.g., carnitine palmitoyltransferase 1
(CPT-1) and enzymes involved in energy storage and consumption.
[0258] The liver represents a major target organ of THs. Microarray
analysis of hepatic gene expression from livers of hypothyroid mice
and mice treated with T3 showed changes in mRNA levels for 55 genes
(14 positively regulated and 41 negatively regulated) (Feng et al.,
Mol. Endocrinol. 14(7): 947-55 (2000). Others have estimated that
approximately 8% of the hepatic genes are regulated by T3. Many of
these genes are important to both fatty acid and cholesterol
synthesis and metabolism. T3 is also known to have other effects in
liver, including effects on carbohydrates through increased
glycogenolysis and gluconeogenesis and decreased insulin
action.
[0259] TH has been used as an antiobesity drug for over 50 years.
In the 1940s TH was used alone, whereas in the 1950s it was used in
combination with diuretics and in the 1960s in combination with
amphetamines. Treating hypothyroidism patients with T3 leads to a
decrease in body weight for most patients. T3 and T3 mimetics are
thought to inhibit atherosclerosis by modulating the levels of
certain lipoproteins known to be independent risk factors or
potential risk factors of atherosclerosis, including low density
lipoprotein (LDL)-cholesterol, high density lipoprotein
(HDL)-cholesterol, apoAI, which is a major apoprotein constituent
of high density lipoprotein (HDL) particles and lipoprotein (a) or
Lp(a).
[0260] Hyperthyroidism worsens glycemic control in type 2
diabetics. TH therapy is reported to stimulate hepatic
gluconeogenesis. Enzymes specific to gluconeogenesis and important
for controlling the pathway and its physiological role of producing
glucose are known to be influenced by TH therapy.
Phosphoenolpyruvate carboxykinase (PEPCK) is upregulated by TH
(Park et al, J. Biol. Chem. 274:211 (1999)) whereas others have
found that glucose 6-phosphatase is upregulated (Feng et al., Mol.
Endocrinol. 14:947 (2000)). TH therapy is also associated with
reduced glycogen levels. TH therapy results in improved non insulin
stimulated and insulin stimulated glucose utilization and decreased
insulin resistance in the muscle of ob/ob mice. (Oh et al., J.
Nutr. 125:125 (1995)).
[0261] Thus, thyromimetics potentially can be used to modulate
cholesterol levels, to treat obesity, and other metabolic disorders
especially with reduced undesirable effects.
[0262] Studies can be used to determine the affinity of T3 and
various thyromimetics for human thyroid hormone receptors
TR.alpha.1 and TR.beta.1, and their resulting efficacy in related
disorders. Binding of compounds to either the TR.alpha.1 or
TR.beta..sub.1 receptors can be performed by means of scintillation
proximity assays (SPA). The SPA assay, a common method used for the
quantitation of receptor-ligand equilibria, makes use of special
beads coated with a scintillant and a capture molecule, copper,
which binds to the histidine-tagged .alpha. or .beta. receptor.
When labeled T3 is mixed with receptor and the SPA beads,
radioactive counts are observed only when the complex of protein
and radiolabeled ligand is captured on the surface of the bead.
Displacement curves are generated with labeled T3 and increasing
concentrations of unlabeled thyromimetics of interest. Subacute
studies can be used in ZDF Rats (Charles River Laboratory) to
demonstrate an improved therapeutic index for T3 Mimetics.
[0263] Subacute studies also can be conducted in cholesterol-fed
rats. The cholesterol-fed rat is an animal model of
hypercholesterolemia generated by feeding the animals a diet with
high cholesterol content. The purpose of these studies is to
evaluate the effects of compounds on serum cholesterol (an efficacy
parameter) and on heart weight and heart mGPDH activity (potential
toxicity parameters). Compounds can be administered, e.g., IP,
e.g., once-a-day for seven days.
[0264] Microsome/primary hepatocyte stability studies can be
conducted using methods available in the art. Prodrug activation in
rat liver microsomes can be conducted to determine the kinetics of
activation of prodrugs of thyromimetics in microsomal preparations.
Microsomes may contain P450 enzyme that may activate a prodrug. The
Km, Vmax, and intrinsic clearance values determined are measures of
prodrug affinity for the microsomal enzymes, the rate at which the
prodrug is activated, and the catalytic efficiency with which the
prodrug is activated, respectively. Prodrugs also can be tested for
conversion to their respective parent compounds by human liver S9.
The S9 fraction is a fraction that contains both cytosolic and
microsomal protein. Uptake and activation of prodrug in isolated
rat hepatocytes also can be conducted using methods known in the
art. Oral bioavailability and liver distribution following oral
administration also can be measured using methods available in the
art.
[0265] Oxygen consumption studies can be conducted. Thermogenesis
is a measurement of energy consumption. Compounds that increase
thermogenesis are likely to increase caloric expenditure and
thereby cause body weight loss and its associated benefits to
metabolic status (e.g., insulin sensitivity). Thermogenesis is
assessed in subcellular fractions of various tissues, isolated
cells, whole tissues, or in whole animals using changes in oxygen
consumption as the endpoint. Oxygen is used up when calories are
burned by various metabolic processes.
[0266] Mitochondrial thermogenesis is measured polarographically
with a Clark-type oxygen electrode using mitochondria isolated from
various tissues, including liver. Mitochondria are isolated by
differential centrifugation. State 3 respiration or cytochrome c
oxidase activity are measured in isolated mitochondria. (Iossa, S,
FEBS Letters, 544: 133-7 (2003)). Oxygen consumption rates are
measured in isolated hepatocytes using a portable Clark-type oxygen
electrode placed in the hepatocyte medium. Hepatocytes are isolated
from liver using a two-step collagenase perfusion (Berry, M. N.,
Friend, D. S. J. Cell Biol. 43: 506-520 (1969)) as modified by
Groen (Groen, A. K. et al., Eur J. Biochem 122: 87-93 (1982)).
Non-parenchymal cells are removed using a Percoll gradient and the
cells are resuspended in tissue culture medium in a spinner flask.
The oxygen consumption of the cells is measured over time once the
system is sealed.
[0267] Oxygen consumption also can be measured in isolated perfused
liver (Fernandez, V., Toxicol Lett. 69:205-10 (1993)). Liver is
perfused in situ and oxygen consumption is calculated by measuring
the difference between the oxygen saturation of the inflow buffer
and the outflow buffer maintained at a constant flow. Whole animal
oxygen consumption can be measured using an indirect calorimeter
(Oxymax, Columbus Instruments, Columbus, Ohio). Animals are removed
from their cages and placed in the chambers. The resting oxygen
consumption is measured in animals during periods of inactivity as
measured by activity monitors. The oxygen consumption is calculated
based on the flow through the chamber and the difference in oxygen
partial pressures at the inflow and outlet ports. Carbon dioxide
efflux is also measured in parallel using a CO2 electrode.
[0268] Tissue distribution and the pharmacokinetics of compounds
can be assessed following IP or oral administration to normal
rats.
[0269] Studies can be conducted to evaluate the effects of a T3
mimetic on serum cholesterol and TSH levels, hepatic and cardiac
gene expression and enzyme activities, heart weight, and clinical
chemistry parameters using methods available in the art. In one
embodiment, rats are made hypercholesterolemic by maintenance on a
diet containing 1.5% cholesterol and 0.5% cholic acid for at least
2 weeks prior to initiation of treatment. Plasma cholesterol values
are assessed prior to and following treatment and the effects of
compound are expressed as a percentage change from the pre-dose
cholesterol levels. Total cholesterol is analyzed using a
commercially available enzymatic kit (Sigma Diagnostics, St. Louis,
Mo.).
[0270] Effects of T3 mimetic compounds (and prodrugs thereof) in
vivo on glucose can be measured in ZDF rats. T3 and T3 mimetic
mediated myosin heavy chain gene transcription in the heart can be
measured. An RT-PCR assay as disclosed in: Sara Danzi, Kaie Ojamaa,
and Irwin Klein Am J Physiol Heart Circ Physiol 284: H2255-H2262,
2003 is used to study both the time course and the mechanism for
the triiodothyronine (T3)-induced transcription of the .alpha.- and
.beta.-myosin heavy chain (MHC) genes in vivo on the basis of the
quantity of specific heterogeneous nuclear RNA (hnRNA). The
temporal relationship of changes in transcriptional activity to the
amount of .alpha.-MHC mRNA and the coordinated regulation of
transcription of more than one gene in response to T3 and T3
mimetics are demonstrated. Analysis of a time course of T3 and T3
mimetics that are not liver specific show mediated induction of
.alpha.-MHC hnRNA and repression of .beta.-MHC hnRNA, whereas no
significant affect is observed with compounds at doses that are
therapeutically useful.
[0271] The effect of T3 on cardiovascular function (heart rate,
inotropic state, and aortic pressure) can be studied in the Sprague
Dawley (SD) rat model using assay methods known in the art.
[0272] Thus, various assays known in the art can be used to assay
for thyroid hormone agonist and its accompanying therapeutic
activity and to establish appropriate dosages.
[0273] Methods of Use
[0274] The phosphoroamidate and phosphonoamidate compounds of a
variety of therapeutic agents can be formed using methods available
in the art and those disclosed herein. Such compounds can be used
in some embodiments to enhance delivery of the drug to the liver.
In one embodiment, the compound comprises a S-acyl-2-thioethyl
phosphoroamidate or S-acyl-2-thioethyl phosphonoamidate, e.g., a
S-pivaloyl-2-thioethyl phosphoroamidate or
S-hydroxypivaloyl-2-thioethyl phosphonoamidate derivative.
Therapeutic agents that can be derivatized to phosphoroamidate or
phosphonoamidate compound form include a therapeutic agent such as
an anti-cancer agent or anti-diabetic agent that includes, or has
been derivatized to include a reactive group for attachment of the
phosphoroamidate or phosphonoamidate moiety.
[0275] Cancer Treatment Methods
[0276] In one embodiment, therapeutic agents for the treatment of
liver cancer can be derivatized to form a phosphoroamidate or
phosphonoamidate compound as described herein, and used for the
treatment of liver cancers. Liver cancers that can be treated
include benign tumors, malignant tumors, hemangioma, hepatic
adenomas, focal nodular hyperplasia, hepatocellular carcinoma,
fibrolamellar carcinoma, cholangiocarcinomas, bile duct cancers,
and other primary and metastatic cancers of the liver.
[0277] Exemplary therapeutic agents include anti-cancer agents
having one or more hydroxy groups that can be derivatized as
compounds described herein by removal of a hydrogen from one of the
hydroxy groups. Exemplary anti-cancer agents include, but are not
limited to aclarubicin, decitabine, daunorubicin,
dihydro-5-azacytidine, doxorubicin, epirubicin, estramustin,
etoposide, fludarabine, 7-hydroxychlorpromazin, neplanocin A,
podophyllotoxin, tezacitabine, troxacitabine, vinblastin,
vincristin, vindesin, etoposide, teniposide, NK-611, camptothecin,
irinotecan, 9-aminocamptothecin, GG-211, topotecan, paclitaxel,
azatoxin, coformycin, pirarubicin, nelarabine and losoxantrone.
Anti-cancer agents known in the art and described herein can be
derivatized to form a phosphoramidate or phosphonoamidate compound
as described herein. Immunosuppressants, such as combretastatin
A-4, mycophenolic, pentostatin, or mitoxantrone, also can be
derivatized to form a phosphoramidate or phosphonoamidate compound
as described herein.
[0278] Such compounds can optionally be used in combination with
another anti-cancer agent that is optionally in prodrug form.
[0279] Methods of Treating Metabolic Diseases
[0280] In certain embodiments, the compounds provided herein are
useful in methods for inhibiting gluconeogenesis, optionally by
inhibiting the enzyme fructose 1,6-bisphosphatase (FBPase).
[0281] In certain embodiments, the compounds provided herein are
useful in methods for inhibiting gluconeogenesis.
[0282] In certain embodiments, the compounds provided herein are
useful in methods for treatment of metabolic diseases. In certain
embodiments, the compounds provided herein are useful in methods
for:
[0283] reducing plasma lipid levels, lowering cholesterol levels,
reducing triglyceride levels, or increasing the ratio of HDL to
LDL;
[0284] lowering blood glucose levels;
[0285] treating hyperlipidemia or hypercholesterolemia;
[0286] treating obesity, reducing fat content, treating fatty
liver, reducing weight or preventing weight gain;
[0287] treating atherosclerosis, coronary heart disease, heart
failure, nephrotic syndrome, or chronic renal failure;
[0288] lowering blood glucose levels, treating diabetes, impaired
glucose tolerance, metabolic syndrome x, insulin resistance or
hyperinsulinemia;
[0289] increasing levels of genes associated with
gluconeogenesis;
[0290] decreasing hepatic glycogen levels or maintaining or
improving glycemic control;
[0291] amelioration of hyperinsulinemia and/or decrease of glucose
levels in diabetic subjects at doses that optionally do not affect
cardiac function, e.g., heart rate, force of systolic contraction,
duration of diastolic relaxation, vascular tone, or heart
weight;
[0292] treating thyroid disease, thyroid cancer, depression,
glaucoma, cardiac arrhythmias, heart failure, or osteoporosis;
[0293] increasing mitochondrial biogenesis, or increasing
expression of PGC-1, AMP activated protein kinase or nuclear
respiratory factor;
[0294] inhibiting hepatic gluconeogenesis; or
[0295] modulating expression of certain genes in the liver
resulting in effects on lipids (e.g., cholesterol), glucose,
lipoproteins, and triglycerides, or modulation of T3-responsive
genes.
[0296] In certain embodiments, the compounds provided herein are
useful in methods for lowering blood glucose levels, treating
diabetes, impaired glucose tolerance, metabolic syndrome x, insulin
resistance or hyperinsulinemia.
[0297] Second Agents Useful in the Methods
[0298] In certain embodiments, the compounds and compositions
provided herein are useful in methods of treatment of a liver
disorder, that comprises further administration of a second agent
effective for the treatment of the disorder, such as liver cancer
in a subject in need thereof. The second agent can be any agent
known to those of skill in the art to be effective for the
treatment of the disorder, including those currently approved by
the FDA.
[0299] In certain embodiments, a compound provided herein is
administered in combination with one second agent. In further
embodiments, a second agent is administered in combination with two
second agents. In still further embodiments, a second agent is
administered in combination with two or more second agents.
[0300] As used herein, the term "in combination" includes the use
of more than one therapy (e.g., one or more prophylactic and/or
therapeutic agents). The use of the term "in combination" does not
restrict the order in which therapies (e.g., prophylactic and/or
therapeutic agents) are administered to a subject with a disorder.
A first therapy (e.g., a prophylactic or therapeutic agent such as
a compound provided herein) can be administered prior to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks before), concomitantly with, or subsequent to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks after) the administration of a second therapy (e.g., a
prophylactic or therapeutic agent) to a subject with a
disorder.
[0301] As used herein, the term "synergistic" includes a
combination of a compound provided herein and another therapy
(e.g., a prophylactic or therapeutic agent) which has been or is
currently being used to prevent, manage or treat a disorder, which
is more effective than the additive effects of the therapies. A
synergistic effect of a combination of therapies (e.g., a
combination of prophylactic or therapeutic agents) permits the use
of lower dosages of one or more of the therapies and/or less
frequent administration of said therapies to a subject with a
disorder. The ability to utilize lower dosages of a therapy (e.g.,
a prophylactic or therapeutic agent) and/or to administer said
therapy less frequently reduces the toxicity associated with the
administration of said therapy to a subject without reducing the
efficacy of said therapy in the prevention or treatment of a
disorder). In addition, a synergistic effect can result in improved
efficacy of agents in the prevention or treatment of a disorder.
Finally, a synergistic effect of a combination of therapies (e.g.,
a combination of prophylactic or therapeutic agents) may avoid or
reduce adverse or unwanted side effects associated with the use of
either therapy alone.
[0302] In certain embodiments, the active compounds provided herein
can be administered in combination or alternation with another
therapeutic agent, for example an anti-cancer agent. In certain
embodiments, the active compounds provided herein can be
administered in combination or alternation with second agents
useful in treating metabolic disorders such as diabetes, obesity,
atherosclerosis, heart disease, metabolic syndrome x, nephrotic
syndrome, thyroid disease, and symptoms associated therewith. In
combination therapy, effective dosages of two or more agents are
administered together, whereas in alternation or sequential-step
therapy, an effective dosage of each agent is administered serially
or sequentially. The dosages given will depend on absorption,
inactivation and excretion rates of the drug as well as other
factors known to those of skill in the art. It is to be noted that
dosage values will also vary with the severity of the condition to
be alleviated. It is to be further understood that for any
particular subject, specific dosage regimens and schedules should
be adjusted over time according to the individual need and the
professional judgment of the person administering or supervising
the administration of the compositions.
[0303] The second agent can be one of the agents disclosed herein.
In certain embodiments, contemplated additional pharmaceutically
active substances include drugs commonly used as chemotherapy for
treatment of cancer and immune modulator substances. For example,
chemotherapeutic agents include anti-metabolites (e.g.,
Pentostatin.RTM.), DNA polymerase inhibitors (e.g, Gemzar.RTM.),
RNA polymerase inhibitors (e.g., ECyd.RTM.), platinum derivatives
(e.g., Paraplatin.RTM.), anti-estrogens (e.g., Nolvadex.RTM.),
Taxanes (e.g., Taxotere.RTM.), GnRH analogs (e.g., Lupron.RTM.),
DNA polymerase inhibitors (e.g., Gemzar.RTM.), topoisomerase
inhibitors (e.g., Hycamptin.RTM.), biphosphonates (e.g.,
Aredia.RTM.), somatostatins (e.g., Sandostatin.RTM.), nucleoside
analogs (e.g., Ribavirin.RTM.), and IMPDH-inhibitors (e.g.,
Tiazofurin.RTM.). Contemplated immunomodulatory substances include
cytokines (e.g., interferon .alpha. and .gamma., IL2, IL4, IL6,
IL8, IL10, and IL12), cytokinins (e.g., kinetin), and chemokines
(e.g., MIP-1).
[0304] In certain embodiments, the second agents for use in
combination with the compounds provided herein include other agents
useful in the treatment, prevention, suppression or amelioration of
the diseases or conditions for which compounds provided herein are
useful, such as treating metabolic diseases, including diabetes,
obesity, atherosclerosis, heart disease, metabolic syndrome x,
nephrotic syndrome, thyroid disease, and symptoms associated
therewith. Such second agents include, but are not limited to:
sulfonylureas, for example, glibenclamide (DAONIL.RTM.),
glimepiride (AMARYL.RTM.), glipizide (GLUCOTROL or MINODIAB),
glyburide (MICRONASE.RTM.), tolbutamide (ORINASE.RTM.),
acetohexamide (DYMELOR.RTM.), tolazamide (TOLINSE.RTM.) and
chlorpropamide, (DIABINESE.RTM.); insulin and insulin mimetics;
biguanides such as metformin (GLUCOPHAGE.RTM.); .alpha.-glucosidase
inhibitors including acarbose (PRECOSE.RTM.) and miglitol
(GLYSET.RTM.); meglitinides, for example, nateglinide
(STARLIX.RTM.) and repaglinide (PRANDIN.RTM.); thiozolidinediones,
for example, ciglitazone, englitazone, rosiglitazone
(AVANDIA.RTM.), pioglitazone (ACTOS.RTM.) and troglitazone
(REZULIN.RTM.); incretin mimetics such as exenatide (BYETTA.TM.);
cholesterol lowering agents such as HMG-CoA reductase inhibitors
(e.g., lovastatin, simvastatin, pravastatin, fluvastatin,
atorvastatin and other statins), bile acid sequestrants (e.g.,
cholestyramine and colestipol), vitamin B.sub.3 (also known as
nicotinic acid, or niacin), vitamin B.sub.6 (pyridoxine), vitamin
B.sub.12 (cyanocobalamin), fibric acid derivatives (e.g.,
gemfibrozil, clofibrate, fenofibrate and benzafibrate), probucol,
and inhibitors of cholesterol absorption (e.g., beta-sitosterol and
acylCoA-cholesterol acyltransferase (ACAT) inhibitors such as
melinamide), HMG-CoA synthase inhibitors, squalene epoxidase
inhibitors and squalene synthetase inhibitors; antithrombotic
agents, such as thrombolytic agents (e.g., streptokinase,
alteplase, anistreplase and reteplase), heparin, hirudin and
warfarin derivatives, .beta.-blockers (e.g., atenolol),
.beta.-adrenergic agonists (e.g., isoproterenol) and ACE inhibitors
and vasodilators (e.g., sodium nitroprusside, nicardipine
hydrochloride, nitroglycerin and enaloprilat).
[0305] Pharmaceutical Compositions and Methods of
Administration
[0306] Phosphoroamidate and phosphonoamidate compounds of a variety
of therapeutic agents can be formulated into pharmaceutical
compositions using methods available in the art and those disclosed
herein. Such compounds can be used in some embodiments to enhance
delivery of the drug to the liver. In one embodiment, the compound
comprises a S-acyl-2-thioethyl phosphoroamidate or
S-acyl-2-thioethyl phosphonoamidate, e.g., a S-pivaloyl-2-thioethyl
phosphoroamidate or S-hydroxypivaloyl-2-thioethyl phosphonoamidate
derivative. In certain embodiments, therapeutic agents that can be
derivatized to phosphoroamidate or phosphonoamidate compound form
include any anti-cancer agent that includes, or has been
derivatized to include a reactive group for attachment of the
phosphoroamidate or phosphonoamidate moiety, including but not
limited to nucleosides and nucleoside analogues including acyclic
nucleosides. In certain embodiments, therapeutic agents that can be
derivatized to phosphoroamidate or phosphonoamidate compound form
include any thyroid harmone receptor effector that includes, or has
been derivatized to include a reactive group for attachment of the
phosphoroamidate or phosphonoamidate moiety. Any of the
phosphoroamidate or phosphonoamidate compounds disclosed herein can
be provided in the appropriate pharmaceutical composition and be
administered by a suitable route of administration.
[0307] The methods provided herein encompass administering
pharmaceutical compositions containing at least one compound as
described herein, including a compound of general formula I, Ia,
IIb, IIIa, IVa, IXa or IXb if appropriate in the salt form, either
used alone or in the form of a combination with one or more
compatible and pharmaceutically acceptable carriers, such as
diluents or adjuvants, or with other therapeutic agents, such as
another anti-cancer or anti-diabetic agent.
[0308] In certain embodiments, the second agent can be formulated
or packaged with the compound provided herein. Of course, the
second agent will only be formulated with the compound provided
herein when, according to the judgment of those of skill in the
art, such co-formulation should not interfere with the activity of
either agent or the method of administration. In certain
embodiments, the compound provided herein and the second agent are
formulated separately. They can be packaged together, or packaged
separately, for the convenience of the practitioner of skill in the
art.
[0309] In clinical practice the active agents provided herein may
be administered by any conventional route, in particular orally,
parenterally, rectally or by inhalation (e.g. in the form of
aerosols). In certain embodiments, the compound provided herein is
administered orally.
[0310] Use may be made, as solid compositions for oral
administration, of tablets, pills, hard gelatin capsules, powders
or granules. In these compositions, the active product is mixed
with one or more inert diluents or adjuvants, such as sucrose,
lactose or starch.
[0311] These compositions can comprise substances other than
diluents, for example a lubricant, such as magnesium stearate, or a
coating intended for controlled release.
[0312] Use may be made, as liquid compositions for oral
administration, of solutions which are pharmaceutically acceptable,
suspensions, emulsions, syrups and elixirs containing inert
diluents, such as water or liquid paraffin. These compositions can
also comprise substances other than diluents, for example wetting,
sweetening or flavoring products.
[0313] The compositions for parenteral administration can be
emulsions or sterile solutions. Use may be made, as solvent or
vehicle, of propylene glycol, a polyethylene glycol, vegetable
oils, in particular olive oil, or injectable organic esters, for
example ethyl oleate. These compositions can also contain
adjuvants, in particular wetting, isotonizing, emulsifying,
dispersing and stabilizing agents. Sterilization can be carried out
in several ways, for example using a bacteriological filter, by
radiation or by heating. They can also be prepared in the form of
sterile solid compositions which can be dissolved at the time of
use in sterile water or any other injectable sterile medium.
[0314] The compositions for rectal administration are suppositories
or rectal capsules which contain, in addition to the active
principle, excipients such as cocoa butter, semi-synthetic
glycerides or polyethylene glycols.
[0315] The compositions can also be aerosols. For use in the form
of liquid aerosols, the compositions can be stable sterile
solutions or solid compositions dissolved at the time of use in
apyrogenic sterile water, in saline or any other pharmaceutically
acceptable vehicle. For use in the form of dry aerosols intended to
be directly inhaled, the active principle is finely divided and
combined with a water-soluble solid diluent or vehicle, for example
dextran, mannitol or lactose.
[0316] In one embodiment, a composition provided herein is a
pharmaceutical composition or a single unit dosage form.
Pharmaceutical compositions and single unit dosage forms provided
herein comprise a prophylactically or therapeutically effective
amount of one or more prophylactic or therapeutic agents (e.g., a
compound provided herein, or other prophylactic or therapeutic
agent), and a typically one or more pharmaceutically acceptable
carriers or excipients. In a specific embodiment and in this
context, the term "pharmaceutically acceptable" includes approval
by a regulatory agency of the Federal or a state government or
listed in the U.S. Pharmacopeia or other generally recognized
pharmacopeia for use in animals, and more particularly in humans.
The term "carrier" includes a diluent, adjuvant (e.g., Freund's
adjuvant (complete and incomplete)), excipient, or vehicle with
which the therapeutic is administered. Such pharmaceutical carriers
can be sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like. Water can
be used as a carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Examples of suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin.
[0317] Typical pharmaceutical compositions and dosage forms
comprise one or more excipients. Suitable excipients are well-known
to those skilled in the art of pharmacy, and non-limiting examples
of suitable excipients include starch, glucose, lactose, sucrose,
gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,
glycerol monostearate, talc, sodium chloride, dried skim milk,
glycerol, propylene, glycol, water, ethanol and the like. 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 subject and
the specific active ingredients in the dosage form. The composition
or single unit dosage form, if desired, can also contain minor
amounts of wetting or emulsifying agents, or pH buffering
agents.
[0318] Lactose free compositions provided herein can comprise
excipients that are well known in the art and are listed, for
example, in the U.S. Pharmocopia (USP) SP (XXI)/NF (XVI). In
general, lactose free compositions comprise an active ingredient, a
binder/filler, and a lubricant in pharmaceutically compatible and
pharmaceutically acceptable amounts. Exemplary lactose free dosage
forms comprise an active ingredient, microcrystalline cellulose,
pre gelatinized starch, and magnesium stearate.
[0319] Further encompassed herein are anhydrous 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.
[0320] Anhydrous pharmaceutical compositions and dosage forms
provided herein 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 can be anhydrous if substantial contact with
moisture and/or humidity during manufacturing, packaging, and/or
storage is expected.
[0321] An anhydrous pharmaceutical composition should be prepared
and stored such that its anhydrous nature is maintained.
Accordingly, anhydrous compositions can be 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.
[0322] Further provided are 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.
[0323] The pharmaceutical compositions and single unit dosage forms
can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules, powders, sustained-release formulations and the
like. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Such compositions and dosage forms will contain a prophylactically
or therapeutically effective amount of a prophylactic or
therapeutic agent, in certain embodiments, in purified form,
together with a suitable amount of carrier so as to provide the
form for proper administration to the subject. The formulation
should suit the mode of administration. In a certain embodiment,
the pharmaceutical compositions or single unit dosage forms are
sterile and in suitable form for administration to a subject, for
example, an animal subject, such as a mammalian subject, for
example, a human subject.
[0324] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include, but are not limited to, parenteral, e.g.,
intravenous, intradermal, subcutaneous, intramuscular,
subcutaneous, oral, buccal, sublingual, inhalation, intranasal,
transdermal, topical, transmucosal, intra-tumoral, intra-synovial
and rectal administration. In a specific embodiment, the
composition is formulated in accordance with routine procedures as
a pharmaceutical composition adapted for intravenous, subcutaneous,
intramuscular, oral, intranasal or topical administration to human
beings. In an embodiment, a pharmaceutical composition is
formulated in accordance with routine procedures for subcutaneous
administration to human beings. Typically, compositions for
intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lignocamne to
ease pain at the site of the injection.
[0325] 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 subject, 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 subject;
and sterile solids (e.g., crystalline or amorphous solids) that can
be reconstituted to provide liquid dosage forms suitable for
parenteral administration to a subject.
[0326] The composition, shape, and type of dosage forms provided
herein will typically vary depending on their use. For example, a
dosage form used in the initial treatment of liver cancer may
contain larger amounts of one or more of the active ingredients it
comprises than a dosage form used in the maintenance treatment of
the same liver cancer. 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 herein will vary from one another will be readily
apparent to those skilled in the art. See, e.g., Remington's
Pharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa.
(2000).
[0327] Generally, the ingredients of compositions are supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the composition is
to be administered by infusion, it can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the composition is administered by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0328] Typical dosage forms comprise a compound provided herein, or
a pharmaceutically acceptable salt, solvate or hydrate thereof lie
within the range of from about 0.1 mg to about 1000 mg per day,
given as a single once-a-day dose in the morning or as divided
doses throughout the day taken with food. Particular dosage forms
can have about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0,
15.0, 20.0, 25.0, 50.0, 100, 200, 250, 500 or 1000 mg of the active
compound.
[0329] Oral Dosage Forms
[0330] Pharmaceutical compositions 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, 20th ed.,
Mack Publishing, Easton Pa. (2000).
[0331] In certain embodiments, the oral dosage forms are solid and
prepared under anhydrous conditions with anhydrous ingredients, as
described in detail in the sections above. However, the scope of
the compositions provided herein extends beyond anhydrous, solid
oral dosage forms. As such, further forms are described herein.
[0332] Typical oral dosage forms 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.
[0333] 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.
[0334] 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.
[0335] Examples of excipients that can be used in oral dosage forms
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.
[0336] 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 is typically present in from about 50 to about 99
weight percent of the pharmaceutical composition or dosage
form.
[0337] 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.
[0338] Disintegrants are used in the compositions 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. 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.
[0339] Disintegrants that can be used in pharmaceutical
compositions and dosage forms 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.
[0340] Lubricants that can be used in pharmaceutical compositions
and dosage forms 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.
[0341] Delayed Release Dosage Forms
[0342] Active ingredients such as the compounds provided herein 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; 5,639,480; 5,733,566; 5,739,108; 5,891,474; 5,922,356;
5,972,891; 5,980,945; 5,993,855; 6,045,830; 6,087,324; 6,113,943;
6,197,350; 6,248,363; 6,264,970; 6,267,981; 6,376,461; 6,419,961;
6,589,548; 6,613,358; 6,699,500 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 provided herein. Thus
encompassed herein are 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.
[0343] All controlled release pharmaceutical products have a common
goal of improving 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 subject 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.
[0344] 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.
[0345] In certain embodiments, the drug may be administered using
intravenous infusion, an implantable osmotic pump, a transdermal
patch, liposomes, or other modes of administration. In one
embodiment, a pump may be used (see, Sefton, CRC Crit. Ref Biomed.
Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric materials can be used. In yet another embodiment, a
controlled release system can be placed in a subject at an
appropriate site determined by a practitioner of skill, i.e., thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
Medical Applications of Controlled Release, vol. 2, pp. 115-138
(1984)). Other controlled release systems are discussed in the
review by Langer (Science 249:1527-1533 (1990)). The active
ingredient can be dispersed in a solid inner matrix, e.g.,
polymethylmethacrylate, polybutylmethacrylate, plasticized or
unplasticized polyvinylchloride, plasticized nylon, plasticized
polyethyleneterephthalate, natural rubber, polyisoprene,
polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate
copolymers, silicone rubbers, polydimethylsiloxanes, silicone
carbonate copolymers, hydrophilic polymers such as hydrogels of
esters of acrylic and methacrylic acid, collagen, cross-linked
polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl
acetate, that is surrounded by an outer polymeric membrane, e.g.,
polyethylene, polypropylene, ethylene/propylene copolymers,
ethylene/ethyl acrylate copolymers, ethylene/vinylacetate
copolymers, silicone rubbers, polydimethyl siloxanes, neoprene
rubber, chlorinated polyethylene, polyvinylchloride, vinylchloride
copolymers with vinyl acetate, vinylidene chloride, ethylene and
propylene, ionomer polyethylene terephthalate, butyl rubber
epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,
ethylene/vinyl acetate/vinyl alcohol terpolymer, and
ethylene/vinyloxyethanol copolymer, that is insoluble in body
fluids. The active ingredient then diffuses through the outer
polymeric membrane in a release rate controlling step. The
percentage of active ingredient in such parenteral compositions is
highly dependent on the specific nature thereof, as well as the
needs of the subject.
[0346] Parenteral Dosage Forms
[0347] In one embodiment, provided are parenteral dosage forms.
Parenteral dosage forms can be administered to subjects by various
routes including, but not limited to, subcutaneous, intravenous
(including bolus injection), intramuscular, and intraarterial.
Because their administration typically bypasses subjects' natural
defenses against contaminants, parenteral dosage forms are
typically, sterile or capable of being sterilized prior to
administration to a subject. 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.
[0348] Suitable vehicles that can be used to provide parenteral
dosage forms 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.
[0349] Compounds that increase the solubility of one or more of the
active ingredients disclosed herein can also be incorporated into
the parenteral dosage forms.
[0350] Transdermal, Topical & Mucosal Dosage Forms
[0351] Also provided are transdermal, topical, and mucosal dosage
forms. Transdermal, topical, and mucosal dosage forms include, but
are not limited to, ophthalmic solutions, sprays, aerosols, 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, 16.sup.th, 18th and 20.sup.th
eds., Mack Publishing, Easton Pa. (1980, 1990 & 2000); 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. Further, 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.
[0352] Suitable excipients (e.g., carriers and diluents) and other
materials that can be used to provide transdermal, topical, and
mucosal dosage forms encompassed herein 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, 16.sup.th, 18th and 20.sup.th eds., Mack Publishing,
Easton Pa. (1980, 1990 & 2000).
[0353] 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 provided. 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).
[0354] 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.
[0355] Dosage and Unit Dosage Forms
[0356] In human therapeutics, the doctor will determine the
posology which he considers most appropriate according to a
preventive or curative treatment and according to the age, weight,
stage of the disease, for example, cancer and other factors
specific to the subject to be treated. In certain embodiments,
doses are from about 1 to about 1000 mg per day for an adult, or
from about 5 to about 250 mg per day or from about 10 to 50 mg per
day for an adult. In certain embodiments, doses are from about 5 to
about 400 mg per day or 25 to 200 mg per day per adult. In certain
embodiments, dose rates of from about 50 to about 500 mg per day
are also contemplated.
[0357] In further aspects, provided are methods of treating or
preventing liver cancer in a subject by administering, to a subject
in need thereof, an effective amount of a compound provided herein,
or a pharmaceutically acceptable salt thereof. In other aspects,
provided are methods of treating or preventing metabolic diseases
in a subject by administering, to a Subject in need thereof, an
effective amount of a compound provided herein, or a
pharmaceutically acceptable salt thereof. The amount of the
compound or composition which will be effective in the prevention
or treatment of a disorder or one or more symptoms thereof will
vary with the nature and severity of the disease or condition, and
the route by which the active ingredient is administered. The
frequency and dosage will also vary according to factors specific
for each subject depending on the specific therapy (e.g.,
therapeutic or prophylactic agents) administered, the severity of
the disorder, disease, or condition, the route of administration,
as well as age, body, weight, response, and the past medical
history of the subject. Effective doses may be extrapolated from
dose-response curves derived from in vitro or animal model test
systems.
[0358] In certain embodiments, exemplary doses of a composition
include milligram or microgram amounts of the active compound per
kilogram of subject or sample weight (e.g., about 10 micrograms per
kilogram to about 50 milligrams per kilogram, about 100 micrograms
per kilogram to about 25 milligrams per kilogram, or about 100
microgram per kilogram to about 10 milligrams per kilogram). For
compositions provided herein, in certain embodiments, the dosage
administered to a subject is 0.140 mg/kg to 3 mg/kg of the
subject's body weight, based on weight of the active compound. In
certain embodiments, the dosage administered to a subject is
between 0.20 mg/kg and 2.00 mg/kg, or between 0.30 mg/kg and 1.50
mg/kg of the subject's body weight.
[0359] In certain embodiments, the recommended daily dose range of
a composition provided herein for the conditions described herein
lie within the range of from about 0.1 mg to about 1000 mg per day,
given as a single once-a-day dose or as divided doses throughout a
day. In one embodiment, the daily dose is administered twice daily
in equally divided doses. In certain embodiments, a daily dose
range should be from about 10 mg to about 200 mg per day, in other
embodiments, between about 10 mg and about 150 mg per day, in
further embodiments, between about 25 and about 100 mg per day. 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 subject
response.
[0360] 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 prevent, manage, treat or ameliorate such
disorders, but insufficient to cause, or sufficient to reduce,
adverse effects associated with the composition provided herein are
also encompassed by the above described dosage amounts and dose
frequency schedules. Further, when a subject is administered
multiple dosages of a composition provided herein, not all of the
dosages need be the same. For example, the dosage administered to
the subject may be increased to improve the prophylactic or
therapeutic effect of the composition or it may be decreased to
reduce one or more side effects that a particular subject is
experiencing.
[0361] In certain embodiment, the dosage of the composition
provided herein, based on weight of the active compound,
administered to prevent, treat, manage, or ameliorate a disorder,
or one or more symptoms thereof in a subject is 0.1 mg/kg, 1 mg/kg,
2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 10 mg/kg, or 15 mg/kg
or more of a subject's body weight. In another embodiment, the
dosage of the composition or a composition provided herein
administered to prevent, treat, manage, or ameliorate a disorder,
or one or more symptoms thereof in a subject is a unit dose of 0.1
mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg,
0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5
mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg,
0.25 to 12 mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg,
0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg
to 10 mg, 1 mg to 7.5 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.
[0362] In certain embodiments, treatment or prevention can be
initiated with one or more loading doses of a compound or
composition provided herein followed by one or more maintenance
doses. In such embodiments, the loading dose can be, for instance,
about 60 to about 400 mg per day, or about 100 to about 200 mg per
day for one day to five weeks. The loading dose can be followed by
one or more maintenance doses. In certain embodiments, each
maintenance does is, independently, about from about 10 mg to about
200 mg per day, between about 25 mg and about 150 mg per day, or
between about 25 and about 80 mg per day. Maintenance doses can be
administered daily and can be administered as single doses, or as
divided doses.
[0363] In certain embodiments, a dose of a compound or composition
provided herein can be administered to achieve a steady-state
concentration of the active ingredient in blood or serum of the
subject. The steady-state concentration can be determined by
measurement according to techniques available to those of skill or
can be based on the physical characteristics of the subject such as
height, weight and age. In certain embodiments, a sufficient amount
of a compound or composition provided herein is administered to
achieve a steady-state concentration in blood or serum of the
subject of from about 300 to about 4000 ng/mL, from about 400 to
about 1600 ng/mL, or from about 600 to about 1200 ng/mL. In some
embodiments, loading doses can be administered to achieve
steady-state blood or serum concentrations of about 1200 to about
8000 ng/mL, or about 2000 to about 4000 ng/mL for one to five days.
In certain embodiments, maintenance doses can be administered to
achieve a steady-state concentration in blood or serum of the
subject of from about 300 to about 4000 ng/mL, from about 400 to
about 1600 ng/mL, or from about 600 to about 1200 ng/mL.
[0364] In certain embodiments, administration of the same
composition may be repeated and the administrations may be
separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15
days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.
In other embodiments, administration of the same prophylactic or
therapeutic agent may be repeated and the administration may be
separated by at least at least 1 day, 2 days, 3 days, 5 days, 10
days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6
months.
[0365] In certain aspects, provided herein are unit dosages
comprising a compound, or a pharmaceutically acceptable salt
thereof, in a form suitable for administration. Such forms are
described in detail above. In certain embodiments, the unit dosage
comprises 1 to 1000 mg, 5 to 250 mg or 10 to 50 mg active
ingredient. In particular embodiments, the unit dosages comprise
about 1, 5, 10, 25, 50, 100, 125, 250, 500 or 1000 mg active
ingredient. Such unit dosages can be prepared according to
techniques familiar to those of skill in the art.
[0366] The dosages of the second agents are to be used in the
combination therapies provided herein. In certain embodiments,
dosages lower than those which have been or are currently being
used to prevent or treat the diseases described herein, for
example, liver cancer and diabetes, are used in the combination
therapies provided herein. The recommended dosages of second agents
can be obtained from the knowledge of those of skill. For those
second agents that are approved for clinical use, recommended
dosages are described in, for example, Hardman et al., eds., 1996,
Goodman & Gilman's The Pharmacological Basis Of Basis Of
Therapeutics 9th Ed, Mc-Graw-Hill, New York; Physician's Desk
Reference (PDR) 57.sup.th Ed., 2003, Medical Economics Co., Inc.,
Montvale, N.J., which are incorporated herein by reference in its
entirety.
[0367] In various embodiments, the therapies (e.g., a compound
provided herein and the second agent) are administered less than 5
minutes apart, less than 30 minutes apart, less than 1 hour apart,
1 hour apart, at about 1 hour apart, at about 1 to about 2 hours
apart, at about 2 hours to about 3 hours apart, at about 3 hours to
about 4 hours apart, at about 4 hours to about 5 hours apart, at
about 5 hours to about 6 hours apart, at about 6 hours to about 7
hours apart, at about 7 hours to about 8 hours apart, at about 8
hours to about 9 hours apart, at about 9 hours to about 10 hours
apart, at about 10 hours to about 11 hours apart, at about 11 hours
to about 12 hours apart, at about 12 hours to 18 hours apart, 18
hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48
hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours
apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84
hours to 96 hours apart, or 96 hours to 120 hours part. In various
embodiments, the therapies are administered no more than 24 hours
apart or no more than 48 hours apart. In certain embodiments, two
or more therapies are administered within the same patient visit.
In other embodiments, the compound provided herein and the second
agent are administered concurrently.
[0368] In certain embodiments, a compound provided herein and a
second agent are administered to a patient, for example, a mammal,
such as a human, in a sequence and within a time interval such that
the compound provided herein can act together with the other agent
to provide an increased benefit than if they were administered
otherwise. For example, the second active agent can be administered
at the same time or sequentially in any order at different points
in time; however, if not administered at the same time, they should
be administered sufficiently close in time so as to provide the
desired therapeutic or prophylactic effect. In one embodiment, the
compound provided herein and the second active agent exert their
effect at times which overlap. Each second active agent can be
administered separately, in any appropriate form and by any
suitable route. In other embodiments, the compound provided herein
is administered before, concurrently or after administration of the
second active agent.
[0369] In other embodiments, the compound provided herein and the
second agent are administered at about 2 to 4 days apart, at about
4 to 6 days apart, at about 1 week part, at about 1 to 2 weeks
apart, or more than 2 weeks apart.
[0370] In certain embodiments, the compound provided herein and the
second agent are cyclically administered to a patient. Cycling
therapy involves the administration of a first agent (e.g., a first
prophylactic or therapeutic agents) for a period of time, followed
by the administration of a second agent and/or third agent (e.g., a
second and/or third prophylactic or therapeutic agents) for a
period of time and repeating this sequential administration.
Cycling therapy can reduce the development of resistance to one or
more of the therapies, avoid or reduce the side effects of one of
the therapies, and/or improve the efficacy of the treatment.
[0371] In certain embodiments, the compound provided herein and the
second active agent are administered in a cycle of less than about
3 weeks, about once every two weeks, about once every 10 days or
about once every week. One cycle can comprise the administration of
a compound provided herein and the second agent by infusion over
about 90 minutes every cycle, about 1 hour every cycle, about 45
minutes every cycle. Each cycle can comprise at least 1 week of
rest, at least 2 weeks of rest, at least 3 weeks of rest. The
number of cycles administered is from about 1 to about 12 cycles,
more typically from about 2 to about 10 cycles, and more typically
from about 2 to about 8 cycles.
[0372] In other embodiments, courses of treatment are administered
concurrently to a patient, i.e., individual doses of the second
agent are administered separately yet within a time interval such
that the compound provided herein can work together with the second
active agent. For example, one component can be administered once
per week in combination with the other components that can be
administered once every two weeks or once every three weeks. In
other words, the dosing regimens are carried out concurrently even
if the therapeutics are not administered simultaneously or during
the same day.
[0373] The second agent can act additively or synergistically with
the compound provided herein. In one embodiment, the compound
provided herein is administered concurrently with one or more
second agents in the same pharmaceutical composition. In another
embodiment, a compound provided herein is administered concurrently
with one or more second agents in separate pharmaceutical
compositions. In still another embodiment, a compound provided
herein is administered prior to or subsequent to administration of
a second agent. Also contemplated are administration of a compound
provided herein and a second agent by the same or different routes
of administration, e.g., oral and parenteral. In certain
embodiments, when the compound provided herein is administered
concurrently with a second agent that potentially produces adverse
side effects including, but not limited to, toxicity, the second
active agent can advantageously be administered at a dose that
falls below the threshold that the adverse side effect is
elicited.
[0374] Kits
[0375] Also provided are kits for use in methods of treatment of a
liver disorder such as cancer or metabolic diseases, such as
diabetes, hyperlipidemia, atherosclerosis, and obesity. The kits
can include a compound or composition provided herein, a second
agent or composition, and instructions providing information to a
health care provider regarding usage for treating the disorder.
Instructions may be provided in printed form or in the form of an
electronic medium such as a floppy disc, CD, or DVD, or in the form
of a website address where such instructions may be obtained. A
unit dose of a compound or composition provided herein, or a second
agent or composition, can include a dosage such that when
administered to a subject, a therapeutically or prophylactically
effective plasma level of the compound or composition can be
maintained in the subject for at least 1 days. In some embodiments,
a compound or composition can be included as a sterile aqueous
pharmaceutical composition or dry powder (e.g., lyophilized)
composition.
[0376] In some embodiments, suitable packaging is provided. As used
herein, "packaging" includes a solid matrix or material customarily
used in a system and capable of holding within fixed limits a
compound provided herein and/or a second agent suitable for
administration to a subject. Such materials include glass and
plastic (e.g., polyethylene, polypropylene, and polycarbonate)
bottles, vials, paper, plastic, and plastic-foil laminated
envelopes and the like. If e-beam sterilization techniques are
employed, the packaging should have sufficiently low density to
permit sterilization of the contents.
[0377] The following Examples illustrate the synthesis of
representative compounds provided herein. These examples are not
intended, nor are they to be construed, as limiting the scope of
the claimed subject matter. It will be clear that the scope of
claimed subject matter may be practiced otherwise than as
particularly described herein. Numerous modifications and
variations of the subject matter are possible in view of the
teachings herein and, therefore, are within the scope the claimed
subject matter.
EXAMPLES
Example 1
Preparation of Hydroxy-tBuSATE N-benzylphosphoroamidate derivative
A550 of L-2',3'-dideoxyadenosine L-ddA
##STR00070##
[0378] Synthetic Scheme:
##STR00071##
[0379] Synthesis of Carboxylic Acid 2:
##STR00072##
[0381] 2,2-Dimethyl-3-hydroxypropanoic acid methyl ester (965
.mu.L, 7.57 mmol) was added dropwise to a stirring solution of
4,4'-dimethoxytrityl chloride (2.82 g, 8.33 mmol) in anhydrous
pyridine (7.6 mL) at room temperature. The reaction mixture turned
to a red solution quickly, then to an orange suspension (ca. 30
min), and this was left stirring overnight. The mixture was poured
carefully over saturated aqueous NaHCO.sub.3 solution (30 mL) and
the product was extracted with Et.sub.2O (3.times.20 mL). The
combined organic extracts were washed with brine (20 mL), dried
(Na.sub.2SO.sub.4) and the volatiles were removed under reduced
pressure. The resulting oil was co-evaporated with toluene and the
residue was quickly purified by flash column chromatography
(SiO.sub.2, O=4 cm, H=20 cm) eluting with
5.fwdarw.10.fwdarw.20.fwdarw.30% Et.sub.2O in petroleum ether
(40-60). Evaporation of the fractions (R.sub.f=0.25, 30% Et.sub.2O
in petroleum ether (40-60)) afforded ether 1 as a yellow oil (3.11
g, 95%). This compound (3.00 g, 6.91 mmol) was dissolved in THF (35
mL) and an aqueous solution of NaOH (10%, 3.5 g in 35 mL H.sub.2O)
was then added at room temperature. The solution turned instantly
dark orange and this was stirred for 2 days. The medium was then
carefully neutralized by dropwise addition of HCl (1M). The product
was extracted with Et.sub.2O (4.times.50 mL) and the combined
organic extracts were washed with brine (50 mL), dried
(Na.sub.2SO.sub.4) and the volatiles were removed under reduced
pressure. The crude yellow oil was quickly purified by flash column
chromatography (SiO.sub.2, O=2 cm, H=10 cm) eluting with 50%
Et.sub.2O in petroleum ether (40-60). Evaporation of the fractions
afforded carboxylic acid 2 as a white foam (2.23 g, 77%).
R.sub.f=0.50 (50% Et.sub.2O in petroleum ether (40-60));
.sup.1H-NMR (300 MHz, CDCl.sub.3) 1.10 (s, 6H, 2.times.CH.sub.3),
3.06 (s, 2H, CH.sub.2O), 3.65 (s, 6H, 2.times.OCH.sub.3), 6.62-6.79
(m, 4H, PhCH), 7.02-7.46 (stack, 8H, PhCH); .sup.13C-NMR (75 MHz,
CDCl.sub.3) 22.6 (2.times.CH.sub.3), 43.5 (C(CH.sub.3).sub.2), 55.1
(2.times.OCH.sub.3), 85.9 (CPh.sub.3), [125.3, 126.7, 127.7, 128.2,
129.1, 130.0, 136.0, 144.9, 158.4 (Ph), some overlap], 182.2
(C.dbd.O).
Synthesis of Thioester 3:
##STR00073##
[0383] 1,1'-carbonyldiimidazole (830 mg, 5.12 mmol) was added to a
stirring solution of carboxylic acid 2 in anhydrous PhMe/DMF (2/1,
v/v, 2.7 mL) at room temperature and the reaction mixture turned
turbid instantly. After 30 min, the medium was diluted by adding
anhydrous PhMe/DMF (93/7, v/v, 17 mL) and cooled to -10.degree. C.
2-Mercaptoethanol (359 .mu.L, 5.12 mmol) was then added dropwise
and the solution was stirred for 1 h at this temperature. The
reaction mixture was diluted with H.sub.2O (60 mL) and the product
was extracted with Et.sub.2O (3.times.15 mL). The combined organic
extracts were washed with brine (15 mL), dried (Na.sub.2SO.sub.4)
and the volatiles were removed under reduced pressure (bath
temperature not exceeding 20.degree. C.). The residue was purified
by flash column chromatography (SiO.sub.2, O=4 cm, H=15 cm, 1%
Et.sub.3N) eluting with 60-70% Et.sub.2O in petroleum ether
(40-60). Evaporation of the fractions afforded thioester 3 as a
white syrup (1.74 g, 92%) that solidified upon storage at 4.degree.
C. R.sub.f=0.35 (70% Et.sub.2O in petroleum ether (40-60));
.sup.1H-NMR (300 MHz, CDCl.sub.3) 1.16 (s, 6H, 2.times.CH.sub.3),
3.02 (t, J 6.0, 2H, CH.sub.2S), 3.09 (s, 2H, CH.sub.2O), 3.66 (t, J
6.0, 2H, CH.sub.2OH), 3.72 (s, 6H, 2.times.OCH.sub.3), 6.74-6.78
(m, 4H, PhCH), 7.09-7.36 (stack, 8H, PhCH); .sup.13C-NMR (75 MHz,
CDCl.sub.3) 22.9 (CH.sub.3, 2.times.CH.sub.3), 31.7 (CH.sub.2,
CH.sub.2S), 51.0 (quat. C, C(CH.sub.3).sub.2), 55.2 (CH.sub.3,
2.times.OCH.sub.3), 61.9 (CH.sub.2, CH.sub.2OH), 70.0 (CH.sub.2,
CH.sub.2O), 85.8 (quat. C., CPh.sub.3), [113.0 (CH, Ph), 126.7 (CH,
Ph), 127.7 (CH, Ph), 128.2 (CH, Ph), 130.1 (CH, Ph), some overlap],
[135.9 (quat. C, Ph), 144.8 (quat. C, Ph), 158.4 (quat. C, Ph),
some overlap], 205.0 (quat. C, C.dbd.O).
Synthesis of H-Phosphonate Monoester 4:
##STR00074##
[0385] .beta.-L-ddA (1.00 g, 4.25 mmol) was co-evaporated with
anhydrous pyridine (3.times.10 mL) and then dissolved in anhydrous
pyridine/DMF (1/1, v/v, 21 mL). Diphenyl phosphite (5.76 mL, 29.8
mmol) was then added dropwise to this solution at room temperature.
The reaction mixture was stirred for 20 min upon which a mixture of
Et.sub.3N/H.sub.2O (1/1, v/v, 8.5 mL) was added dropwise, and
stirring was pursued for an additional 20 min. The reaction mixture
was concentrated under reduced pressure to approximately 15-20 mL
and this residue was directly purified by flash column
chromatography (SiO.sub.2, O=4 cm, H=15 cm, 1% Et.sub.3N) eluting
slowly with CH.sub.2Cl.sub.2 (150 mL) then 5% (200 mL) .delta. 10%
(200 mL) .delta. 15% (300 mL) MeOH in CH.sub.2Cl.sub.2. Evaporation
of the fractions afforded H-phosphonate monoester 4 as a white foam
(1.36 g, 80%) that could be kept for several weeks at 4.degree. C.
R.sub.f=0.10 (Et.sub.3N/MeOH/CH.sub.2Cl.sub.2, Jan. 10, 1989);
.sup.1H-NMR (300 MHz, CDCl.sub.3) 1.21 (t, J 7.4, 9H,
3.times.NCH.sub.2CH.sub.3), 1.92-2.50 (stack, 4H, 2.times.2'-H,
2.times.3'-H), 3.02 (q, J 7.4, 6H, 3.times.NCH.sub.2CH.sub.3),
[3.96-4.03 and 4.18-4.30 (stacks, 3H, 4'-H, 2.times.5'-H), 6.28 (m,
1'-H), 6.91 (d, J 623, 1H, P--H), 7.05 (br s, 2H, NH.sub.2), 8.21
(s, 1H), 8.54 (br s, 1H, OH), 8.57 (s, 1H).
Synthesis of Phosphoroamidate Diester 5:
##STR00075##
[0387] H-Phosphonate monoester 4 (1.03 g, 2.57 mmol) and alcohol 3
(1.66 g, 3.45 mmol) were co-evaporated with anhydrous pyridine
(3.times.5 mL) and then dissolved in anhydrous pyridine (5 mL).
PyBOP (1H-benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate, 1.60 g, 3.08 mmol) was then added in one
portion and the reaction mixture was stirred for 15 min at room
temperature. The solution was poured over saturated aqueous
NaHCO.sub.3 solution (30 mL) and the product was extracted with
CH.sub.2Cl.sub.2 (4.times.15 mL). The combined organic extracts
were washed with brine (10 mL), dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure to leave the corresponding
H-phosphonate diester as a slightly yellow oil (1.84 g, assuming
2.41 mmol). This was co-evaporated with anhydrous pyridine
(3.times.5 mL; note: do not evaporate to dryness in order to help
further solubilization), and the residue was dissolved in anhydrous
CCl.sub.4 (24 mL). Benzylamine (791 .mu.L, 7.23 mmol) was added
dropwise and the reaction mixture turned cloudy instantly (slight
heat development was observed). The milky solution was stirred for
1 h at room temperature and poured over saturated aqueous
NaHCO.sub.3 solution (30 mL) and the product was extracted with
CH.sub.2Cl.sub.2 (4.times.15 mL). The combined organic extracts
were washed with brine (15 mL), dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure to afford phosphoroamidate
diester 5 as a yellow oil (2.00 g, assuming 2.31 mmol). This was
used in the next step without any further purification.
R.sub.f=0.29 (4% MeOH in CH.sub.2Cl.sub.2); .sup.1H-NMR (300 MHz,
CDCl.sub.3) 1.11 (s, 6H, 2.times.CH.sub.3), 1.91-2.05 (m, 2H),
2.31-2.59 (m, 2H), 3.06 (m, 2H, CH.sub.2S), 3.08 (s, 2H,
CH.sub.2ODMTr), 3.69 (s, 6H, 2.times.OCH.sub.3), 3.83-4.28 (stacks,
7H, CH.sub.2O, NCH.sub.2Ph, 4'-H, 2.times.5'-H), 5.71 (br s, 1H,
NH), 6.18 (m, 1H, 1'-H), 6.69-6.80 (m, 4H, PhCH), 7.02-7.31 (stack,
13H, PhCH), 7.90 (s, 1H), 8.01 (s, 1H), 8.23 (s, 2H, NH.sub.2);
.sup.13P-NMR (61 MHz, CDCl.sub.3) 8.82, 8.99.
Synthesis of Hydroxy-tBuSATE N-benzylphosphoroamidate Derivative of
L-ddA:
##STR00076##
[0389] Crude phosphoroamidate diester 5 (2.00 g, assuming 2.31
mmol) was dissolved in dioxane/AcOH/H.sub.2O (25/17/25, v/v/v, 462
mL) and the solution was stirred for 3 d at room temperature.
Evaporation of the volatiles under reduced pressure left a residue
that was purified by flash column chromatography (SiO.sub.2, O=3
cm, H=15 cm) eluting with CH.sub.2Cl.sub.2 (100 mL) then 2% (100
mL).fwdarw.4% (100 mL).fwdarw.6% (100 mL).fwdarw.8% (150 mL) MeOH
in CH.sub.2Cl.sub.2. Evaporation of the fractions left NM 204 as a
white foam that was dissolved in MeCN (5 mL). Upon addition of
H.sub.2O (5 mL), the solution turned turbid and required sonication
before lyophilization. The resulting white powder was dried at room
temperature (using P.sub.2O.sub.5 as a desiccant) under vacuum for
1 d. The title compound was obtained as a highly hygroscopic white
powder (1:1 mixture of diastereoisomers as judged by .sup.31P-NMR;
499 mg, 35% over 3 steps). [.alpha.].sup.20.sub.D=+4.2.degree. (c
1.0, CHCl.sub.3); R.sub.f=0.29 (4% MeOH in CH.sub.2Cl.sub.2);
.sup.1H-NMR (300 MHz, DMSO-d6) 1.10 (s, 6H, 2.times.CH.sub.3),
2.02-2.14 (m, 2H, 2.times.3'-H), 2.41-2.55 (m, 2H, 2.times.2'-H),
3.01 (t, J 6.4, 2H, CH.sub.2S), 3.43 (d, J 5.0, 2H, CH.sub.2OH),
3.75-4.07 and 4.18-4.29 (stacks, 7H, CH.sub.2O, NCH.sub.2Ph, 4'-H,
2.times.5'-H), 5.02 (t, J 5.0, 1H, OH), 5.62 (m, 1H, NH), 6.25 (t,
J 5.1, 1H, 1'-H), 7.16-7.36 (stack, 7H, PhH, NH.sub.2), 8.14 (s,
1H), 8.26 (s, 1H); .sup.13C-NMR (75 MHz, DMSO-d6) 21.8
(2.times.CH.sub.3), 25.9 and 26.0 (CH.sub.2, 3'-C), 28.2 and 28.3
(CH.sub.2, CH.sub.2S), 30.9 and 31.0 (CH.sub.2, 2'-C), 44.2
(CH.sub.2, NCH.sub.2Ph), 51.7 (quat. C, C(CH.sub.3).sub.2), 63.7
and 63.8 (CH.sub.2, CH.sub.2O), 66.8 (CH.sub.2, m, 5'-C), 68.3
(CH.sub.2, CH.sub.2OH), 78.9 (CH, m, 4'-C), 84.2 (CH, 1'-C), 118.9
(quat. C), [126.5 (CH, Ph), 127.2 (CH, Ph), 128.1 (CH, Ph), some
overlap], 138.8 and 138.9 (CH), 140.5 and 140.6 (quat. C), 148.9
(quat. C), 152.3 (CH), 155.0 (quat. C), 204.0 (quat. C, C.dbd.O);
.sup.13P-NMR (61 MHz, DMSO-d6) 9.86, 9.95; m/z (FAB.sup.-) 563 (2),
306 (76), 153 (100); HRMS 565.2034 ([M+H].sup.+.
C.sub.24H.sub.34O.sub.6N.sub.6PS requires 565.1998); HPLC
t.sub.R=3.52 min (20% TEAC 20 mM in MeCN); UV (EtOH 95%)
.lamda..sub.max=259 (.epsilon..sub.max 15900), .lamda..sub.min=224
(.epsilon..sub.min 7200).
Example 2
Preparation of Hydroxy-tBuSATE N-Benzylphosphoroamidate Derivative
of 2'-C-methylcytidine
##STR00077##
[0390] Synthesis of H-Phosphonate Monoester 5
##STR00078##
[0391] Synthesis of Carboxylic Acid 3:
[0392] To a stirred solution of 2,2-dimethyl-3-hydroxypropanoic
acid methyl ester (1, 15 ml, 117.6 mmol) in a mixture of anhydrous
methylene chloride (590 ml) and triethylamine (23 ml), were added
triphenylmethylene chloride (1.2 eq, 39.3 g) and
4-dimethylaminopyridine (0.1 eq, 1.44 g). The reaction mixture was
left refluxing overnight. The mixture was poured carefully over a
saturated aqueous NaHCO.sub.3 solution and the product was
extracted with methylene chloride and washed with water. The
combined organic extracts were evaporated under reduced pressure to
give crude compound 2 which will be used for the next step without
further purification. The resulting oil was dissolved in a mixture
of dioxan (350 ml) and an aqueous solution of NaOH (30%, 350 ml).
The heterogene mixture was refluxed for 16 hours. The reaction
mixture was allowed to cool down to room temperature, the two
phases were separated, and the organic phase carefully neutralized
by dropwise addition of HCl (1M). The product was extracted with
methylene chloride and the organic phases were evaporated under
reduced pressure. The crude orange oil was recrystallized from
methylene chloride to afford carboxylic acid 3 as white crystals
(92%). R.sub.f=0.50 (70% diethyl ether in petroleum ether);
.sup.1H-NMR (400 MHz, CDCl.sub.3) 1.24 (s, 6H, 2.times.CH.sub.3),
3.19 (s, 2H, CH.sub.2O), 7.2-7.5 (m, 15H, C.sub.6H.sub.5).
Synthesis of H-Phosphonate Monoester 5:
[0393] 1,1'-carbonyldiimidazole (1.3 eq, 1.17 g) was added to a
stirring solution of carboxylic acid 3 (2 g, 5.56 mmol) in an
anhydrous mixture of toluene and dimethylformamide (2/1, v/v, 4.5
ml) at room temperature, and the reaction mixture turned turbid
instantly. After 30 min, the reaction mixture was diluted with a
mixture of toluene and dimethylformamide (93/7, v/v, 28 ml), cooled
to -10.degree. C., and 2-mercaptoethanol (1.3 eq, 500 .mu.L) was
added. The solution was stirred for 3 h at this temperature. The
volatiles were removed under reduced pressure (bath temperature not
exceeding 25.degree. C.). The residue was dissolved in methylene
chloride and washed with water. The organic phases were combined,
dried over sodium sulphate (Na.sub.2SO.sub.4), filtered and
evaporated to dryness to give compound 4 as a yellow oil. This
compound will be coevaporated with anhydrous pyridine and used for
the next step without further purification. R.sub.f=0.71 (70%
Et.sub.2O in petroleum ether); .sup.1H-NMR (400 MHz, CDCl.sub.3)
1.20 (s, 6H, 2.times.CH.sub.3), 3.05 (t, J=6.4 Hz, 2H, CH.sub.2S),
3.15 (s, 2H, CH.sub.2OTr), 3.69 (t, J=6.4 Hz, 2H, CH.sub.2OH),
7.3-7.9 (m, 15H, C.sub.6H.sub.5).
[0394] Phosphorus acid (10 eq, 4.1 g) was coevaporated two times
with anhydrous pyridine, dissolved in that solvent (25 ml) and
added to crude 4. The reaction mixture was stirred at room
temperature and a white precipitate appeared after few minutes. The
reaction mixture was cooled down to 0.degree. C. and pivaloyl
chloride (5.5 eq, 3.4 ml) was added. The reaction mixture was
allowed to warm to room temperature and stirred for 3 h. The
reaction was stopped by addition of a solution of triethylammonium
bicarbonate (TEAB 1M, 10 ml) and diluted with ethyl acetate
(EtOAc). After extraction with EtOAc and TEAB 0.5M, the organic
phases were combined, dried over sodium sulphate, filtered and
evaporated to dryness (bath temperature not exceeding 30.degree.
C.). The residue was purified by flash column chromatography
eluting with 10% of methanol in methylene chloride+1%
triethylamine. Evaporation of the fractions afforded the
H-phosphonate monoester 5 as a white syrup (90%). R.sub.f=0.25 (70%
Et.sub.2O in petroleum ether); .sup.1H-NMR (400 MHz, CDCl.sub.3)
1.17 (m, 2.times.CH.sub.3+excess (CH.sub.3CH.sub.2).sub.3N), 2.9
(m, excess (CH.sub.3CH.sub.2).sub.3N), 3.12 (t, J=6.8 Hz, 2H,
CH.sub.2S), 3.37 (s, 2H, CH.sub.2OTr), 3.90 (m, 2H, CH.sub.2OP),
7.2-7.6 (m, 15H, C.sub.6H.sub.5), 9.9 (m, excess
(CH.sub.3CH.sub.2).sub.3NH); .sup.31P-NMR (161 MHz, CDCl.sub.3)
3.85 (s).
Synthesis of Hydroxy-tBuSATE N-benzylphosphoroamidate derivative of
2'-C-methylcytidine: The following two strategies were used for the
synthesis:
Strategy A
Synthesis of the Protected Nucleoside 7
##STR00079##
[0396] A mixture of 2'C-methylcytidine (NM107) (10 g, 39.0 mmol),
triethyl orthoformate (8.3 eq, 54 ml) and p-toluenesulfonic acid
monohydrate (1 eq, 7.4 g) in anhydrous acetone (650 ml), was
refluxed overnight under nitrogen atmosphere. The reaction mixture
was neutralized with an aqueous ammonia solution (26%) and the
precipitate filtered. The filtrate was evaporated under reduced
pressure and coevaporated with ethanol. Purification of the crude
mixture on silica gel column chromatography (eluant: stepwise
gradient [0-10%] of methanol in methylene chloride) led to compound
6 as a pale-yellow solid (86%). R.sub.f=0.30 (20% MeOH in methylene
chloride), .sup.1H-NMR (400 MHz, DMSO-d.sub.6) 1.06 (s, 3H,
CH.sub.3), 1.33 (s, 3H, CH.sub.3), 1.47 (s, 3H, CH.sub.3), 3.6 (m,
2H, H-5', H-5''), 4.1 (m, 1H, H-4'), 4.41 (d, 1H, H-3', J=3.2 Hz),
5.16 (t, 1H, OH-5', J=4.0 Hz, D.sub.2O exchangeable), 5.69 (d, 1H,
H-5, J=8.0 Hz), 6.04 (s, 1H, H-1'), 7.14-7.19 (bd, 2H, NH.sub.2,
D.sub.2O exchangeable), 7.74 (d, 1H, H-6, J=8.0 Hz); LC/MS Scan ES-
296 (M-H).sup.-, Scan ES+ 298 (M+H).sup.+, .lamda..sub.max=280.7
nm.
[0397] Compound 6 (4.4 g, 14.8 mmol) was dissolved in anhydrous
pyridine (74 ml) and chlorotrimethylsilane (3 eq, 5.4 ml) was
added. The reaction mixture was stirred at room temperature under
nitrogen atmosphere for 2 h, then 4,4'-dimethoxytrityl chloride
(1.5 eq, 7.5 g) and 4-dimethylaminopyridine (0.5 eq, 900 mg) were
successively added. The reaction mixture was stirred overnight at
room temperature, then quenched with a saturated aqueous
NaHCO.sub.3 solution. The crude product was extracted with
methylene chloride, washed with saturated aq NaHCO.sub.3 solution,
and water. The combined organic phases were concentrated under
reduced pressure, then dissolved in a mixture of dioxan (160 ml)
and aqueous ammonia (28%, 29 ml). The solution was heated at
70.degree. C. for 3 h and evaporated to dryness. The crude mixture
was purified on silica gel column chromatography (eluant: stepwise
gradient of methanol [1-5%] in methylene chloride) to give
protected nucleoside 7 as a yellow solid (81%). R.sub.f=0.16 (30%
EtOAc in CH.sub.2Cl.sub.2) .sup.1H-NMR (400 MHz, DMSO-d.sub.6) 1.03
(s, 3H, CH.sub.3), 1.30 (s, 3H, CH.sub.3), 1.42 (s, 3H, CH.sub.3),
3.5 (m, 2H, H-5', H-5''), 3.71 (s, 6H, 2.times.OCH.sub.3), 4.0 (d,
1H, H-4', J=3.2 Hz), 4.36 (d, 1H, H-3', J=2.8 Hz), 5.1 (m, 1H,
OH-5', D.sub.2O exchangeable), 5.90 (s, 1H, H-1'), 6.2 (m, 1H,
H-5), 6.8-7.2 (m, 13H, DMTr), 7.6 (m, 1H, H-6), 8.32 (s, 1H, NH,
D.sub.2O exchangeable); LC/MS Scan ES- 598 (M-H).sup.-,
.lamda..sub.max1=231.7 nm, .lamda..sub.max2=283.7 mm.
Synthesis of the Pronucleotide 10
##STR00080##
[0399] Compounds 7 (2.0 g, 3.34 mmol) and 5 (2.2 eq, 4.3 g) were
coevaporated together with anhydrous pyridine and dissolved in this
solvent (50 ml). Pivaloyl chloride (2.5 eq, 1 ml) was added
dropwise and the solution stirred at room temperature for 2h30. The
reaction mixture was diluted with methylene chloride and
neutralized with an aqueous solution of ammonium chloride
(NH.sub.4Cl 0.5M). After extraction with methylene chloride/aq
NH.sub.4Cl 0.5M, the organic phases were combined, evaporated under
reduced pressure (bath temperature not exceeding 30.degree. C.) and
coevaporated with toluene. The crude mixture was purified on silica
gel column chromatography (eluant: stepwise gradient [0-5%] of
methanol in methylene chloride+2.Salinity. acetic acid) to afford
the desired product 8 which was coevaporated with toluene to give a
beige foam (94%). R.sub.f=0.63 (5% MeOH in CH.sub.2Cl.sub.2);
.sup.1H-NMR (400 MHz, CDCl.sub.3) 1.21 (m, 9H, 3 CH.sub.3), 1.42
(s, 3H, CH.sub.3), 1.60 (s, 3H, CH.sub.3), 3.13 (m, 2H, CH.sub.2S),
3.17 (m, 2H, CH.sub.2OTr), 3.79 (s, 6H, 2.times.OCH.sub.3), 4.1 (m,
2H, CH.sub.2OP), 4.2-4.3 (m, 3H, H-5', H-5'', H-4'), 5.09 (d, 1H,
H-3', J=7.6 Hz), 5.89 (d, 1H, H-5, J=5.6 Hz), 6.0 (m, 1H, H-1'),
6.8-7.7 (m, 29H, Tr, DMTr, H-6); .sup.13P-NMR (161 MHz, CDCl.sub.3)
7.92, 8.55; LC/MS Scan ES+ 1066 (M+H).sup.+, Scan ES- 1064
(M-H).sup.-.
[0400] To a solution of compound 8 (3.4 g, 3.15 mmol) in anhydrous
carbon tetrachloride (30 ml) was added dropwise benzylamine (10 eq,
3.4 ml). The reaction mixture was stirred at room temperature for
1h30. A white precipitate appeared. The solution was diluted with
methylene chloride and neutralized with an aqueous solution of
hydrogen chloride (HCl 1M). After successive extractions with
CH.sub.2Cl.sub.2/HCl 1M and CH.sub.2Cl.sub.2/aq NaHCO.sub.3, the
organic phases were combined, dried over Na.sub.2SO.sub.4, filtered
and evaporated to dryness. The crude mixture was purified on silica
gel column chromatography (eluant: stepwise gradient [0-5%] of
methanol in methylene chloride) to give 2 as a yellow foam (87%).
Rf=0.35 (5% MeOH in methylene chloride); .sup.1H-NMR (400 MHz,
CDCl.sub.3) 1.1-1.2 (m, 9H, 3 CH.sub.3), 1.40 (s, 3H, CH.sub.3),
1.59 (s, 3H, CH.sub.3), 2.9-3.2 (m, 4H, CH.sub.2OTr, CH.sub.2OS),
3.76 (s, 6H, 2.times.OCH.sub.3), 3.9-4.4 (m, 8H, CH.sub.2OP,
CH.sub.2N, H-3', H-4', H-5', H-5''), 5.0 (m, 1H, H-5), 6.0 (2s, 1H,
H-1'), 6.7-7.7 (m, 34H, Tr, DMTr, C.sub.6H.sub.5CH.sub.2, H-6);
.sup.13P-NMR (161 MHz, CDCl.sub.3) 8.40, 8.8.68; LC/MS Scan ES+
1171 (M+H).sup.+.
[0401] Finally, compound 9 (2.39 g, 2.04 mmol) was dissolved in a
mixture of methylene chloride (10 ml) and an aqueous solution of
trifluoroacetic acid (90%, 10 ml). The reaction mixture was stirred
at 35-40.degree. C. for 2 h, then diluted with ethanol (140 ml).
The volatiles were evaporated under reduced pressure and
coevaporated with ethanol. The crude mixture was purified by silica
gel column chromatography (eluant: stepwise gradient of methanol
[0-30%] in methylene chloride), followed by a purification on
reverse phase chromatography (eluant: stepwise gradient of
acetonitrile [0-50%] in water), to give the desired product 10
(B102) (1:1 mixture of diastereoisomers as judged by .sup.31P-NMR,
36%) which was lyophilized from a mixture of dioxan/water. Rf=0.34
(15% MeOH in methylene chloride); .sup.1H-NMR (400 MHz, DMSO-d6)
0.92 (s, 3H, CH.sub.3), 1.10 (s, 6H, 2.times.CH.sub.3), 3.0 (m, 2H,
CH.sub.2S), 3.33 (m, 1H, H-3'), 3.56 (s, 2H, CH.sub.2OH), 3.8-4.0
and 4.05-4.25 (stacks, 7H, CH.sub.2OP, NCH.sub.2Ph, H-4', H-5' and
H-5''), 4.9 (m, 1H, OH-3', J=5.4 Hz, D.sub.2O exchangeable), 5.07
(s, 1H, OH-2', D.sub.2O exchangeable), 5.3 (m, 1H, CH.sub.2OH,
D.sub.2O exchangeable), 5.6-5.7 (m, 2H, H-5 and NH, D.sub.2O
exchangeable), 5.91 (s, 1H, H-1'), 7.3-7.4 (stack, 7H, PhH,
NH.sub.2, D.sub.2O exchangeable), 7.6 (m, 1H, H-6); .sup.13P-NMR
(161 MHz, DMSO-d6) 9.71, 9.91; HPLC t.sub.R=4.67 min (0-100%
acetonitrile over a period of 8 min), .lamda..sub.max=274.9; LC/MS
Scan ES+ 587 (M+H).sup.+.
Strategy B
[0402] Synthesis of Protected Nucleoside 11
##STR00081##
[0403] NM107 (10 g, 38.87 mmol) was dissolved in anhydrous pyridine
(194 ml) and chlorotrimethylsilane (4.5 eq, 21.6 ml) was added. The
reaction mixture was stirred at room temperature under nitrogen
atmosphere for 2h30, then 4,4'-dimethoxytrityl chloride (1.5 eq,
19.8 g) and 4-dimethylaminopyridine (0.5 eq, 2.37 g) were
successively added. The reaction mixture was stirred overnight at
room temperature, then quenched with a saturated aqueous
NaHCO.sub.3 solution. The crude product was extracted with
methylene chloride, washed with saturated aq NaHCO.sub.3 solution,
and water. The combined organic phases were concentrated under
reduced pressure, then dissolved in tetrahydrofuran (110 ml). To
that solution was added tetrabutylammonium fluoride 1M in THF (1
eq, 38.87 ml) and the reaction mixture was stirred for 30 min at
room temperature. After extraction with EtOAc and water, the
organic phases were collected and evaporated to dryness. The crude
mixture was purified on silica gel column chromatography (eluant:
stepwise gradient of methanol [0-10%] in methylene chloride) to
give protected nucleoside 11 as a yellow solid (93%). R.sub.f=0.32
(10% MeOH in CH.sub.2Cl.sub.2) .sup.1H-NMR (400 MHz, DMSO-d.sub.6)
0.79 (s, 3H, CH.sub.3), 3.56 (m, 2H, H-5', H-5''), 3.71 (s, 7H,
2.times.OCH.sub.3, H-4'), 5.0 (m, 4H, H-3', OH-2', OH-3', OH-5',
D.sub.2O exchangeable), 5.72 (s, 1H, H-1'), 6.16 (m, 1H, H-5),
6.8-7.2 (m, 13H, DMTr), 7.82 (m, 1H, H-6), 8.24 (m, 1H, NH D.sub.2O
exchangeable); LC/MS Scan ES- 560 (M+H).sup.+, ES- 558 (M-H).sup.-,
.lamda..sub.max=284.7 nm.
Synthesis of Protected Phosphoroamidate Pronucleotide 13, Precursor
of 10
##STR00082##
[0405] Compound 11 (7 g, 12.5 mmol) and 5 (1.5 eq, 11.0 g) were
coevaporated together with anhydrous pyridine and dissolved in this
solvent (187 ml). Pivaloyl chloride (2.0 eq, 3.08 ml) was added
dropwise at -15.degree. C. and the solution stirred at this
temperature for 1h30. The reaction mixture was diluted with
methylene chloride and neutralized with an aqueous solution of
ammonium chloride (NH.sub.4Cl 0.5M). After extraction with
methylene chloride/aq NH.sub.4Cl 0.5M, the organic phases were
combined, evaporated under reduced pressure (bath temperature not
exceeding 30.degree. C.) and coevaporated with toluene. The crude
mixture was purified on silica gel column chromatography (eluant:
stepwise gradient [0-5%] of methanol in methylene chloride+0.2%
acetic acid) to afford the desired product 12 which was
coevaporated with toluene to give a white foam (3.5 g, 27%).
R.sub.f=0.44 (5% MeOH in CH.sub.2Cl.sub.2); .sup.1H-NMR (400 MHz,
DMSO) 0.8 (m, 3H, CH.sub.3), 1.14 and 1.06 (2s, 6H, 2 CH.sub.3),
3.06 (m, 2H, CH.sub.2S), 3.16 (m, 2H, CH.sub.2OTr), 3.5 (m, 1H,
H-3'), 3.70 (m, 6H, 2 OCH.sub.3), 3.90 (m, 1H, H-4'), 4.03 (m, 2H,
CH.sub.2OP), 4.24 (m, 2H, H-5', H-5''), 5.30 and 5.04 (2 ms, 2H,
OH-2' and OH-3', D.sub.2O exchangeable), 5.78 (m, 1H, H-1'), 5.98
(m, 1H, P--H), 6.22 (m, 1H, H-5), 7.0-7.5 (m, 16H, Tr), 8.32 (m,
1H, H-6); .sup.13P-NMR (161 MHz, DMSO) 9.17, 9.65; LC/MS Scan ES+
1026 (M+H).sup.+, .lamda..sub.max=282.7 nm.
[0406] To a solution of compound 12 (500 mg, 0.49 mmol) in
anhydrous carbon tetrachloride (4.9 ml) was added dropwise
benzylamine (5 eq, 0.266 ml). The reaction mixture was stirred at
room temperature for 3 h and the solvent removed under reduced
pressure. The crude mixture was purified on silica gel column
chromatography (eluant: stepwise gradient [0-5%] of methanol in
methylene chloride) to afford compound 13 as a foam (75%). Rf=0.25
(3% MeOH in methylene chloride); .sup.1H-NMR (400 MHz, DMSO) 0.79
(s, 3H, CH.sub.3), 1.13 and 1.06 (2s, 6H, 2 CH.sub.3), 3.05 (m, 4H,
CH.sub.2OTr, CH.sub.2OS), 3.51 (m, 1H, H-3'), 3.69 (s, 6H,
2.times.OCH.sub.3), 3.87 (m, 3H, CH.sub.2OP, CH.sub.2N, H-3'), 4.08
(m, 2H, H-5', H-5''), 5.19 and 5.0 (2m, 2H, OH-2' and OH-3',
D.sub.2O exchangeable), 5.67 (m, 1H, NH, D.sub.2O exchangeable),
5.75 (2s, 1H, H-1'), 6.21 (m, 1H, H-5), 6.7-7.5 (m, 34H, Tr, DMTr,
C.sub.6H.sub.5CH.sub.2, H-6); .sup.13P-NMR (161 MHz, DMSO) 9.84,
9.69; LC/MS Scan ES+ 1132 (M+H).sup.+.
[0407] Compound 13 can be converted into the phosphoroamidate
prodrug 10 (B102) following experimental conditions described for
the last step of NM108- and NM105-OH-SATE phosphoroamidate
synthesis, in Examples 3 and 4, respectively.
Example 3
Preparation of Hyrdoxy-t-BuSATE-N-Benzylphosphoramidate Derivative
of 2'-C-Methylguanosine
##STR00083##
[0408] Synthetic Strategy:
##STR00084## ##STR00085##
[0410] 2'-C-methylguanidine (NM108) (3 g, 10.10 mmol) and compound
5 (6.48 g, 11.10 mmol) were coevaporated together with anhydrous
pyridine and dissolved in this solvent (152 mL). Pivaloyl chloride
(2.48 mL, 20.18 mmol) was added dropwise at -15.degree. C. and the
solution was stirred at the same temperature for 2 h. The reaction
mixture was diluted with methylene chloride and neutralized with an
aqueous solution of ammonium chloride (NH.sub.4Cl 0.5M). After
extraction with methylene chloride/aq NH.sub.4Cl 0.5M, the organic
phases were combined, dried over Na.sub.2SO.sub.4 evaporated under
reduce pressure (bath temperature not exceeding 30.degree. C.) and
coevaporated twice with toluene. The crude mixture was purified on
silica gel flash column chromatography (eluant: stepwise gradient
[0-10%] of methanol in methylene chloride+0.2% acetic acid) to
afford the desired product 6 (2.5 g, 32%). R.sub.f=0.34 (15% MeOH
in CH.sub.2Cl.sub.2); .sup.1H-NMR (400 MHz, DMSO-d.sub.6 0.80 (s,
3H, CH.sub.3), 1.13 (s, 6H, 2.times.CH.sub.3), 3.04 (m, 2H,
CH.sub.2OTr), 3.14 (m, 2H, CH.sub.2S), 3.97-4.08 (m, 4H, H-3',
H-4', CH.sub.2OP), 4.28-4.38 (m, 2H, H-5', H-5''), 5.10-5.35 (m,
2H, OH-2', OH-3', D.sub.2O exchangeable), 5.77 (s, 1H, H-1'), 6.52
(bs, 2H, NH.sub.2, D.sub.2O exchangeable), 7.11-7.42 (m, 15H, Tr),
7.75 (s, 1H, H-8), 10.67 (bs, 1H, NH, D.sub.2O exchangeable);
.sup.13P-NMR (161 MHz, DMSO-d.sub.6) 9.47, 9.20; LC/MS Scan ES+ 764
(M+H).sup.+, Scan ES- 762 (M-H).sup.-.
[0411] To a solution of compound 6 (2.5 g, 3.27 mmol) in anhydrous
carbon tetrachloride (33 mL) was added dropwise benzylamine (5 eq,
1.79 mL). The reaction mixture was stirred at room temperature for
1 h and evaporated under reduced pressure (bath temperature not
exceeding 30.degree. C.). The crude mixture was purified on silica
gel flash column chromatography (eluant: stepwise gradient [0-10%]
of methanol in methylene chloride) to give compound 7 as a white
foam (2.9 g, quantitative yield). R.sub.f=0.27 (10% MeOH in
methylene chloride); .sup.1H-NMR (400 MHz, DMSO-d.sub.6) 0.81 (s,
3H, CH.sub.3), 1.10 (s, 6H, 2.times.CH.sub.3), 2.99-3.08 (m, 4H,
CH.sub.2OTr, CH.sub.2S), 3.87-4.30 (m, 8H, H-3', H-4', H-5', H-5''
CH.sub.2OP, NCH.sub.2Ph), 5.66 (m, 1H, NH, D.sub.2O exchangeable),
5.76 (s, 1H, H-1'), 6.60 (bs, 2H, NH.sub.2, D.sub.2O exchangeable),
7.17-7.39 (m, 20H, Tr, C.sub.6H.sub.5CH.sub.2), 7.77 (s, 1H, H-8);
.sup.13P-NMR (161 MHz, DMSO-d.sub.6) 9.93, 9.78; LC/MS Scan ES+ 869
(M+H).sup.+, Scan ES- 867 (M-H).sup.-.
[0412] Compound 7 (2.84 g, 3.27 mmol) was dissolved in a mixture of
trifluoroacetic acid (1.1 mL) and methylene chloride (11.4 mL). The
reaction mixture was stirred 0.5 h at room temperature. The
solution was diluted with ethanol, evaporated under reduce pressure
(bath temperature not exceeding 30.degree. C.) and coevaporated
twice with toluene. The crude mixture was purified on silica gel
flash column chromatography (eluant: stepwise gradient [0-30%] of
methanol in methylene chloride) and then, on reverse phase column
chromatography (eluant: stepwise gradient [0-100%] of acetonitrile
in water) to give the desired product 8 (B184) (1:1 mixture of
diastereoisomers according to .sup.31P-NMR, 800 mg, 39%) which was
lyophilized from a mixture of dioxan/water. Rf=0.57 (20% MeOH in
methylene chloride); .sup.1H-NMR (400 MHz, DMSO-d6) 0.82 (s, 3H,
CH.sub.3), 1.09 (s, 6H, 2.times.CH.sub.3), 3.01 (m, 2H, CH.sub.2S),
3.42 (d, 2H, CH.sub.2OH, J=8.0 Hz), 3.81-4.00 (m, 6H, H-3', H-4'
CH.sub.2OP, NCH.sub.2Ph), 4.11-4.27 (m, 2H, H-5', H-5''), 4.92 (t,
1H, CH.sub.2OH, J=8.0 Hz, D.sub.2O exchangeable), 5.16 (s, 1H,
OH-2', D.sub.2O exchangeable), 5.40 (m, 1H, OH-3', D.sub.2O
exchangeable), 5.64 (m, 1H, NH, D.sub.2O exchangeable), 5.75 (s,
1H, H-1'), 6.50 (bs, 2H, NH.sub.2 D.sub.2O exchangeable), 7.19-7.32
(m, 5H, PhH), 7.77 (s, 1H, H-8), 10.61 (bs, 1H, NH, D.sub.2O
exchangeable); 13P-NMR (161 MHz, DMSO-d6) 9.91, 9.78; HPLC
t.sub.R=3.67 min (0-100% acetonitrile over a period of 8 min),
.lamda..sub.max=251.3; LC/MS Scan ES+ 627 (M+H).sup.+, Scan ES- 625
(M-H).sup.-.
Example 4
[0413] A anti-cancer drug, R--OH, such as an antiviral nucleoside,
having a free OH group, is derivatized to form a phosphoramidate
compound according to the following scheme. Reactive groups on the
molecule, such as other hydroxyl groups, are protected using
methods known in the art.
##STR00086##
Example 5
[0414] A phosphoroamidate of 5-azacytidine is prepared as
follows:
##STR00087##
[0415] Examples 6-10 illustrate by way of example the effect of the
phosphoroamidate group on an antiviral compound to promote liver
specific delivery of an active agent to liver cells.
Example 6
Preparation of Calibration Curve
[0416] Measurements of the concentration of
2'-3'-dideoxyadenosine-5'-triphosphate (ddATP) (the triphosphate
nucleotide of 2'-3'-dideoxyadenosine (ddA) are performed by liquid
chromatography tandem mass spectrometry (LC/MS/MS), e.g., of
methanolic extracts of hepatocytes.
[0417] The concentration of ddATP is measured by comparison to a
standard curve. Working stock solutions of TP-ddA are prepared from
a 100 .mu.mol/.mu.l stock solution in de-ionized water of ddATP
(tetrasodium salt of >91% purity) purchased from Sigma Chemical
Co as follows:
ddATP Working Stock Solutions and Preparation of Standard Curve for
ddATP.
TABLE-US-00003 Stock Vol DIH.sub.2O Total conc taken vol vol Conc
mol per pmol/.mu.l .mu.L .mu.L .mu.L pmol/.mu.l 10 .mu.l 1. Working
stock#1 Test compound TP-ddA 100 2000 2000 4000 50.0 500 2. Working
stock#2 Test article TP-ddA 100 1000 3000 4000 25.0 250 3. Working
stock#4 (prepared from stock#1) TP-ddA 100 500 3500 4000 12.5 125
4. Working stock#5 (prepared from stock#1) TP-ddA 100 200 3800 4000
5.0 50 5. Working stock#6 (prepared from stock#1) TP-ddA 100 100
3900 4000 2.5 25 6. Working stock#7 (prepared from stock#1) TP-ddA
100 40 3960 4000 1.0 10
[0418] Internal standard (ISTD) working stock are prepared from a
0.50 mg/mL stock solution of 2-deoxyadenosine 5-triphosphate
purchased from Sigma Chemical Co.
TABLE-US-00004 Stock conc Vol taken MeOH vol Total vol Conc Conc
ISTD .mu.g/mL .mu.L .mu.L .mu.L .mu.g/mL pmol/mL dATP 500 200 9800
10000 10 500
[0419] In some embodiments, calibration standards are prepared as
follows using liver
TABLE-US-00005 Preparation of cal stds: std conc working working
stock con working stock vol ISTD vol MeOH vol total vol cal std#
pmol/ml liver wt G stock# pmol/.mu.L uL uL uL uL Blk 0 0.1 0 50 940
990 #1 50 0.1 #5 5.0 10 50 940 1000 #2 125 0.1 #4 12.5 10 50 940
1000 #3 250 0.1 #3 25.0 10 50 940 1000 #4 500 0.1 #2 50.0 10 50 940
1000 #5 1000 0.1 #1 100.0 10 50 940 1000
samples:
[0420] In some embodiments the following HPLC conditions are used
for the HPLC MS, e.g. HPLC Tandem MS analysis instrument
method:
[0421] HPLC is conducted on Phenomenex Luna Amino 3 .mu.m 100 A,
30.times.2 mm column, with a mobile phase: A: 70% 10 mM NH.sub.4OAc
30% ACN pH 6.0; and B: 70% 1 mM NH.sub.4OAc 30% ACN pH 10.5 as
follows:
TABLE-US-00006 Time Flow Step (min) (.mu.l/min) A (%) B (%)
Gradient elution program: 0 0 400 60 40 1 1.1 400 60 40 2 1.11 400
40 60 3 2.11 400 30 70 4 2.6 400 20 80 5 3.1 400 0 100 6 5.5 400 0
100 7 5.51 400 60 40 8 10 400 60 40 Injection volume: 50 ul Flow
rate to MS: 0.400 mL/min, no splitting of flow Multiple Reaction
Monitoring (MRM) conditions: (API3000) Ionization Mode: Positive
Ion Electrospray (ESI+) IonSpray Voltage (IS): 5000 V Temperature
(TEM): 550.degree. C. Turbo IS gas 8 L/min Nebulizer (NEB): 14 CAD
Gas Setting (CAD): 6 Declustering potential (DP) 68 V Collision
energy (CE) 27 eV Entrance/Exit potentials 10 V/11 V (EP/CXP)
Compound Precursor ion => Product Ion ddA triphosphate 476.2
=> 135.9 ddA diphosphate 396.2 => 135.9 dA triphosphate
(ISTD) 460.2 => 135.9
*Luna Amino column is directly connected on the inlet end to a
"Security Guard" cartridge holder suitable for 2.1 mm Phenomenex
columns, containing a C18 cartridge.
Example 7
In Vitro Phosphorylation in Hepatocytes
[0422] Primary hepatocytes (Rat, Cynomolgus Monkey or human) were
seeded at 0.8.times.10.sup.6 in a collagen-coated 12-well plate and
allowed to attach 4-6 hours after which time the seeding medium was
replaced with serum-free culture medium and cells allowed to
acclimatize to the new medium overnight. On the next day, cells
were exposed for 1, 4, 8 and 24 hours to test article A550 (NM204)
at 10 and 50 .mu.M prepared in fresh culture medium from stock
solution in DMSO (final DMSO concentration was 0.1%). At each time
point, an aliquot (500 .mu.l) was collected and immediately added
to 500 .mu.l of acetonitrile and stored at -20.degree. C. until
analysis. The remaining exposure medium was removed and the cell
monolayer (stuck to dish) washed 2 times with ice-cold PBS. Any
remaining PBS was carefully removed by aspiration and cells were
harvested by scraping in 1 mL 70% ice-cold methanol. Cell samples
were placed overnight at -20.degree. C. and cellular debris removed
by centrifugation on the next day. The supernatants were removed
and filtered prior to analysis by LC/MS. A standard curve was
prepared by using untreated cells processed similarly except that
prior to harvesting in 70% methanol, 10 .mu.l of LddATP standard
solutions prepared in methanol were added to the washed monolayers.
These control samples were then processed and analyzed as described
for test samples.
[0423] The results are shown below:
TABLE-US-00007 LddA-TP formation in hepatocytes LddA TP Levels
(pmol/million cells) Rat Monkey Human A550 (Ex 1) 10 .mu.M Time
(hour) 1 159.5 287.5 161.5 4 388.0 978.0 312.5 8 468.5 1230.0 352.5
24 422.0 344.0 366.0 A550 (Ex 1) 50 .mu.M Time (hour) 1 393.0
2085.0 682.5 4 1212.0 5690.0 1480.0 8 1590.0 6030.0 1930.0 24
1505.0 3030.0 2062.5
[0424] As indicated from the data, significant levels of L-ddATP
were detected in the hepatocytes. In monkeys, the levels appear to
reach a maximum level at 8 hours followed by a rapid decline. In
contrast, levels in both rat and human hepatocyte appear to level
off after 8 hours.
Example 8
In Vivo Studies in Rat
[0425] Distribution of A550 (NM-204) (the compound of Example 1
(Hydroxy-tBuSATE N-benzylphosphoroamidate derivative of L-ddA) in
the rat liver was evaluated following a single intravenous (I.V.)
or oral administration of A550 (NM-204) at a dose of 20 (oral) or
10 (I.V.) mg/Kg body weight. The dose solutions were prepared on
the same day prior to dose administration.
[0426] At the specified time point (1 and 3 hours for IV animals or
1, 3 and 8 hours for oral animals), each animal was euthanized by
CO.sub.2 gas followed by exsanguination via the abdominal vein.
Livers were collected immediately after sacrifice, flash frozen in
liquid nitrogen, placed on dry ice, and later stored at -70.degree.
C., before being analyzed.
Preparation of Calibration Standards from Control Liver
Extracts:
[0427] Control rat liver samples were taken from whole frozen
livers (Bioreclamation, Inc. Hicksville, N.Y.) with the aid of a
tissue coring utensil (Harris Unicore, 8.0 mm, VWR). Each
.about.0.1 g sample was placed in individual 2 mL poly vials with
0.940 mL of 80% MeOH/20% DIH.sub.2O and homogenates were prepared
using a mechanical tissue disruptor (Tissue Master,
Omni-International, Inc, Marietta Ga.). The vials received a 10
.mu.l aliquot of a working stock solution and a 50 .mu.l aliquot of
the ISTD before vortexing for .about.30 sec. The mixtures were
stored overnight at -20.degree. C. and the next day were removed
for 10 min of centrifugation in a benchtop centrifuge. Each
supernatant was transferred to individual centrifugation filtration
units (0.45 .mu.m) and the resulting filtrates were transferred to
HPLC vials for the LC/MS/MS analysis. The final concentrations of
ddATP in the calibration standards was 1000, 500, 250, 125, 50, and
0 pmol/ml. Each calibration standard was directly injected in a 50
.mu.L volume onto the ion-exchange column for analysis. Standard
curve analysis of calibration standards from control liver extracts
was conducted.
[0428] Analysis of ddATP was done by an ion-exchange chromatography
method with on-line positive ionization ESI-MS/MS detection in
multiple reaction monitoring (MRM) detection mode. The peak areas
obtained for 4 of the 5 calibrants allowed for construction of a
standard curve that demonstrated good linearity (R.sup.2=0.9996)
over a 50-1000 .mu.mol/ml concentration range. This is equivalent
to a range of 5-100 .mu.mol per gram liver by the sample
preparation employed. The HPLC MS MS conditions described in
Example 5 were utilized. The lower limit of quantitation
demonstrated by the LC/MS/MS method is e.g., .about.0.2 pmol/mL for
hepatocyte cellular extracts which contain much less salt.
[0429] The results showing intracellular levels of A550 (NM204)
(showing the compound entered the liver cells) and LddATP (showing
cleaving of the phosphoroamidate moiety and triphosphorylation of
the ddA to the active triphosphate in the liver) are shown
below:
TABLE-US-00008 A550 (Ex 1) and LddATP measured in livers of male
rats dosed IV or O with A550 (Ex 1) Concentration Concentration.
Compound ddA-TP (Ex 1) Timepoint (pmol/ (pmol/10.sup.6 Animal
Number (pmol/g liver) (hrs) g liver) cells)* IV dose (10 mg/kg) 1M1
65.8 1 2025 17.8 1M2 89.1 1 1930 16.9 1M3 85.1 1 1355 11.9 Mean
80.0 1770 15.5 IV dose (10 mg/kg) 2M1 28.3 3 1345 11.8 2M2 26.0 3
1940 17.0 2M3 29.3 3 2990 26.2 Mean 27.9 2092 18.3 Oral dose (20
mg/kg) 3M1 411 1 210 1.8 3M2 272 1 575 5.0 3M3 70.2 1 400 3.5 Mean
251 395 3.5 Oral dose (20 mg/kg) 4M1 360 3 200 1.8 4M2 92.1 3 330
2.9 4M3 161 3 405 3.6 Mean 204 312 2.7 Oral dose (20 mg/kg) 5M1
16.4 8 280 2.5 5M2 28 8 805 5.2 5M3 16.2 8 275 2.4 Mean 20.1 382
3.3 *Hepatocellularity number for rat was 114 .times. 106 cells per
gram liver (Toxicology in Vitro 20 (2005) 1582-1586.
[0430] Thus, these results show that the compound can be used to
enhance concentration of the drug in the liver. These results also
show the enhanced concentration of the active triphosphate which is
formed in the liver cells.
Example 9
Determination of Total Metabolism in Liver Subcellular
Fractions
Depletion of Parent
[0431] NADPH Incubations. Microsomal or S9 incubations were
conducted in a final volume of 0.5 mL. Pooled liver microsomal or
S9 protein (1.0 mg/mL), suspended in incubation buffer (100 mM
potassium phosphate, pH 7.4, 5 mM MgCl.sub.2, and 0.1 mM EDTA) was
preincubated for 5 min at 37.degree. C. with 10-50 .mu.M OHSATE
phosphoroamidate compound from a stock solution in DMSO (final DMSO
concentration was 0.1%); the reaction was initiated by the addition
of NADPH (3 mM final concentration). Incubations with no NADPH
served as controls. At specific times (0-120 min), 0.1 mL samples
were taken and the reaction terminated by the addition of 1 volume
of stop solution (acetonitrile). The samples were vortex for 30 sec
and then centrifuged at 1500 g for 10 min. The supernatant was
transferred to HPLC glass vials and analyzed without further
processing by HPLC. FIGS. 1 and 2 depict depletion of NM108 SATE
and NM107 SATE, respectively, after incubation with NADPH in monkey
liver S9.
HPLC System for Medium Samples-Unchanged Prodrug
TABLE-US-00009 [0432] HPLC: Agilent 1100 Column: Phenomenex Luna
C18(2), 20 .times. 2 mm, Mobile phases (MP): MP(A) 10 mM
K.sub.2HPO.sub.4 pH5, MP(B) ACN Gradient elution: 20 to 63% MP(B)
run from 0 to 30 min Runtime: 20 min Flow rate: 1 mL/min Injection
volume: 10-20 .mu.L UV: 252 nm-NM108SATE 272 nm-NM107SATE
[0433] Thus, without being limited to any theory, since the
metabolism is NADPH dependent, it is possible that the
phosphoroamidate compound is preferentially activated by Cytochrome
P450 in the liver.
Example 10
Determination of Triphosphate Levels in Cells
[0434] Preparation of Primary Hepatocyte Cultures
[0435] Freshly isolated cells from animal and human liver were
obtained in suspension on ice. Following receipt, cells were
pelleted by centrifugation at 500 rpm (rat) or 700 rpm (monkey and
human) and resuspended at 0.8 million cells per mL of platting
medium (HPM). Multi-well collagen-coated plates (12-well) were then
seeded by addition of 1 mL of cell suspention (0.8 million
cells/mL). The plates were gently shaken to evenly distribute the
cells and placed in an incubator at 37.degree. C. for approximately
4 to 6 hours to allow cells to attach. Once cells have attached,
the platting medium was removed and replaced with hepatocyte
culture medium (HCM). Cells were left overnight in an incubator at
37.degree. C. to acclimatize to culture and the medium.
[0436] Incubations with Test Article
[0437] Hepatocyte incubations were conducted in a final volume of
1.0 mL HCM/well (0.8 million cells/mL). HCM from overnight
incubation of cells was removed and replaced with fresh HCM,
pre-warmed to 37.degree. C., containing 10 .mu.M test article from
a stock solution in DMSO (final DMSO concentration was 0.1%). At
specific times (up to 24 hrs), incubation medium was discarded and
the cell monolayers were carefully washed two times with ice-cold
PBS. Following the last wash, all PBS was carefully removed and 1
mL of extraction buffer (ice-cold 70% methanol) was added. Each
well was sealed with parafilm immediately following addition of
methanol. Once the entire plate was processed, additional parafilm
was placed on entire plate forming a double seal to prevent
evaporation during the extraction process. The cover lid was then
placed on the plate and sealed with tape. The plates were then
stored at -20.degree. C. for a minimum of 24 hrs to allow for
extraction of intracellular contents.
[0438] Preparation of Huh7 or HepG2 Cultures
[0439] HepG2s or Huh7 cells were plated at 0.4.times.10.sup.6
cells/well in collagen-coated 12-well plates. Cells were allowed to
attach overnight. Culture medium from overnight incubation of cells
was removed and replaced with fresh culture medium, pre-warmed to
37.degree. C., containing 10 .mu.M test article from a stock
solution in DMSO (final DMSO concentration was 0.1%). After 24-72
hours, incubation medium was discarded and the cell monolayers were
carefully washed two times with ice-cold PBS. Following the last
wash, all PBS was carefully removed and 1 mL of extraction buffer
(ice-cold 70% methanol) was added. Each well was sealed with
parafilm immediately following addition of methanol. Once the
entire plate was processed, additional parafilm was placed on
entire plate forming a double seal to prevent evaporation during
the extraction process. The cover lid was then placed on the plate
and sealed with tape. The plates were then stored at -20.degree. C.
for a minimum of 24 hrs to allow for extraction of intracellular
contents.
[0440] Sample Preparation for Analysis
[0441] Cellular extracts were prepared by transferring 0.9 mL of
extract into 2 mL microfuge tubes followed by centrifugation for 5
min at 14,000 rpm. Approximately 100 .mu.L of the supernatant was
transferred to HPLC vials and triphosphate levels determined by
LCMS/MS as described below.
[0442] HPLC conditions: NM107-triphosphate
TABLE-US-00010 HPLC: Column: Phenomenex Luna Amino 3 .mu.m 100A, 30
.times. 2 mm, Mobile phases (MP): (A) 70% 10 mM NH.sub.4OAc 30% ACN
pH 6.0 (B) 70% 1 mM NH.sub.4OAc 30% ACN pH 10.5 Step Time Flow A B
Gradient elution: 0 0.00 400 80 20 1 0.10 400 80 20 2 0.11 400 40
60 3 0.21 400 40 60 4 2.60 400 10 90 5 2.61 400 0 100 6 5.60 400 0
100 7 5.61 400 80 20 8 9.00 400 80 20 Flow rate to MS: 0.400
mL/min, no split Injection volume: 10 .mu.L Compound Precursor ion
Product ion NM107 triphosphate 498.0 112.0
[0443] HPLC conditions: NM108-triphosphate
TABLE-US-00011 HPLC: Column: Phenomenex Luna Amino 3 pm 100A, 30
.times. 2 mm, Mobile phases (MP): (A) 70% 10 mM NH.sub.4OAc 30% ACN
pH 6.0 (B) 70% 1 mM NH.sub.4OAc 30% ACN pH 10.5 Step Time Flow A B
Gradient elution: 0 0.00 400 60 40 1 0.10 400 60 40 2 0.11 400 40
60 3 0.21 400 40 60 4 2.60 400 10 90 5 2.61 400 0 100 6 5.61 400 0
100 7 5.61 400 60 40 8 9.00 400 60 40 Flow rate to MS: 0.400
mL/min, no split Injection volume: 10 .mu.L Compound Precursor ion
Product ion NM108 triphosphate 538.0 152.0
##STR00088##
[0444] NM107 triphosphate levels and B102 in cell extracts were
observed as follows:
TABLE-US-00012 Intracellular Triphosphate (pmol/million cells) drug
in culture Human Monkey HepG2* Huh7* B102 (Ex. 2) 991 1838 1.5 9.2
NM107 19 10 17 37 24 hr incubation in 10 .mu.M drug *72 hr
incubation in 10 .mu.M drug
[0445] As seen from the data levels of intracellular triphosphate
for B102 (Ex. 2) were much higher as compared to those for
NM107.
[0446] All publications and patent, applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. While the
claimed subject matter has been described in terms of various
embodiments, the skilled artisan will
* * * * *