U.S. patent application number 12/298652 was filed with the patent office on 2009-12-31 for formulations containing pyridazine compounds.
Invention is credited to Wenhui Hu, Linda Van Eldik, D. Martin Watterson.
Application Number | 20090325973 12/298652 |
Document ID | / |
Family ID | 38626233 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090325973 |
Kind Code |
A1 |
Watterson; D. Martin ; et
al. |
December 31, 2009 |
FORMULATIONS CONTAINING PYRIDAZINE COMPOUNDS
Abstract
The invention relates to chemical compounds, compositions and
methods of making and using the same. In particular, the invention
provides selected pyridazine compounds of the formula I
##STR00001## are independently hydrogen, hydroxyl, alkyl, alkenyl,
alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl,
cycloalkenyl, cycloalkynyl, cycloalkoxy, aryl, aryloxy, arylalkoxy,
aroyl, heteroaryl, heterocyclic, acyl, acyloxy, amino, imino,
azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano, halo,
sulfate, sulfenyl, sulfinyl, sulfonyl, sulfonate, sulfoxide, silyl,
silyloxy, silylalkyl, silylthio, .dbd.O, .dbd.S, phosphonate,
ureido, carboxyl, carbonyl, carbamoyl, or carboxamide; and X is
optionally substituted pyrimidinyl or pyridazinyl, an isomer, a
pharmaceutically acceptable salt, or derivative thereof. The
invention additional relates to compositions comprising the
compounds, and methods of using the compounds and compositions for
modulation of cellular pathways, for treatment or prevention of
inflammatory diseases, for research, drug screening, and
therapeutic applications.
Inventors: |
Watterson; D. Martin;
(Chicago, IL) ; Van Eldik; Linda; (Chicago,
IL) ; Hu; Wenhui; (Guangzhou, CN) |
Correspondence
Address: |
MILLEN, WHITE, ZELANO & BRANIGAN, P.C.
2200 CLARENDON BLVD., SUITE 1400
ARLINGTON
VA
22201
US
|
Family ID: |
38626233 |
Appl. No.: |
12/298652 |
Filed: |
April 27, 2007 |
PCT Filed: |
April 27, 2007 |
PCT NO: |
PCT/US07/10248 |
371 Date: |
March 13, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60796328 |
Apr 28, 2006 |
|
|
|
Current U.S.
Class: |
514/252.02 ;
544/238 |
Current CPC
Class: |
A61P 31/04 20180101;
A61P 25/06 20180101; A61K 31/506 20130101; A61P 29/00 20180101;
A61P 27/12 20180101; A61P 11/02 20180101; A61P 25/14 20180101; A61P
1/04 20180101; A61P 19/02 20180101; A61P 37/02 20180101; C07D
403/12 20130101; A61K 31/501 20130101; A61P 9/10 20180101; C07D
401/12 20130101; A61P 3/10 20180101; A61P 25/02 20180101; A61P
17/00 20180101; A61P 25/16 20180101; C07D 403/14 20130101; C07D
237/20 20130101; A61P 25/28 20180101; C07D 401/14 20130101; A61P
25/00 20180101; A61P 25/04 20180101; A61P 5/24 20180101; A61P 17/06
20180101; A61P 1/18 20180101 |
Class at
Publication: |
514/252.02 ;
544/238 |
International
Class: |
A61K 31/501 20060101
A61K031/501; C07D 403/04 20060101 C07D403/04 |
Claims
1. A composition effective to provide lower risk of side effects
and/or a beneficial pharmacokinetic profile following treatment of
a subject suffering from a neuroinflammatory disease comprising a
therapeutically effective amount of a compound of formula I:
##STR00041## wherein R.sup.1, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, R.sup.9, R.sup.12, R.sup.13, and R.sup.14 are
independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl,
alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl,
cycloalkynyl, cycloalkoxy, aryl, aryloxy, arylalkoxy, aroyl,
heteroaryl, heterocyclic, acyl, acyloxy, amino, imino, azido,
thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano, halo,
sulfate, sulfenyl, sulfinyl, sulfonyl, sulfonate, sulfoxide, silyl,
silyloxy, silylalkyl, silylthio, .dbd.O, .dbd.S, phosphonate,
ureido, carboxyl, carbonyl, carbamoyl, or carboxamide; and X is
optionally substituted pyrimidinyl or pyridazinyl, an isomer, a
pharmaceutically acceptable salt, or derivative thereof.
2. A composition according to claim 1 wherein R.sup.1, R.sup.4,
R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.12, R.sup.13,
and R.sup.14 are independently hydrogen, C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl,
C.sub.1-C.sub.6alkoxy, C.sub.2-C.sub.6 alkenyloxy, C.sub.3-C.sub.10
cycloalkyl, C.sub.4-C.sub.10cycloalkenyl, C3-C.sub.10cycloalkoxy,
C.sub.6-C.sub.10aryl, C.sub.6-C.sub.10aryloxy,
C.sub.6-C.sub.10aryl-C.sub.1-C.sub.3alkoxy, C.sub.6-C.sub.10aroyl,
C.sub.6-C.sub.10heteroaryl, C.sub.3-C.sub.10heterocyclic,
C.sub.1-C.sub.6acyl, C.sub.1-C.sub.6acyloxy, --NH.sub.2,
--NHR.sup.28, --NR.sup.28R.sup.29, .dbd.NR.sup.28,
--S(O).sub.2R.sup.28, --SH, --SO.sub.3H, nitro, cyano, halo,
haloalkyl, haloalkoxy, hydroxyalkyl, --CO.sub.2H,
--CO.sub.2R.sup.28, --NHC(O)R.sup.28, --C(O)NH.sub.2,
--C(O)NHR.sup.28, --C(O)NR.sup.28R.sup.29, --NHS(O).sub.2R.sup.28,
wherein R.sup.28 and R.sup.29 are independently selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.3-C.sub.10cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10 aryl C.sub.1-C.sub.3alkyl, C.sub.6-C.sub.10
heteroaryl and C.sub.3-C.sub.10heterocyclic.
3. A composition according to claim 1 comprising a therapeutically
effective amount of a compound of the formula II: ##STR00042##
wherein R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15,
R.sup.16, and R.sup.17 are independently hydrogen, hydroxyl, alkyl,
alkenyl, alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy,
cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkoxy, aryl, aryloxy,
arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl, acyloxy, amino,
imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano,
halo, sulfoxide, sulfenyl, sulfinyl, sulfonyl, sulfonate, sulfate,
silyl, silyloxy, silylalkyl, silylthio, .dbd.O, .dbd.S,
phosphonate, ureido, carboxyl, carbonyl, carbamoyl, or carboxamide;
or an isomer, a pharmaceutically acceptable salt, or derivative
thereof.
4. A composition according to claim 3 wherein R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12,
R.sup.13, R.sup.14, R.sup.15R.sup.16, and R.sup.17 are
independently selected from hydrogen, C.sub.1-C.sub.6alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl,
C.sub.1-C.sub.6alkoxy, C.sub.2-C.sub.6 alkenyloxy, C.sub.3-C.sub.10
cycloalkyl, C.sub.4-C.sub.10cycloalkenyl,
C.sub.3-C.sub.10cycloalkoxy, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10aryloxy,
C.sub.6-C.sub.10aryl-C.sub.1-C.sub.3alkoxy, C.sub.6-C.sub.10aroyl,
C.sub.6-C.sub.10heteroaryl, C.sub.3-C.sub.10heterocyclic,
C.sub.1-C.sub.6acyl, C.sub.1-C.sub.6acyloxy, --NH.sub.2,
--NHR.sup.28, --NR.sup.28R.sup.29, .dbd.NR.sup.28,
--S(O).sub.2R.sup.28, --SH, --SO.sub.3H, nitro, cyano, halo,
haloalkyl, haloalkoxy, hydroxyalkyl, --CO.sub.2H,
--CO.sub.2R.sup.28, --NHC(O)R.sup.28, --C(O)NH.sub.2,
--C(O)NHR.sup.28, --C(O)NR.sup.28R.sup.29, --NHS(O).sub.2R.sup.28,
wherein R.sup.28 and R.sup.29 are independently selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.3-C.sub.10cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10 aryl C.sub.1-C.sub.3alkyl, C.sub.6-C.sub.10
heteroaryl and C.sub.3-C.sub.10heterocyclic.
5. A composition according to claim 1 wherein R.sup.1 is alkyl,
cycloalkyl, or heteroaryl.
6. A composition according to claim 1 wherein R.sup.1 is:
##STR00043## wherein R.sup.15, R.sup.16 and R.sup.17 are
independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl,
alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl,
cycloalkoxy, cycloalkynyl, aryl, aryloxy, arylalkoxy, aroyl,
heteroaryl, heterocyclic, acyl, acyloxy, amino, imino, azido,
thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano, halo,
sulfoxide, sulfate, sulfonyl, sulfenyl, sulfinyl, sulfonate, silyl,
silyloxy, silylalkyl, silylthio, .dbd.O, .dbd.S, phosphonate,
ureido, carboxyl, carbonyl, carbamoyl, or carboxamide.
7. A composition according to claim 6, wherein R.sup.15, R.sup.16
and R.sup.17 are independently selected from hydrogen,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.1-C.sub.6alkoxy, C.sub.2-C.sub.6 alkenyloxy,
C.sub.3-C.sub.10 cycloalkyl, C.sub.4-C.sub.10cycloalkenyl,
C.sub.3-C.sub.10cycloalkoxy, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10aryloxy,
C.sub.6-C.sub.10aryl-C.sub.1-C.sub.3alkoxy, C.sub.6-C.sub.10aroyl,
C.sub.6-C.sub.10heteroaryl, C.sub.3-C.sub.10heterocyclic,
C.sub.1-C.sub.6acyl, C.sub.1-C.sub.6acyloxy, --NH.sub.2,
--NHR.sup.28, --NR.sup.28R.sup.29, .dbd.NR.sup.28,
--S(O).sub.2R.sup.28--SH, --SO.sub.3H, nitro, cyano, halo,
haloalkyl, haloalkoxy, hydroxyalkyl, --CO.sub.2H,
--CO.sub.2R.sup.28, --NHC(O)R.sup.28, --C(O)NH.sub.2,
--C(O)NHR.sup.28, --C(O)NR.sup.28R.sup.29, --NHS(O).sub.2R.sup.28,
wherein R.sup.28 and R.sup.29 are independently selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.3-C.sub.10cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10aryl C.sub.1-C.sub.3alkyl,
C.sub.6-C.sub.10heteroaryl and C.sub.3-C.sub.10heterocyclic.
8. A composition effective to provide lower risk of side effects
and/or a beneficial pharmacokinetic profile following treatment in
a subject suffering from a neuroinflammatory disease comprising a
therapeutically effective amount of a compound of the formula III:
##STR00044## wherein R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14,
R.sup.15, R.sup.16, and R.sup.17 hydrogen, hydroxyl, alkyl,
alkenyl, alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy,
cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkoxy, aryl, aryloxy,
arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl, acyloxy, amino,
imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano,
halo, sulfoxide, sulfate, sulfonyl, sulfenyl, sulfinyl, sulfonate,
silyl, silyloxy, silylalkyl, silylthio, .dbd.O, .dbd.S,
phosphonate, ureido, carboxyl, carbonyl, carbamoyl, or carboxamide;
with the proviso that R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14,
R.sup.15, R.sup.16, and R.sup.17 cannot all be hydrogen, or an
isomer, a pharmaceutically acceptable salt, or derivative thereof,
or an isomer, a pharmaceutically acceptable salt, or derivative
thereof.
9. A composition according to claim 8 wherein R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12,
R.sup.13, R.sup.14, R.sup.15, R.sup.16, and R.sup.17 are
independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6alkoxy,
C.sub.2-C.sub.6 alkenyloxy, C.sub.3-C.sub.10 cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.3-C.sub.10cycloalkoxy,
C.sub.6-C.sub.10aryl, C.sub.6-C.sub.10aryloxy,
C.sub.6-C.sub.10aryl-C.sub.1-C.sub.3alkoxy, C.sub.6-C.sub.10aroyl,
C.sub.6-C.sub.10heteroaryl, C.sub.3-C.sub.10heterocyclic,
C.sub.1-C.sub.6acyl, C.sub.1-C.sub.6acyloxy, --NH.sub.2,
--NHR.sup.28, --NR.sup.28R.sup.29.dbd.NR.sup.28,
--S(O).sub.2R.sup.28, --SH, --SO.sub.3H, nitro, cyano, halo,
haloalkyl, haloalkoxy, hydroxyalkyl, --CO.sub.2H,
--CO.sub.2R.sup.28, --NHC(O)R.sup.28, --C(O)NH.sub.2,
--C(O)NHR.sup.28, --C(O)NR.sup.28R.sup.29, --NHS(O).sub.2R.sup.28
wherein R.sup.28 and R.sup.29 are independently selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.3-C.sub.10cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10aryl C.sub.1-C.sub.3alkyl, C.sub.6-C.sub.10
heteroaryl and C.sub.3-C.sub.10heterocyclic.
10. A composition according to claim 8 wherein in the compound of
the formula III R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, and
R.sup.17 are hydrogen, hydroxyl, alkyl, and one or both of R.sup.10
and R.sup.11 are independently substituted or unsubstituted
hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene,
alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy,
arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl, acyloxy,
sulfonyl, sulfinyl, sulfenyl, amino, imino, azido, thiol,
thioalkyl, thioalkoxy, thioaryl, nitro, ureido, cyano, halo, silyl,
silyalkyl, silyloxy, silylthio, .dbd.O, .dbd.S, carboxyl, carbonyl,
or carbamoyl, or an isomer or a pharmaceutically acceptable salt
thereof.
11. A composition according to claim 8 wherein in the compound of
the formula III one of R.sup.10 and R.sup.11 is alkyl, in
particular C.sub.1-C.sub.6 alkyl and the other of R.sup.10 and
R.sup.11 is hydrogen.
12. A composition according to claim 8 wherein in the compound of
the formula III one of R.sup.10 and R.sup.11 is aryl, and the other
of R.sup.10 and R.sup.11 is hydrogen.
13. A composition according to claim 8 wherein in the compound of
the formula III one of R.sup.10 and R.sup.11 is a heteroaryl in
particular an unsaturated 5 to 6 membered heteromonocyclyl group
containing 1 to 4 nitrogen atoms, and the other of R.sup.10 and
R.sup.11 is hydrogen.
14. A composition according to claim 8 wherein in the compound of
the formula III R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.12, R.sup.13, R.sup.14, R.sup.15,
R.sup.16, and R.sup.17 are hydrogen, and R.sup.11 is alkyl,
alkenyl, alkynyl, alkylene, alkoxy, aryl, or an unsaturated 5 to 6
membered heteromonocyclyl group containing 1 to 4 nitrogen
atoms.
15. A composition according to claim 8 wherein in the compound of
the formula III R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.12, R.sup.13, R.sup.14, R.sup.15,
R.sup.16, and R.sup.17 are hydrogen and R.sup.11 is alkyl or
pyridinyl.
16. A composition according to claim 8 wherein the compound of the
formula III is
4-methyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine.
17. A composition according to claim 1 comprising a therapeutically
effective amount of a compound to selectively reduce or block
up-regulation of IL-1.beta. and S100B, and/or reduce or prevent
loss of PSD-95 and/or synaptophysin.
18. A composition according to claim 1 comprising a therapeutically
effective amount of compound of the formula I, II or III to treat a
neuroinflammatory disease while reducing inhibitory activity at
hERG potassium channel.
19. A composition according to claim 1 comprising a therapeutically
effective amount of a compound of the formula I, II or III to treat
a neuroinflammatory disease while reducing hERG inhibition.
20. A composition according to claim 1 wherein the therapeutically
effective amount is effective to selectively reduce or block
up-regulation of IL-1.beta. and S100B, reduce or prevent loss of
PSD-95 and/or synaptophysin over a dosing period.
21. A composition according to claim 1 comprising a therapeutically
effective amount of a compound of the formula I, II or III suitable
for administration to a subject to provide effective concentrations
of the compound in an environment of use or an effective dose that
results in therapeutic effects in the prevention, treatment, or
control of symptoms of a disease disclosed herein.
22. A composition according to claim 21 wherein the disease is a
neuroinflammatory disease.
23. A composition according to claim 1 comprising a dose of
compound of formula I, II or III of about 0.1 to 100 mg/kg, 0.1 to
50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 20 mg/kg, 0.1 to 15 mg/kg, 0.1 to
10 mg/kg, 0.1 to 5 mg/kg, 0.1 to 4 mg/kg, 0.1 to 3 mg/kg, 0.1 to 2
mg/kg, or 0.1 to 1 mg/kg.
24. A method of treating a neuroinflammatory disease in a subject
comprising administering a composition of claim 1 to the
subject.
25. Use of at least one compound of the formula I, II, or III as
defined in claim 1 for the preparation of a medicament for
providing lower risks of side effects and/or a beneficial
pharmacokinetic profile in treating a neuroinflammatory
disease.
26. A kit comprising one or more composition of claim 1, a
container, and instructions for use.
Description
FIELD OF INVENTION
[0001] The invention relates to chemical compounds, compositions
and methods of making and using the same. In particular, the
invention provides selected pyridazine compounds, compositions
comprising the compounds, and methods of using the compounds and
compositions for modulation of cellular pathways, for treatment or
prevention of inflammatory diseases, for treatment or prevention of
neurological conditions, for research, drug screening, and
therapeutic applications.
BACKGROUND OF INVENTION
[0002] The treatment of neurological conditions and disorders is of
great importance in medicine and there is a need for new drugs and
treatments to prevent progression and reverse the impairments of
these conditions and disorders. Neuroinflammation is recognized as
a prominent feature in the pathology of many neurological
conditions and diseases. Neuroinflammation is a process that
results primarily from abnormally high or chronic activation of
glia (microglia and astrocytes). This overactive state of glia
results in increased levels of inflammatory and oxidative stress
molecules, which can lead to neuron damage or death. Neuronal
damage or death can also induce glial activation, facilitating the
propagation of a localized, detrimental cycle of neuroinflammation
(Griffin, W S T et al, Brain Pathol 8: 65-72, 1998). The
inflammation cycle has been proposed as a potential therapeutic
target in the development of new approaches to treat inflammatory
disease. However, most anti-inflammatory therapeutics developed to
date are palliative and provide minimal, short-lived, symptomatic
relief with limited effects on inflammatory disease progression.
Thus, there is a need for anti-inflammatory therapeutics that
impact disease progression or prevention.
SUMMARY OF INVENTION
[0003] The present invention provides certain pyridazine compounds,
compositions comprising the compounds, and methods of using the
compounds and compositions for modulation of cellular pathways
(e.g., signal transduction pathways), for treatment or prevention
of inflammatory diseases, for treatment or prevention of
neurological diseases and conditions, for research, drug screening,
and therapeutic applications. In particular, the invention
generally provides dosage forms, formulations and methods that
provide lower risk of side effects and/or produce beneficial
pharmacokinetic profiles, in particular in neuroinflammatory
diseases.
[0004] The invention contemplates a composition, in particular a
formulation or dosage form, effective to provide lower risk of side
effects and/or a beneficial pharmacokinetic profile following
treatment comprising a compound of the formula I:
##STR00002##
wherein R.sup.1, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.12, R.sup.13, and R.sup.14 are independently
hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene,
alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, cycloalkynyl,
cycloalkoxy, aryl, aryloxy, arylalkoxy, aroyl, heteroaryl,
heterocyclic, acyl, acyloxy, amino, imino, azido, thiol, thioalkyl,
thioalkoxy, thioaryl, nitro, cyano, halo, sulfate, sulfenyl,
sulfinyl, sulfonyl, sulfonate, sulfoxide, silyl, silyloxy,
silylalkyl, silylthio, .dbd.O, .dbd.S, phosphonate, ureido,
carboxyl, carbonyl, carbamoyl, or carboxamide; and X is optionally
substituted pyrimidinyl or pyridazinyl, an isomer, a
pharmaceutically acceptable salt, or derivative thereof.
[0005] In aspects of the invention, a compound of the formula I is
provided wherein R.sup.1, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, R.sup.9, R.sup.12, R.sup.13, and R.sup.14 are
independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl,
alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl,
aryl, aryloxy, arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl,
acyloxy, amino, imino, azido, thiol, thioalkyl, thioalkoxy,
thioaryl, nitro, cyano, halo, silyl, silyloxy, silylalkyl,
silylthio, .dbd.O, .dbd.S, phosphonate, carboxyl, carbonyl,
carbamoyl, or carboxamide; and X is pyrimidinyl or pyridazinyl, an
isomer, a pharmaceutically acceptable salt, or derivative
thereof.
[0006] In an aspect, a composition, formulation or dosage form is
provided which is effective to provide lower risk of side effects
and/or a beneficial pharmacokinetic profile following treatment
comprising a compound of the formula II:
##STR00003##
wherein R.sup.1, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, and R.sup.14 are
independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl,
alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl,
cycloalkynyl, cycloalkoxy, aryl, aryloxy, arylalkoxy, aroyl,
heteroaryl, heterocyclic, acyl, acyloxy, amino, imino, azido,
thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano, halo,
sulfenyl, sulfinyl, sulfonyl, sulfonate, sulfate, sulfoxide, silyl,
silyloxy, silylalkyl, silylthio, .dbd.O, .dbd.S, phosphonate,
ureido, carboxyl, carbonyl, carbamoyl, or carboxamide; or an
isomer, a pharmaceutically acceptable salt, or derivative
thereof.
[0007] In an aspect of the invention, a compound of the formula II
is provided wherein R.sup.1, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, and
R.sup.14 are independently hydrogen, hydroxyl, alkyl, alkenyl,
alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl,
cycloalkenyl, aryl, aryloxy, arylalkoxy, aroyl, heteroaryl,
heterocyclic, acyl, acyloxy, amino, imino, azido, thiol, thioalkyl,
thioalkoxy, thioaryl, nitro, cyano, halo, silyl, silyloxy,
silylalkyl, silylthio, .dbd.O, .dbd.S, carboxyl, carbonyl,
carbamoyl, or carboxamide; or an isomer, a pharmaceutically
acceptable salt, or derivative thereof.
[0008] In aspects of the invention, R.sup.1 in a compound of the
formula I or II is substituted or unsubstituted alkyl, cyclohexyl,
aryl, arylalkoxy, aroyl, or heteroaryl.
[0009] In a particular aspect, R.sup.1 in a compound of the formula
I or II is substituted or unsubstituted aryl, arylalkoxy, aroyl, or
heteroaryl.
[0010] In certain aspects of the invention R.sup.1 in a compound of
the formula I or II is:
##STR00004##
wherein R.sup.15, R.sup.16 and R.sup.17 are independently hydrogen,
hydroxyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkoxy,
alkenyloxy, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkoxy,
aryl, aryloxy, arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl,
acyloxy, amino, imino, azido, thiol, thioalkyl, thioalkoxy,
thioaryl, nitro, cyano, halo, sulfoxide, sulfate, sulfonyl,
sulfenyl, sulfinyl, sulfonate, silyl, silyloxy, silylalkyl,
silylthio, .dbd.O, .dbd.S, phosphonate, ureido, carboxyl, carbonyl,
carbamoyl, or carboxamide.
[0011] Therefore, certain aspects of the invention contemplate a
composition, in particular a formulation or dosage form, effective
to provide lower risk of side effects and/or a beneficial
pharmacokinetic profile following treatment comprising an amount of
a compound of the formula III:
##STR00005##
wherein R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15,
R.sup.16, and R.sup.17 are independently hydrogen, hydroxyl, alkyl,
alkenyl, alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy,
cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkoxy, aryl, aryloxy,
arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl, acyloxy, amino,
imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano,
halo, sulfoxide, sulfate, sulfonyl, sulfenyl, sulfinyl, sulfonate,
silyl, silyloxy, silylalkyl, silylthio, .dbd.O, .dbd.S, ureido,
phosphonate, carboxyl, carbonyl, carbamoyl, or carboxamide.
[0012] In general, R.sup.4R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14,
R.sup.15, R.sup.16, and R.sup.17 in a compound of the formula III
cannot all be hydrogen.
[0013] The invention relates to compounds of the formula I, II or
III disclosed herein, in particular pure or substantially pure
compounds of the formula I, II or III.
[0014] The invention also contemplates utilizing in compositions
and methods of the invention a compound in FIG. 1, in particular
MW01-4-179LKM, MW01-7-084WH, MW01-7-085WH, MW01-7-133WH,
MW01-2-151SRM, MW01-5-188WH or MW01-7-057, or isomers,
pharmaceutically acceptable salts or derivatives thereof.
[0015] A composition of the invention, in particular a formulation
or dosage form, may be further characterized by its ability to
selectively reduce or block up-regulation of IL-1.beta. and S100B,
and/or reduce or prevent loss of PSD-95 and/or synaptophysin.
[0016] In aspects, a composition of the invention, in particular a
formulation or dosage form, may provide a lower risk of QT-related
side effects.
[0017] In particular aspects, the invention further provides a
composition, in particular a formulation or dosage form, comprising
a compound of the formula I, II or III in a therapeutically
effective amount to treat a disease disclosed herein while reducing
inhibitory activity at hERG potassium channel.
[0018] In another particular aspect, the invention provides a
composition, in particular a formulation or dosage form, comprising
a compound of the formula I, II or III in a therapeutically
effective amount to treat a disease disclosed herein while reducing
hERG inhibition.
[0019] In another particular aspect the invention provides a
composition, in particular a formulation or dosage form, comprising
a compound of the formula I, II or III in a therapeutically
effective amount to treat a disease disclosed herein in a subject
receiving a therapeutic or treatment that prolongs QT interval.
[0020] The invention contemplates a formulation for the treatment
of a disease disclosed herein comprising a therapeutically
effective amount of a compound of the formula I, II or III, to
provide a beneficial pharmacokinetic profile, in particular a
sustained pharmacokinetic profile, in a pharmaceutically acceptable
carrier, excipient, or vehicle. In an aspect, a formulation
comprising a compound of the formula I, II or III is provided which
is in a form or which has been adapted for administration to a
subject to provide a beneficial pharmacokinetic profile to treat a
disease disclosed herein. In an embodiment, a dosage form is
provided such that administration of the dosage form to a subject
suffering from a disease disclosed herein provides a beneficial
pharmacokinetic profile resulting in therapeutic effects including
selectively reducing or blocking up-regulation of IL-1.beta. and
S100B, and/or reducing or preventing loss of PSD-95 and/or
synaptophysin over a dosing period. In particular, the composition
is in a form adapted to provide a beneficial pharmacokinetic
profile that results in one or more of the following in a subject
for a sustained time over a dosing period: selective reduction of
up-regulation of IL-1.beta. and S100B, and/or reduction of loss of
PSD-95 and/or synaptophysin.
[0021] In another aspect, the invention relates to a dosage form
comprising amounts of a compound of the formula I, II or III
suitable for administration to a subject to provide effective
concentrations of the compound in an environment of use or an
effective dose that results in therapeutic effects in the
prevention, treatment, or control of symptoms of a disease
disclosed herein, in particular a neuroinflammatory disease. In
aspects of the invention, the environment of use is the brain or
plasma.
[0022] In a further aspect, the invention is directed to a
formulation or dosage form suitable for once, twice- or three-times
a day administration to treat a disease disclosed herein comprising
one or more compound of the formula I, II or III in an amount
effective to provide lower risk of side effects and/or a beneficial
pharmacokinetic profile in a dosing period.
[0023] In a still further aspect, the invention contemplates a
dosage form comprising one or more compound of the formula I, II or
III in an amount effective to maintain the compound within an
effective plasma and/or brain drug concentration that results in
therapeutic effects in the subject.
[0024] The invention additionally relates to a method of preparing
a stable formulation or dosage form of a compound of the formula I,
II or III adapted to provide lower risks of side effects and/or
beneficial pharmacokinetic profiles following treatment.
Formulations may be placed in an appropriate container and labelled
for treatment of an indicated disease. For administration of a
formulation of the invention, such labelling would include amount,
frequency, and method of administration.
[0025] The invention also provides methods to make commercially
available formulations which contain a compound of the formula I,
II or III that provides lower risk of side effects and/or a
beneficial pharmacokinetic profile in the treatment of a disease
disclosed herein.
[0026] The invention relates to the use of at least one compound of
the formula I, II or III for the preparation of a medicament for
providing lower risks of side effects and/or a beneficial
pharmacokinetic profile in treating a disease disclosed herein. The
invention additionally relates to uses of a pharmaceutical
composition of the invention in the preparation of medicaments for
providing lower risks of side effects and/or a beneficial
pharmacokinetic profile in the prevention and/or treatment of a
disease disclosed herein.
[0027] Commercially available formulations or medicaments may be
pills, tablets, caplets, soft and hard gelatin capsules, lozenges,
sachets, cachets, vegicaps, liquid drops, elixirs, suspensions,
emulsions, solutions, syrups, aerosols (as a solid or in a liquid
medium) suppositories, sterile injectable solutions, and/or sterile
packaged powders, which contain a compound of the formula I, II or
III.
[0028] Compounds of the formula I, II or III and compositions of
the invention may be administered therapeutically or
prophylactically to treat a disease disclosed herein, in particular
neuroinflammatory disease. Therefore the invention provides a
method for treating a disease disclosed herein, in particular a
neuroinflammatory disease, comprising administering a
therapeutically effective amount or prophylactically effective
amount of a compound of the formula I, II or III. In an aspect, the
invention provides a method for treating a disease disclosed herein
in particular a neuroinflammatory disease comprising administering
a compound of the formula I, II or III in an amount effective to
lower risks of side effects and/or provide a beneficial
pharmacokinetic profile. In an aspect, a method is provided for
treating a disease disclosed herein, in particular a
neuroinflammatory disease, comprising administering a compound of
the formula I, II or III in an amount effective to selectively
inhibit up-regulation of IL-1.beta. and S100B, reduce or prevent
loss of PSD-95 and/or synaptophysin, and/or prevent behavioral
deficit.
[0029] Aspects of the invention provide methods for treating a
disease disclosed herein, in particular a neuroinflammatory
disease, comprising administering to a subject a compound of the
formula I, II or III in an amount effective to lower risk of
QT-related side effects in the subject. Certain aspects of the
invention provide methods for treating a disease disclosed herein,
in particular a neuroinflammatory disease, comprising administering
to a subject a therapeutically effective amount of a compound of
the formula I, II or III to treat the disease while reducing
inhibitory activity at hERG potassium channel. Other aspects of the
invention provide methods for treating a disease disclosed herein,
in particular a neuroinflammatory disease, comprising administering
to a subject a therapeutically effective amount of a compound of
the formula I, II or III to treat the disease while reducing hERG
inhibition. Further aspects of the invention provide methods for
treating a disease disclosed herein in a subject suffering from a
disease disclosed herein and receiving a therapeutic or treatment
that prolongs QT interval comprising administering to the subject a
therapeutically effective amount of a compound of the formula I to
reduce the QT-related side effects.
[0030] The invention also provides a method for treating and/or
preventing a disease disclosed herein in a subject comprising
administering to the subject one or more, in particular two, three
or four dosages of a formulation comprising one or more compound of
the formula I, II or III in an amount effective to maintain the
compound within the effective brain and/or plasma drug
concentration that results in therapeutic effects in the
subject.
[0031] In particular aspects of the invention, a method is provided
for treating in a subject a disease involving or characterized by
inflammation, in particular neuroinflammation, comprising
administering to the subject a compound of the formula I, II or III
in a therapeutically effective amount that provides beneficial
pharmacokinetic profiles, in a pharmaceutically acceptable carrier,
excipient, or vehicle.
[0032] In a further aspect, the invention provides a method
involving administering to a subject a therapeutic compound of the
formula I, II or III or a pharmaceutically acceptable salt thereof,
or a composition comprising a compound of the formula I, II or III
and a pharmaceutically acceptable carrier, excipient, or vehicle
which inhibit or reduce neuroflammation, activation of glia,
activation of astrocytes, activation of microglia, proimflammatory
cytokines, oxidative stress-related enzymes, acute phase proteins
and/or components of the complement cascade, and provide lower risk
of QT-related side effects and/or a beneficial pharmacokinetic
profile.
[0033] The invention also provides a kit comprising one or more
compound of the formula I, II or III, or a composition of the
invention adapted to provide lower risk of side effects and/or a
beneficial pharmacokinetic profile. In an aspect, the invention
provides a kit for preventing and/or treating a disorder and/or
disease disclosed herein, comprising a formulation or dosage form
of the invention, a container, and instructions for use.
[0034] These and other aspects, features, and advantages of the
present invention should be apparent to those skilled in the art
from the following drawings and detailed description.
DESCRIPTION OF THE FIGURES
[0035] FIG. 1 shows the structures of MW01-2-151SRM, MW01-6-189WH,
MW01-7-107WH, MW01-4-179LKM, MW01-7-084WH, MW01-7-085WH,
MW01-7-133WH, and MW01-7-057.
[0036] FIG. 2 depicts a synthetic scheme for MW01-3-183WH.
[0037] FIG. 3 depicts a synthetic scheme for MW01-2-151SRM.
[0038] FIG. 4 depicts a synthetic scheme for MW01-2-151SRM.
[0039] FIG. 5 depicts a synthetic scheme for MW01-2-151SRM.
[0040] FIG. 6 depicts a synthetic scheme for MW01-2-151SRM.
[0041] FIG. 7 depicts a synthetic scheme for MW01-5-188WH.
[0042] FIG. 8 depicts a synthetic scheme for MW01-5-188WH.
[0043] FIG. 9 depicts a synthetic scheme for MW01-5-188WH.
[0044] FIGS. 10A and 10B depict synthetic schemes for
MW01-6-189WH
[0045] FIG. 11 depicts a synthetic scheme for MW01-7-084WH.
[0046] FIG. 12 depicts a synthetic scheme for MW01-7-085WH.
[0047] FIG. 13 depicts a synthetic scheme for MW01-7-133WH.
[0048] FIG. 14 depicts a synthetic scheme for MW01-7-107WH.
[0049] FIG. 15 depicts a synthetic scheme for MW01-7-057.
[0050] FIG. 16 show graphs and micrographs illustrating
proinflammatory cytokine production by MW01-5-151SRM. (A)
Concentration dependent inhibition by MW01-5-151SRM of LPS-induced
increases of IL-1.beta. in the BV2 microglial cell line. (B)
LPS-stimulated accumulation of the NO metabolite, nitrite, was not
inhibited by MW01-5-1151SRM at concentrations up to 33 .mu.M. (C)
MW01-5-1151SRM does not suppress LPS-induced production of iNOS or
COX-2 in activated BV-2 cells.
[0051] FIG. 17 show graphs and micrographs illustrating
proinflammatory cytokine production by MW01-5-189WH. (A)
Concentration dependent inhibition by MW01-5-189WH of LPS-induced
increases of IL-1.beta. in the BV2 microglial cell line. (B)
LPS-stimulated accumulation of the NO metabolite, nitrite, was not
inhibited by MW01-5-189WH at concentrations up to 33 .mu.M. (C)
MW01-5-189WH does not suppress LPS-induced production of iNOS or
COX-2 in activated BV-2 cells.
[0052] FIG. 18 show graphs and micrographs illustrating
proinflammatory cytokine production by MW01-5-107WH. (A)
Concentration dependent inhibition by MW01-5-107WH of LPS-induced
increases of IL-1.beta. in the BV2 microglial cell line. (B)
LPS-stimulated accumulation of the NO metabolite, nitrite, was
inhibited by MW01-5-107WH. (C) MW01-5-107WH also inhibited
LPS-induced production of iNOS or COX-2 in activated BV-2
cells.
[0053] FIG. 19 show graphs and micrographs illustrating
proinflammatory cytokine production by MW01-5-179WH. (A)
Concentration dependent inhibition by MW01-5-179WH of LPS-induced
increases of IL-1.beta. in the BV2 microglial cell line. (B)
LPS-stimulated accumulation of the NO metabolite, nitrite, was not
inhibited by MW01-5-179WH at concentrations up to 33 .mu.M. (C)
MW01-5-179WH does not suppress LPS-induced production of iNOS or
COX-2 in activated BV-2 cells.
[0054] FIG. 20 show graphs and micrographs illustrating
proinflammatory cytokine production by MW01-5-084WH. (A)
Concentration dependent inhibition by MW01-5-084WH of LPS-induced
increases of IL-1.beta. in the BV2 microglial cell line. (B)
LPS-stimulated accumulation of the NO metabolite, nitrite, was not
inhibited by MW01-5-084WH at concentrations up to 33 .mu.M. (C)
MW01-5-084WH does not suppress LPS-induced production of iNOS or
COX-2 in activated BV-2 cells.
[0055] FIG. 21 show graphs and micrographs illustrating
proinflammatory cytokine production by MW01-5-085WH. (A)
Concentration dependent inhibition by MW01-5-085WH of LPS-induced
increases of IL-1.beta. in the BV2 microglial cell line. (B)
LPS-stimulated accumulation of the NO metabolite, nitrite, was not
inhibited by MW01-5-085WH at concentrations up to 33 .mu.M. (C)
MW01-5-085WH does not suppress LPS-induced production of iNOS or
COX-2 in activated BV-2 cells.
[0056] FIG. 22 show graphs and micrographs illustrating
proinflammatory cytokine production by MW01-5-0133WH. (A)
Concentration dependent inhibition by MW01-5-133WH of LPS-induced
increases of IL-1.beta. in the BV2 microglial cell line. (B)
LPS-stimulated accumulation of the NO metabolite, nitrite, was not
inhibited by MW01-5-133WH at concentrations up to 33 .mu.M. (C)
MW01-5-133WH does not suppress LPS-induced production of iNOS or
COX-2 in activated BV-2 cells.
[0057] FIG. 23 show graphs and micrographs illustrating
proinflammatory cytokine production by MW01-5-057WH. (A)
Concentration dependent inhibition by MW01-5-057WH of LPS-induced
increases of IL-1.beta. in the BV2 microglial cell line. (B)
LPS-stimulated accumulation of the NO metabolite, nitrite, was not
inhibited by MW01-5-057WH at concentrations up to 33 .mu.M. (C)
MW01-5-057WH does not suppress LPS-induced production of iNOS or
COX-2 in activated BV-2 cells.
[0058] FIG. 24 A-H shows graphs illustrating in vivo activity of
MW01-2-151SRM in the A.beta. infusion mouse model. Graphs are of
MW01-2-151SRM suppression of A.beta.-induced neuroinflammation and
synaptic damage and activity in the Y-maze. Hippocampal sections or
extracts from vehicle-infused mice (control), A.beta.-infused mice
injected with solvent, and A.beta.-infused mice injected with
MW01-2-151SRM were evaluated for neuroinflammation by measurement
of the levels of the pro-inflammatory cytokines IL-1.beta. (A),
TNF.alpha. (B), and S100B (C), and the number of GFAP-positive
astrocytes (D), F4/80 (E), the presynaptic marker, synaptophysin
(F), and evaluated for synaptic damage by analysis of the levels of
the post-synaptic density protein 95 (PSD-95) (G), and Y-maze (H).
Data are from one of two independent experiments, and are the
mean.+-.SEM for 4-5 mice per experimental group.
[0059] FIG. 25 A-E shows graphs illustrating in vivo activity of
MW01-2-189SRM in the A.beta. infusion mouse model. Graphs are of
MW01-2-189SRM suppression of A.beta.-induced neuroinflammation and
synaptic damage and activity in the Y-maze. Hippocampal sections or
extracts from vehicle-infused mice (control), A.beta.-infused mice
injected with solvent, and A.beta.-infused mice injected with
MW01-2-189SRM were evaluated for neuroinflammation by measurement
of the levels of the pro-inflammatory cytokines IL-1.beta. (A), and
S100B (B), the presynaptic marker, synaptophysin (C), and evaluated
for synaptic damage by analysis of the levels of the post-synaptic
density protein 95 (PSD-95) (D), and Y-maze (E). Data are from
three samples in the MW01-2-189SRM were analyzed.
[0060] FIG. 26 A-E shows graphs illustrating in vivo activity of
MW01-2-084SRM in the A.beta. infusion mouse model. Graphs are of
MW01-2-084SRM suppression of A.beta.-induced neuroinflammation and
synaptic damage and activity in the Y-maze. Hippocampal sections or
extracts from vehicle-infused mice (control), A.beta.-infused mice
injected with solvent, and A.beta.-infused mice injected with
MW01-2-084SRM were evaluated for neuroinflammation by measurement
of the levels of the pro-inflammatory cytokines IL-1.beta. (A), and
S100B (B), the presynaptic marker, synaptophysin (C), and evaluated
for synaptic damage by analysis of the levels of the post-synaptic
density protein 95 (PSD-95) (D), and Y-maze (E). Data are from five
samples per group analyzed.
[0061] FIG. 27 A-E shows graphs illustrating in vivo activity of
MW01-2-085SRM in the A.beta. infusion mouse model. Graphs are of
MW01-2-085SRM suppression of A.beta.-induced neuroinflammation and
synaptic damage and activity in the Y-maze. Hippocampal sections or
extracts from vehicle-infused mice (control), A.beta.-infused mice
injected with solvent, and A.beta.-infused mice injected with
MW01-2-085SRM were evaluated for neuroinflammation by measurement
of the levels of the pro-inflammatory cytokines IL-1.beta. (A), and
S100B (B), the presynaptic marker, synaptophysin (C), and evaluated
for synaptic damage by analysis of the levels of the post-synaptic
density protein 95 (PSD-95) (D), and Y-maze (E). Data are from
three samples in the MW01-2-085SRM were analyzed.
[0062] FIG. 28 A-E shows graphs illustrating in vivo activity of
MW01-2-057WH in the A.beta. infusion mouse model. Graphs are of
MW01-2-057WH suppression of A.beta.-induced neuroinflammation and
synaptic damage and activity in the Y-maze. Hippocampal sections or
extracts from vehicle-infused mice (control), A.beta.-infused mice
injected with solvent, and A.beta.-infused mice injected with
MW01-2-057WH were evaluated for neuroinflammation by measurement of
the levels of the pro-inflammatory cytokines IL-1.beta. (A), and
S100B (B), the presynaptic marker, synaptophysin (C), and evaluated
for synaptic damage by analysis of the levels of the post-synaptic
density protein 95 (PSD-95) (D), and Y-maze (E). Data are from
three samples in the MW01-2-057SRM were analyzed. There was no
significant effect on PSD-95.
[0063] FIG. 29 is a graph showing QTc interval of MW01-2-151SRM (15
mg/10 ml/kg/po) (Bazett's). Changes in QTc following oral
administration of MW01-2-151SRM at 15 mg/kg in guinea pigs. QT
intervals were corrected for heart rate changes using Bazett's
formula. The broken lines represent 95% confidence limits
(mean.+-.2SD) for QTc changes in the vehicle (2% Tween 80 in
Distilled Water)-treated control. The five treated animals are
represented by individual symbols.
[0064] FIG. 30 is a graph showing QTc interval of Sotalol (0.3
mg/kg/iv) (Bazett's). Changes in QTc following intravenous
administration of Sotalol at 0.3 mg/kg in guinea pigs. QT intervals
were corrected for heart rate changes using Bazett's formula. The
broken lines represent 95% confidence limits (mean.+-.2SD) for QTc
changes in the vehicle (0.9% NaCl)-treated control. The five
treated animals are represented by individual symbols.
[0065] FIG. 31 is a graph showing QTc interval of MW01-2-151SRM (15
mg/10 ml/kg/po) (Fredericia's). Changes in QTc following oral
administration of MW01-2-151SRM at 15 mg/kg in guinea pigs. QT
intervals were corrected for heart rate changes using Federicia's
formula. The broken lines represent 95% confidence limits
(mean.+-.2SD) for QTc changes in the vehicle (2% Tween 80 in
Distilled Water)-treated control. The five treated animals are
represented by individual symbols.
[0066] FIG. 32 is a graph showing QTc interval of Sotalol (0.3
mg/kg/iv) (Fredericia's). Changes in QTc following intravenous
administration of Sotalol at 0.3 mg/kgin guinea pigs. QT intervals
were corrected for heart rate changes using Fredericia's formula.
The broken lines represent 95% confidence limits (mean.+-.2SD) for
QTc changes in the vehicle (0.9% NaCl)-treated control. The five
treated animals are represented by individual symbols.
[0067] FIG. 33 is a graph showing QTc interval of oral
administration of MW01-5-188WH (15 mg/kg p.o.) in guinea pig.
[0068] FIG. 34 are graphs of results of liver toxicity studies with
MW01-5-188WH, MW01-2-151SRM, and MW01-6-189WH. Compounds were
administered to C57Bl/6 mice by oral gavage (2.5 mg/kg/day, once
daily for 2 weeks). Histological liver toxicity was assessed by
examination of tissue architecture, cell necrosis, and inflammatory
infiltrate. The scoring scale ranges from 0 (best) to 9 (worst).
MW01-5-188WH, MW01-2-151SRM, and MW01-6-189 show no significant
differences in liver toxicity score from the control mice receiving
either no gavage or vehicle gavage.
[0069] FIG. 35 shows that MW01-5-188WH is readily detected in the
plasma and the brain after a single oral dose administration and
does not suppress peripheral tissue inflammatory responses or cause
liver injury after chronic oral administration. C57BL/6 mice were
administered MW01-5-188WH (2.5 mg/kg) by oral gavage, blood and
brain processed at different times after administration, and
compound levels in plasma and brain determined as described herein.
MW01-5-188WH rapidly appears in plasma (A) and brain (B), reaches a
peak at 15 min, and then slowly declines to basal levels by 120
min. Data are the mean_SEM from three to six mice at each time
point. MW01-5-188WH does not inhibit increased production of
IL-1.beta. (C) and TNF-.alpha. (D) in the serum but does suppress
the cytokine response in the brains from the same mice (E, F). Mice
(n=3-6 per group) were administered by oral gavage either diluent
or MW01-5-188WH (2.5 mg/kg) once daily for 2 weeks and then
challenged with LPS (10 mg/kg, i.p.) for 6 h. Control mice were
injected with saline. IL-1.beta. and TNF-.alpha. levels in the
serum and in brain supernatants were determined. Data represent
mean.+-.SEM. ***p.sub.--0.001, significantly different from
diluent. Daily oral administration of diluent (G) or
MW01-5-188WH(H) (2.5 mg/kg) does not result in any histological
liver toxicity. Representative liver sections from mice treated as
in C--F were stained with hematoxylin and eosin. Scale bar, 125
.mu.m. 188, were stained with hematoxylin and eosin. Scale bar, 125
.mu.m. 188, MW01-5-188WH.
[0070] FIG. 36 are graphs of stability data using human (A, B) and
rat (C, D) microsomes with MW01-2-151SRM in two different amounts,
for two time periods. E and F show human (E) and (F) rat microsomes
with MW01-2-151SRM stability for different time periods compared to
minaprine.
[0071] FIG. 37 shows a synthetic scheme for synthesis of compounds
of the formula I where R.sup.11 is benzyl, 4-pyridyl, iso-butyl, or
methyl. Reagents and conditions: a) PhCH.sub.2NH.sub.2NH.sub.2,
CH.sub.3COONa, ethanol, reflux, 29 h; b) POCl.sub.3, PCL.sub.5,
120.degree. C., 12 h; c) CH.sub.3COOH, reflux, 5 h; d)
1-(2-pyrimidyl)piperazine, 1-butanol, 130.degree. C., 41 h; e)
POCl.sub.3, 100.degree. C., 3 h; f) boronic acid, Pd(0). 2
R=benzyl; 3 R=4-pyridyl; 4 R=iso-butyl; 5 R=methyl.
[0072] FIG. 38 shows a synthetic scheme for synthesis of compounds
of the formula I where R.sup.1 is methyl.
[0073] FIG. 39 shows a synthetic scheme for synthesis of pyrazine
analogs of the invention. a) NaOH, -41.degree. C., MeOH; b)
Tf.sub.2O, DMAP, Pyridine, rt; c) 1-(2-pyrimidyl)piperazine, DMSO,
60.degree. C.
DETAILED DESCRIPTION OF EMBODIMENTS
[0074] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0075] Numerical ranges recited herein by endpoints include all
numbers and fractions subsumed within that range (e.g. 1 to 5
includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be
understood that all numbers and fractions thereof are presumed to
be modified by the term "about." The term "about" means plus or
minus 0.1 to 50%, 5-50%, or 10-40%, preferably 10-20%, more
preferably 10% or 15%, of the number to which reference is being
made. Further, it is to be understood that "a," "an," and "the"
include plural referents unless the content clearly dictates
otherwise. Thus, for example, reference to a composition comprising
"a compound" includes a mixture of two or more compounds.
[0076] As used herein the terms "administering" and
"administration" refer to a process by which a therapeutically
effective amount of a compound of the formula I, II or III or
composition contemplated herein is delivered to a subject for
prevention and/or treatment purposes. Compositions are administered
in accordance with good medical practices taking into account the
subject's clinical condition, the site and method of
administration, dosage, patient age, sex, body weight, and other
factors known to physicians.
[0077] As used herein, the term "co-administration" of
"co-administered" refers to the administration of at least two
compounds or agent(s) or therapies to a subject. In some
embodiments, the co-administration of two or more agents/therapies
is concurrent. In other embodiments, a first agent/therapy is
administered prior to a second agent/therapy. In this aspect, each
component may be administered separately, but sufficiently close in
time to provide the desired effect, in particular a beneficial,
additive, or synergistic effect. Those of skill in the art
understand that the formulations and/or routes of administration of
the various agents/therapies used may vary. The appropriate dosage
for co-administration can be readily determined by one skilled in
the art. In some embodiments, when agents/therapies are
co-administered, the respective agents/therapies are administered
at lower dosages than appropriate for their administration alone.
Thus, co-administration is especially desirable in embodiments
where the co-administration of the agents/therapies lowers the
requisite dosage of a known potentially harmful (e.g., toxic)
agent(s).
[0078] The term "treating" refers to reversing, alleviating, or
inhibiting the progress of a disease, or one or more symptoms of
such disease, to which such term applies. Depending on the
condition of the subject, the term also refers to preventing a
disease, and includes preventing the onset of a disease, or
preventing the symptoms associated with a disease. A treatment may
be either performed in an acute or chronic way. The term also
refers to reducing the severity of a disease or symptoms associated
with such disease prior to affliction with the disease. Such
prevention or reduction of the severity of a disease prior to
affliction refers to administration of a compound or composition of
the present invention to a subject that is not at the time of
administration afflicted with the disease. "Preventing" also refers
to preventing the recurrence of a disease or of one or more
symptoms associated with such disease. "Treatment" and
"therapeutically," refer to the act of treating, as "treating" is
defined above. The purpose of prevention and intervention is to
combat the disease, condition, or disorder and includes the
administration of an active compound to prevent or delay the onset
of the symptoms or complications, or alleviating the symptoms or
complications, or eliminating the disease, condition, or
disorder.
[0079] The terms "subject", "individual", or "patient" are used
interchangeably herein and refer to an animal preferably a
warm-blooded animal such as a mammal. Mammal includes without
limitation any members of the Mammalia. A mammal, as a subject or
patient in the present disclosure, can be from the family of
Primates, Carnivora, Proboscidea, Perissodactyla, Artiodactyla,
Rodentia, and Lagomorpha. Among other specific embodiments a mammal
of the present invention can be Canis familiaris (dog), Felis catus
(cat), Elephas maximus (elephant), Equus caballus (horse), Sus
domesticus (pig), Camelus dromedarious (camel), Cervus axis (deer),
Giraffa camelopardalis (giraffe), Bos taurus (cattle/cows), Capra
hircus (goat), Ovis aries (sheep), Mus musculus (mouse), Lepus
brachyurus (rabbit), Mesocricetus auratus (hamster), Cavia
porcellus (guinea pig), Meriones unguiculatus (gerbil), or Homo
sapiens (human). In a particular embodiment, the mammal is a human.
In other embodiments, animals can be treated; the animals can be
vertebrates, including both birds and mammals. In aspects of the
invention, the terms include domestic animals bred for food or as
pets, including equines, bovines, sheep, poultry, fish, porcines,
canines, felines, and zoo animals, goats, apes (e.g. gorilla or
chimpanzee), and rodents such as rats and mice.
[0080] Typical subjects for treatment include persons afflicted
with or suspected of having or being pre-disposed to a disease
disclosed herein, or persons susceptible to, suffering from or that
have suffered a disease disclosed herein. A subject may or may not
have a genetic predisposition for a disease disclosed herein. In
the context of certain aspects of the invention, the term "subject"
generally refers to an individual who will receive or who has
received treatment (e.g., administration of a compound of the
formula I, II or III, and optionally one or more other agents) for
a condition characterized by inflammation, the dysregulation of
protein kinase activity, and/or dysregulation of apototic
processes. In certain aspects, a subject may be a healthy
subject.
[0081] In particular aspects, a subject shows signs of cognitive
deficits and Alzheimer's disease neuropathology. In embodiments of
the invention the subjects are suspectible to, or suffer from
Alzheimer's disease.
[0082] As utilized herein, the term "healthy subject" means a
subject, in particular a mammal, having no diagnosed disease,
disorder, infirmity, or ailment, more particularly a disease,
disorder, infirmity or ailment known to impair or otherwise
diminish memory.
[0083] The term "diagnosed," as used herein, refers to the
recognition of a disease by its signs and symptoms (e.g.,
resistance to conventional therapies), or genetic analysis,
pathological analysis, histological analysis, and the like.
[0084] As used herein, the term "modulate" refers to the activity
of a compound (e.g., a compound of the formula I, II or III) to
affect (e.g., to promote or retard) an aspect of cellular function,
including, but not limited to, cell growth, proliferation,
apoptosis, and the like.
[0085] "Therapeutically effective amount" relates to the amount or
dose of an active compound of the formula I, II or III or
composition comprising the same, that will lead to one or more
desired effects, in particular, one or more therapeutic effects or
beneficial pharmacokinetic profiles. A therapeutically effective
amount of a substance can vary according to factors such as the
disease state, age, sex, and weight of the subject, and the ability
of the substance to elicit a desired response in the subject. A
dosage regimen may be adjusted to provide the optimum therapeutic
response or pharmacokinetic profile. For example, several divided
doses may be administered daily or the dose may be proportionally
reduced as indicated by the exigencies of the therapeutic
situation.
[0086] The term "prophylactically effective amount" refers to an
amount effective, at dosages and for periods of time necessary, to
achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount.
[0087] The term "beneficial pharmacokinetic profile" refers to
amounts or doses of a compound of the formula I, II or III that
provide levels of the compound in plasma and/or brain or a required
dose resulting in therapeutic effects in the prevention, treatment,
or control of symptoms of a disease disclosed herein, in particular
a neuroinflammatory disease, more particularly Alzheimer's disease.
The term "sustained pharmacokinetic profile" as used herein refers
to a length of time efficacious levels of a biologically active
compound of the formula I, II or III is in its environment of use.
A sustained pharmacokinetic profile can be such that a single or
twice daily administration adequately prevents, treats, or controls
symptoms of a disease disclosed herein. A beneficial
pharmacokinetic profile may provide therapeutically effective
amounts of the compound of the formula I, II or III in the plasma
and/or brain for about 12 to about 48 hours, 12 hours to about 36
hours, or 12 hours to about 24 hours.
[0088] A "therapeutic effect" refers to an effect of a composition,
in particular a formulation or dosage form, or method disclosed
herein, including improved biological activity, efficacy, and/or
lower risk of side effects (e.g., lower risk of QT-related side
effects). A therapeutic effect may be a sustained therapeutic
effect that correlates with a continuous plasma and/or brain
concentration of a compound of the formula I, II or III over a
dosing period, in particular a sustained dosing period. A
therapeutic effect may be a statistically significant effect in
terms of statistical analysis of an effect of a compound of the
formula I, II or III versus the effects without the compound.
[0089] "Statistically significant" or "significantly different"
effects or levels may represent levels that are higher or lower
than a standard. In aspects of the invention, the difference may be
1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 50 times higher or
lower compared with the effect obtained without a compound of the
formula I, II or III.
[0090] In an embodiment, where the disease is neuroinflammatory
disease such as Alzheimer's disease, therapeutic effects of a
compound or composition or treatment of the invention can manifest
as one, two, three, four, five, six, seven, eight, or all of the
following, in particular five or more, more particularly seven or
more of the following: [0091] a) A reduction in protein kinase
activity (e.g. DAPK), in particular at least about a 0.05%, 0.1%,
0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, 60%,
70%, 80%, 90%, 95%, or 99% decrease in protein kinase activity.
[0092] b) A reduction in glial activation response, in particular,
at least about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%,
33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction
in glial activation response. [0093] c) A reduction in glial
activity in the brain, relative to the levels determined in the
absence of a compound of the formula I, II or III in subjects with
symptoms of a neuroinflammatory disease. In particular, the
compounds induce at least about a 2%, 5%, 10%, 15%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, or 90% decrease in glial activity. [0094] d) A
reduction in astrocyte activation response, in particular, at least
about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%,
35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in
astrocyte activation response. [0095] e) A reduction in astrocyte
activity in the brain, relative to the levels determined in the
absence of a compound or treatment according to the invention. In
particular, the compounds induce at least about a 2%, 5%, 10%, 15%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease in astrocyte
activity. [0096] f) A reduction in microglial activation, in
particular, at least about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%,
15%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or
99% reduction in microglial activation. [0097] g) A reduction in
microglial activation response, in particular, at least about a
0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%,
45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in microglial
activation response. [0098] h) A reduction in loss of synaptophysin
and/or PSD-95, in particular at least about a 0.05%, 0.1%, 0.5%,
1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%,
80%, 90%, 95%, or 99% reduction in loss of synaptophysin and/or
PSD-95. [0099] i) A reduction in oxidative stress-related responses
(e.g., nitric oxide synthase production and/or nitric oxide
accumulation), in particular at least about a 0.05%, 0.1%, 0.5%,
1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%,
80%, 90%, 95%, or 99% reduction in oxidative stress-related
responses such as nitric oxide synthase production and nitric oxide
accumulation. [0100] j) A reduction in cellular apoptosis and/or
death associated protein kinase activity, in particular a 0.05%,
0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%, 45%,
50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in cellular
apoptosis and/or death associated protein kinase activity. [0101]
k) A reduction in proinflammatory cytokine responses in particular
a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%,
45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in
proinflammatory cytokine responses. [0102] l) A reduction in
interleukin-1.beta. and/or tumor necrosis factor a production in
particular a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%,
33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction
in interleukin-1.beta. and/or tumor necrosis factor .alpha.
production. [0103] m) A slowing of the rate of disease progression
in a subject with a neuroinflammatory disease (e.g., Alzheimer's
disease). [0104] n) Increase in survival in a subject with symptoms
of a neuroinflammatory disease (e.g., Alzheimer's disease).
[0105] In particular aspects of the invention therapeutic effects
of compounds, compositions or treatments of the invention can
manifest as (a) and (b); (a), (b) and (c); (a) through (d); (a)
through (e); (a) through (f); (a) through (g); (a) through (h); (a)
through (i), (a) through (j), and (a) through (k), (a) through (l),
(a) through (m), or (a) through (n).
[0106] The term "pharmaceutically acceptable carrier, excipient, or
vehicle" refers to a medium which does not interfere with the
effectiveness or activity of an active ingredient and which is not
toxic to the hosts to which it is administered. A carrier,
excipient, or vehicle includes diluents, binders, adhesives,
lubricants, disintegrates, bulking agents, wetting or emulsifying
agents, pH buffering agents, and miscellaneous materials such as
absorbants that may be needed in order to prepare a particular
composition. Examples of carriers etc. include but are not limited
to saline, buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. The use of such media and agents for an
active substance is well known in the art.
[0107] The compounds of the formula I, II or III disclosed herein
also include "pharmaceutically acceptable salt(s)". By
pharmaceutically acceptable salts is meant those salts which are
suitable for use in contact with the tissues of a subject or
patient without undue toxicity, irritation, allergic response and
the like, and are commensurate with a reasonable benefit/risk
ratio. Pharmaceutically acceptable salts are described for example,
in S. M. Berge, et al., J. Pharmaceutical Sciences, 1977, 66:1.
Suitable salts include salts that may be formed where acidic
protons in the compounds are capable of reacting with inorganic or
organic bases. Suitable inorganic salts include those formed with
alkali metals, e.g. sodium and potassium, magnesium, calcium, and
aluminum. Suitable organic salts include those formed with organic
bases such as the amine bases, e.g. ethanolamine, diethanolamine,
triethanolamine, tromethamine, N-methylglucamine, and the like.
Suitable salts also include acid addition salts formed with
inorganic acids (e.g. hydrochloric and hydrobromic acids) and
organic acids (e.g. acetic acid, citric acid, maleic acid, and the
alkane- and arene-sulfonic acids such as methanesulfonic acid and
benezenesulfonic acid). When there are two acidic groups present, a
pharmaceutically acceptable salt may be a mono-acid-mono-salt or a
di-salt; and similarly where there are more than two acidic groups
present, some or all of such groups can be salified.
[0108] A compound of the formula I, II or III can contain one or
more asymmetric centers and may give rise to enantiomers,
diasteriomers, and other stereoisomeric forms which may be defined
in terms of absolute stereochemistry as (R)- or (S)-. Thus,
compounds of the formula I, II or III include all possible
diasteriomers and enantiomers as well as their racemic and
optically pure forms. Optically active (R)- and (S)-isomers may be
prepared using chiral synthons or chiral reagents, or resolved
using conventional techniques. When a compound of the formula I, II
or III contains centers of geometric asymmetry, and unless
specified otherwise, it is intended that the compounds include both
E and A geometric isomers. All tautomeric forms are also included
within the scope of a compound of the formula I, II or III.
[0109] A compound of the formula I, II or III includes crystalline
forms which may exist as polymorphs. Solvates of the compounds
formed with water or common organic solvents are also intended to
be encompassed within the term. In addition, hydrate forms of the
compounds and their salts are encompassed within this invention.
Further prodrugs of compounds of the formula I, II or III are
encompassed within the term.
[0110] The term "solvate" means a physical association of a
compound with one or more solvent molecules or a complex of
variable stoichiometry formed by a solute (for example, a compound
of the invention) and a solvent, for example, water, ethanol, or
acetic acid. This physical association may involve varying degrees
of ionic and covalent bonding, including hydrogen bonding. In
certain instances, the solvate will be capable of isolation, for
example, when one or more solvent molecules are incorporated in the
crystal lattice of the crystalline solid. In general, the solvents
selected do not interfere with the biological activity of the
solute. Solvates encompass both solution-phase and isolatable
solvates. Representative solvates include hydrates, ethanolates,
methanolates, and the like. Dehydrate, co-crystals, anhydrous, or
amorphous forms of the compounds of the invention are also
included. The term "hydrate" means a solvate wherein the solvent
molecule(s) is/are H.sub.2O, including, mono-, di-, and various
poly-hydrates thereof. Solvates can be formed using various methods
known in the art.
[0111] Crystalline compounds of the formula I, II or III can be in
the form of a free base, a salt, or a co-crystal. Free base
compounds can be crystallized in the presence of an appropriate
solvent in order to form a solvate. Acid salt compounds of the
formula I, II or III (e.g. HCl, HBr, benzoic acid) can also be used
in the preparation of solvates. For example, solvates can be formed
by the use of acetic acid or ethyl acetate. The solvate molecules
can form crystal structures via hydrogen bonding, van der Waals
forces, or dispersion forces, or a combination of any two or all
three forces.
[0112] The amount of solvent used to make solvates can be
determined by routine testing. For example, a monohydrate of a
compound of the formula I, II or III would have about 1 equivalent
of solvent (H.sub.2O) for each equivalent of a compound of the
invention. However, more or less solvent may be used depending on
the choice of solvate desired.
[0113] Compounds of the formula I, II or III may be amorphous or
may have different crystalline polymorphs, possibly existing in
different solvation or hydration states. By varying the form of a
drug, it is possible to vary the physical properties thereof. For
example, crystalline polymorphs typically have different
solubilities from one another, such that a more thermodynamically
stable polymorph is less soluble than a less thermodynamically
stable polymorph. Pharmaceutical polymorphs can also differ in
properties such as shelf-life, bioavailability, morphology, vapor
pressure, density, color, and compressibility.
[0114] The term "prodrug" means a covalently-bonded derivative or
carrier of the parent compound or active drug substance which
undergoes at least some biotransformation prior to exhibiting its
pharmacological effect(s). In general, such prodrugs have
metabolically cleavable groups and are rapidly transformed in vivo
to yield the parent compound, for example, by hydrolysis in blood,
and generally include esters and amide analogs of the parent
compounds. The prodrug is formulated with the objectives of
improved chemical stability, improved patient acceptance and
compliance, improved bioavailability, prolonged duration of action,
improved organ selectivity, improved formulation (e.g., increased
hydrosolubility), and/or decreased side effects (e.g., toxicity).
In general, prodrugs themselves have weak or no biological activity
and are stable under ordinary conditions. Prodrugs can be readily
prepared from the parent compounds using methods known in the art,
such as those described in A Textbook of Drug Design and
Development, Krogsgaard-Larsen and H. Bundgaard (eds.), Gordon
& Breach, 1991, particularly Chapter 5: "Design and
Applications of Prodrugs"; Design of Prodrugs, H. Bundgaard (ed.),
Elsevier, 1985; Prodrugs: Topical and Ocular Drug Delivery, K. B.
Sloan (ed.), Marcel Dekker, 1998; Methods in Enzymology, K. Widder
et al. (eds.), Vol. 42, Academic Press, 1985, particularly pp. 309
396; Burger's Medicinal Chemistry and Drug Discovery, 5th Ed., M.
Wolff (ed.), John Wiley & Sons, 1995, particularly Vol. I and
pp. 172 178 and pp. 949 982; Pro-Drugs as Novel Delivery Systems,
T. Higuchi and V. Stella (eds.), Am. Chem. Soc., 1975; and
Bioreversible Carriers in Drug Design, E. B. Roche (ed.), Elsevier,
1987.
[0115] Examples of prodrugs include, but are not limited to esters
(e.g., acetate, formate, and benzoate derivatives), carbamates
(e.g. N,N-dimethylaminocarbonyl) of hydroxy functional groups on
compounds of the formula I, II or III, and the like
[0116] A compound of the formula I, II or III can include a
pharmaceutically acceptable co-crystal or a co-crystal salt. A
pharmaceutically acceptable co-crystal includes a co-crystal that
is suitable for use in contact with the tissues of a subject or
patient without undue toxicity, irritation, allergic response and
has the desired pharmacokinetic properties.
[0117] The term "co-crystal" as used herein means a crystalline
material comprised of two or more unique solids at room
temperature, each containing distinctive physical characteristics,
such as structure, melting point, and heats of fusion. Co-crystals
can be formed by an active pharmaceutical ingredient (API) and a
co-crystal former either by hydrogen bonding or other non-covalent
interactions, such as pi stacking and van der Waals interactions.
An aspect of the invention provides for a co-crystal wherein the
co-crystal former is a second API. In another aspect, the
co-crystal former is not an API. In another aspect, the co-crystal
comprises more than one co-crystal former. For example, two, three,
four, five, or more co-crystal formers can be incorporated in a
co-crystal with an API. Pharmaceutically acceptable co-crystals are
described, for example, in "Pharmaceutical co-crystals," Journal of
Pharmaceutical Sciences, Volume 95 (3) Pages 499-516, 2006. The
methods producing co-crystals are discussed in the United States
Patent Application 20070026078.
[0118] A co-crystal former which is generally a pharmaceutically
acceptable compound, may be, for example, benzoquinone,
terephthalaldehyde, saccharin, nicotinamide, acetic acid, formic
acid, butyric acid, trimesic acid, 5-nitroisophthalic acid,
adamantane-1,3,5,7-tetracarboxylic acid, formamide, succinic acid,
fumaric acid, tartaric acid, malic acid, tartaric acid, malonic
acid, benzamide, mandelic acid, glycolic acid, fumaric acid, maleic
acid, urea, nicotinic acid, piperazine, p-phthalaldehyde,
2,6-pyridinecarboxylic acid, 5-nitroisophthalic acid, citric acid,
and the alkane- and arene-sulfonic acids such as methanesulfonic
acid and benezenesulfonic acid.
[0119] In general, all physical forms of compounds of the formula
I, II or III are intended to be within the scope of the present
invention.
[0120] A compound of the formula I, II or III may be pure or
substantially pure. As used herein, the term "pure" in general
means better than 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
pure, and "substantially pure" means a compound synthesized such
that the compound, as made or as available for consideration into a
composition or therapeutic dosage described herein, has only those
impurities that can not readily nor reasonably be removed by
conventional purification processes.
[0121] "Optional" or "optionally" means that the subsequently
described event or circumstance may but need not occur, and that
the description includes instances where the event or circumstance
occurs and instances in which it does not occur. For example,
"alkyl group optionally substituted with a halo group" means that
the halo may but need not be present, and the description includes
situations where the alkyl group is substituted with a halo group
and situations where the alkyl group is not substituted with the
halo group.
[0122] A compound of the formula I, II or III includes derivatives.
As used herein the term "derivative" of a compound of the formula
I, II or III refers to a chemically modified compound wherein the
chemical modification takes place either at a functional group or
ring of the compound. Non-limiting examples of derivatives of
compounds of the formula I, II or III may include N-acetyl,
N-methyl, N-hydroxy groups at any of the available nitrogens in the
compound. Derivative groups that may be used to modify the
compounds of the formula I, II or III can be found in U.S. Patent
Application No. 20030176437 (herein incorporated by reference in
its entirety for all purposes).
[0123] In aspects of the invention, a compound of the formula I, II
or III is a pharmaceutically functional derivative. A
"pharmaceutically functional derivative" includes any
pharmaceutically acceptable derivative of a compound of the formula
I, II or III, for example, an ester or an amide, which upon
administration to a subject is capable of providing (directly or
indirectly) a compound of the formula I, II or III or an active
metabolite or residue thereof. Such derivatives are recognizable to
those skilled in the art, without undue experimentation (see for
example Burger's Medicinal Chemistry and Drug Discovery, 5.sup.th
Edition, Vol 1: Principles and Practice, which has illustrative
pharmaceutically functional derivatives).
[0124] A compound of the formula I, II or III may include a
carrier. Suitable carriers include a polymer, carbohydrate, or a
peptide.
[0125] A "polymer" refers to molecules comprising two or more
monomer subunits that may be identical repeating subunits or
different repeating subunits. A monomer generally comprises a
simple structure, low-molecular weight molecule containing carbon.
Polymers may optionally be substituted. Polymers that can be used
in the present invention include without limitation vinyl, acryl,
styrene, carbohydrate derived polymers, polyethylene glycol (PEG),
polyoxyethylene, polymethylene glycol, poly-trimethylene glycols,
polyvinylpyrrolidone, polyoxyethylene-polyoxypropylene block
polymers, and copolymers, salts, and derivatives thereof. In
aspects of the invention, the polymer is
poly(2-acrylamido-2-methyl-1-propanesulfonic acid);
poly(2-acrylamido-2-methyl-1-propanesulfonic acid-coacrylonitrile,
poly(2-acrylamido-2-methyl-1-propane sulfonic acid-co-styrene),
poly(vinylsulfonic acid); poly(sodium 4-styrenesulfonic acid); and
sulfates and sulfonates derived therefrom; poly(acrylic acid),
poly(methylacrylate), poly(methyl methacrylate), and poly(vinyl
alcohol).
[0126] A "carbohydrate" as used herein refers to a
polyhydroxyaldehyde, or polyhydroxyketone and derivatives thereof.
The term includes monosaccharides such as erythrose, arabinose,
allose, altrose, glucose, mannose, threose, xylose, gulose, idose,
galactose, talose, aldohexose, fructose, ketohexose, ribose, and
aldopentose. The term also includes carbohydrates composed of
monosaccharide units, including disaccharides, oligosaccharides, or
polysaccharides. Examples of disaccharides are sucrose, lactose,
and maltose. Oligosaccharides generally contain between 3 and 9
monosaccharide units and polysaccharides contain greater than 10
monosaccharide units. A carbohydrate group may be substituted at
one two, three or four positions, other than the position of
linkage to a compound of the formula I, II or III. For example, a
carbohydrate may be substituted with one or more alkyl, amino,
nitro, halo, thiol, carboxyl, or hydroxyl groups, which are
optionally substituted. Illustrative substituted carbohydrates are
glucosamine, or galactosamine. In aspects of the invention, the
carbohydrate is a sugar, in particular a hexose or pentose and may
be an aldose or a ketose. A sugar may be a member of the D or L
series and can include amino sugars, deoxy sugars, and their uronic
acid derivatives. In embodiments of the invention where the
carbohydrate is a hexose, the hexose is glucose, galactose, or
mannose, or substituted hexose sugar residues such as an amino
sugar residue such as hexosamine, galactosamine, glucosamine, in
particular D-glucosamine (2-amino-2-doexy-D-glucose) or
D-galactosamine (2-amino-2-deoxy-D-galactose). Illustrative pentose
sugars include arabinose, fucose, and ribose.
[0127] A sugar residue may be linked to a compound of the formula
I, II or III from a 1,1 linkage, 1,2 linkage, 1,3 linkage, 1,4
linkage, 1,5 linkage, or 1,6 linkage. A linkage may be via an
oxygen atom of a compound of the formula I, II or III. An oxygen
atom can be replaced one or more times by --CH.sub.2-- or --S--
groups.
[0128] The term "carbohydrate" also includes glycoproteins such as
lectins (e.g. concanavalin A, wheat germ agglutinin,
peanutagglutinin, seromucoid, and orosomucoid) and glycolipids such
as cerebroside and ganglioside.
[0129] A "peptide" carrier for use in the practice of the present
invention includes one, two, three, four, or five or more amino
acids covalently linked through a peptide bond. A peptide can
comprise one or more naturally occurring amino acids, and analogs,
derivatives, and congeners thereof. A peptide can be modified to
increase its stability, bioavailability, solubility, etc. "Peptide
analogue" and "peptide derivative" as used herein include molecules
which mimic the chemical structure of a peptide and retain the
functional properties of the peptide. A carrier for use in the
present invention can be an amino acid such as alanine, glycine,
proline, methionine, serine, threonine, histidine, asparagine,
alanyl-alanyl, prolyl-methionyl, or glycyl-glycyl. A carrier can be
a polypeptide such as albumin, antitrypsin, macroglobulin,
haptoglobin, caeruloplasm, transferring, .alpha.- or
.beta.-lipoprotein, .beta.- or .gamma.-globulin or fibrinogen.
[0130] Approaches to designing peptide analogues, derivatives and
mimetics are known in the art. For example, see Farmer, P. S. in
Drug Design (E. J. Ariens, ed.) Academic Press, New York, 1980,
vol. 10, pp. 119-143; Ball. J. B. and Alewood, P. F. (1990) J. Mol.
Recognition. 3:55; Morgan, B. A. and Gainor, J. A. (1989) Ann. Rep.
Med. Chem. 24:243; and Freidinger, R. M. (1989) Trends Pharmacol.
Sci. 10:270. See also Sawyer, T. K. (1995) "Peptidomimetic Design
and Chemical Approaches to Peptide Metabolism" in Taylor, M. D. and
Amidon, G. L. (eds.) Peptide-Based Drug Design: Controlling
Transport and Metabolism, Chapter 17; Smith, A. B. 3rd, et al.
(1995) J. Am. Chem. Soc. 117:11113-11123; Smith, A. B. 3rd, et al.
(1994) J. Am. Chem. Soc. 116:9947-9962; and Hirschman, R., et al.
(1993) J. Am. Chem. Soc. 115:12550-12568.
[0131] A peptide can be attached to a compound of the formula I, II
or III through a functional group on the side chain of certain
amino acids (e.g. serine) or other suitable functional groups. A
carrier may comprise four or more amino acids with groups attached
to three or more of the amino acids through functional groups on
side chains. In an aspect, the carrier is one amino acid, in
particular a sulfonate derivative of an amino acid, for example
cysteic acid.
[0132] The term "alkyl", either alone or within other terms such as
"thioalkyl" and "arylalkyl", means a monovalent, saturated
hydrocarbon radical which may be a straight chain (i.e. linear) or
a branched chain. An alkyl radical for use in the present invention
generally comprises from about 1 to 20 carbon atoms, particularly
from about 1 to 10, 1 to 8 or 1 to 7, more particularly about 1 to
6 carbon-atoms, or 3 to 6. Illustrative alkyl radicals include
methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl,
isobutyl, isopentyl, amyl, sec-butyl, tert-butyl, tert-pentyl,
n-heptyl, n-octyl, n-nonyl, n-decyl, undecyl, n-dodecyl,
n-tetradecyl, pentadecyl, n-hexadecyl, heptadecyl, n-octadecyl,
nonadecyl, eicosyl, dosyl, n-tetracosyl, and the like, along with
branched variations thereof. In certain aspects of the invention an
alkyl radical is a C.sub.1-C.sub.6 lower alkyl comprising or
selected from the group consisting of methyl, ethyl, n-propyl,
n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, isopentyl, amyl,
tributyl, sec-butyl, tert-butyl, tert-pentyl, and n-hexyl. An alkyl
radical may be optionally substituted with substituents as defined
herein at positions that do not significantly interfere with the
preparation of compounds of the formula I, II or III and do not
significantly reduce the efficacy of the compounds. In certain
aspects of the invention, an alkyl radical is substituted with one
to five substituents including halo, lower alkoxy, lower aliphatic,
a substituted lower aliphatic, hydroxy, cyano, nitro, thio, amino,
keto, aldehyde, ester, amide, substituted amino, carboxyl,
sulfonyl, sulfinyl, sulfenyl, sulfate, sulfoxide, substituted
carboxyl, halogenated lower alkyl (e.g. CF.sub.3), halogenated
lower alkoxy, hydroxycarbonyl, lower alkoxycarbonyl, lower
alkylcarbonyloxy, lower alkylcarbonylamino, cycloaliphatic,
substituted cycloaliphatic, or aryl (e.g., phenylmethyl (i.e.
benzyl)), heteroaryl (e.g., pyridyl), and heterocyclic (e.g.,
piperidinyl, morpholinyl). Substituents on an alkyl group may
themselves be substituted.
[0133] In aspects of the invention, "substituted alkyl" includes an
alkyl group substituted by, for example, one to five substituents,
and preferably 1 to 3 substituents, such as alkyl, alkoxy, oxo,
alkanoyl, aryl, aralkyl, aryloxy, alkanoyloxy, cycloalkyl, acyl,
amino, hydroxyamino, alkylamino, arylamino, alkoxyamino,
aralkylamino, cyano, halogen, hydroxyl, carboxyl, carbamyl,
carboxylalkyl, keto, thioketo, thiol, alkylthiol, arylthio,
aralkylthio, sulfonamide, thioalkoxy, and nitro.
[0134] In respect to certain aspects of the invention, the term
"substituted aliphatic" refers to an alkyl or an alkane possessing
less than 10 carbons. The term "substituted aliphatic" refers to an
alkyl or an alkane possessing less than 10 carbons where at least
one of the aliphatic hydrogen atoms has been replaced by a halogen,
an amino, a hydroxy, a nitro, a thio, a ketone, an aldehyde, an
ester, an amide, a lower aliphatic, a substituted lower aliphatic,
or a ring (aryl, substituted aryl, cycloaliphatic, or substituted
cycloaliphatic, etc.). Examples of such groups include, but are not
limited to, 1-chloroethyl and the like.
[0135] As used herein in respect to certain aspects of the
invention, the term "lower-alkyl-substituted-amino" refers to any
alkyl unit containing up to and including eight carbon atoms where
one of the aliphatic hydrogen atoms is replaced by an amino group.
Examples of such include, but are not limited to, ethylamino and
the like.
[0136] As used herein in respect to certain aspects of the
invention, the term "lower-alkyl-substituted-halogen" refers to any
alkyl chain containing up to and including eight carbon atoms where
one of the aliphatic hydrogen atoms is replaced by a halogen.
Examples of such include, but are not limited to, chlorethyl and
the like.
[0137] As used herein, the term "acetylamino" shall mean any
primary or secondary amino that is acetylated. Examples of such
include, but are not limited to, acetamide and the like.
[0138] As used herein the term "alkenyl" refers to an unsaturated,
acyclic branched or straight-chain hydrocarbon radical comprising
at least one double bond. An alkenyl radical may contain from about
2 to 24 or 2 to 10 carbon atoms, in particular from about 3 to 8
carbon atoms and more particularly about 3 to 6 or 2 to 6 carbon
atoms. Suitable alkenyl radicals include without limitation
ethenyl, propenyl (e.g., prop-1-en-1-yl, prop-1-en-2-yl,
prop-2-en-1-yl (allyl), and prop-2-en-2-yl), buten-1-yl,
but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,
but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, hexen-1-yl,
3-hydroxyhexen-1-yl, hepten-1-yl, and octen-1-yl, and the like. An
alkenyl radical may be optionally substituted similar to alkyl.
[0139] In aspects of the invention, "substituted alkenyl" includes
an alkenyl group substituted by, for example, one to three
substituents, preferably one to two substituents, such as alkyl,
alkoxy, haloalkoxy, alkylalkoxy, haloalkoxyalkyl, alkanoyl,
alkanoyloxy, cycloalkyl, cycloalkoxy, acyl, acylamino, acyloxy,
amino, alkylamino, alkanoylamino, aminoacyl, aminoacyloxy, cyano,
halogen, hydroxyl, carboxyl, carboxylalkyl, carbamyl, keto,
thioketo, thiol, alkylthio, sulfonyl, sulfonamido, thioalkoxy,
aryl, nitro, and the like.
[0140] As used herein, the term "alkynyl" refers to an unsaturated,
branched or straight-chain hydrocarbon radical comprising one or
more triple bonds. An alkynyl radical may contain about 1 to 20, 1
to 15, or 2 to 10 carbon atoms, particularly about 3 to 8 carbon
atoms and more particularly about 3 to 6 carbon atoms. Suitable
alkynyl radicals include without limitation ethynyl, such as
prop-1-yn-1-yl and prop-2-yn-1-yl, butynyls such as but-1-yn-1-yl,
but-1-yn-3-yl, and but-3-yn-1-yl, pentynyls such as pentyn-1-yl,
pentyn-2-yl, 4-methoxypentyn-2-yl, and 3-methylbutyn-1-yl, hexynyls
such as hexyn-1-yl, hexyn-2-yl, hexyn-3-yl, and
3,3-dimethylbutyn-1-yl radicals and the like. In aspects of the
invention, alkenyl groups include ethenyl (--CH.dbd.CH.sub.2),
n-propenyl (--CH.sub.2CH.dbd.CH.sub.2), iso-propenyl
(--C(CH.sub.3).dbd.CH.sub.2), and the like. An alkynyl may be
optionally substituted similar to alkyl. The term "cycloalkynyl"
refers to cyclic alkynyl groups.
[0141] In aspects of the invention, "substituted alkynyl" includes
an alkynyl group substituted by, for example, a substituent, such
as, alkyl, alkoxy, alkanoyl, alkanoyloxy, cycloalkyl, cycloalkoxy,
acyl, acylamino, acyloxy, amino, alkylamino, alkanoylamino,
aminoacyl, aminoacyloxy, cyano, halogen, hydroxyl, carboxyl,
carboxylalkyl, carbamyl, keto, thioketo, thiol, alkylthio,
sulfonyl, sulfonamido, thioalkoxy, aryl, nitro, and the like.
[0142] As used herein the term "alkylene" refers to a linear or
branched radical having from about 1 to 10, 1 to 8, 1 to 6, or 2 to
6 carbon atoms and having attachment points for two or more
covalent bonds. Examples of such radicals are methylene, ethylene,
propylene, butylene, pentylene, hexylene, ethylidene,
methylethylene, and isopropylidene. When an alkenylene radical is
present as a substituent on another radical it is typically
considered to be a single substituent rather than a radical formed
by two substituents.
[0143] As used herein the term "alkenylene" refers to a linear or
branched radical having from about 2 to 10, 2 to 8 or 2 to 6 carbon
atoms, at least one double bond, and having attachment points for
two or more covalent bonds. Examples of alkenylene radicals include
1,1-vinylidene (--CH.sub.2.dbd.C--), 1,2-vinylidene
(--CH.dbd.CH--), and 1,4-butadienyl (--CH.dbd.CH--CH.dbd.CH--).
[0144] As used herein the term "halo" refers to a halogen such as
fluorine, chlorine, bromine or iodine atoms.
[0145] As used herein the term "hydroxyl" or "hydroxy" refers to an
--OH group.
[0146] As used herein the term "cyano" refers to a carbon radical
having three of four covalent bonds shared by a nitrogen atom, in
particular --C--N. A cyano group may be substituted with
substituents described herein.
[0147] As used herein the term "alkoxy" refers to a linear or
branched oxy-containing radical having an alkyl portion of one to
about ten carbon atoms, such as a methoxy radical, which may be
substituted. In aspects of the invention an alkoxy radical may
comprise about 1-10, 1-8, 1-6 or 1-3 carbon atoms. In embodiments
of the invention, an alkoxy radical comprises about 1-6 carbon
atoms and includes a C.sub.1-C.sub.6 alkyl-O-radical wherein
C.sub.1-C.sub.6 alkyl has the meaning set out herein. Examples of
alkoxy radicals include without limitation methoxy, ethoxy,
propoxy, butoxy, isopropoxy and tert-butoxy alkyls. An "alkoxy"
radical may optionally be substituted with one or more
substitutents disclosed herein including alkyl atoms to provide
"alkylalkoxy" radicals; halo atoms, such as fluoro, chloro or
bromo, to provide "haloalkoxy" radicals (e.g. fluoromethoxy,
chloromethoxy, trifluoromethoxy, difluoromethoxy, trifluoroethoxy,
fluoroethoxy, tetrafluoroethoxy, pentafluoroethoxy, and
fluoropropox) and "haloalkoxyalkyl" radicals (e.g.
fluoromethoxymethyl, chloromethoxyethyl, trifluoromethoxymethyl,
difluoromethoxyethyl, and trifluoroethoxymethyl).
[0148] As used herein the term "alkenyloxy" refers to linear or
branched oxy-containing radicals having an alkenyl portion of about
2 to 10 carbon atoms, such as an ethenyloxy or propenyloxy radical.
An alkenyloxy radical may be a "lower alkenyloxy" radical having
about 2 to 6 carbon atoms. Examples of alkenyloxy radicals include
without limitation ethenyloxy, propenyloxy, butenyloxy, and
isopropenyloxy alkyls. An "alkenyloxy" radical may be substituted
with one or more substitutents disclosed herein including halo
atoms, such as fluoro, chloro or bromo, to provide "haloalkenyloxy"
radicals (e.g. trifluoroethenyloxy, fluoroethenyloxy,
difluoroethenyloxy, and fluoropropenyloxy).
[0149] A "carbocylic" includes radicals derived from a saturated or
unsaturated, substituted or unsubstituted 5 to 14 member organic
nucleus whose ring forming atoms (other than hydrogen) are solely
carbon. Examples of carbocyclic radicals are cycloalkyl,
cycloalkenyl, aryl, in particular phenyl, naphthyl, norbornanyl,
bicycloheptadienyl, toluoyl, xylenyl, indenyl, stilbenzyl,
terphenylyl, diphenylethylenyl, phenylcyclohexyl, acenapththylenyl,
anthracenyl, biphenyl, bibenzylyl, and related bibenzylyl homologs,
octahydronaphthyl, tetrahydronaphthyl, octahydroquinolinyl,
dimethoxytetrahydronaphthyl and the like.
[0150] As used herein, the term "cycloalkyl" refers to radicals
having from about 3 to 15, 3 to 10, 3 to 8, or 3 to 6 carbon atoms
and containing one, two, three, or four rings wherein such rings
may be attached in a pendant manner or may be fused. In aspects of
the invention, "cycloalkyl" refers to an optionally substituted,
saturated hydrocarbon ring system containing 1 to 2 rings and 3 to
7 carbons per ring which may be further fused with an unsaturated
C.sub.3-C.sub.7 carbocylic ring. Examples of cycloalkyl groups
include single ring structures such as cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl,
cyclodecyl, cyclododecyl, and the like, or multiple ring structures
such as adamantanyl, and the like. In certain aspects of the
invention the cycloalkyl radicals are "lower cycloalkyl" radicals
having from about 3 to 10, 3 to 8, 3 to 6, or 3 to 4 carbon atoms,
in particular cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and
cycloheptyl. The term "cycloalkyl" also embraces radicals where
cycloalkyl radicals are fused with aryl radicals or heterocyclyl
radicals. A cycloalkyl radical may be optionally substituted with
groups as disclosed herein.
[0151] In aspects of the invention, "substituted cycloalkyl"
includes cycloalkyl groups having from 1 to 5 (in particular 1 to
3) substituents including without limitation alkyl, alkenyl,
alkoxy, cycloalkyl, substituted cycloalkyl, acyl, acylamino,
acyloxy, amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano,
halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol,
thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, hydroxyamino,
alkoxyamino, and nitro.
[0152] As used herein in respect to certain aspects of the
invention, the term "cycloaliphatic" refers to a cycloalkane
possessing less than 8 carbons or a fused ring system consisting of
no more than three fused cycloaliphatic rings. Examples of such
groups include, but are not limited to, decalin and the like.
[0153] As used herein in respect to certain aspects of the
invention, the term "substituted cycloaliphatic" refers to a
cycloalkane possessing less than 8 carbons or a fused ring system
consisting of no more than three fused rings, and where at least
one of the aliphatic hydrogen atoms has been replaced by a halogen,
a nitro, a thio, an amino, a hydroxy, a ketone, an aldehyde, an
ester, an amide, a lower aliphatic, a substituted lower aliphatic,
or a ring (aryl, substituted aryl, cycloaliphatic, or substituted
cycloaliphatic). Examples of such groups include, but are not
limited to, 1-chlorodecalyl and the like.
[0154] A used herein, the term "cycloalkenyl" refers to radicals
comprising about 4 to 16, 2 to 15, 2 to 10, 2 to 8, 4 to 10, 3 to
8, 3 to 7, 3 to 6, or 4 to 6 carbon atoms, one or more
carbon-carbon double bonds, and one, two, three, or four rings
wherein such rings may be attached in a pendant manner or may be
fused. In certain aspects of the invention the cycloalkenyl
radicals are "lower cycloalkenyl" radicals having three to seven
carbon atoms. Examples of cycloalkenyl radicals include without
limitation cyclobutenyl, cyclopentenyl, cyclohexenyl and
cycloheptenyl. A cycloalkenyl radical may be optionally substituted
with groups as disclosed herein, in particular 1, 2, or 3
substituents which may be the same or different.
[0155] As used herein the term "cycloalkoxy" refers to cycloalkyl
radicals (in particular, cycloalkyl radicals having 3 to 15, 3 to 8
or 3 to 6 carbon atoms) attached to an oxy radical. Examples of
cycloalkoxy radicals include cyclohexoxy and cyclopentoxy. A
cycloalkoxy radical may be optionally substituted with groups as
disclosed herein.
[0156] As used herein, the term "aryl", alone or in combination,
refers to a carbocyclic aromatic system containing one, two or
three rings wherein such rings may be attached together in a
pendant manner or may be fused. In aspects of the invention an aryl
radical comprises 4 to 24 carbon atoms, in particular 4 to 10, 4 to
8, or 4 to 6 carbon atoms. Illustrative "aryl" radicals includes
without limitation aromatic radicals such as phenyl, benzyl,
naphthyl, indenyl, benzocyclooctenyl, benzocycloheptenyl,
pentalenyl, azulenyl, tetrahydronaphthyl, indanyl, biphenyl,
acephthylenyl, fluorenyl, phenalenyl, phenanthrenyl, and
anthracenyl, preferably phenyl.
[0157] An aryl radical may be optionally substituted with groups as
disclosed herein, in particular hydroxyl, alkyl, carbonyl,
carboxyl, thiol, amino, and/or halo, in particular a substituted
aryl includes without limitation arylamine and arylalkylamine.
[0158] As used herein in respect to certain aspects of the
invention, the term "substituted aryl" includes an aromatic ring,
or fused aromatic ring system consisting of no more than three
fused rings at least one of which is aromatic, and where at least
one of the hydrogen atoms on a ring carbon has been replaced by a
halogen, an amino, a hydroxy, a nitro, a thio, an alkyl, a ketone,
an aldehyde, an ester, an amide, a lower aliphatic, a substituted
lower aliphatic, or a ring (aryl, substituted aryl, cycloaliphatic,
or substituted cycloaliphatic). Examples of such include, but are
not limited to, hydroxyphenyl, chlorophenyl and the like.
[0159] In aspects of the invention, an aryl radical may be
optionally substituted with one to four substituents such as alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, aralkyl, halo,
trifluoromethoxy, trifluoromethyl, hydroxy, alkoxy, alkanoyl,
alkanoyloxy, aryloxy, aralkyloxy, amino, alkylamino, arylamino,
aralkylamino, dialkylamino, alkanoylamino, thiol, alkylthio,
ureido, nitro, cyano, carboxy, carboxyalkyl, carbamyl,
alkoxycarbonyl, alkylthiono, arylthiono, arylsulfonylamine,
sulfonic acid, alkysulfonyl, sulfonamido, aryloxy and the like. A
substituent may be further substituted by hydroxy, halo, alkyl,
alkoxy, alkenyl, alkynyl, aryl or aralkyl. In aspects of the
invention an aryl radical is substituted with hydroxyl, alkyl,
carbonyl, carboxyl, thiol, amino, and/or halo. The term "aralkyl"
refers to an aryl or a substituted aryl group bonded directly
through an alkyl group, such as benzyl. Other particular examples
of substituted aryl radicals include chlorobenyzl, and amino
benzyl.
[0160] As used herein, the term "aryloxy" refers to aryl radicals,
as defined above, attached to an oxygen atom. Exemplary aryloxy
groups include napthyloxy, quinolyloxy, isoquinolizinyloxy, and the
like.
[0161] As used herein the term "arylalkoxy," refers to an aryl
group attached to an alkoxy group. Representative examples of
arylalkoxy groups include, but are not limited to, 2-phenylethoxy,
3-naphth-2-ylpropoxy, and 5-phenylpentyloxy.
[0162] As used herein, the term "aroyl" refers to aryl radicals, as
defined above, attached to a carbonyl radical as defined herein,
including without limitation benzoyl and toluoyl. An aroyl radical
may be optionally substituted with groups as disclosed herein.
[0163] As used herein the term "heteroaryl" refers to fully
unsaturated heteroatom-containing ring-shaped aromatic radicals
having at least one heteroatom selected from carbon, nitrogen,
sulfur and oxygen. A heteroaryl radical may contain one, two or
three rings and the rings may be attached in a pendant manner or
may be fused. In aspects of the invention the term refers to fully
unsaturated heteroatom-containing ring-shaped aromatic radicals
having from 3 to 15, 3 to 10, 3 to 8, 5 to 15, 5 to 10, or 5 to 8
ring members selected from carbon, nitrogen, sulfur and oxygen,
wherein at least one ring atom is a heteroatom. Examples of
"heteroaryl" radicals, include without limitation, an unsaturated 5
to 6 membered heteromonocyclyl group containing 1 to 4 nitrogen
atoms, in particular, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazolyl, tetrazolyl and the like; an unsaturated
condensed heterocyclic group containing 1 to 5 nitrogen atoms, in
particular, indolyl, isoindolyl, indolizinyl, benzimidazolyl,
quinolyl, isoquinolyl, indazolyl, quinazolinyl, pteridinyl,
quinolizidinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,
cinnolinyl, phenanthridinyl, acridinyl, phenanthrolinyl,
phenazinyl, carbazolyl, purinyl, benzimidazolyl, quinolinyl,
isoquinolinyl, benzotriazolyl, tetrazolopyridazinyl and the like;
an unsaturated 3 to 6-membered heteromonocyclic group containing an
oxygen atom, in particular, 2-furyl, 3-furyl, pyranyl, and the
like; an unsaturated 5 to 6-membered heteromonocyclic group
containing a sulfur atom, in particular, thienyl, 2-thienyl,
3-thienyl, and the like; unsaturated 5 to 6-membered
heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3
nitrogen atoms, in particular, furazanyl, benzofurazanyl, oxazolyl,
isoxazolyl, and oxadiazolyl; an unsaturated condensed heterocyclic
group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, in
particular benzoxazolyl, benzoxadiazolyl and the like; an
unsaturated 5 to 6-membered heteromonocyclic group containing 1 to
2 sulfur atoms and 1 to 3 nitrogen atoms, for example, thiazolyl,
isothiazolyl, thiadiazolyl and the like; an unsaturated condensed
heterocyclic group containing 1 to 2 sulfur atoms and 1 to 3
nitrogen atoms such as benzothiazolyl, benzothiadiazolyl and the
like. The term also includes radicals where heterocyclic radicals
are fused with aryl radicals, in particular bicyclic radicals such
as benzofuranyl, benzothiophenyl, phthalazinyl, chromenyl,
xanthenyl, and the like. A heteroaryl radical may be optionally
substituted with groups as disclosed herein, for example with an
alkyl, amino, halogen, etc., in particular a heteroarylamine.
[0164] In aspects of the invention, the term refers to an
unsaturated 5 to 6 membered heteromonocyclyl group containing 1 to
4 nitrogen atoms, in particular, pyrrolyl, pyrrolinyl, imidazolyl,
pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl,
pyrazinyl, pyridazinyl, triazolyl, tetrazolyl and the like.
[0165] A heteroaryl radical may be optionally substituted with
groups disclosed herein, for example with an alkyl, amino, halogen,
etc., in particular a substituted heteroaryl radical is a
heteroarylamine.
[0166] The term "heterocyclic" refers to saturated and partially
saturated heteroatom-containing ring-shaped radicals having at
least one heteroatom selected from carbon, nitrogen, sulfur and
oxygen. A heterocylic radical may contain one, two or three rings
wherein such rings may be attached in a pendant manner or may be
fused. In an aspect, the term refers to a saturated and partially
saturated heteroatom-containing ring-shaped radicals having from
about 3 to 15, 3 to 10, 5 to 15, 5 to 10, or 3 to 8 ring members
selected from carbon, nitrogen, sulfur and oxygen, wherein at least
one ring atom is a heteroatom. Examplary saturated heterocyclic
radicals include without limitiation a saturated 3 to 6-membered
heteromonocylic group containing 1 to 4 nitrogen atoms [e.g.
pyrrolidinyl, imidazolidinyl, and piperazinyl]; a saturated 3 to
6-membered heteromonocyclic group containing 1 to 2 oxygen atoms
and 1 to 3 nitrogen atoms [e.g. morpholinyl; sydnonyl]; and, a
saturated 3 to 6-membered heteromonocyclic group containing 1 to 2
sulfur atoms and 1 to 3 nitrogen atoms [e.g., thiazolidinyl] etc.
Examples of partially saturated heterocyclyl radicals include
without limitation dihydrothiophene, dihydropyranyl, dihydrofuranyl
and dihydrothiazolyl. Illustrative heterocyclic radicals include
without limitation aziridinyl, azetidinyl, 2-pyrrolinyl,
3-pyrrolinyl, pyrrolidinyl, azepinyl, 1,3-dioxolanyl, 2H-pyranyl,
4H-pyranyl, piperidinyl, 1,4-dioxanyl, morpholinyl, pyrazolinyl,
1,4-dithianyl, thiomorpholinyl, 1,2,3,6-tetrahydropyridinyl,
oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,
tetrahydropyridinyl, tetrahydrothiopyranyl, thioxanyl, indolinyl,
2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl,
dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl,
imidazolinyl, imidazolidinyl, 3H-indolyl, quinuclidinyl,
quinolizinyl, and the like. In certain compounds of the formula II,
when R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, and
R.sup.17 are hydrogen, R.sup.11 cannot be piperidinyl.
[0167] As used herein in respect to certain aspects of the
invention, the term "heterocyclic" refers to a cycloalkane and/or
an aryl ring system, possessing less than 8 carbons, or a fused
ring system consisting of no more than three fused rings, where at
least one of the ring carbon atoms is replaced by oxygen, nitrogen
or sulfur. Examples of such groups include, but are not limited to,
morpholino and the like.
[0168] As used herein in respect to certain aspects of the
invention, the term "substituted heterocyclic" refers to a
cycloalkane and/or an aryl ring system, possessing less than 8
carbons, or a fused ring system consisting of no more than three
fused rings, where at least one of the ring carbon atoms is
replaced by oxygen, nitrogen or sulfur, and where at least one of
the aliphatic hydrogen atoms has been replaced by a halogen,
hydroxy, a thio, nitro, an amino, a ketone, an aldehyde, an ester,
an amide, a lower aliphatic, a substituted lower aliphatic, or a
ring (aryl, substituted aryl, cycloaliphatic, or substituted
cycloaliphatic). Examples of such groups include, but are not
limited to 2-chloropyranyl.
[0169] The foregoing heteroaryl and heterocyclic groups may be
C-attached or N-attached (where such is possible).
[0170] As used herein the term "sulfonyl", used alone or linked to
other terms such as alkylsulfonyl or arylsulfonyl, refers to the
divalent radicals --SO.sub.2--. In aspects of the invention, the
sulfonyl group may be attached to a substituted or unsubstituted
hydroxyl, alkyl group, ether group, alkenyl group, alkynyl group,
aryl group, cycloalkyl group, cycloalkenyl group, cycloalkynyl
group, heterocyclic group, carbohydrate, peptide, or peptide
derivative.
[0171] The term "sulfinyl", used alone or linked to other terms
such as alkylsulfinyl (i.e.--S(O)-alkyl) or arylsulfinyl, refers to
the divalent radicals --S(O)--.
[0172] The term "sulfonate" is art recognized and includes a group
represented by the formula:
##STR00006##
wherein R.sup.18 is an electron pair, hydrogen, alkyl, cycloalkyl,
aryl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, heterocyclic,
carbohydrate, peptide, or peptide derivative.
[0173] The term "sulfate", used alone or linked to other terms, is
art recognized and includes a group that can be represented by the
formula:
##STR00007##
wherein R.sup.19 is an electron pair, hydrogen, alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heterocyclic, carbohydrate, peptide or peptide derivative.
[0174] The term "sulfoxide" refers to the radical --S.dbd.O.
[0175] As used herein the term "amino", alone or in combination,
refers to a radical where a nitrogen atom (N) is bonded to three
substituents being any combination of hydrogen, hydroxyl, alkyl,
cycloalkyl, alkenyl, alkynyl, aryl, silyl, heterocyclic, or
heteroaryl which may or may not be substituted. Generally an "amino
group" has the general chemical formula --NR.sup.20R.sup.21 where
R.sup.20 and R.sup.21 can be any combination of hydrogen, hydroxyl,
alkyl, cycloalkyl, alkoxy, alkenyl, alkynyl, aryl, carbonyl
carboxyl, amino, silyl, heteroaryl, or heterocyclic which may or
may not be substituted. Optionally one substituent on the nitrogen
atom may be a hydroxyl group (--OH) to provide an amine known as a
hydroxylamine. Illustrative examples of amino groups are amino
(--NH.sub.2), alkylamino, acylamino, cycloamino, acycloalkylamino,
arylamino, arylalkylamino, and lower alkylsilylamino, in particular
methylamino, ethylamino, dimethylamino, 2-propylamino, butylamino,
isobutylamino, cyclopropylamino, benzylamino, allylamino,
hydroxylamino, cyclohexylamino, piperidinyl, hydrazinyl,
benzylamino, diphenylmethylamino, tritylamino, trimethylsilylamino,
and dimethyl-tert.-butylsilylamino, which may or may not be
substituted.
[0176] As used herein the term "thiol" means --SH. A thiol may be
substituted with a substituent disclosed herein, in particular
alkyl (thioalkyl), aryl (thioaryl), alkoxy (thioalkoxy) or
carboxyl.
[0177] The term "sulfenyl" used alone or linked to other terms such
as alkylsulfenyl, refers to the radical --SR.sup.22 wherein
R.sup.22 is not hydrogen. In aspects of the invention R.sup.22 is
substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl,
aryl, silyl, silylalkyl, heterocyclic, heteroaryl, carbonyl,
carbamoyl, alkoxy, or carboxyl.
[0178] As used herein, the term "thioalkyl", alone or in
combination, refers to a chemical functional group where a sulfur
atom (S) is bonded to an alkyl, which may be substituted. Examples
of thioalkyl groups are thiomethyl, thioethyl, and thiopropyl. A
thioalkyl may be substituted with a substituted or unsubstituted
carboxyl, aryl, heterocylic, carbonyl, or heterocyclic.
[0179] As used herein the term "thioaryl", alone or in combination,
refers to a chemical functional group where a sulfur atom (S) is
bonded to an aryl group with the general chemical formula
--SR.sup.23 where R.sup.23 is aryl which may be substituted.
Illustrative examples of thioaryl groups and substituted thioaryl
groups are thiophenyl, chlorothiophenyl, para-chlorothiophenyl,
thiobenzyl, 4-methoxy-thiophenyl, 4-nitro-thiophenyl, and
para-nitrothiobenzyl.
[0180] As used herein the term "thioalkoxy", alone or in
combination, refers to a chemical functional group where a sulfur
atom (S) is bonded to an alkoxy group with the general chemical
formula --SR.sup.24 where R.sup.24 is an alkoxy group which may be
substituted. A "thioalkoxy group" may have 1-6 carbon atoms i.e. a
--S--(O)--C.sub.1-C.sub.6 alkyl group wherein C.sub.1-C.sub.6 alkyl
have the meaning as defined above. Illustrative examples of a
straight or branched thioalkoxy group or radical having from 1 to 6
carbon atoms, also known as a C.sub.1-C.sub.6 thioalkoxy, include
thiomethoxy and thioethoxy.
[0181] A thiol may be substituted with a substituted or
unsubstituted heteroaryl or heterocyclic, in particular a
substituted or unsubstituted saturated 3 to 6-membered
heteromonocylic group containing 1 to 4 nitrogen atoms [e.g.
pyrrolidinyl, imidazolidinyl, piperidinyl, and piperazinyl] or a
saturated 3 to 6-membered heteromonocyclic group containing 1 to 2
oxygen atoms and 1 to 3 nitrogen atoms [e.g. morpholinyl;
sydnonyl], especially a substituted morpholinyl or piperidinyl.
[0182] As used herein, the term "carbonyl" refers to a carbon
radical having two of the four covalent bonds shared with an oxygen
atom.
[0183] As used herein, the term "carboxyl", alone or in
combination, refers to --C(O)OR.sup.25-- or --C(.dbd.O)OR.sup.25
wherein R.sup.25 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, amino, thiol, aryl, heteroaryl, thioalkyl, thioaryl,
thioalkoxy, a heteroaryl, or a heterocyclic, which may optionally
be substituted. In aspects of the invention, the carboxyl groups
are in an esterified form and may contain as an esterifying group
lower alkyl groups. In particular aspects of the invention,
--C(O)OR.sup.25 provides an ester or an amino acid derivative. An
esterified form is also particularly referred to herein as a
"carboxylic ester". In aspects of the invention a "carboxyl" may be
substituted, in particular substituted with alkyl which is
optionally substituted with one or more of amino, amine, halo,
alkylamino, aryl, carboxyl, or a heterocyclic. Examples of carboxyl
groups are methoxycarbonyl, butoxycarbonyl, tert.alkoxycarbonyl
such as tert.butoxycarbonyl, arylmethyoxycarbonyl having one or two
aryl radicals including without limitation phenyl optionally
substituted by for example lower alkyl, lower alkoxy, hydroxyl,
halo, and/or nitro, such as benzyloxycarbonyl,
methoxybenzyloxycarbonyl, diphenylmethoxycarbonyl,
2-bromoethoxycarbonyl, 2-iodoethoxycarbonyltert.butylcarbonyl,
4-nitrobenzyloxycarbonyl, diphenylmethoxy-carbonyl,
benzhydroxycarbonyl, di-(4-methoxyphenyl-methoxycarbonyl,
2-bromoethoxycarbonyl, 2-iodoethoxycarbonyl,
2-trimethylsilylethoxycarbonyl, or 2-triphenylsilylethoxycarbonyl.
Additional carboxyl groups in esterified form are silyloxycarbonyl
groups including organic silyloxycarbonyl. The silicon substituent
in such compounds may be substituted with lower alkyl (e.g.
methyl), alkoxy (e.g. methoxy), and/or halo (e.g. chlorine).
Examples of silicon substituents include trimethylsilyl and
dimethyltert.butylsilyl. In aspects of the invention, the carboxyl
group may be an alkoxy carbonyl, in particular methoxy carbonyl,
ethoxy carbonyl, isopropoxy carbonyl, t-butoxycarbonyl,
t-pentyloxycarbonyl, or heptyloxy carbonyl, especially methoxy
carbonyl or ethoxy carbonyl.
[0184] As used herein, the term "carbamoyl", alone or in
combination, refers to amino, monoalkylamino, dialkylamino,
monocycloalkylamino, alkylcycloalkylamino, and dicycloalkylamino
radicals, attached to one of two unshared bonds in a carbonyl
group.
[0185] As used herein, the term "carboxamide" refers to the group
--CONH--.
[0186] As used herein, the term "nitro" means --NO.sub.2--.
[0187] As used herein, the term "acyl", alone or in combination,
means a carbonyl or thiocarbonyl group bonded to a radical selected
from, for example, optionally substituted, hydrido, alkyl (e.g.
haloalkyl), alkenyl, alkynyl, alkoxy ("acyloxy" including
acetyloxy, butyryloxy, iso-valeryloxy, phenylacetyloxy, benzoyloxy,
p-methoxybenzoyloxy, and substituted acyloxy such as alkoxyalkyl
and haloalkoxy), aryl, halo, heterocyclyl, heteroaryl, sulfinyl
(e.g. alkylsulfinylalkyl), sulfonyl (e.g. alkylsulfonylalkyl),
cycloalkyl, cycloalkenyl, thioalkyl, thioaryl, amino (e.g
alkylamino or dialkylamino), and aralkoxy. Illustrative examples of
"acyl" radicals are formyl, acetyl, 2-chloroacetyl, 2-bromacetyl,
benzoyl, trifluoroacetyl, phthaloyl, malonyl, nicotinyl, and the
like.
[0188] In aspects of the invention, "acyl" refers to a group
--C(O)R.sup.26, where R.sup.26 is hydrogen, alkyl, cycloalkyl,
cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, and
heteroarylalkyl. Examples include, but are not limited to formyl,
acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl,
benzylcarbonyl and the like.
[0189] As used herein the term "phosphonate" refers to a
C--PO(OH).sub.2 or C--PO(OR.sup.27).sub.2 group wherein R.sup.27 is
alkyl or aryl which may be substituted.
[0190] As used herein, "ureido" refers to the group "--NHCONH--". A
ureido radical includes an alkylureido comprising a ureido
substituted with an alkyl, in particular a lower alkyl attached to
the terminal nitrogen of the ureido group. Examples of an
alkylureido include without limitation N'-methylureido,
N'-ethylureido, N'-n-propylureido, N'-i-propylureido and the like.
A ureido radical also includes a N',N'-dialkylureido group
containing a radical --NHCON where the terminal nitrogen is
attached to two optionally substituted radicals including alkyl,
aryl, heterocylic, and heteroaryl.
[0191] The terms used herein for radicals including "alkyl",
"alkoxy", "alkenyl", "alkynyl", "hydroxyl" etc. refer to both
unsubstituted and substituted radicals. The term "substituted," as
used herein, means that any one or more moiety on a designated atom
(e.g., hydrogen) is replaced with a selection from a group
disclosed herein, provided that the designated atom's normal
valency is not exceeded, and that the substitution results in a
stable compound. Combinations of substituents and/or radicals are
permissible only if such combinations result in stable compounds.
"Stable compound" refers to a compound that is sufficiently robust
to survive isolation to a useful degree of purity from a reaction
mixture, and formulation into an efficacious therapeutic agent.
[0192] A functional group or ring of a compound of the formula I,
II or III may be modified with, or a radical in a compound of the
formula I, II or III may be substituted with one or more groups or
substituents apparent to a person skilled in the art including
without limitation alkyl, alkoxy, alkenyl, alkynyl, alkanoyl,
alkylene, alkenylene, hydroxyalkyl, haloalkyl, haloalkylene,
haloalkenyl, alkoxy, alkenyloxy, alkenyloxyalkyl, alkoxyalkyl,
aryl, alkylaryl, haloalkoxy, haloalkenyloxy, heterocyclic,
heteroaryl, alkylsulfonyl, sulfinyl, sulfonyl, sulfenyl,
alkylsulfinyl, aralkyl, heteroaralkyl, cycloalkyl, cycloalkenyl,
cycloalkoxy, cycloalkenyloxy, amino, oxy, halo, azido, thio,
.dbd.O, .dbd.S, cyano, hydroxyl, phosphonato, phosphinato,
thioalkyl, alkylamino, arylamino, arylsulfonyl, alkylcarbonyl,
arylcarbonyl, heteroarylcarbonyl, heteroarylsulfinyl,
heteroarylsulfonyl, heteroarylamino, heteroaryloxy,
heteroaryloxylalkyl, arylacetamidoyl, aryloxy, aroyl, aralkanoyl,
aralkoxy, aryloxyalkyl, haloaryloxyalkyl, heteroaroyl,
heteroaralkanoyl, heteroaralkoxy, heteroaralkoxyalkyl, thioaryl,
arylthioalkyl, alkoxyalkyl, and acyl groups. These groups or
substitutents may themselves be substituted. Derivative groups that
may be used to modify compounds of the Formula I can also be found
in U.S. Patent Application No. 20030176437.
[0193] A chemical substituent is "pendant" from a radical if it is
bound to an atom of the radical. In this context, the substituent
can be pending from a carbon atom of a radical, a carbon atom
connected to a carbon atom of the radical by a chain extender, or a
heteroatom of the radical. The term "fused" means that a second
ring is present (i.e, attached or formed) by having two adjacent
atoms in common or shared with the first ring.
[0194] A "dosage form" refers to a composition or device comprising
a compound of the formula I, II or III and optionally
pharmaceutically acceptable carrier(s), excipient(s), or vehicles.
A dosage form may be an immediate release dosage form or a
sustained release dosage form.
[0195] An "immediate release dosage form" refers to a dosage form
which does not include a component for sustained release i.e., a
component for slowing disintegration or dissolution of an active
compound. These dosage forms generally rely on the composition of
the drug matrix to effect the rapid release of the active
ingredient agent.
[0196] By "sustained release dosage form" is meant a dosage form
that releases active compound for many hours. In an aspect, a
sustained dosage form includes a component for slowing
disintegration or dissolution of the active compound. A dosage form
may be a sustained release formulation, engineered with or without
an initial delay period. Sustained release dosage forms may
continuously release drug for sustained periods of at least about 4
hours or more, about 6 hours or more, about 8 hours or more, about
12 hours or more, about 15 hours or more, or about 20 hours to 24
hours. A sustained release dosage form can be formulated into a
variety of forms, including tablets, lozenges, gelcaps, buccal
patches, suspensions, solutions, gels, etc. In aspects of the
invention the sustained release form results in administration of a
minimum number of daily doses.
[0197] A "disease" that can be treated and/or prevented using a
compound, composition, or method of the invention includes a
condition associated with or requiring modulation of one or more of
inflammation (e.g. neuroinflammation); signaling pathways involved
in inflammation (e.g., neuroinflammation); cell signaling molecule
production; activation of glia or glial activation pathways and
responses; proinflammatory cytokines or chemokines (e.g.,
interleukin (IL), in particular IL-1.beta.) or tumor necrosis
factor (TNF, in particular TNF.alpha.); activation of astrocytes or
astrocyte activation pathways and responses; activation of
microglia or microglial activation pathways and responses;
oxidative stress-related responses such as nitric oxide synthase
production and nitric oxide accumulation; acute phase proteins;
loss of synaptophysin and/or 95; components of the complement
cascade; loss or reduction of synaptic function; protein kinase
activity (e.g., death associated protein kinase activity); cell
damage (e.g., neuronal cell damage); cell death (e.g., neuronal
cell death); amyloid .beta. deposition of amyloid plaques; and
behavioral deficits.
[0198] In particular a disease is a neurological disease or
condition including without limitation, dementing disorder, a
neurodegenerative disorder, a CNS demyelinating disorder, a pain
disorder, an autoimmune disorder, or a peripheral inflammatory
disease.
[0199] A disease may be characterized by an inflammatory process
due to the presence of macrophages activated by an amyloidogenic
protein or peptide. Thus, a method of the invention may involve
inhibiting macrophage activation and/or inhibiting an inflammatory
process. A method may comprise decreasing, slowing, ameliorating,
or reversing the course or degree of macrophage invasion or
inflammation in a patient.
[0200] Examples of diseases that can be treated and/or prevented
using the compounds, compositions and methods of the invention
include Alzheimer's disease and related disorders, presenile and
senile forms; amyloid angiopathy; mild cognitive impairment;
Alzheimer's disease-related dementia (e.g., vascular dementia or
Alzheimer dementia); AIDS related dementia, tauopathies (e.g.,
argyrophilic grain dementia, corticobasal degeneration, dementia
pugilistica, diffuse neurofibrillary tangles with calcification,
frontotemporal dementia with parkinsonism, Prion-related disease,
Hallervorden-Spatz disease, myotonic dystrophy, Niemann-Pick
disease type C, non-Guamanian Motor Neuron disease with
neurofibrillary tangles, Pick's disease, postencephalitic
parkinsonism, cerebral amyloid angiopathy, progressive subcortical
gliosis, progressive supranuclear palsy, subacute sclerosing
panencephalitis, and tangle only dementia), alpha-synucleinopathy
(e.g., dementia with Lewy bodies, multiple system atrophy with
glial cytoplasmic inclusions), multiple system atrophies,
Shy-Drager syndrome, spinocerebellar ataxia (e.g., DRPLA or
Machado-Joseph Disease); striatonigral degeneration,
olivopontocerebellar atrophy, neurodegeneration with brain iron
accumulation type I, olfactory dysfunction, and amyotrophic lateral
sclerosis); Parkinson's disease (e.g., familial or non-familial);
Amyotrophic Lateral Sclerosis; Spastic paraplegia (e.g., associated
with defective function of chaperones and/or triple A proteins);
Huntington's Disease, spinocerebellar ataxia, Freidrich's Ataxia;
cerebrovascular diseases including stroke, hypoxia, ischemia,
infarction, intracerebral hemorrhage; traumatic brain injury;
Down's syndrome; head trauma with post-traumatic accumulation of
amyloid beta peptide; Familial British Dementia; Familial Danish
Dementia; Presenile Dementia with Spastic Ataxia; Cerebral Amyloid
Angiopathy, British Type; Presenile Dementia With Spastic Ataxia
Cerebral Amyloid Angiopathy, Danish Type; Familial encephalopathy
with neuroserpin inclusion bodies (FENIB); Amyloid Polyneuropathy
(e.g., senile amyloid polyneuropathy or systemic Amyloidosis);
Inclusion Body myositis due to amyloid beta peptide; Familial and
Finnish Type Amyloidosis; Systemic amyloidosis associated with
multiple myeloma; Familial Mediterranean Fever; multiple sclerosis,
optic neuritis; Guillain-Barre Syndrome; chronic inflammatory
demyelinating polyneuropathy; chronic infections and inflammations;
acute disseminated encephalomyelitis (ADEM); autoimmune inner ear
disease (AIED); diabetes; myocardial ischemia and other
cardiovascular disorders; pancreatitis; gout; inflammatory bowel
disease; ulcerative colitis, Crohn's disease, rheumatoid arthritis,
osteoarthritis; artheriosclerosis, inflammatory aortic aneurysm;
asthma; adult respiratory distress syndrome; restenosis;
ischemia/reperfusion injury; glomerulonephritis; sacoidosis cancer;
restenosis; rheumatic fever; systemic lupus erythematosus; Reiter's
syndrome; psoriatic arthritis; ankylosing spondylitis;
coxarthritis; pelvic inflammatory disease; osteomyelitis; adhesive
capsulitis; oligoarthritis; periarthritis; polyarthritis;
psoriasis; Still's disease; synovitis; inflammatory dermatosis;
and, wound healing.
[0201] In aspects of the invention, the disease is Alzheimer's
disease, vascular dementia, dementia associated with Parkinson's
disease, visuospatial deficits, Williams syndrome, encephalitis,
meningitis, fetal alcohol syndrome, Korsakoffs syndrome, anoxic
brain injury, cardiopulmonary resuscitation injuries, diabetes,
Sjogren's syndrome, strokes, ocular diseases such as cataracts and
macular degeneration, sleep disorders, and cognitive impairments
caused by high cholesterol levels.
[0202] In aspects of the invention, a compound, composition, or
method disclosed herein may be utilized to prevent and/or treat a
disease involving neuroinflammation (i.e., neuroinflammatory
disease). Neuroinflammation is a characteristic feature of disease
pathology and progression in a diverse array of neurodegenerative
disorders that are increasing in their societal impact (for a
recent review, see, e.g., Prusiner, S. B. (2001) New Engl. J. Med.
344, 1516-1526). These neuroinflammation-related disorders include
Alzheimer's disease (AD), amyotrophic lateral sclerosis, autoimmune
disorders, priori diseases, stroke and traumatic brain injury.
Neuroinflammation is brought about by glial cell (e.g., astrocytes
and microglia) activation, which normally serves a beneficial role
as part of an organism's homeostatic response to injury or
developmental change. However, disregulation of this process
through chronic or excessive activation of glia contributes to the
disease process through the increased production of proinflammatory
cytokines and chemokines, oxidative stress-related enzymes, acute
phase proteins, and various components of the complement cascades.
(See, e.g., Akiyama et al., (2000) Neurobiol. Aging 21, 383-421).
The direct linkage of glial activation to pathology that is a
hallmark of disease underscores the importance of understanding the
signal transduction pathways that mediate these critical glial
cellular responses and the discovery of cell permeable ligands that
can modulate these disease relevant pathways.
[0203] In certain selected aspects of the invention, the disease is
a neurodegenerative disease or neurodegenerative disorder including
such diseases and impairments as Alzheimer's disease, dementia,
MCI, Huntington's disease, Parkinson's disease, amyotrophic lateral
sclerosis, and other similar diseases and disorders disclosed
herein.
[0204] For Alzheimer's disease (AD) in particular, the deposition
of .beta.-amyloid (A.beta.) and neurofibrillary tangles are
associated with glial activation, neuronal loss and cognitive
decline. On a molecular level, Alzheimer's disease is characterized
by; increased expression of nitric oxide synthase (NOS) in glial
cells surrounding amyloid plaques; neuropathological evidence of
peroxynitrite-mediated neuronal damage; and nitric oxide (NO)
overproduction involved in A.beta.-induced brain dysfunction. NOSH
(iNOS) is induced as part of the glial activation response and is
an oxidative stress-related enzyme that generates NO. When NO is
present in high levels along with superoxide, the highly reactive
NO-derived molecule peroxynitrite is generated, leading to neuronal
cell death. The pro-inflammatory cytokine IL-1.beta. is also
overexpressed in activated glia in AD brain and polymorphisms in
IL-1.beta. genes are associated with an increased risk of early
onset sporadic AD (See, e.g., Du et al., (2000) Neurology 55,
480-483). IL-1.beta. can also influence amyloid plaque development
and is involved in additional glial inflammatory and neuronal
dysfunction responses (See, e.g., Griffin, et al., (1998) Brain
Pathol. 8, 65-72; and Sheng, et al., (1996) Neurobiol. Aging 17,
761-766). Therefore, because glial activation and specific glial
products are associated with neurodegenerative disorders (e.g.,
Alzheimer's disease), the compounds and compositions disclosed
herein that are capable of modulating cell signaling pathways
(e.g., glial activation pathways) will have particular application
in the treatment and prevention of inflammatory disease.
[0205] In aspects of the invention, a compound, composition, or
method disclosed herein may be utilized to prevent and/or treat a
disease involving disregulation of protein kinase signaling.
Disregulation of protein kinase signaling often accompanies
disregulation of cell signaling pathways (e.g., glial cell
activation pathways). Protein kinases are a large family of
proteins that play a central role in regulating a number of
cellular functions including cell growth, differentiation and
death. There are thought to be more than 500 protein kinases and
130 protein phosphatases exerting tight control on protein
phosphorylation. Each protein kinase transfers the
.gamma.-phosphate of ATP to a specific residue(s) of a protein
substrate. Protein kinases can be further categorized as tyrosine,
serine/threonine or dual specific based on acceptor residue.
Examples of serine/threonine kinases include MAP kinase, MAPK
kinase (MEK), Akt/PKB, Jun kinase (INK), CDKs, protein kinase A
(PRA), protein kinase C(PKC), and calmodulin (CaM)-dependent
kinases (CaMKs). Disregulated protein kinase activity (e.g., hyper-
or hypo-active) leads to abnormal protein phosphorylation,
underlying a great number of diseases including diabetes,
rheumatoid arthritis, inflammation, hypertension, and proliferative
diseases such as cancer. Therefore, because aberrant kinase
activity is associated with inflammatory disease (e.g.,
neurodegenerative disorders like Alzheimer's disease), the
compounds and compositions that are disclosed herein that are
capable of modulating kinases involved in cell signaling pathways
will have particular application for treatment and prevention of
inflammatory disease.
[0206] Diseases that may also be treated and/or prevented according
to the invention include Demyelinating Diseases. "Demyelinating
Diseases" refers to diseases in which myelin is the primary target.
These diseases can be divided into two groups: Acquired Diseases
and Hereditary Metabolic Disorders. Acquired Demyelinating Diseases
include Multiple sclerosis (MS) including its alternating
relapsing/remitting phases. Hereditary Metabolic Disorders includes
the leukodystrophies such as metachromatic leukodystrophy, Refsum's
disease, adrenoleukodystrophy, Krabbe's disease, phenylketonuria,
Canavan disease, Pelizaeus-Merzbacher disease and Alexander's
disease.
[0207] Diseases that may also be treated and/or prevented according
to the invention include "Demyelinating Conditions". The term
refers to conditions that result in deficient myelination. Such
conditions include, but are not limited to, Spinal Cord Injury,
Traumatic Brain Injury and Stroke.
[0208] The term "Spinal Cord Injury (SCI)" refers to an injury to
the spinal cord which results in loss of function such as mobility
or feeling.
[0209] The term "Traumatic Brain Injury (TBI)" refers to an injury
which results in damage to the brain. A head injury may be a closed
head injury or penetrating head injury. A closed head injury may
occur when the head is hit by a blunt object causing the brain to
interact with the hard bony surface inside the skull. A closed head
injury may also occur without direct external trauma to the head if
the brain undergoes a rapid forward or backward movement, (e.g.
whiplash). A penetrating head injury may occur when a fast moving
object such as a bullet pierces the skull. A closed or penetrating
head injury may result in localized and widespread, or diffuse,
damage to the brain which may manifest as memory loss, emotional
disturbances, motor difficulties, including paralysis, damage to
the senses, and death. The term also includes secondary damage that
follows an injury including swelling and fluid buildup and the
accumulation of substances toxic to surrounding neurons such as the
neurotransmitter glutamate.
[0210] The term "Stroke" refers to a sudden loss of brain function
caused by the interruption of the flow of blood to the brain (an
ischemic stroke) or the rupture of blood vessels in the brain (a
hemorrhagic stroke). The interruption of the blood flow or the
rupture of blood vessels causes neurons in the affected area to
die. The term also includes stroke rehabilitation which refers to
the intervention resulting in the full or partial recovery of
functions that have been lost due to stroke.
[0211] A pain disorder may also be treated and/or prevented
according to the invention. A "pain disorder" refers to a disorder
or condition involving pain and includes without limitation acute
pain, persistent pain, chronic pain, inflammatory pain, neuropathic
pain, neurogenic pain, and chemokine-induced pain. In aspects of
the invention, a pain disorder includes without limitation pain
resulting from soft tissue and peripheral damage such as acute
trauma; complex regional pain syndrome also referred to as reflex
sympathetic dystrophy; postherpetic neuralgia, occipital neuralgia,
trigeminal neuralgia, segmental or intercostal neuralgia and other
neuralgias; pain associated with osteoarthritis and rheumatoid
arthritis; musculo-skeletal pain such as pain associated with
strains, sprains and trauma such as broken bones; spinal pain,
central nervous system pain such as pain due to spinal cord or
brain stem damage; lower back pain, sciatica, dental pain,
myofascial pain syndromes, episiotomy pain, gout pain, and pain
resulting from burns; deep and visceral pain, such as heart pain;
muscle pain, eye pain, inflammatory pain, orofacial pain, for
example, odontalgia; abdominal pain, and gynecological pain, for
example, dysmenorrhoea, labour pain and pain associated with
endometriosis; somatogenic pain; pain associated with nerve and
root damage, such as pain associated with peripheral nerve
disorders, for example, nerve entrapment, brachial plexus
avulsions, and peripheral neuropathies; pain associated with limb
amputation, tic douloureux, neuroma, or vasculitis; diabetic
neuropathy, chemotherapy-induced-neuropathy, acute herpetic and
postherpetic neuralgia; atypical facial pain, nerve root damage,
neuropathic lower back pain, HIV related neuropathic pain, cancer
related neuropathic pain, diabetes related neuropathic pain and
arachnoiditis, trigeminal neuralgia, occipital neuralgia, segmental
or intercostal neuralgia, HIV related neuralgias and AIDS related
neuralgias and other neuralgias; allodynia, hyperalgesia,
idiopathic pain, pain caused by chemotherapy; occipital neuralgia,
psychogenic pain, brachial plexus avulsion, pain associated with
restless legs syndrome; pain associated with gallstones; pain
caused by chronic alcoholism or hypothyroidism or uremia or vitamin
deficiencies; neuropathic and non-neuropathic pain associated with
carcinoma, often referred to as cancer pain, phantom limb pain,
functional abdominal pain, headache, including migraine with aura,
migraine without aura and other vascular headaches, acute or
chronic tension headache, sinus headache and cluster headache;
temperomandibular pain and maxillary sinus pain; pain resulting
from ankylosing spondylitis and gout; pain caused by increased
bladder contractions; pain associated with gastrointestinal (GI)
disorders, disorders caused by helicobacter pylori and diseases of
the GI tract such as gastritis, proctitis, gastroduodenal ulcers,
peptic ulcers, dyspepsia, disorders associated with the neuronal
control of viscera, ulcerative colitis, chronic pancreatitis,
Crohn's disease and emesis; post operative pain, scar pain, and
chronic non-neuropathic pain such as pain associated with HIV,
anthralgia and myalgia, vasculitis and fibromyalgia.
[0212] The term "Neuropathic pain" refers to pain initiated or
caused by a primary lesion or dysfunction in the nervous system.
For the purpose of this invention included under this heading or to
be treated as synonymous is "Neurogenic Pain" which is defined as
pain initiated or caused by a primary lesion, dysfunction or
transitory perturbation in the peripheral or central nervous
system. In aspects, the uses of the present invention include
central or peripheral neuropathic pain or neurogenic pain. In other
aspects, neuropathic pain includes the pain caused by either
mononeuropathy or polyneuropathy. Neuropathic pain also includes
Chemokine-Induced Pain.
[0213] "Peripheral neuropathic pain" refers to a pain initiated or
caused by a primary lesion or dysfunction in the peripheral nervous
system and "peripheral neurogenic pain" refers to a pain initiated
or caused by a primary lesion, dysfunction or transitory
perturbation in the peripheral nervous system. A peripheral
neuropathic pain can be allodynia (i.e., a pain due to a stimulus
which does not normally provoke pain); causalgia (i.e., a syndrome
of sustained burning pain, allodynia and hyperpathia after a
traumatic nerve lesion, often combined with vasomotor and sudomotor
dysfunction and later trophic changes); hyperalgesia (i.e., an
increased response to a stimulus which is normally painful);
hyperesthesia (i.e., increased sensitivity to stimulation,
excluding the senses); hyperpathia. (i.e., a painful syndrome
characterized by an abnormally painful reaction to a stimulus,
especially a repetitive stimulus, as well as an increased
threshold); neuritis (i.e., inflammation of a nerve or nerves); or
neuropathy (i.e., a disturbance of function or pathological change
in a nerve). [See IASP, Classification of chronic pain, 2nd
Edition, IASP Press (2002), for detailed definitions of these
categories of neuropathic pain and neurogenic pain).
[0214] Exemplary types of neuropathic pain include infective (e.g.,
post herpetic neuralgia and HIV neuropathy), metabolic (e.g.,
diabetic neuropathy and Fabry's disease), toxic (e.g., from lead or
chemotherapy), traumatic/stretch injury (e.g., post incisional,
trauma, phantom limb pain, and reflex sympathetic dystrophy/complex
regional pain syndrome/causalgia), and idiopathic (e.g., trigeminal
neuralgia/tic douloureux).
[0215] Particular examples of Neuropathic Pain include
post-herpetic neuralgia, painful diabetic neuropathy, phantom limb
pain, central post-stroke pain, HIV neuropathy, Fabry's disease,
peripheral neuropathy, trigeminal neuralgia, post incisional
neuropathic pain, phantom limb pain, reflex sympathetic dystrophy,
causalgia, anesthesia dolorosa, intercoastal neuralgia,
post-traumatic localized pain, atypical facial neuralgia pain after
tooth extraction and the like, complex regional pain syndrome,
neuropathic pain caused by trauma, lead, or chemotherapy, cancer
pain resistant to narcotic analgesics such as morphine.
[0216] Treatment of neuropathic pain may be defined as
administration of a therapeutic dose of a compound of the formula
I, II or III to reduce and preferably eliminate pain that results
from nerve injury. Treatment of nerve injury may be defined as
administration of a therapeutic dose of a compound of the formula
I, II or III to ameliorate injury and to increase the rate of
recovery. An increased rate of recovery is defined as a reduction
of indications of pain from peripheral nerve injury, such as
thermal hyperalgesia and mechanical allodynia, more quickly than
would be accomplished without pharmacological or other medical
intervention.
[0217] "Chemokine-Induced Pain" refers to pain that occurs in
response, in whole or in part, to chemokines, in particular
pro-inflammatory cytokines (e.g. fractalkine, CCL2, and CCL5). An
example of chemokine-induced pain is arthritic pain.
Compounds and Processes
[0218] The invention contemplates the use of isolated and pure, in
particular, substantially pure, compounds of the formula I wherein
R.sup.1, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.12, R.sup.13, and R.sup.14 are independently hydrogen,
hydroxyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkoxy,
alkenyloxy, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkoxy,
aryl, aryloxy, arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl,
acyloxy, amino, imino, azido, thiol, thioalkyl, thioalkoxy,
thioaryl, nitro, cyano, halo, sulfate, sulfenyl, sulfinyl,
sulfonyl, sulfonate, sulfoxide, silyl, silyloxy, silylalkyl,
silylthio, .dbd.O, .dbd.S, phosphonate, ureido, carboxyl, carbonyl,
carbamoyl, or carboxamide; and X is optionally substituted
pyrimidinyl or pyridazinyl, an isomer, a pharmaceutically
acceptable salt, or derivative thereof.
[0219] The invention also contemplates the use of isolated and
pure, in particular, substantially pure, compounds of the formula I
wherein R.sup.1, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.12, R.sup.13, and R.sup.14 are independently
hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6alkoxy, C.sub.2-C.sub.6
alkenyloxy, C.sub.3-C.sub.10 cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.3-C.sub.10cycloalkoxy,
C.sub.6-C.sub.10aryl, C.sub.6-C.sub.10aryloxy,
C.sub.6-C.sub.10aryl-C.sub.1-C.sub.3alkoxy, C.sub.6-C.sub.10aroyl,
C.sub.6-C.sub.10heteroaryl, C.sub.3-C.sub.10heterocyclic,
C.sub.1-C.sub.6acyl, C.sub.1-C.sub.6acyloxy, --NH.sub.2,
--NHR.sup.28, --NR.sup.28R.sup.28, .dbd.NR.sup.28,
--S(O).sub.2R.sup.28, --SH, --SO.sub.3H, nitro, cyano, halo,
haloalkyl, haloalkoxy, hydroxyalkyl, --CO.sub.2H,
--CO.sub.2R.sup.28, --NHC(O)R.sup.28, --C(O)NR.sup.28,
--C(O)NHR.sup.28, --C(O)NR.sup.28R.sup.29, --NHS(O).sub.2R.sup.X,
wherein R.sup.28 and R.sup.29 are independently selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.3-C.sub.10cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10 aryl C.sub.1-C.sub.3alkyl, C.sub.6-C.sub.10
heteroaryl and C.sub.3-C.sub.10heterocyclic, and X is pyrimidinyl
or pyridazinyl, an isomer, a pharmaceutically acceptable salt, or
derivative thereof.
[0220] The invention further contemplates the use of isolated and
pure, in particular, substantially pure, compounds of the formula
II wherein R.sup.1, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, and R.sup.14 are
independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl,
alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl,
cycloalkynyl, cycloalkoxy, aryl, aryloxy, arylalkoxy, aroyl,
heteroaryl, heterocyclic, acyl, acyloxy, sulfoxide, sulfate,
sulfonyl, sulfenyl, sulfinyl, sulfonate, amino, imino, azido,
thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano, halo, silyl,
silyloxy, silylalkyl, silylthio, .dbd.O, .dbd.S, phosphonate,
ureido, carboxyl, carbonyl, carbamoyl, or carboxamide; or an
isomer, a pharmaceutically acceptable salt, or derivative
thereof.
[0221] The invention still further contemplates the use of isolated
and pure, in particular, substantially pure, compounds of the
formula II wherein R.sup.1, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, and
R.sup.14 are independently selected from hydrogen, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl,
C.sub.1-C.sub.6alkoxy, C.sub.2-C.sub.6 alkenyloxy, C.sub.3-C.sub.10
cycloalkyl, C.sub.4-C.sub.10cycloalkenyl,
C.sub.3-C.sub.10cycloalkoxy, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10aryloxy,
C.sub.6-C.sub.10aryl-C.sub.1-C.sub.3alkoxy, C.sub.6-C.sub.10aroyl,
C.sub.6-C.sub.10heteroaryl, C.sub.3-C.sub.10heterocyclic,
C.sub.1-C.sub.6acyl, C.sub.1-C.sub.6acyloxy, --NH.sub.2,
--NHR.sup.28, --NR.sup.28R.sup.29, .dbd.NR.sup.28,
--S(O).sub.2R.sup.9, --SH, --SO.sub.3H, nitro, cyano, halo,
haloalkyl, haloalkoxy, hydroxyalkyl, --CO.sub.2H,
--CO.sub.2R.sup.28, --NHC(O)R.sup.28, --C(O)NH.sub.2,
--C(O)NHR.sup.X, --C(O)NR.sup.XR.sup.X, --NHS(O).sub.2R.sup.X,
wherein R.sup.28 and R.sup.29 are independently selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.3-C.sub.10cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10 aryl C.sub.1-C.sub.3alkyl, C.sub.6-C.sub.10
heteroaryl and C.sub.3-C.sub.10heterocyclic, or an isomer, a
pharmaceutically acceptable salt, or derivative thereof.
[0222] In aspects of the invention, R.sup.1 in a compound of the
formula I or II is alkyl, alkenyl, alkynyl, alkoxy, or cycloalkyl.
In certain aspects of the invention, R.sup.1 in a compound of the
formula I or II is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6alkoxy, or C.sub.3-C.sub.10
cycloalkyl. In embodiments, R.sup.1 is lower alkyl. In another
embodiment, R.sup.1 is cyclohexyl.
[0223] In aspects of the invention, R.sup.1 in a compound of the
formula I or II is aryl, in particular phenyl, benzyl, naphthyl,
indenyl, benzocyclooctenyl, benzocycloheptenyl, pentalenyl,
azulenyl, tetrahydronaphthyl, indanyl, biphenyl, acephthylenyl,
fluorenyl, phenalenyl, phenanthrenyl, and anthracenyl. In aspects
of the invention R.sup.1 is aryl substituted with one or more of
hydroxyl, alkyl, carbonyl, carboxyl, thiol, amino, nitro, ketone,
aldehyde, ester, amide, lower aliphatic, aryl, cycloalkyl, and
halo. In aspects of the invention R.sup.1 in a compound of the
formula I or II comprises two fused aromatic rings.
[0224] In aspects of the invention, R.sup.1 in a compound of the
formula I or II is aryloxy, in particular C.sub.6-C.sub.10aryloxy.
In embodiments of the invention, R.sup.1 in a compound of the
formula I or II is napthyloxy, quinolyloxy, isoquinolizinyloxy, and
the like.
[0225] In aspects of the invention, R.sup.1 in a compound of the
formula I or II is arylalkoxy, in particular
C.sub.6-C.sub.10aryloxy or
C.sub.6-C.sub.10aryl-C.sub.1-C.sub.3alkoxy. In embodiments, R.sup.1
in a compound of the formula I or II is 2-phenylethoxy,
3-naphth-2-ylpropoxy, and 5-phenylpentyloxy.
[0226] In aspects of the invention, R.sup.1 in a compound of the
formula I or II is aroyl, in particular C.sub.6-C.sub.10aroyl. In
embodiments of the invention R.sup.1 in a compound of the formula I
or II is benzoyl or toluoyl.
[0227] In aspects of the invention, R.sup.1 in a compound of the
formula I or II is a heteroaryl, in particular
C.sub.6-C.sub.10heteroaryl. In aspects, R.sup.1 in a compound of
the formula I or II comprises one or two rings attached in a
pendant manner or fused. In certain aspects of the invention,
R.sup.1 in a compound of the formula I or II is: (a) an unsaturated
5 to 6 membered heteromonocyclyl group containing 1 to 4 nitrogen
atoms, most particularly, pyrrolyl, pyrrolinyl, imidazolyl,
pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl,
pyrazinyl, pyridazinyl, triazolyl, tetrazolyl and the like; (b) an
unsaturated condensed heterocyclic group containing 1 to 5 nitrogen
atoms, in particular, indolyl, isoindolyl, indolizinyl, indazolyl,
quinazolinyl, pteridinyl, quinolizidinyl, phthalazinyl,
naphthyridinyl, quinoxalinyl, cinnolinyl, phenanthridinyl,
acridinyl, phenanthrolinyl, phenazinyl, carbazolyl, purinyl,
benzimidazolyl, quinolyl, isoquinolyl, quinolinyl, isoquinolinyl,
indazolyl, benzotriazolyl, tetrazolopyridazinyl and the like; (c)
an unsaturated 3 to 6-membered heteromonocyclic group containing an
oxygen atom, in particular, 2-furyl, 3-furyl, pyranyl, and the
like; (d) an unsaturated 5 to 6-membered heteromonocyclic group
containing a sulfur atom, in particular, thienyl, 2-thienyl,
3-thienyl, and the like; (e) an unsaturated 5 to 6-membered
heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3
nitrogen atoms, in particular, furazanyl, benzofurazanyl, oxazolyl,
isoxazolyl, and oxadiazolyl; (f) an unsaturated condensed
heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3
nitrogen atoms, in particular benzoxazolyl, benzoxadiazolyl and the
like; (g) an unsaturated 5 to 6-membered heteromonocyclic group
containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, for
example, thiazolyl, isothiazolyl, thiadiazolyl and the like; or (h)
an unsaturated condensed heterocyclic group containing 1 to 2
sulfur atoms and 1 to 3 nitrogen atoms such as benzothiazolyl,
benzothiadiazolyl and the like.
[0228] In certain aspects of the invention, R.sup.1 in a compound
of the formula I or II is a heterocyclic fused with an aryl, in
particular benzofuranyl, benzothiophenyl, phthalazinyl, chromenyl,
xanthenyl, and the like.
[0229] In particular aspects of the invention R.sup.1 in a compound
of the formula I or II is:
##STR00008##
wherein R.sup.15, R.sup.16 and R.sup.17 are independently hydrogen,
hydroxyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkoxy,
alkenyloxy, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkoxy,
aryl, aryloxy, arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl,
acyloxy, amino, imino, azido, thiol, thioalkyl, thioalkoxy,
thioaryl, nitro, cyano, halo, sulfoxide, sulfate, sulfonyl,
sulfenyl, sulfinyl, sulfonate, silyl, silyloxy, silylalkyl,
silylthio, .dbd.O, .dbd.S, phosphonate, ureido, carboxyl, carbonyl,
carbamoyl, or carboxamide.
[0230] In embodiments of the invention, R.sup.15, R.sup.16 and
R.sup.17 are independently hydrogen, hydroxyl, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl,
C.sub.1-C.sub.6alkoxy, C.sub.2-C.sub.6 alkenyloxy, C.sub.3-C.sub.10
cycloalkyl, C.sub.4-C.sub.10cycloalkenyl,
C.sub.3-C.sub.10cycloalkoxy, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10aryloxy,
C.sub.6-C.sub.10aryl-C.sub.1-C.sub.3alkoxy, C.sub.6-C.sub.10aroyl,
C.sub.6-C.sub.10heteroaryl, C.sub.3-C.sub.10heterocyclic,
C.sub.1-C.sub.6acyl, C.sub.1-C.sub.6acyloxy, --NH.sub.2,
--NHR.sup.28, --NR.sup.28R.sup.29, .dbd.NR.sup.28, .dbd.O, .dbd.S,
nitro, cyano, halo, haloalkyl, haloalkoxy, hydroxyalkyl,
--CO.sub.2H, --CO.sub.2R.sup.28, --NHC(O)R.sup.28, --C(O)NH.sub.2,
--C(O)NHR.sup.28, --C(O)NR.sup.28R.sup.29, --NHS(O).sub.2R.sup.28,
wherein R.sup.28 and R.sup.29 are independently selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.3-C.sub.10cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10 aryl C.sub.1-C.sub.3alkyl, C.sub.6-C.sub.10
heteroaryl and C.sub.3-C.sub.10heterocyclic.
[0231] In other particular aspects of the invention a compound of
the formula III is employed.
##STR00009##
wherein R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15,
R.sup.16 and R.sup.17 are independently hydrogen, hydroxyl, alkyl,
alkenyl, alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy,
cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkoxy, aryl, aryloxy,
arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl, acyloxy, amino,
imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano,
halo, sulfoxide, sulfate, sulfonyl, sulfenyl, sulfinyl, sulfonate,
silyl, silyloxy, silylalkyl, silylthio, .dbd.O, .dbd.S,
phosphonate, ureido, carboxyl, carbonyl, carbamoyl, or
carboxamide.
[0232] In aspects of the invention R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13,
R.sup.14, R.sup.15, R.sup.16, and R.sup.17 are independently
selected from hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, Cl C.sub.6alkoxy, C.sub.2-C.sub.6
alkenyloxy, C.sub.3-C.sub.10 cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.3-C.sub.10cycloalkoxy,
C.sub.6-C.sub.10aryl, C.sub.6-C.sub.10aryloxy,
C.sub.6-C.sub.10aryl-C.sub.1-C.sub.3alkoxy, C.sub.6-C.sub.10aroyl,
C.sub.6-C.sub.10heteroaryl, C.sub.3C.sub.10heterocyclic,
C.sub.1-C.sub.6acyl, C.sub.1-C.sub.6acyloxy, --NH.sub.2,
--NHR.sup.28, --NR.sup.28R.sup.29, .dbd.NR.sup.28,
--S(O).sub.2R.sup.28, --SH, --SO.sub.3H, nitro, cyano, halo,
haloalkyl, haloalkoxy, hydroxyalkyl, --CO.sub.2H,
--CO.sub.2R.sup.28, --NHC(O)R.sup.28, --C(O)NH.sub.2,
--C(O)NHR.sup.28, --C(O)NR.sup.28R.sup.29, --NHS(O).sub.2R.sup.28,
wherein R.sup.28 and R.sup.29 are independently selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.3-C.sub.10cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10 aryl C.sub.1-C.sub.3alkyl, C.sub.6-C.sub.10
heteroaryl and C.sub.3-C.sub.10heterocyclic.
[0233] In general, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14,
R.sup.15, R.sup.16, and R.sup.17 in a compound of the formula III
cannot all be hydrogen. In aspects of the invention a compound of
the formula III is provided wherein both of R.sup.10 and R.sup.11
are not hydrogen. In other aspects of the invention a compound of
the formula II is provided wherein R.sup.11 is not hydrogen.
[0234] In further aspects of the invention, pure, in particular,
substantially pure, compounds of the formula III are employed
wherein R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, and
R.sup.17 are independently hydrogen, hydroxyl, alkyl, alkenyl,
alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl,
cycloalkenyl, aryl, aryloxy, arylalkoxy, aroyl, heteroaryl,
heterocyclic, acyl, acyloxy, amino, imino, azido, thiol, thioalkyl,
thioalkoxy, thioaryl, nitro, cyano, halo, silyl, silyloxy,
silylalkyl, silylthio, .dbd.O, .dbd.S, carboxyl, carbonyl,
carbamoyl, or carboxamide, and R.sup.11 is alkyl, alkenyl, alkynyl,
alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl,
aryl, aryloxy, arylalkoxy, aroyl, heteroaryl, acyl, acyloxy, amino,
imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano,
halo, silyl, silyloxy, silylalkyl, silylthio, .dbd.O, .dbd.S,
carboxyl, carbonyl, carbamoyl, or carboxamide; or an isomer, a
pharmaceutically acceptable salt, or derivative thereof. In aspects
of the invention R.sup.11 is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6alkoxy,
C.sub.2-C.sub.6 alkenyloxy, C.sub.3-C.sub.10 cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.3-C.sub.10cycloalkoxy,
C.sub.6-C.sub.10aryl, C.sub.6-C.sub.10aryloxy,
C.sub.6-C.sub.10aryl-C.sub.1-C.sub.3alkoxy, C.sub.6-C.sub.10aroyl,
C.sub.6-C.sub.10heteroaryl, C.sub.3-C.sub.10heterocyclic,
C.sub.1-C.sub.6acyl, C.sub.1-C.sub.6acyloxy, --NH.sub.2,
--NHR.sup.28--NR.sup.28R.sup.29, .dbd.NR.sup.28,
--S(O).sub.2R.sup.28, --SH, --SO.sub.3H, nitro, cyano, halo,
haloalkyl, haloalkoxy, hydroxyalkyl, --CO.sub.2H,
--CO.sub.2R.sup.28, --NHC(O)R.sup.28, --C(O)NH.sub.2,
--C(O)NHR.sup.28, --C(O)NR.sup.28R.sup.29, --NHS(O).sub.2R.sup.28,
wherein R.sup.28 and R.sup.29 are independently selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.3-C.sub.10cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10 aryl C.sub.1-C.sub.3alkyl, C.sub.6-C.sub.10
heteroaryl and C.sub.3-C.sub.10heterocyclic.
[0235] In certain aspects a compound of the formula III is employed
wherein R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, and R.sup.17 are
hydrogen, hydroxyl, alkyl, and one or both of R.sup.10 and R.sup.11
are independently substituted or unsubstituted hydrogen, hydroxyl,
alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy,
cycloalkyl, cycloalkenyl, aryl, aryloxy, arylalkoxy, aroyl,
heteroaryl, heterocyclic, acyl, acyloxy, sulfonyl, sulfinyl,
sulfenyl, amino, imino, azido, thiol, thioalkyl, thioalkoxy,
thioaryl, nitro, ureido, cyano, halo, silyl, silylalkyl, silyloxy,
silylthio, .dbd.O, .dbd.S, carboxyl, carbonyl, or carbamoyl, or an
isomer or a pharmaceutically acceptable salt thereof. In aspects of
the invention one or both of R.sup.10 and R.sup.11 are
independently C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6alkoxy, C.sub.2-C.sub.6
alkenyloxy, C.sub.3-C.sub.10 cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.3-C.sub.10cycloalkoxy,
C.sub.6-C.sub.10aryl, C.sub.6-C.sub.10aryloxy,
C.sub.6-C.sub.10aryl-C.sub.1-C.sub.3alkoxy, C.sub.6-C.sub.10aroyl,
C.sub.6-C.sub.10heteroaryl, C.sub.3-C.sub.10heterocyclic,
C.sub.1-C.sub.6acyl, C.sub.1-C.sub.6acyloxy, --NH.sub.2,
--NHR.sup.28, --NR.sup.28R.sup.29, .dbd.NR.sup.28,
--S(O).sub.2R.sup.28, --SH, --SO.sub.3H, nitro, cyano, halo,
haloalkyl, haloalkoxy, hydroxyalkyl, --CO.sub.2H,
--CO.sub.2R.sup.28, --NHC(O)R.sup.28, --C(O)NH.sub.2,
--C(O)NHR.sup.28, --C NR.sup.28R.sup.29, --NHS(O).sub.2R.sup.28,
wherein R.sup.28 and R.sup.29 are independently selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.3-C.sub.10cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10aryl C.sub.1-C.sub.3alkyl, C.sub.6-C.sub.10
heteroaryl and C.sub.3-C.sub.10heterocyclic.
[0236] In certain aspects a compound of the formula III is employed
wherein R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, and R.sup.17 are
hydrogen; and R.sup.10 is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6alkoxy,
C.sub.2-C.sub.6 alkenyloxy, C.sub.3-C.sub.10 cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.3-C.sub.10cycloalkoxy,
C.sub.6-C.sub.10aryl, C.sub.6-C.sub.10aryloxy,
C.sub.6-C.sub.10aryl-C.sub.1-C.sub.3alkoxy, C.sub.6-C.sub.10aroyl,
C.sub.6-C.sub.10heteroaryl, C.sub.3-C.sub.10heterocyclic,
C.sub.1-C.sub.6acyl, C.sub.1-C.sub.6acyloxy, --NH.sub.2,
--NHR.sup.28, --NR.sup.28R.sup.29,
.dbd.NR.sup.28--S(O).sub.2R.sup.28, --SH, --SO.sub.3H, nitro,
cyano, halo, haloalkyl, haloalkoxy, hydroxyalkyl, --CO.sub.2H,
--CO.sub.2R.sup.28, --NHC(O)R.sup.28, --C(O)NH.sub.2,
--C(O)NHR.sup.28, --C(O)NR.sup.28R.sup.29, --NHS(O).sub.2R.sup.28
wherein R.sup.28 and R.sup.29 are independently selected from
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.3-C.sub.10cycloalkyl,
C.sub.4-C.sub.10cycloalkenyl, C.sub.6-C.sub.10aryl,
C.sub.6-C.sub.10 aryl C.sub.1-C.sub.3alkyl, C.sub.6-C.sub.10
heteroaryl and C.sub.3-C.sub.10heterocyclic.
[0237] In certain aspects a compound of the formula III is employed
wherein R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, and R.sup.17 are
hydrogen; R.sup.10 is hydrogen, hydroxyl, alkyl (e.g.,
C.sub.1-C.sub.6 alkyl), aryl [e.g., C.sub.6-C.sub.10aryl, in
particular, phenyl which is optionally substituted (e.g., with
halide)], C.sub.3-C.sub.10heterocyclic (e.g., piperazinyl which may
be substituted, for example substituted with a pyrimidinyl; or
morpholinyl which may be substituted), --NR.sup.3OR.sup.31 wherein
R.sup.30 is hydrogen or alkyl, and R.sup.31 is phenyl which may be
substituted or alkyl (e.g., C.sub.1-C.sub.6 alkyl) which may be
substituted [e.g. with amino, in particular
--CH.sub.2CH.sub.2NH.sub.2;
CH.sub.2CH.sub.2NHCOOC(CH.sub.3).sub.3], or --SR.sup.32 wherein
R.sup.32 is phenyl which may be substituted; and R.sup.11 is
hydrogen, alkyl, or aryl (e.g., C6-C.sub.10aryl, in particular,
e.g. phenyl) which may be substituted.
[0238] In aspects of the invention R.sup.11 is alkyl, halo, aryl,
substituted aryl (e.g. alkylaryl), or an unsaturated 5 to 6
membered heteromonocyclyl group containing 1 to 4 nitrogen atoms In
an embodiment R.sup.11 is lower alkyl (e.g., C.sub.1-C.sub.6 alkyl)
or a branched alkyl. In another embodiment, R.sup.11 is
C.sub.6-C.sub.10aryl, in particular phenyl. In another embodiment,
R.sup.11 is halo. In a still further embodiment, R.sup.11 is an
unsaturated 5 to 6 membered heteromonocyclyl group containing 1 to
4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl,
pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl,
pyrazinyl, pyridazinyl, triazolyl, tetrazolyl and the like. In a
particular embodiment, R.sup.11 is pyridinyl.
[0239] In certain aspects of the invention a compound of the
formula III is employed wherein R.sup.10 is hydrogen, halo,
optionally substituted hydroxyl, alkyl, pyridinyl, phenyl, benzyl,
piperazinyl, amino, morpholinyl, or --SR.sup.33 wherein R.sup.33 is
alkyl or aryl. In an embodiment, R.sup.10 is
--NH[CH.sub.2].sub.mNR.sup.34R.sup.35 wherein m is 1 to 6, in
particular 2 to 4, R.sup.34 is hydrogen, R.sup.35 is a carboxyl, in
particular --COOC(CH.sub.3).sub.3.
[0240] In particular embodiments of the invention, one of R.sup.10
and R.sup.11 in a compound of the formula III is a heteroaryl in
particular an unsaturated 5 to 6 membered heteromonocyclyl group
containing 1 to 4 nitrogen atoms, more particularly pyridinyl, and
the other of R.sup.10 and R.sup.11 is hydrogen.
[0241] In an aspect of the invention a compound of the formula III
is employed wherein R.sup.11 is hydrogen, halo, optionally
substituted alkyl, pyridinyl, piperidinyl, morpholinyl,
piperazinyl, or phenyl.
[0242] In aspects of the invention, a compound of the formula III
is used wherein R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.12, R.sup.13, R.sup.14, R.sup.15,
R.sup.16, and R.sup.17 are hydrogen, alkyl, alkoxy, sulfonyl,
sulfinyl, halo, thiol, or carboxyl, and R.sup.11 is alkyl, alkenyl,
alkoxy, alkenyloxy, aryl, heteroaryl, acyl, acyloxy, amino, imino,
azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano, halo,
silyl, .dbd.O, .dbd.S, carboxyl, carbonyl, carbamoyl, or
carboxamide; or an isomer or a pharmaceutically acceptable salt
thereof. In particular aspects, R.sup.11 is C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6alkoxy, C.sub.2-C.sub.6
alkenyloxy, C.sub.6-C.sub.10aryl, C.sub.6-C.sub.10heteroaryl,
C.sub.1-C.sub.6acyl, C.sub.1-C.sub.6acyloxy, --NH.sub.2,
--NHR.sup.28, --NR.sup.28R.sup.29, .dbd.NR.sup.28,
--S(O).sub.2R.sup.28, --SH, --SO.sub.3H, nitro, cyano, halo,
haloalkyl, haloalkoxy, --CO.sub.2H, --CO.sub.2R.sup.28,
--NHC(O)R.sup.28, --C(O)NH.sub.2, --C(O)NHR.sup.28,
--C(O)NR.sup.28R.sup.29, --NHS(O).sub.2R.sup.28, wherein R.sup.28
and R.sup.29 are independently selected from C.sub.1-C.sub.6alkyl,
C.sub.2-C.sub.6alkenyl, C.sub.2-C.sub.6alkynyl,
C.sub.3-C.sub.10cycloalkyl, C.sub.4-C.sub.10cycloalkenyl,
C.sub.6-C.sub.10aryl, C.sub.6-C.sub.10 aryl C.sub.1-C.sub.3alkyl,
C.sub.6-C.sub.10 heteroaryl and C.sub.3-C.sub.10heterocyclic.
[0243] In aspects of the invention, a compound of the formula III
is employed wherein R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.12, R.sup.13, R.sup.14, R.sup.15,
R.sup.16, and R.sup.17 are hydrogen, and R.sup.11 is alkyl,
alkenyl, alkynyl, alkylene, alkoxy, aryl, or an unsaturated 5 to 6
membered heteromonocyclyl group containing 1 to 4 nitrogen atoms.
In particular aspects, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.12, R.sup.13, R.sup.14, R.sup.15,
R.sup.16, and R.sup.17 are hydrogen and R.sup.11 is alkyl or
pyridinyl, more particularly R.sup.11 is alkyl.
[0244] In other aspects of the invention, one of R.sup.10 and
R.sup.11 in a compound of the formula III is alkyl, in particular
C.sub.1-C.sub.6 alkyl and the other of R.sup.10 and R.sup.11 is
hydrogen.
[0245] In particular embodiments of the invention, one of R.sup.10
and R.sup.11 in a compound of the formula III is aryl in particular
C.sub.6-C.sub.10aryl, more particularly phenyl or benzyl, and the
other of R.sup.10 and R.sup.11 is hydrogen.
[0246] In embodiments of the invention, the compound of the formula
II is a compound in Table 1 or 2.
[0247] In particular embodiments of the invention, the compound of
the formula III is MW01-6-189WH, MW01-5-188WH, or MW01-2-151SRM,
and/or a salt or derivatives thereof.
[0248] In more particular embodiments, the compound of the formula
II is
4-methyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-2-151SRM), and/or a salt or derivative thereof.
[0249] In more particular embodiments, the compound of the formula
II is 4,6-diphenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-5-188WH), and/or a salt or derivative thereof.
[0250] A compound of the formula I, II or III may be in the form of
a prodrug that is converted in vivo to an active compound. For
example, in a compound of the formula II one or more of R.sup.10
and R.sup.11 may comprise a cleavable group that is cleaved after
administration to a subject to provide an active (e.g.,
therapeutically active) compound, or an intermediate compound that
subsequently yields the active compound. A cleavable group can be
an ester that is removed either enzymatically or
non-enzymatically.
[0251] A compound of the formula I, II or III may comprise a
carrier, such as one or more of a polymer, carbohydrate, peptide or
derivative thereof, which may be directly or indirectly covalently
attached to the compound. A carrier may be substituted with
substituents described herein including without limitation one or
more alkyl, amino, nitro, halogen, thiol, thioalkyl, sulfate,
sulfonyl, sulfinyl, sulfoxide, hydroxyl groups. In aspects of the
invention the carrier is an amino acid including alanine, glycine,
praline, methionine, serine, threonine, asparagine, alanyl-alanyl,
prolyl-methionyl, or glycyl-glycyl. A carrier can also include a
molecule that targets a compound of the formula I, II or III to a
particular tissue or organ. Thus, a carrier may facilitate or
enhance transport of a compound of the formula I, II or III to the
brain.
[0252] Compounds of the formula I, II or III can be prepared using
reactions and methods generally known to the person of ordinary
skill in the art, having regard to that knowledge and the
disclosure of this application including the Examples. The
reactions are performed in a solvent appropriate to the reagents
and materials used and suitable for the reactions being effected.
It will be understood by those skilled in the art of organic
synthesis that the functionality present on the compounds should be
consistent with the proposed reaction steps. This will sometimes
require modification of the order of the synthetic steps or
selection of one particular process scheme over another in order to
obtain a desired compound of the invention. It will also be
recognized that another major consideration in the development of a
synthetic route is the selection of the protecting group used for
protection of the reactive functional groups present in the
compounds described in this invention. An authoritative account
describing the many alternatives to the skilled artisan is Greene
and Wuts (Protective Groups In Organic Synthesis, Wiley and Sons,
1991).
[0253] The starting materials and reagents used in preparing
compounds or the invention are either available from commercial
suppliers or are prepared by methods well known to a person of
ordinary skill in the art, following procedures described in such
references as Fieser and Fieser's Reagents for Organic Synthesis,
vols. 1-17, John Wiley and Sons, New York, N.Y., 1991; Rodd's
Chemistry of Carbon Compounds, vols. 1-5 and supps., Elsevier
Science Publishers, 1989; Organic Reactions, vols. 1-40, John Wiley
and Sons, New York, N.Y., 1991; March J.: Advanced Organic
Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.; and
Larock: Comprehensive Organic Transformations, VCH Publishers, New
York, 1989.
[0254] The starting materials, intermediates, and compounds of the
formula I, II or III may be isolated and purified using
conventional techniques, such as precipitation, filtration,
distillation, crystallization, chromatography, and the like. The
compounds of the formula I, II or III may be characterized using
conventional methods, including physical constants and
spectroscopic methods, in particular HPLC.
[0255] The compounds of the formula I, II or III which are basic in
nature can form a wide variety of different salts with various
inorganic and organic acids. In practice is it desirable to first
isolate a compound of the formula I, II or III from the reaction
mixture as a pharmaceutically unacceptable salt and then convert
the latter to the free base compound by treatment with an alkaline
reagent and subsequently convert the free base to a
pharmaceutically acceptable acid addition salt. The acid addition
salts of the base compounds of the formula I, II or III are readily
prepared by treating the base compound with a substantially
equivalent amount of the chosen mineral or inorganic or organic
acid in an aqueous solvent medium or in a suitable organic solvent
such as methanol or ethanol. Upon careful evaporation of the
solvent, the desired solid salt is obtained.
[0256] Compounds of the formula I, II or III which are acidic in
nature are capable of forming base salts with various
pharmacologically acceptable cations. These salts may be prepared
by conventional techniques by treating the corresponding acidic
compounds with an aqueous solution containing the desired
pharmacologically acceptable cations and then evaporating the
resulting solution to dryness, preferably under reduced pressure.
Alternatively, they may be prepared by mixing lower alkanolic
solutions of the acidic compounds and the desired alkali metal
alkoxide together and then evaporating the resulting solution to
dryness in the same manner as before. In either case,
stoichiometric quantities of reagents are typically employed to
ensure completeness of reaction and maximum product yields.
[0257] In particular aspects, the present invention provides
methods of making the compounds disclosed herein, comprising the
steps provided (See, for example, the Figures and Examples).
[0258] In an aspect, the invention provides a process for preparing
a compound of the formula III wherein R.sup.11 is hydrogen and
R.sup.10 is an unsaturated 5 to 6 membered heteromonocyclyl group
containing 1 to 4 nitrogen atoms, in particular, pyrrolyl,
pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, or
tetrazolyl, more particularly pyridinyl, which comprises reacting a
compound of the formula III wherein R.sup.10 is halo, in particular
chloro, and R.sup.11 is hydrogen with boronic acid substituted with
an unsaturated 5 to 6 membered heteromonocyclyl group containing 1
to 4 nitrogen atoms, in particular, pyrrolyl, pyrrolinyl,
imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridinyl,
pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, or tetrazolyl, more
particularly pyridinyl, under suitable conditions to prepare a
compound of the formula III wherein R.sup.11 is hydrogen and
R.sup.10 is an unsaturated 5 to 6 membered heteromonocyclyl group
containing 1 to 4 nitrogen atoms, in particular, pyrrolyl,
pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, or
tetrazolyl, more particularly pyridinyl.
[0259] In another aspect, the invention provides a process for
preparing a compound of the formula III wherein R.sup.11 is
hydrogen and R.sup.10 is a substituted aryl which comprises
reacting a compound of the formula III wherein R.sup.10 is halo, in
particular chloro, and R.sup.11 is hydrogen, with a substituted
aryl boronic acid under suitable conditions to prepare a compound
of the formula III wherein R.sup.11 is hydrogen and R.sup.10 is a
substituted aryl.
[0260] In another aspect, the invention provides a process for
preparing a compound of the formula III wherein R.sup.10 is
hydrogen and R.sup.11 is alkyl which comprises reacting a compound
of the formula III wherein R.sup.11 is halo, in particular chloro,
and R.sup.10 is hydrogen, with an alkyl boronic acid under suitable
conditions to prepare a compound of the formula III wherein
R.sup.10 is hydrogen and R.sup.11 is alkyl. In an embodiment,
R.sup.11 is lower alkyl, in particular methyl or ethyl, and a
compound of the formula III wherein R.sup.11 is chloro is reacted
with lower alkyl boronic acid, in particular methyl or ethyl
boronic acid under suitable conditions.
[0261] In another aspect, the invention provides a process for
preparing a compound of the formula III wherein R.sup.10 is
hydrogen and R.sup.11 is aryl which comprises reacting a compound
of the formula III wherein R.sup.10 is hydrogen and R.sup.11 is
halo (e.g., chloro), with pyridazine substituted at the C3 position
with halo (e.g., chloro), at the C4 position with aryl, and at the
6 position with phenyl, with 2-(piperidin-4-yloxy)pyrimidine under
suitable conditions to prepare a compound of the formula III
wherein R.sup.10 is hydrogen and R.sup.11 is aryl. In an
embodiment, R.sup.11 is phenyl.
[0262] In another aspect, the invention provides a process for
preparing a compound of the formula III wherein R.sup.10 is
hydrogen and R.sup.11 is an unsaturated 5 to 6 membered
heteromonocyclyl group containing 1 to 4 nitrogen atoms, in
particular, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl,
3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazolyl, or tetrazolyl, more particularly pyridinyl
which comprises reacting a compound of the formula III wherein
R.sup.11 is halo, in particular chloro, and R.sup.10 is hydrogen,
with a boronic acid substituted with an unsaturated 5 to 6 membered
heteromonocyclyl group containing 1 to 4 nitrogen atoms, in
particular, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl,
3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazolyl, or tetrazolyl, more particularly pyridinyl,
under suitable conditions to prepare a compound of the formula III
wherein R.sup.10 is hydrogen and R.sup.11 is an unsaturated 5 to 6
membered heteromonocyclyl group containing 1 to 4 nitrogen atoms,
in particular, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazolyl, or tetrazolyl, more particularly
pyridinyl.
[0263] In an embodiment, the invention provides a process for
preparing a compound of the formula III wherein R.sup.10 is
hydrogen and R.sup.11 is pyridinyl which comprises reacting a
compound of the formula III wherein R.sup.11 is halo, in particular
chloro, and R.sup.10 is hydrogen, with a pyridinyl boronic acid
under suitable conditions to prepare a compound of the formula III
wherein R.sup.10 is hydrogen and R.sup.11 is pyridinyl.
[0264] In another aspect, the invention provides a process for
preparing a compound of the formula III wherein R.sup.10 is
hydrogen and R.sup.11 is an unsaturated 5 to 6 membered
heteromonocyclyl group containing 1 to 4 nitrogen atoms, in
particular, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl,
3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazolyl, or tetrazolyl, more particularly pyridinyl
which comprises reacting a pyridazine substituted at the C3
position with halo, at the C4 position with an unsaturated 5 to 6
membered heteromonocyclyl group containing 1 to 4 nitrogen atoms,
in particular, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazolyl, or tetrazolyl, more particularly pyridinyl,
and at the 6 position with phenyl, with
2-(piperidin-4-yloxy)pyrimidine under suitable conditions to
prepare a compound of the formula III wherein R.sup.10 is hydrogen
and R.sup.11 is an unsaturated 5 to 6 membered heteromonocyclyl
group containing 1 to 4 nitrogen atoms, in particular, pyrrolyl,
pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, or
tetrazolyl, more particularly pyridinyl.
[0265] In an embodiment, the invention provides a process for
preparing a compound of the formula III wherein R.sup.10 is
hydrogen and R.sup.11 is pyridinyl which comprises reacting a
pyridazine substituted at the C3 position with halo, at the C4
position with pyridinyl, and at the 6 position with phenyl, with
2-(piperidin-4-yloxy)pyrimidine under suitable conditions to
prepare a compound of the formula III wherein R.sup.10 is hydrogen
and R.sup.11 is pyridinyl.
[0266] In another aspect, the invention provides a process for
preparing a compound of the formula III wherein R.sup.10 is
hydrogen and R.sup.11 is piperidinyl or substituted piperidinyl
which comprises reacting a compound of the formula II wherein
R.sup.11 is halo, in particular chloro, and R.sup.10 is hydrogen
with piperazinyl or substituted piperazinyl under suitable
conditions to prepare a compound of the formula II wherein R.sup.10
is hydrogen and R.sup.11 is piperidinyl or substituted
piperidinyl.
[0267] In another aspect, the invention provides a process for
preparing a compound of the formula III wherein R.sup.10 is
hydrogen and R.sup.11 is an alkyl which comprises reacting a
pyridazine substituted at the C3 position with halo (e.g., chloro),
at the C4 position with alkyl, and at the 6 position with phenyl,
with 2-(piperidin-4-yloxy)pyrimidine under suitable conditions to
prepare a compound of the formula III wherein R.sup.10 is hydrogen
and R.sup.11 is an alkyl. In an embodiment, R.sup.11 is methyl or
ethyl.
[0268] In a particular aspect, the invention provides a process for
preparing a compound of the formula III wherein R.sup.10 is
hydrogen and R.sup.11 is alkyl comprising reacting a compound of
the formula IV
##STR00010##
wherein R.sup.1' is halo, in particular chloro or bromo, more
particularly chloro and R.sup.2' is alkyl with
2-(piperazin-1-yl)pyrimidine under suitable conditions, in
particular under reflux conditions to produce a compound of the
formula III wherein R.sup.10 is hydrogen and R.sup.11 is alkyl.
[0269] Therapeutic efficacy and toxicity of compounds, compositions
and methods of the invention may be determined by standard
pharmaceutical procedures in cell cultures or with experimental
animals such as by calculating a statistical parameter such as the
ED.sub.50 (the dose that is therapeutically effective in 50% of the
population) or LD.sub.50 (the dose lethal to 50% of the population)
statistics. The therapeutic index is the dose ratio of therapeutic
to toxic effects and it can be expressed as the ED.sub.50/LD.sub.50
ratio. Pharmaceutical compositions which exhibit large therapeutic
indices are preferred. By way of example, one or more of the
therapeutic effects can be demonstrated in a subject or disease
model, for example, a TgCRND8 mouse with symptoms of Alzheimer's
disease.
[0270] Biological investigations were done with compounds disclosed
herein that were >95% homogenous as determined by HPLC/MS
analysis. As part of a hierarchal, cell-based screening protocol,
the compounds were screened for their ability to block IL-1.beta.
and TNF.alpha. production by BV-2 mouse microglial cells stimulated
with LPS.
Compositions, Methods and Kits
[0271] The invention provides dosage forms, formulations, and
methods that provide advantages, in particular lower risk of side
effects (e.g. lower risk of QT-related side effects) and/or
beneficial pharmacokinetic profiles, more particularly sustained
pharmacokinetic profiles. A compound of the formula I, II or III
can be utilized in dosage forms in pure or substantially pure form,
in the form of its pharmaceutically acceptable salts, and also in
other forms including anhydrous or hydrated forms.
[0272] A beneficial pharmacokinetic profile may be obtained by
administering a formulation or dosage form suitable for once, twice
a day, or three times a day or more administration comprising one
or more compound of the formula I, II or III present in an amount
sufficient to provide the required concentration or dose of the
compound to an environment of use to treat a disease disclosed
herein, in particular a neuroinflammatory disease. In an aspect,
the environment of use is the brain and/or plasma.
[0273] Embodiments of the invention relate to a dosage form
comprising one or more compound of the formula I, II or III that
provides peak plasma concentrations of the compound, C.sub.max, of
between about 0.001 to 2 mg/ml, 0.001 to 1 mg/ml, 0.002 to 2 mg/ml,
0.005 to 2 mg/ml, 0.01 to 2 mg/ml, 0.05 to 2 mg/ml, 0.1 to 2 mg/ml,
0.001 to 0.5 mg/ml, 0.002 to 1 mg/ml, 0.005 to 1 mg/ml, 0.01 to 1
mg/ml, 0.05 to 1 mg/ml, or 0.1 to 1 mg/ml.
[0274] In further aspects, the invention provides a formulation or
dosage form comprising one or more compound of the formula I, II or
III that provides an elimination t.sub.1/2 of 0.5 to 20 hours, 0.5
to 15 hours, 0.5 to 10 hours, 0.5 to 6 hours, 1 to 20 hours, 1 to
15 hours, 1 to 10 hours, or 1 to 6 hours.
[0275] Further aspects of the invention relate to a formulation or
dosage form comprising one or more compound of the formula I, II or
III that provides an AUC for plasma of about 3 to 2000 ngh/ml, 3 to
3000 ngh/ml, 3 to 4000 ngh/ml, 2 to 2000 ngh/ml, 2 to 3000 ngh/ml,
2 to 4000 ngh/ml, 1 to 2000 ngh/ml, 1 to 3000 ngh/ml, 1 to 4000
ngh/ml, 1, and in particular 3 to 3000 ngh/ml
[0276] A subject may be treated with a compound of the formula I,
II or III or composition or unit dosage thereof on substantially
any desired schedule. They may be administered one or more times
per day, in particular 1 or 2 times per day, once per week, once a
month or continuously. However, a subject may be treated less
frequently, such as every other day or once a week, or more
frequently. A compound or composition may be administered to a
subject for about or at least about 24 hours, 2 days, 3 days, 1
week, 2 weeks to 4 weeks, 2 weeks to 6 weeks, 2 weeks to 8 weeks, 2
weeks to 10 weeks, 2 weeks to 12 weeks, 2 weeks to 14 weeks, 2
weeks to 16 weeks, 2 weeks to 6 months, 2 weeks to 12 months, 2
weeks to 18 months, 2 weeks to 24 months, or for more than 24
months, periodically or continuously.
[0277] In an aspect, a beneficial pharmacokinetic profile can be
obtained by the administration of a formulation or dosage form
suitable for once, twice, or three times a day administration,
preferably twice a day administration comprising one or more
compound of the formula I, II or III present in an amount
sufficient to provide the required dose of the compound. In an
aspect, the required dose of a compound of the formula I, II or III
administered once twice, three times or more daily is about 0.01 to
3000 mg/kg, 0.01 to 2000 mg/kg, 0.5 to 2000 mg/kg, about 0.5 to
1000 mg/kg, 0.1 to 1000 mg/kg, 0.1 to 500 mg/kg, 0.1 to 400 mg/kg,
0.1 to 300 mg/kg, 0.1 to 200 mg/kg, 0.1 to 100 mg/kg, 0.1 to 50
mg/kg, 0.1 to 20 mg/kg, 0.1 to 10 mg/kg, 0.1 to 6 mg/kg, 0.1 to 5
mg/kg, 0.1 to 3 mg/kg, 0.1 to 2 mg/kg, 0.1 to 1 mg/kg, 1 to 1000
mg/kg, 1 to 500 mg/kg, 1 to 400 mg/kg, 1 to 300 mg/kg, 1 to 200
mg/kg, 1 to 100 mg/kg, 1 to 50 mg/kg, 1 to 20 mg/kg, 1 to 10 mg/kg,
1 to 6 mg/kg, 1 to 5 mg/kg, or 1 to 3 mg/kg, or 1 to 2.5 mg/kg, or
less than or about 10 mg/kg, 5 mg/kg, 2.5 mg/kg, 1 mg/kg, or 0.5
mg/kg twice daily or less
[0278] Certain dosage forms and formulations may minimize the
variation between peak and trough plasma and/or brain levels of
compounds of the formula I, II or III and in particular provide a
sustained therapeutically effective amount of the compounds.
[0279] The invention also contemplates a formulation or dosage form
comprising amounts of one or more compound of the formula I, II or
III that results in therapeutically effective amounts of the
compound over a dosing period, in particular a 24 hour dosing
period. In aspects of the invention the therapeutically effective
amounts of a compound of the formula I, II or III are between about
0.1 to 1000 mg/kg, 0.1 to 500 mg/kg, 0.1 to 400 mg/kg, 0.1 to 300
mg/kg, 0.1 to 200 mg/kg, 0.1 to 100 mg/kg, 0.1 to 75 mg/kg, 0.1 to
50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 20 mg/kg, 0.1 to 15 mg/kg, 0.1 to
10 mg/kg, 0.1 to 9 mg/kg, 0.1 to 8 mg/kg, 0.1 to 7 mg/kg, 0.1 to 6
mg/kg, 0.1 to 5 mg/kg, 0.1 to 4 mg/kg, 0.1 to 3 mg/kg, 0.1 to 2
mg/kg, or 0.1 to 1 mg/kg.
[0280] A medicament or treatment of the invention may comprise a
unit dosage of at least one compound of the formula I, II or III to
provide therapeutic effects. A "unit dosage" or "dosage unit"
refers to a unitary, i.e. a single dose, which is capable of being
administered to a patient, and which may be readily handled and
packed, remaining as a physically and chemically stable unit dose
comprising either the active agents as such or a mixture with one
or more solid or liquid pharmaceutical excipients, carriers, or
vehicles.
[0281] A formulation or dosage form of the invention may be an
immediate release dosage form or a non-immediate release delivery
system, including without limitation a delayed-release or
sustained-release dosage form.
[0282] In aspects, the invention provides a sustained-release
dosage form of a compound of the formula I, II or III which
advantageously achieves a more sustained drug plasma and/or brain
level response while mitigating or eliminating drug concentration
spikes by providing a substantially steady release of the compound
over time. A substantially constant plasma concentration preferably
correlates with one or more therapeutic effects disclosed herein.
In embodiments, the sustained-release dosage form is for oral
administration.
[0283] A composition, in particular a dosage form or formulation,
may be in any form suitable for administration to a subject,
including without limitation, a form suitable for oral, parenteral,
intravenous (bolus or infusion), intraperitoneal, subcutaneous, or
intramuscular administration. A dosage form or formulation may be a
pill, tablet, caplet, soft and hard gelatin capsule, lozenge,
sachet, cachet, vegicap, liquid drop, elixir, suspension, emulsion,
solution, syrup, aerosol (as a solid or in a liquid medium)
suppository, sterile injectable solution, and/or sterile packaged
powder.
[0284] In an aspect of the invention a dosage form or formulation
is an oral dosage form or formulation such as tablets, caplets,
soft and hard gelatin capsules, pills, powders, granules, elixirs,
tinctures, suspensions, syrups, and emulsions. In another aspect
the dosage form or formulation is a parenteral dosage form such as
an active substance in a sterile aqueous or non-aqueous solvent,
such as water, isotonic saline, isotonic glucose solution, buffer
solution, or other solvents conveniently used for parenteral
administration.
[0285] A compound of the formula I, II or III of the invention may
be formulated into a pharmaceutical composition for administration
to a subject by appropriate methods known in the art.
Pharmaceutical compositions of the present invention or fractions
thereof comprise suitable pharmaceutically acceptable carriers,
excipients, and vehicles selected based on the intended form of
administration, and consistent with conventional pharmaceutical
practices. Suitable pharmaceutical carriers, excipients, and
vehicles are described in the standard text, Remington: The Science
and Practice of Pharmacy (21.sup.st Edition. 2005, University of
the Sciences in Philadelphia (Editor), Mack Publishing Company),
and in The United States Pharmacopeia: The National Formulary (USP
24 NF19) published in 1999. By way of example for oral
administration in the form of a capsule or tablet, the active
components can be combined with an oral, non-toxic pharmaceutically
acceptable inert carrier such as lactose, starch, sucrose, methyl
cellulose, magnesium stearate, glucose, calcium sulfate, dicalcium
phosphate, mannitol, sorbital, and the like. For oral
administration in a liquid form, the drug components may be
combined with any oral, non-toxic, pharmaceutically acceptable
inert carrier such as ethanol, glycerol, water, and the like.
Suitable binders (e.g. gelatin, starch, corn sweeteners, natural
sugars including glucose; natural and synthetic gums, and waxes),
lubricants (e.g. sodium oleate, sodium stearate, magnesium
stearate, sodium benzoate, sodium acetate, and sodium chloride),
disintegrating agents (e.g. starch, methyl cellulose, agar,
bentonite, and xanthan gum), flavoring agents, and coloring agents
may also be combined in the compositions or components thereof.
Compositions as described herein can further comprise wetting or
emulsifying agents, or pH buffering agents.
[0286] A composition of the invention can be a liquid solution,
suspension, emulsion, tablet, pill, capsule, sustained release
formulation, or powder. The compositions can be formulated as a
suppository, with traditional binders and carriers such as
triglycerides. Oral formulations can include standard carriers such
as pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Various delivery systems are known and can be used to administer a
composition of the invention, e.g. encapsulation in liposomes,
microparticles, microcapsules, and the like.
[0287] Formulations for parenteral administration may include
aqueous solutions, syrups, aqueous or oil suspensions and emulsions
with edible oil such as cottonseed oil, coconut oil or peanut oil.
Dispersing or suspending agents that can be used for aqueous
suspensions include synthetic or natural gums, such as tragacanth,
alginate, acacia, dextran, sodium carboxymethylcellulose, gelatin,
methylcellulose, and polyvinylpyrrolidone.
[0288] Compositions for parenteral administration may include
sterile aqueous or non-aqueous solvents, such as water, isotonic
saline, isotonic glucose solution, buffer solution, or other
solvents conveniently used for parenteral administration of
therapeutically active agents. A composition intended for
parenteral administration may also include conventional additives
such as stabilizers, buffers, or preservatives, e.g. antioxidants
such as methylhydroxybenzoate or similar additives.
[0289] A composition of the invention may be sterilized by, for
example, filtration through a bacteria retaining filter, addition
of sterilizing agents to the composition, irradiation of the
composition, or heating the composition. Alternatively, the
compounds or compositions of the present invention may be provided
as sterile solid preparations e.g. lyophilized powder, which are
readily dissolved in sterile solvent immediately prior to use.
[0290] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of a composition of
the invention, such labeling would include amount, frequency, and
method of administration.
[0291] According to the invention, a kit is provided. In an aspect,
the kit comprises a compound of the formula I, II or III or a
formulation of the invention in kit form. The kit can be a package
which houses a container which contains compounds of the formula I,
II or III or formulations of the invention and also houses
instructions for administering the compounds or formulations to a
subject. The invention further relates to a commercial package
comprising compounds of the formula I, II or III or formulations of
the invention together with instructions for simultaneous, separate
or sequential use. In particular a label may include amount,
frequency, and method of administration.
[0292] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of a composition of the invention to provide a
therapeutic effect. Associated with such container(s) can be
various written materials such as instructions for use, or a notice
in the form prescribed by a governmental agency regulating the
labeling, manufacture, use or sale of pharmaceuticals or biological
products, which notice reflects approval by the agency of
manufacture, use, or sale for human administration.
[0293] The invention also relates to articles of manufacture and
kits containing materials useful for treating a disease disclosed
herein. An article of manufacture may comprise a container with a
label. Examples of suitable containers include bottles, vials, and
test tubes which may be formed from a variety of materials
including glass and plastic. A container holds compounds of the
formula I, II or III or formulations of the invention which are
effective for treating a disease disclosed herein. The label on the
container indicates that the compounds of the formula I, II or III
or formulations of the invention are used for treating a disease
disclosed herein and may also indicate directions for use. In
aspects of the invention, a medicament or formulation in a
container may comprise any of the medicaments or formulations
disclosed herein.
[0294] The invention also contemplates kits comprising one or more
of compounds of the formula I, II or III. In aspects of the
invention, a kit of the invention comprises a container described
herein. In particular aspects, a kit of the invention comprises a
container described herein and a second container comprising a
buffer. A kit may additionally include other materials desirable
from a commercial and user standpoint, including, without
limitation, buffers, diluents, filters, needles, syringes, and
package inserts with instructions for performing any methods
disclosed herein (e.g., methods for treating a disease disclosed
herein). A medicament or formulation in a kit of the invention may
comprise any of the formulations or compositions disclosed
herein.
[0295] In aspects of the invention, the kits may be useful for any
of the methods disclosed herein, including, without limitation
treating a subject suffering from Alzheimer's disease. Kits of the
invention may contain instructions for practicing any of the
methods described herein.
[0296] The compositions and methods described herein are indicated
as therapeutic agents or methods either alone or in conjunction
with other therapeutic agents or other forms of treatment. They may
be co-administered, combined or formulated with one or more
therapies or agents used to treat a condition described herein.
Compositions of the invention may be administered concurrently,
separately, or sequentially with other therapeutic agents or
therapies. Therefore, compounds of the formula I, II or III may be
co-administered with one or more additional therapeutic agents for
treating diseases disclosed herein including without limitation
beta-secretase inhibitors, alpha-secretase inhibitors, and
epsilon-secretase inhibitors, acetylcholinesterase inhibitors,
agents that are used for the treatment of complications resulting
from or associated with a disease disclosed herein, or general
medications that treat or prevent side effects.
[0297] The invention will be described in greater detail by way of
specific examples. The following examples are offered for
illustrative purposes, and are not intended to limit the invention
in any manner.
EXAMPLES
Example 1
Synthesis of Pyridazine Compounds
[0298] The structures of MW01-2-151SRM, MW01-6-189WH, MW01-7-107WH,
MW01-4-179LKM, WM01-7-084WH, MH01-7-085WH, MW01-7-133WH, and
MW01-7-057 are shown in FIG. 1 and synthetic schemes for producing
the compounds are described below.
A. Preparation of
2-(4-(6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine
(MW01-3-183WH)
[0299] FIG. 2 depicts a synthetic scheme for the preparation of
2-(4-(6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine
(MW01-3-183WH). Reagents and conditions: (a) 1-BuOH, NH.sub.4Cl,
and 2-(piperazin-1-yl)pyrimidine. A typical reaction mixture
comprising about 0.01 mol of 3-chloro-6-phenylpyridazine by
2-(piperazin-1-yl)pyrimidine, about 0.05 mol of
2-(piperazin-1-yl)pyrimidine and about 0.01 mol of ammonium
hydrochloride was prepared in about 15 ml of 1-BuOH. The mixture
was stirred at about 130.degree. C. for about 48 h, and then the
solvent was removed under reduced pressure. The remaining residue
was then extracted with ethyl acetate, washed with water and brine,
dried over anhydrous Na.sub.2SO.sub.4. Removal of solvent followed
by recrystallization from 95% ethanol yielded light yellow
crystals, yield 96.4%; HPLC: 97.4% purity; HRMS calculated
318.1587, found 318.1579; 1H NMR (CDCl.sub.3): d 8.356 (d, J=4.5,
2H), 8.011 (d, J=7.5, 11 2H), 7.692 (d, J=9.5, 1H), 7.468 (t,
J=6.0, 2H), 7.417 (d, J=7.5, 1H), 7.047 (d, J=9.5, 1H), 6.546 (t,
J=4.5, 1H), 4.013 (t, J=5.0, 4H), 3.826 (t, J=5.0, 4H).
B. Preparation of
4-methyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-2-151SRM)
[0300]
4-methyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-2-151 SRM) was prepared by several synthetic schemes as
depicted in FIG. 3 (Scheme 1), FIG. 4 (Scheme 2), FIG. 5 (Scheme
3), and FIG. 6 (Scheme 4), which were carried out as described in
detail herein. The various reaction schemes (Schemes 1, 2, and 3)
are generally applicable to the compounds of the present invention
and are not restricted in utility only to the preparation of
MW01-2-151 SRM.
Scheme 1 (FIG. 3)
4,5-dihydro-4-methyl-6-phenylpyridazin-3(2H)-one (2)
[0301] A 250 mL three-neck round bottom flask fit with a
temperature probe and condenser is charged with 7.7 g (40 mmole) of
2-methyl-4-oxo-4-phenylbutanoic acid 1 and 20 ml of ethanol (95%).
The suspension is cooled to below 10.degree. C. and 2.2 ml (42
mmole, 1.05 equiv.) of hydrazine monohydrate in 10 mL of ethanol is
added dropwise at a rate that maintains the solution temperature at
below 20.degree. C. Upon addition, the suspension changes to a pale
yellow solution. After addition, the reaction mixture is heated to
reflux and stirred for 2 h, and after 20 minutes of heating, a
solid is seen in the mixture. Once the reaction is completed, the
flask is removed from the oil bath and cooled to ambient
temperature. Upon cooling, white crystals form in the flask, which
are collected by filtration. The solid is washed first with 30 mL
of 2N NaHCO.sub.3, followed by 60 mL Milli-Q water three times, and
dried over a medium frit sintered glass funnel in vacuo to give the
desired product 2 in 96.1% yield. [See Hansen, K B et al. Organic
process research & development, 2005, 9, 634-639; Nelson, D A.
US 20050137397A1. Coudert, P et al. Journal of Heterocyclic
Chemistry, 1988, 25(3), 799-802.]
4-methyl-6-phenylpyridazin-3(2H)-one (3)
[0302] 7.0 g (35 mmole) of 2 is placed in a 250 ml single-necked
round bottom flask followed by 30 mL of acetonitrile. The mixture
is stirred to allow 2 to dissolve. 11.3 g (84 mmole, 2.4 equiv.) of
anhydrous copper (II) chloride is added to the solution to give a
green-yellow suspension. A reflux condenser is connected to the
flask and a dry tube filled with anhydrous CaCl.sub.2 is fitted to
the top of the condenser. To control the HCl gas that forms during
the course of the reaction, a NaOH solution is used to absorb the
HCl that escapes from dry tube. The reaction mixture is heated to
reflux, and the color of the reaction suspension changes to dark
green upon heating. When the reaction is complete (after refluxing
for 2 h), the flask is removed from the oil bath and cooled to
ambient temperature. The reaction is cooled in an ice-water bath
and 150 mL of ice-water is added to quench the reaction. The
mixture is stirred vigorously for 10 minutes to give a gray
precipitate and blue liquid containing copper (I) chloride. The
precipitate is collected by filtration (pH of the filtrate is 0-1)
and washed with 100 mL of 1N HCl solution, then 100 mL of water 5
times. To remove remaining copper by-products that are trapped in
the solid, the filter cake is stirred in 150 mL of 1N HCl solution
for 0.5 h and filtered. The filter cake is subsequently washed with
Milli-Q water until the filtrate is at pH 7 (approximately 7
washes). The solid is dried over a medium frit sintered glass
funnel in vacuo to give 3 as a light gray powder in 93.8% yield.
[See Eddy, S et al. Synthetic Communications, 2000, 30(1), 1-7.
Csende, F et al. Synthesis, 1995, 1240-1242.]
3-chloro-4-methyl-6-phenylpyridazine (4)
[0303] 6.0 g (32 mmole) of 3 is placed in a 250 mL single neck
round bottom flask and 30 mL of acetonitrile is added to create a
pale yellow slurry. 6.0 ml (64 mmole, 2 equiv.) of phosphorus
oxychloride is added changing the slurry to a darker color. The
flask is fitted with a reflux condenser and a dry tube filled with
anhydrous CaCl.sub.2 is fitted to the top of the condenser. The
reaction mixture is heated at reflux and becomes a dark red liquid.
After the reaction is completed (2.5 h), the mixture is cooled to
ambient temperature and placed in an ice water bath. Ice water (150
mL) is slowly poured into the reaction mixture with stirring to
decompose the phosphorus oxychloride into IC.sub.1 and
H.sub.3PO.sub.4, resulting in formation of a pink solid. The solid
is collected by filtration and washed three times with 50 mL of
Milli-Q water. The solid is transferred to a 250 mL beaker,
followed by addition of 100 mL of water to form a suspension.
Subsequently, 1N NaOH is added until the aqueous suspension is at
pH=8, and the mixture is stirred for 5 minutes to remove all trace
of starting material contaminants. The solid is filtered and washed
3 times with 100 mL of water to wash out the excess base. The solid
is dried over a medium frit sintered glass funnel in vacuo to
provide 4 as a light pink powder in 96% yield. [See Contreras, J M
et al. Journal of Medicinal Chemistry, 2001, 44(17), 2707-2718;
Nelson, D A. US 20050137397A1.]
2-(4-(4-methyl-6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine
(5)
[0304] 7.5 g (36.6 mmole) of 4 is placed in a 250 mL single neck
round bottom flask and suspended in 125 mL of water. 60.17 g (366.0
mmole, 10 equiv.) of 2-(piperazine-1-yl)pyrimidine is added and the
flask fit with a condenser. The reaction mixture is heated at
reflux with rapid stirring for 60 h, with continuous amine addition
possible to boost reaction rates. When complete, the reaction
mixture is cooled to ambient temperature and two layers are
observed in the flask consisting of an orange aqueous layer and a
brown oil that settles to the bottom of the flask. The water is
decanted off, leaving the oil, which is the product 5. The oil is
then dissolved in minimal volume of isopropanol and heated to
reflux. After 10 minutes of reflux, the solution is cooled to
ambient temperature, and cooled to 0.degree. C. to induce
crystallization. Pale yellow crystals are filtered from isopropanol
and rinsed with minimal cold ether to provide 5. Recovery of the
crystals is 50%, but may be increased by recursive crystallization
of compound. [Contreras, J M et al. Journal of Medicinal Chemistry,
1999, 42(4), 730-741. Chayer, S et al. Tetrahedron Letters, 1998,
39, 841-844.]
Scheme 2 (FIG. 4)
[0305] 3-chloro-6-phenylpyridazin-4-ol was synthesized according to
the procedure described by Coudert, P., et al., supra.
6-phenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazin-4-ol
(MW01-1-7-121 WH)
[0306] This compound was prepared from
3-chloro-4-hydroxy-6-phenylpyridazine (14 g, 68 mmol) in the same
manner as described below, yielding white solid (22.1 g, 66 mmol,
97.3%). ESI-MS: m/z 335.2 (M+H+). 1H NMR (DMSO): 1H NMR (DMSO): d
8.406 (d, J=6.5, 2H), 7.740 (d, J=4.0, 2H), 7.558 (s, 3H), 6.686
(t, J=4.8, J=4.4, 1H), 6.841 (s, 1H), 3.881 (s, 4H), 3.620 (s, 4H),
3.776 (s, 4H).
4-chloro-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-6-127WH)
[0307] 6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazin-4-ol
(22.0 g, 66 mmol) was suspended in 75 ml phosphorus oxychloride and
heated with stirring at 100.degree. C. for 3 h. After cooling to
room temperature the mixture was poured onto crushed ice. The
mixture was then neutralized with NaOH solution to give white
suspension. The precipitation was filtered off, washed with water,
dried over filter funnel to provide white solid (21.3 g, 60.3 mmol,
91.4%). ESI-MS: m/z 353.4 (M+H+). 1H NMR (CDCl.sub.3): d 8.377 (d,
J=4.5, 2H), 8.036 (d, J=7.5, 2H), 7.833 (s, 1H), 7.508 (m, 3H),
6.564 (t, J=4.5, 1H), 4.073 (t, J=4.0, J=4.5, 4H), 3.672 (t, J=4.0,
J=4.5, 4H).
4-methyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-2-151 SRM)
[0308] Into a reaction tube were added MW01-6-127WH (1.4 g, 4.0
mmol), K.sub.2CO.sub.3 powder (1.7 g, 12.4 mmol), Pd(dppf)Cl.sub.2
(326 mg, 0.4 mmol), silver oxide (2.3 g, 10 mmol), methylboronic
acid (324 mg, 5.4 mmol) and 20 ml of THF. Argon was then flushed
through the tube for 3 min. The tube was then sealed tightly and
heated with stirring at 80 degree for 12 h. After cooled down, the
mixture was quenched with 10% NaOH solution and extracted with
ethyl acetate. The organic phase was concentrated in vacuo and the
residue was purified by column chromatography eluting with 1:4,
Ethyl Acetate:Petroleum ether. White powder solid was obtained
(0.60 g, 1.8 mmol, yield 45.2%). ESI-MS: m/z 333.4 (M+H+). 1H NMR
(CDCl.sub.3): d 8.380 (d, J=5.0, 2H), 7.065 (d, J=7.0, 2H), 7.626
(s, 1H), 7.473 (m, 3H), 6.567 (t, J=4.5, J=5.0, 1H), 4.056 (t,
J=5.0, 4H), 3.475 (t, J=5.0, 4H), 2.456 (s, 3H).
Scheme 3 (FIG. 5)
[0309] Into a reaction tube were added MW01-6-127WH (1.4 g, 4.0
mmol), K.sub.2CO.sub.3 powder (1.7 g, 12.4 mmol),
Pd(PPh.sub.3).sub.4 (240 mg, 0.2 mmol), silver oxide (2.3 g, 10
mmol), methylboronic acid (324 mg, 5.4 mmol) and 20 ml of DME.
Argon was then flushed through the tube for 3 min. The tube was
then sealed tightly and heated with stirring at 120.degree. C. for
24 h. After cooled down, the mixture was filter through acelite
earth, the filtrate was then concentrated and the residue was
purified by column chromatography eluting with 1:4, Ethyl
Acetate:Petroleum ether. White powder solid was obtained (0.64 g,
1.93 mmol, yield 48.1%). ESI-MS: m/z 333.4 (M+H+). 1H NMR
(CDCl.sub.3): d 8.380 (d, J=5.0, 2H), 7.065 (d, J=7.0, 2H), 7.626
(s, 1H), 7.473 (m, 3H), 6.567 (t, J=4.5, J=5.0, 1H), 4.056 (t,
J=5.0, 4H), 3.475 (t, J=5.0, 4H), 2.456 (s, 3H).
Scheme 4 (FIG. 6)
4,5-dihydro-4-methyl-6-phenylpyridazin-3(2H)-one (MW01-8-004WH)
[0310] 7.7 g (40 mmole) of 2-methyl-4-oxo-4-phenylbutanoic acid was
added to a 100 ml single-necked round bottom flask followed by 3.0
ml (60 mmole) of hydrazine monohydrate and then 20 ml of reagent
grade ethanol (100%, 95% of ethanol should be fine also). The flask
was fitted with a reflux condenser and the reaction mixture was
heated to reflux in an oil bath at 110.degree. C. (temperature of
oil bath) and stirred for 2 h. The flask was then removed from the
oil bath and the reaction mixture cooled to ambient temperature.
The stir bar was removed and the solvent was evaporated in vacuo in
a water bath at 45.degree. C. The residue was then treated with 50
ml of Milli-Q water and stirred for 10 minutes to give a
suspension. The precipitate was collected by filtering, washed with
100 ml of 2N NaHCO.sub.3, then washed with 60 ml Milli-Q water
three times, and dried over a medium frit sintered glass funnel in
vacuo to give 7.15 g of white crystals (Syn. ID, WH-8-004). Yield,
95%, confirmed by ESI-MS. ESI-MS: m/z 189.2 (M+H+).
4-methyl-6-phenylpyridazin-3(2H)-one (MW01-8-008WH)
[0311] 7.0 g (35 mmole) of MW01-8-004WH was placed in a 100 ml
single-necked round bottom flask followed by 9.4 g (70 mmole) of
anhydrous copper (II) chloride and then 30 ml of acetonitrile to
give a brown yellow suspension. A reflux condenser was connected to
the flask and a dry tube filled with CaCl.sub.2 was fitted to the
top of the condenser. The reaction mixture was heated to reflux in
an oil bath (110.degree. C.) for 3 h. The color of the reaction
suspension changed to dark yellow once the reflux started. After
the completion of the reaction (monitored by HPLC), the flask was
removed from the oil bath and cooled to ambient temperature. The
mixture was poured on to 300 g of crushed ice and stirred
vigorously for 10 minutes to give a gray precipitate and blue
liquid. The precipitate was then collected by filtering (pH of the
filtrate was 1.5-2.0), and washed with 100 ml of a 1N HCl solution
to rid the solid of any remaining copper byproducts. This is
followed by washing with 100 ml of Milli-Q water to get rid of the
acid in the solid, and is monitored by checking the pH value of the
filtrate. The solid was washed until the filtrate shows a pH of 7,
after approximately 5 washes. The solid was dried over a medium
frit sintered glass funnel in vacuo to give 6.3 g of a blue gray
solid. Yield was 96.7% and confirmed by ESI-MS. ESI-MS: m/z 187.3
(M+H+).
3-chloro-4-methyl-6-phenylpyridazine(MW01-8-012WH)
[0312] 6.0 g (32 mmole) of MW01-8-008WH and 30 ml (320 mmole) of
phosphorus oxychloride were placed in a 100 ml single-necked round
bottom flask. The flask was connected with a reflux condenser and a
dry tube filled with anhydrous CaCl.sub.2 was fitted to the top of
the condenser. (HCl gas is formed in the reaction so a basic
solution such as NaOH may be needed to absorb HCl in a large-scale
synthesis). The reaction mixture was stirred in an oil bath
(90.degree. C.) for 2 h, then cooled to ambient temperature and
poured onto crushed ice (phosphorus oxychloride can be decomposed
by water to give HCl and H.sub.3PO.sub.4). The mixture was then
stirred vigorously for 10 minutes to give a white suspension. The
suspension was neutralized with a 2N NaOH solution until the pH of
the suspension was pH=7. The precipitate was filtered, washed three
times with 100 ml of Milli-Q water and dried over a medium frit
sintered glass funnel in vacuo to provide 5.9 g of a light pink
powder (Syn. ID, WH-8-012). Yield was 89.4% and confirmed by
ESI-MS. ESI-MS: m/z 205.4 (M+H+).
2-(4-(4-methyl-6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine
(MW01-2-151SRM)
[0313] 0.82 g (4.0 mmole) of WH-8-012 was placed in a 30 ml
pressure vessel followed by addition of 2.6 g (16.0 mmole) of
1-(2-pyrimidyl)piperazine and then 15 ml of 1-BuOH. The vessel was
sealed tightly and placed into an oil bath and stirred at
130.degree. C. (temperature of oil bath) for 2.5 days. The reaction
mixture was then cooled to ambient temperature and transferred to a
single-necked flask for evaporation under reduced pressure. Removal
of solvent gave rise to a brown-red residue that was treated with
30 ml of water to give a brown sticky oil. The mixture was kept at
ambient temperature overnight while the oil solidified gradually.
The formed solid was then broken into small pieces with a steel
spatula. The solid was collected by filtering and washed with 50 ml
of Milli-Q water three times and dried over a filter funnel in
vacuo to provide 1.25 g of light yellow solid (Syn. ID, WH-8-020).
Yield was 94%. (Alternative separation is to use a precipitation
procedure instead of a solidification process. Solidification is a
simple and cheap operation, yet time-consuming. Precipitation is
time efficient, yet more costly than the former one. So it is up to
the process chemist to decide which procedure to pick for the
manufacture. The precipitation process is below: The oil product
was dissolved completely in 10 ml of reagent grade ethanol or
acetone to form a solution. The solution was then added dropwise to
150 ml of ice water under vigorous stirring. Light yellow
suspension was then formed gradually. The solid was collected by
filtering, washed with Milli-Q water, dried over filter funnel in
vacuo to give the desired product.) The final compound was
confirmed by ESI-MS and NMR. ESI-MS: m/z 333.8 (M+H+). 1H NMR
(CDCl.sub.3): d 8.380 (d, J=5.0, 2H), 7.065 (d, J=7.0, 2H), 7.626
(s, 1H), 7.473 (m, 3H), 6.567 (t, J=4.5, J=5.0, 1H), 4.056 (t,
J=5.0, 4H), 3.475 (t, J=5.0, 4H), 2.456 (s, 3H).
C. Preparation of
4,6-diphenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-5-188WH)
[0314] 4,6-diphenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-5-188WH) was prepared by several synthetic schemes as
depicted in FIG. 7 (Scheme 1), FIG. 8 (Scheme 2), and FIG. 9
(Scheme 3), which were carried out as described in detail herein.
The various reaction schemes (Schemes 1, 2, and 3) are generally
applicable to the compounds of the present invention and are not
restricted in utility only to the preparation of MW01-2-188WH.
Scheme 1 (FIG. 7)
[0315] 3-chloro-6-phenylpyridazin-4-ol was synthesized according to
the procedure described by Coudert, P., et al. supra.
6-phenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazin-4-ol
(MW01-7-121WH)
[0316] The compound was prepared from
3-chloro-4-hydroxy-6-phenylpyridazine (14 g, 68 mmol). A mixture of
3-chloro-4,6-diphenylpyridazine (267 mg, 1.0 mmol),
1-(2-pyrimidyl)piperazine (656 mg, 4.0 mmol) in 3 ml of 1-BuOH was
heated with stirring at 130.degree. C. for 3 days. The solvent was
removed by evaporation in vacuo the residue was treated with water
to give a suspension. The solid was then filtered off, washed with
water, dried over filter funnel in vacuo to give light pink solid
yielding white solid (22.1 g, 66 mmol, 97.3%). ESI-MS: m/z 335.2
(M+H+). 1H NMR (DMSO): 1H NMR (DMSO): d 8.406 (d, J=6.5, 2H), 7.740
(d, J=4.0, 2H), 7.558 (s, 3H), 6.686 (t, J=4.8, J=4.4, 1H), 6.841
(s, 1H), 3.881 (s, 4H), 3.620 (s, 4H), 3.776 (s, 4H).
4-chloro-6-phenyl-3-(4-pyrimidin-2-yl)piperazin-1-yl)pyridazine
(MW01-6-127WH)
[0317] 6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazin-4-ol
(22.0 g, 66 mmol) was suspended in 75 ml phosphorus oxychloride and
heated with stirring at 100.degree. C. for 3 h. After cooling to
room temperature the mixture was poured onto crushed ice. The
mixture was then neutralized with NaOH solution to give white
suspension. The precipitation was filtered off, washed with water,
dried over filter funnel to provide white solid (21.3 g, 60.3 mmol,
91.4%). ESI-MS: m/z 353.4 (M+H+). 1H NMR (CDCl.sub.3): d 8.377 (d,
J=4.5, 2H), 8.036 (d, J=7.5, 2H), 7.833 (s, 1H), 7.508 (m, 3H),
6.564 (t, J=4.5, 1H), 4.073 (t, J=4.0, J=4.5, 4H), 3.672 (t, J=4.0,
J=4.5, 4H).
4,6-diphenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-5-188WH)
[0318] A mixture of 3-chloro-4,6-diphenylpyridazine (267 mg, 1.0
mmol), 1-(2-pyrimidyl)piperazine (656 mg, 4.0 mmol) in 3 ml of
1-BuOH was heated with stirring at 130.degree. C. for 3 days. The
solvent was removed by evaporation in vacuo the residue was treated
with water to give a suspension. The solid was then filtered off,
washed with water, dried over filter funnel in vacuo to give light
pink solid. (320 mg, 0.81 mmol, yield 81.1%). ESI-MS: m/z 395.5
(M+H+). HRMS calcd 395.1979, found 395.1973; 1H NMR (CDCl.sub.3): d
8.329 (d, J=5.0, 2H), 8.101 (d, J=7.5, 2H), 7.734 (d, J=7.5, 2H),
7.655 (s, 1H), 7.509 (m, 6H), 6.530 (t, J=4.5, 1H), 3.836 (t,
J=4.5, J=5.0, 4H), 3.394 (t, J=5.0, J=4.5, 4H).
Scheme 2 (FIG. 8)
4,5-dihydro-6-phenyl-4-phenylpyridazin-3(2H)-one
[0319] 135 ml (135 mmole) of a solution of phenylmagnesium bromide
(1M) in THF was added to a hot suspension of 6-phenylpyridazinone
compound 7.8 g (45 mmole) in dry toluene (50 ml). The mixture was
refluxed for 8 h, left overnight at ambient temperature, then
decomposed with a saturated solution of ammonium chloride. The
organic layer was separated, and the aqueous layer was extracted
with 100 ml of ethyl acetate. The solvent was removed and the
residue was crystallized from ethanol. The crystals were collected
by filtering and dried over a medium frit sintered glass funnel in
vacuo to give 5.6 g of white crystals. Yield was 50%, confirmed by
ESI-MS. ESI-MS: m/z 250.1 (M+H+).
6-phenyl-4-phenylpyridazin-3(2H)-one
[0320] 4.4 g (17.5 mmole) of 6-pyridazinone obtained above was
placed in a 50 ml single-necked round bottom flask followed by 4.7
g (35 mmole) of anhydrous copper (II) chloride and then 20 ml of
acetonitrile to give a brown yellow suspension. A reflux condenser
was connected to the flask and a dry tube filled with CaCl.sub.2
was fitted to the top of the condenser. The reaction mixture was
heated to reflux in an oil bath (110.degree. C.) for 3 h. The color
of the reaction suspension changed to dark yellow once the reflux
started. After the completion of the reaction (monitored by HPLC),
the flask was removed from the oil bath and cooled to ambient
temperature. The mixture was poured on to 200 g of crushed ice and
stirred vigorously for 10 minutes to give a gray precipitate and
blue liquid. The precipitate was then collected by filtering (pH of
the filtrate was 1.5-2.0), and washed with 50 ml of a 1N HCl
solution to rid the solid of any remaining copper byproducts. This
is followed by washing with 100 ml of Milli-Q water to get rid of
the acid in the solid, and is monitored by checking the pH value of
the filtrate. The solid was washed until the filtrate shows a pH of
7, after approximately 5 washes. The solid was dried over a medium
frit sintered glass funnel in vacuo to give 3.9 g of a blue gray
solid. Yield was 90%, confirmed by ESI-MS. ESI-MS: m/z 248.1
(M+H+).
3-chloro-6-phenyl-4-phenylpyridazine
[0321] 2.0 g (8 mmole) of 6-phenylpyridazinone obtained above and
10 ml (54 mmole) of phosphorus oxychloride (reagent grade, Aldrich)
were placed in a 50 ml single-necked round bottom flask. The flask
was connected with a reflux condenser and a dry tube filled with
CaCl.sub.2 was fitted to the top of the condenser. (HCl gas is
formed in the reaction so a basic solution such as NaOH may be
needed to absorb HCl in a large-scale synthesis). The reaction
mixture was stirred in an oil bath (90.degree. C.) for 2 h, then
cooled to ambient temperature and poured onto crushed ice.
(Phosphorus oxychloride can be decomposed by water to give HCl and
H.sub.3PO.sub.4). The mixture was then stirred vigorously for 10
minutes to give a white suspension. The suspension was neutralized
with a 2N NaOH solution until the pH of the suspension was pH=7.
The precipitate was filtered, washed three times with 100 ml of
water and dried over a medium frit sintered glass funnel in vacuo
to provide 1.8 g of a light pink powder. Yield was 85%, confirmed
by ESI-MS. ESI-MS: m/z 266.4 (M+H+).
2-(4-(6-phenyl-4-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine
[0322] 1.1 g (4.0 mmole) of 3-chloropyridazine obtained above was
placed in a 30 ml pressure vessel followed by addition of 2.6 g
(16.0 mmole) of 1-(2-pyrimidyl)piperazine and then 15 ml of 1-BuOH
(reagent grade). The vessel was sealed tightly and placed into an
oil bath and stirred at 130.degree. C. (temperature of oil bath)
for 3 days. The reaction mixture was then cooled to ambient
temperature and transferred to a single-necked flask for
evaporation under reduced pressure. Removal of solvent gave rise to
a brown-red residue that was treated with 30 ml of water to give a
brown suspension. The solid was collected by filtering and washed
with 50 mL of water three times and dried over a filter funnel in
vacuo to provide 0.96 g of light yellow solid. Yield was 90%,
ESI-MS: m/z 395.5 (M+H+). HRMS calcd 395.1979, found 395.1973; 1H
NMR (CDCl.sub.3): d 8.329 (d, J=5.0, 2H), 8.101 (d, J=7.5, 2H),
7.734 (d, J=7.5, 2H), 7.655 (s, 1H), 7.509 (m, 6H), 6.530 (t,
J=4.5, 1H), 3.836 (t, J=4.5, J=5.0, 4H), 3.394 (t, J=5.0, J=4.5,
4H).
Scheme 3 (FIG. 9)
[0323] 3-chloro-6-phenylpyridazin-4-ol was synthesized according to
the procedure described by Coudert, P., et al., supra.
4,6-diphenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-5-188WH)
[0324] A mixture of 3-chloro-4,6-diphenylpyridazine (267 mg, 1.0
mmol), 1-(2-pyrimidyl)piperazine (656 mg, 4.0 mmol) in 3 ml of
1-BuOH was heated with stirring at 130.degree. C. for 3 days. The
solvent was removed by evaporation in vacuo the residue was treated
with water to give a suspension. The solid was then filtered off,
washed with water, dried over filter funnel in vacuo to give light
pink solid. (320 mg, 0.81 mmol, yield 81.1%). ESI-MS: m/z 395.5
(M+H+). HRMS calcd 395.1979, found 395.1973; 1H NMR (CDCl.sub.3): d
8.329 (d, J=5.0, 2H), 8.101 (d, J=7.5, 2H), 7.734 (d, J=7.5, 2H),
7.655 (s, 1H), 7.509 (m, 6H), 6.530 (t, J=4.5, 1H), 3.836 (t,
J=4.5, J=5.0, 4H), 3.394 (t, J=5.0, J=4.5, 4H).
D. Preparation of
4-pyridyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-6-189WH)
[0325]
4-pyridyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-6-189WH) was prepared by two synthetic schemes as depicted in
FIGS. 10A and 10B, which were carried out as described in detail
herein. The various reaction schemes (Schemes 1 and 2) are
generally applicable to the compounds of the present invention and
are not restricted in utility only to the preparation of
MW01-2-189WH.
Scheme 1
[0326] 3-chloro-6-phenylpyridazin-4-ol was synthesized according to
the procedure described by Coudert, P., et al., supra.
6-phenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazin-4-ol
(MW01-7-121WH)
[0327] This compound was prepared from
3-chloro-4-hydroxy-6-phenylpyridazine (14 g, 68 mmol). A mixture of
3-chloro-4,6-diphenylpyridazine (267 mg, 1.0 mmol),
1-(2-pyrimidyl)piperazine (656 mg, 4.0 mmol) in 3 ml of 1-BuOH was
heated with stirring at 130.degree. C. for 3 days. The solvent was
removed by evaporation in vacuo the residue was treated with water
to give a suspension. The solid was then filtered off, washed with
water, dried over filter funnel in vacuo to give light pink solid
yielding white solid (22.1 g, 66 mmol, 97.3%). ESI-MS: m/z 335.2
(M+H+). 1H NMR (DMSO): 1H NMR (DMSO): d 8.406 (d, J=6.5, 2H), 7.740
(d, J=4.0, 2H), 7.558 (s, 3H), 6.686 (t, J=4.8, J=4.4, 1H), 6.841
(s, 1H), 3.881 (s, 4H), 3.620 (s, 4H), 3.776 (s, 4H).
4-chloro-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-6-127WH)
[0328] 6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazin-4-ol 1
h (22.0 g, 66 mmol) was suspended in 75 ml phosphorus oxychloride
and heated with stirring at 100.degree. C. for 3 h. After cooling
to room temperature the mixture was poured onto crushed ice. The
mixture was then neutralized with NaOH solution to give white
suspension. The precipitation was filtered off, washed with water,
dried over filter funnel to provide white solid (21.3 g, 60.3 mmol,
91.4%). ESI-MS: m/z 353.4 (M+H+). 1H NMR (CDCl.sub.3): d 8.377 (d,
J=4.5, 2H), 8.036 (d, J=7.5, 2H), 7.833 (s, 1H), 7.508 (m, 3H),
6.564 (t, J=4.5, 1H), 4.073 (t, J=4.0, J=4.5, 4H), 3.672 (t, J=4.0,
J=4.5, 4H).
4-pyridyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-6-189WH)
[0329] Into a reaction tube were added WH-6-127 (1.4 g, 4.0 mmol),
K.sub.2CO.sub.3 powder (1.7 g, 12.4 mmol), Pd(PPh.sub.3).sub.4 (240
mg, 0.2 mmol), 4-pyridineboronic acid (664 mg, 5.4 mmol) and 20 ml
of DME. Argon was then flushed through the tube for 3 min. The tube
was then sealed tightly and heated with stirring at 120 degree for
24 h. After cooled down, the mixture was filter through a celite
earth, the filtrate was then concentrated and the residue was
purified by column chromatography eluting with 1:4, Ethyl
Acetate:Petroleum ether. Light yellow needle crystals were obtained
(0.65 g, 1.65 mmol, yield 41.2%). Confirmed by ESI-MS and NMR.
ESI-MS: m/z 396.2 (M+H+). 1H NMR (CDCl.sub.3): d 8.809 (d, J=6.0,
2H), 8.335 (d, J=5.0, 2H), 8.090 (d, J=7.5, 2H), 7.750 (m, 6H),
6.543 (t, J=4.5, 1H), 3.868 (t, J=5.0, 4H), 3.404 (t, J=5.0,
4H).
Scheme 2
4,5-dihydro-6-phenyl-4-(pyridin-4-yl)pyridazin-3(2H)-one
[0330] To a 200 ml, three-necked, round-bottomed flask equipped
with a magnetic stir bar, 150 ml pressure-equalizing addition
funnel, reflux condenser and a glass stopper, was added 21 g (135
mmole) of 4-bromopyridine and 70 of anhydrous THF. The system was
oven-dried and flushed with argon before use. 135 ml (135 mmole) of
THF solution of phenylmagnesium bromide (1M) was placed in the
pressure-equalizing addition funnel. Then, the Grignard solution
was added dropwise over a period of 10 minutes. After the addition,
the reaction was stirred for 15 minutes for completion. The
solution of Grignard reagent was then obtained. A solution of
4-pyridylmagnesium bromide obtained above was added to a hot
suspension of 6-phenylpyridazinone compound 7.8 g (45 mmole) in dry
toluene (50 ml). The mixture was refluxed for 8 h, left overnight
at ambient temperature, then decomposed with a saturated solution
of ammonium chloride. The organic layer was separated, and the
aqueous layer was extracted with 100 ml of ethyl acetate. The
solvent was removed and the residue was crystallized from ethanol.
The crystals were collected by filtering and dried over a medium
frit sintered glass funnel in vacuo to give 5.6 g of white
crystals. Yield was 50%, confirmed by ESI-MS. ESI-MS: m/z 252.1
(M+H+).
6-phenyl-4-(pyridin-4-yl)pyridazin-3(2H)-one
[0331] 4.4 g (17.5 mmole) of 6-pyridazinone obtained above was
placed in a 50 ml single-necked round bottom flask followed by 4.7
g (35 mmole) of anhydrous copper (II) chloride and then 20 ml of
acetonitrile to give a brown yellow suspension. A reflux condenser
was connected to the flask and a dry tube filled with CaCl.sub.2
was fitted to the top of the condenser. The reaction mixture was
heated to reflux in an oil bath (110.degree. C.) for 3 h. The color
of the reaction suspension changed to dark yellow once the reflux
started. After the completion of the reaction (monitored by HPLC),
the flask was removed from the oil bath and cooled to ambient
temperature. The mixture was poured on to 200 g of crushed ice and
stirred vigorously for 10 minutes to give a gray precipitate and
blue liquid. The precipitate was then collected by filtering (pH of
the filtrate was 1.5-2.0), and washed with 50 ml of a 1N HCl
solution to rid the solid of any remaining copper byproducts. This
is followed by washing with 100 ml of Milli-Q water to get rid of
the acid in the solid, and is monitored by checking the pH value of
the filtrate. The solid was washed until the filtrate shows a pH of
7, after approximately 5 washes. The solid was dried over a medium
frit sintered glass funnel in vacuo to give 3.9 g of a blue gray
solid. Yield was 90%, confirmed by ESI-MS. ESI-MS: m/z 250.1
(M+H+).
3-chloro-6-phenyl-4-(pyridin-4-yl)pyridazine
[0332] 2.0 g (8 mmole) of 6-phenylpyridazinone obtained above and
10 ml (54 mmole) of phosphorus oxychloride (reagent grade, Aldrich)
were placed in a 50 ml single-necked round bottom flask. The flask
was connected with a reflux condenser and a dry tube filled with
CaCl.sub.2 was fitted to the top of the condenser. (HCl gas is
formed in the reaction so a basic solution such as NaOH may be
needed to absorb HCl in a large-scale synthesis). The reaction
mixture was stirred in an oil bath (90.degree. C.) for 2 h, then
cooled to ambient temperature and poured onto crushed ice.
(phosphorus oxychloride can be decomposed by water to give HCl and
H.sub.3PO.sub.4). The mixture was then stirred vigorously for 10
minutes to give a white suspension. The suspension was neutralized
with a 2N NaOH solution until the pH of the suspension was pH=7.
The precipitate was filtered, washed three times with 100 ml of
water and dried over a medium frit sintered glass funnel in vacuo
to provide 1.8 g of a light pink powder. Yield was 85%, confirmed
by ESI-MS. ESI-MS: m/z 268.4 (M+H+).
4-pyridyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-6-189WH)
[0333] 1.1 g (4.0 mmole) of 3-chloropyridazine obtained above was
placed in a 30 ml pressure vessel followed by addition of 2.6 g
(16.0 mmole) of 1-(2-pyrimidyl)piperazine and then 15 ml of 1-BuOH
(reagent grade). The vessel was sealed tightly and placed into an
oil bath and stirred at 130.degree. C. (temperature of oil bath)
for 3 days. The reaction mixture was then cooled to ambient
temperature and transferred to a single-necked flask for
evaporation under reduced pressure. Removal of solvent gave rise to
a brown-red residue that was treated with 30 ml of water to give a
brown suspension. The solid was collected by filtering and washed
with 50 mL of water three times and dried over a filter funnel in
vacuo to provide 0.96 g of light yellow solid. Yield was 90%,
confirmed by ESI-MS and NMR. ESI-MS: m/z 396.2 (M+H+). 1H NMR
(CDCl.sub.3): d 8.809 (d, J=6.0, 2H), 8.335 (d, J=5.0, 2H), 8.090
(d, J=7.5, 2H), 7.750 (m, 6H), 6.543 (t, J=4.5, 1H), 3.868 (t,
J=5.0, 4H), 3.404 (t, J=5.0, 4H).
E. Preparation of
N-(cyclopropylmethyl)-6-phenyl-4-(Pyridin-4-yl)pyridazin-3-amine
(MW01-7-084WH)
[0334] A synthetic scheme for the preparation of
N-(cyclopropylmethyl)-6-phenyl-4-(pyridin-4-yl)pyridazin-3-amine
(MW01-7-084WH) is depicted in FIG. 11, and synthesis was carried
out as described herein.
4-chloro-6-phenylpyridazin-3(2H)-one (MW01-6-093WH)
[0335] 4-chloro-6-phenylpyridazin-3(2H)-one was synthesized
according to the procedure described by Coudert, P. [18].
4-chloro-2-(methoxymethyl)-6-phenylpyridazin-3(2H)-one
(MW01-7-053WH)
[0336] A mixture of chloropyridazinone 1 (25.5 g, 0.12 mol),
4-N,N-dimethylaminopyridine (0.20 g) and i-Pr.sub.2NEt (26.7 g,
0.21 mol) in anhydrous CH.sub.2Cl.sub.2 (300 mL) was stirred at
0.degree. C. (ice bath) for 30 min. Methoxymethyl chloride (25 g,
0.31 mol) was added and the mixture was stirred at 0.degree. C. for
1 h and then allowed to warm to room temperature. The reaction was
stirred at room temperature till complete. The solvent was then
removed in vacuo, the residue was treated with water, washed with
dilute Na.sub.2CO.sub.3 solution and extracted with EtOAc. The
organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered
and evaporated. The residue was then purified by recrystallization
from 95% ethanol to give 20.1 light yellow solid. Yield 66.9%.
6-phenyl-4-(pyridin-4-yl)pyridazin-3(2H)-one (MW01-7-069WH)
[0337] The protected pyridazinone MW01-7-053WH (1.0 equiv.) was
mixed with arylboronic acid (1.37 equiv.), Pd(PPh.sub.3).sub.4
(0.05 equiv.) and K.sub.2CO.sub.3 (3.1 equiv) and 200 mL of DME in
a 350 ml of pressure vessel, flushed with argon for 3 min, and the
mixture was then stirred and refluxed (oil bath, 120.degree. C.)
until the starting material had disappeared. After cooling, the
solution was concentrated to dryness under reduced pressure, the
residue was treated with water and filtered off. The filter cake
was washed with water over filter funnel and then used for next
step directly. The residue obtained above was dissolved in 200 ml
of EtOH, 6 N HCl (200 mL) was added and the reaction mixture was
refluxed (oil bath, 120.degree. C.) for 6 h, then it was allowed to
cool to room temperature, and concentrated to dryness under reduced
pressure. The residue was neutralized with dilute NaOH solution.
The suspension was then filtered off, washed with water and dried
over a filter funnel. Recrystallization from 90% ethanol provided
brown yellow solid. Yield 80.4%. ESI-MS: m/z 294.3 (M+H+)
3-chloro-6-phenyl-4-(pyridin-4-yl)pyridazine (MW0'-7-076WH)
[0338] 3-chloro-6-phenyl-4-(pyridin-4-yl)pyridazine (MW01-7-076WH)
(66 mmol) was suspended in 75 ml phosphorus oxychloride and heated
with stirring at 100.degree. C. for 3 h. After cooling to room
temperature the mixture was poured onto crushed ice. The mixture
was then neutralized with NaOH solution to give white suspension.
The precipitation was filtered off, washed with water, dried over
filter funnel to yielding a light yellow solid. ESI-MS: m/z 268.4
(M+H+).
N-(cyclopropylmethyl)-6-phenyl-4-(pyridin-4-yl)pyridazin-3-amine
(MW01-7-084WH)
[0339] A mixture of
N-(cyclopropylmethyl)-6-phenyl-4-(pyridin-4-yl)pyridazin-3-amine
(MW01-7-084WH) (0.5 mmol), C-Cyclopropyl-methylamine (2.0 mmol) in
3 ml of 1-BuOH was heated with stirring at 130.degree. C. for 7
days. The solvent was removed by evaporation in vacuo, the residue
was treated with water to give a suspension. The solid was then
filtered off, washed with water, then 1:3, Ethyl Acetate:Petroleum
ether, dried over filter funnel in vacuo yielding gray solid.
ESI-MS: f/z 330.4 (M+H+).
F. Preparation of
3-(4-methylpiperazin-1-yl)-6-phenyl-4-(pyridin-4-yl)pyridazine
(MW01-7-085WH)
[0340] A mixture of 3-chloro-6-phenyl-4-(pyridin-4-yl)pyridazine
(MW01-7-076WH) (0.5 mmol), 1-methyl-piperazine (2.0 mmol) in 3 ml
of 1-BuOH was heated with stirring at 130.degree. C. for about 7
days. The solvent was removed by evaporation in vacuo the residue
was treated with water to give a suspension. The solid was then
filtered off, washed with water, then 1:3, Ethyl Acetate:Petroleum
ether, dried over filter funnel in vacuo to yield a brown solid.
ESI-MS: m/z 332.2 (M+H+). A synthetic reaction scheme for the
preparation of
3-(4-methylpiperazin-1-yl)-6-phenyl-4-(pyridin-4-yl)pyridazine
(MW01-7-085WH) is depicted in FIG. 12.
G. Preparation of 4,6-diphenyl-3-piperazinylpyridazine
(MW01-7-133WH)
[0341] A synthetic reaction scheme for the preparation of
4,6-diphenyl-3-piperazinylpyridazine (MW01-7-133WH) is depicted in
FIG. 13, and synthesis was carried out as described herein. The
compound was prepared from 3-chloro-4,6-diphenylpyridazine (533 mg,
20 mmole) in the same manner as described for MW01-7-057WH,
yielding light yellow solid (550 mg, 17.4 mmole, yield 86.9%).
ESI-MS: m/z 317.3 (M+H+). 1H NMR (CDCl.sub.3): d 8.086 (d, J=7.5,
2H), 7.705 (d, J=7.5, 2H), 7.619 (s, 1H), 7.498 (m, 6H), 3.318 (d,
J=4.0, 4H), 2.932 (d, J=4.0, 4H) 1.896 (s, 1H).
H. Preparation of
2-(4-(6-phenyl-4-(Piperidin-1-yl)pyridazin-3-yl)piperazin-1-yl)pyrimidine
(MW01-7-107WH)
[0342] A synthetic reaction scheme for the preparation of
2-(4-(6-phenyl-4-(piperidin-1-yl)pyridazin-3-yl)piperazin-1-yl)pyrimidine
(MW01-7-107WH) is depicted in FIG. 14, and synthesis was carried
out as described herein. The compound was prepared from
MW01-6-127WH (200 mg, 0.57 mmole) in the same manner as described
for MW01-7-057WH, yielding light yellow solid (220 mg, 0.55 mmole,
yield 96.3%). ESI-MS: m/z 402.5 (M+H+).
I. Preparation of
6-methyl-4-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-7-057)
[0343] A synthetic reaction scheme for the preparation of
6-methyl-4-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine
(MW01-7-057) is depicted in FIG. 15, and synthesis was carried out
as described herein. A mixture of
3-chloro-6-methyl-4-phenylpyridazine (100 mg, 0.5 mmol),
1-(2-pyrimidyl)piperazine (400 mg, 2.0 mmol) in 3 ml of 1-BuOH was
heated with stirring at 130.degree. C. for 7 days. The solvent was
removed by evaporation in vacuo the residue was treated with water
to give a suspension. The solid was then filtered off, washed with
water, then 1:3, Ethyl Acetate:Petroleum ether, dried over filter
funnel in vacuo to give light yellow solid (68 mg, 0.20 mmol, yield
41.7%). Purity >95%; ESI-MS: m/z 333.1 (M+H+). 1H NMR
(CDCl.sub.3): d 8.310 (d, J=5.0, 2H), 7.678 (d, J=7.5, 2H), 7.476
(m, 3H), 7.119 (s, H), 6.509 (t, J=4.5, 1H), 3.785 (t, J=4.5,
J=5.0, 4H), 3.277 (t, J=4.5, J=5.0, 4H), 2.669 (s, 3H).
Example 2
Assays for Confirming Activity of Pyridazine Compounds
[0344] The following assays can be used to confirm the activity of
the pyridazine compounds. Cell culture assays. Cell-based assays of
the concentration-dependent activity of a compound of the invention
will be conducted using methods previously described (Mirzoeva et
al., J Med Chem 45:563-566, 2002). BV-2 mouse microglial cells
(1.25.times.10.sup.4 cells/well in a 48-well plate) will be
cultured for one day in .alpha.MEM media containing 10% fetal
bovine serum (FBS), and then treated in serum-free media for 16 hrs
with either control buffer or the standard glial activating
stimulus lipopolysaccharide (LPS, from Salmonella typhimurium; 100
ng/ml final concentration) in the presence of diluent or compound.
Stock solutions (20 mM) of compounds will be prepared in
dimethylsulfoxide (DMSO). Solutions for cell treatments will be
prepared by dilution of stock solutions into serum-free media
immediately before adding to the cells. Control wells will contain
the same final concentration of DMSO as the compound-containing
wells. It has been previously determined that this concentration of
DMSO is not toxic to the cells (Mirzoeva et al., Brain Res.
844:126-134, 1999). The accumulation of nitrite, the stable
metabolite of nitric oxide (NO), will be measured in BV-2
conditioned media by the Griess assay as previously described
(Mirzoeva et al., Brain Res. 844:126-134, 999; Mirzoeva et al., J
Med Chem 45:563-566, 2002). Levels of IL-1.beta. in cell lysates
and TNF.alpha. in conditioned media will be measured by ELISA
(Biosource International) as per the manufacturer's instructions.
Cell lysates will be analyzed by Western blots as described
(Mirzoeva et al., J Med Chem, 2002) to determine the levels of
inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)
and apolipoprotein E (apoE). For apoE measurements, rat primary
mixed glia will be prepared and stimulated with human oligomeric
A.beta..sub.1-42 (10 .mu.M) as previously described (Mirzoeva et
al., 2002, supra). Antibodies and dilutions used for Western blots
will be as follows: anti-COX-2 (1:1000, Santa Cruz), anti-iNOS
(1:1000, Transduction Laboratories), anti-apoE (1:1000). Antibody
against M-actin (1:500,000 dilution, Sigma) will be used to confirm
equal protein loading among the samples. In vivo efficacy studies
in mice. The study design and treatment paradigm for
intracerebroventricular (ICV) infusion of human oligomeric
A.beta..sub.1-42 into the mouse will be as described previously
(Craft et al., Neurobiol Aging 25:1283-1292, 2004b), except that
compound administration will be by mouth. Female C57Bl/6 mice
(Harlan) weighing 20-25 g (3-4 months old) will be housed in a
pathogen free facility under an approximate 12 h/12 h dark and
light cycle and they will have access ad libitum to food and
water.
[0345] Mice will be administered by oral gavage either test
compound (2.5 mg/kg/day) or solvent control (10% DMSO) in a 0.5%
(w/v) carboxymethylcellulose suspension. Once per day treatment
will begin at day 21 after start of A.beta. ICV infusion and
continue for 14 days. Beginning at day 50 after start of A.beta.
ICV infusion, the Y maze test of spontaneous alternation will be
used to evaluate hippocampus-dependent spatial learning as
described previously (Craft et al., J Mol Neurosci 24:115-122,
2004a). Briefly, each mouse will be placed in the "start" arm and
then released to choose one of the two other arms. The mouse will
be blocked from exiting the chosen arm for 30s then they will be
placed back in the start arm and released again to choose one of
the two other arms. If the second choice is different from the
first one, the mouse will be scored as alternating. Mice will be
tested for 10 days with one trial per day, and a mean percent
alternation will be calculated for each mouse. At day 60 after
start of A.beta. ICV infusion, mice will be anesthetized with
pentobarbital (50 mg/kg) and perfused with a HEPES buffer (10 mM,
pH 7.2) containing a protease inhibitor cocktail (1 .mu.g/ml
leupeptin, 1 .mu.M dithithreitol, 2 mM sodium vanadate, 1 .mu.M
phenylmethylsulphonylfluoride). The brain will be removed and
longitudinally bisected as described previously (Craft et al.,
Neurobiolo Aging 25:1283-1292, 2004b). The right half of the brain
will be fixed in 4% (v/v) paraformaldehyde and paraffin-embedded
for histology. The hippocampus will be dissected from the left half
of the brain and snap-frozen for subsequent biochemical evaluation.
Hippocampal extract supernatants will be prepared by dounce and
sonication in the HEPES buffer containing a protease inhibitor
cocktail, followed by centrifugation as described (Craft et al.,
2004b, supra).
[0346] Levels of IL-1.beta. and TNF.alpha. in hippocampal
supernatants will be measured by ELISA (Biosource International)
per the manufacturer's instructions. S100B levels in hippocampal
supernatants will be measured by a europium-based ELISA essentially
as previously described (Van Eldik and Griffin, Biochem Biophys
Acta 1223:398-403, 1994). Synaptophysin levels in hippocampal
supernatants will be quantified by ELISA following the procedure
described previously (Craft et al, 2004b, supra). PSD-95 levels
will be determined by Western blots using anti-PSD-95 antibodies
(1:100,000 dilution; Upstate Biotechnology) as described (Craft et
al., 2004b).
[0347] Immunohistochemical detection of activated astrocytes and
microglia will be performed on 10 .mu.m sections as described
previously (Craft et al, 2004b, supra), with anti-GFAP (1:1500;
Sigma) and anti-F4/80 (1:100; Serotek) antibodies, respectively,
using the mouse on mouse or Vectastain Universal Elite ABC
immunodetection kits (Vector/Novocastra) and development with
diaminobenzidine (DAB) substrate. Cell bodies will be manually
counted in the hippocampus of three GFAP and F4/80 labeled sections
positioned at -1.8, -2.1, and -2.3 mm from bregma. A.beta.
immunohistochemistry will be done with a rabbit anti-human A.beta.
antibody as previously described (Craft et al., 2004b, supra). Cell
counts and amyloid plaque counts will be determined by two blinded
observers and amyloid plaque area will be determined as previously
described (Craft et al., 2004b, supra). Peroxynitrite-mediated
neuronal damage will be measured with an anti-nitrotyrosine
antibody (1:125; Chemicon), using the Vectastain Rabbit Elite ABC
kit. For nitrotyrosine cell counts, all DAB-stained cell bodies in
the neuronal layers of the hippocampus and subiculum will be
counted on three sections roughly adjacent to those used for F4/80
and GFAP analysis, as described (Craft et al., 2004b, supra).
[0348] In vitro stability, oral bioavailability and brain uptake.
The stability of compounds (1 .mu.M) in a standard incubation with
rat liver microsomes (BD Biosciences) and an NADPH-regenerating
system will be done at 37.degree. C. for 30 and 120 min. Reactions
will be stopped by acetonitrile, and the reaction mixture will be
centrifuged at 16 000.times.g for 10 min. 10 .mu.l of the
supernatant will be analyzed by calibrated HPLC to quantify the
percentage of the initial amount of compound remaining after the
incubation. The HPLC system (Dionex Corp., Sunnyvale, Calif.)
includes a Dionex P480 pump, a Phenomenex Luna C18 column
(250.times.2.0 mm, 5 .mu.m) with a guard column (Phenomenex,
Torrance, Calif.) and a Dionex UVD340U Ultraviolet (UV) detector.
The mobile phase will consist of 0.1% formic acid as reagent A and
0.08% formic acid/water in 80% acetonitrile as reagent B, at a flow
rate of 0.2 ml per minute. The gradient will consist of the
following linear and isocratic gradient elution changes in reagent
B: isocratic at 60% from 0 to 5 min, 60% to 90% from 5 to 39 min,
isocratic at 90% until 44 min. Peak quantification will be done
based on absorption measured at 260 nm relative to a standard curve
obtained by using serial dilutions of the compound.
[0349] To estimate oral bioavailability (concentration of compound
in the blood as a function of time after oral administration) and
to gain insight into potential brain uptake, a compound (2.5 mg/kg)
will be administered to mice by oral gavage in a 0.5% (w/v)
carboxymethylcellulose suspension. At 5, 15, 60 and 120 min after
compound administration, the animals will be anesthetized with
pentobarbital (50 mg/kg). Blood will be harvested by intracardiac
puncture, collected in heparinized tubes, and plasma will be
obtained by centrifugation. Mice will be perfused with a HEPES
buffer (10 mM, pH 7.2) containing a protease inhibitor cocktail (1
.mu.g/ml leupeptin, 1 .mu.M dithithreitol, 2 mM sodium vanadate, 1
.mu.M phenylmethylsulphonylfluoride), and brains will be removed
and weighed. Brain homogenates will be prepared by dounce and
sonication in the HEPES buffer containing a protease inhibitor
cocktail. Brain homogenates will be centrifuged at 12000.times.g
for 10 minutes and the supernatant acidified by diluting 1:3 with
0.1% formic acid (Fluka). Solid phase extraction followed by HPLC
analysis will be used to quantify the amount of compound in brain
supernatants. Briefly, cartridges (Sep-Pak.RTM. C18, Waters) will
be conditioned with 1 ml of acetonitrile (HPLC grade, EMD
Biosciences) and equilibrated with 1 ml of water. A structural
analog of the compound will be used as an internal standard. The
acidified brain supernatant will be added to the cartridge followed
by a 1 ml wash with 30% acetonitrile. The compound will be eluted
from the cartridge using 80% acetonitrile. The eluate will be
evaporated to dryness, reconstituted in 0.08% formic acid/water in
80% acetonitrile and analyzed by HPLC using the following gradient
in reagent B: 0% to 60% from 2 to 5 min, isocratic at 65% until 7
min, 65% to 80% from 7 to 12 min, isocratic at 80% until 15 min,
89% to 100% from 15 to 18 min and isocratic at 100% until 23 min.
Plasma samples will be deproteinized in 0.1M perchloric acid and
centrifuged at 12000.times.g for 10 min. The supernatant will be
neutralized with 1M NaOH then extracted with dichloromethane, and
the layers separated at 3000.times.g for 5 min. The organic phases
from three successive extractions will be pooled and then
evaporated to dryness under reduced pressure. The dried residue
will be reconstituted in 50 .mu.l of reagent B, and 10 .mu.l of the
reconstituted material will be analyzed by HPLC using the gradient
described above for brain supernatants.
[0350] Suppression of CNS versus peripheral inflammation. Mice will
be administered by oral gavage of compound (2.5 mg/kg/day) or
diluent (10% DMSO) in a 0.5% (w/v) carboxymethylcellulose
suspension once daily for two weeks. After the last administration,
mice will be injected intraperitoneally (i.p) with 10 mg/kg of LPS.
Control mice will be injected with saline. Six hours after the LPS
challenge, mice will be anesthetized with pentobarbital (50 mg/kg)
and blood will be drawn by intracardiac puncture, allowed to clot,
and centrifuged for serum preparation. Brains will be removed and
processed as described above. Levels of IL-1.beta. and TNF.alpha.
in brain supernatants and serum will be measured using a MSD
multiplex assay per the manufacturer's instructions (Meso Scale
Discovery, Gaithersburg, Md.).
[0351] Liver toxicity after chronic in vivo administration of
Compound. Mice will be administered by oral gavage either test
compound (2.5 mg/kg/day) or diluent (10% DMSO) in a 0.5% (w/v)
carboxymethylcellulose suspension once daily for two weeks. Mice
will be anesthetized and sacrificed as described above. Livers will
be removed, fixed in 4% (v/v) paraformaldehyde and
paraffin-embedded for histology. To assess histological toxicity, 4
.mu.m liver sections will be stained with haematoxylin and eosin.
Two independent observers blinded to the treatment groups will
perform microscopic assessment of the tissue for injury.
[0352] Morris Water Maze. This test is based on the swimming maze
test for spatial memory (Morris, Learn Mot 12:239-260, 1981; J
Neurosci Methods 11:47-60, 1984) and takes advantage of the natural
swimming ability of rodents and the ease of manipulating cues
around the maze. In this task, a mouse is placed in a pool of
liquid that is made opaque by the addition of non-toxic tempera
powdered paint. The mouse then swims until an escape platform
(hidden just under the surface of the water) is found. Finding the
platform enables the mouse to escape from the water and therefore
is positively reinforced. When the platform is kept in the same
position, the animal quickly learns to use distal cues to locate
the position of the platform, even if the mouse is placed in the
pool at different starting positions. The experimental protocol for
the Morris maze test is as described in Ohno et al, (Eur. J.
Neurosci. 2006, 23(8): 2235-40; Learn Mem 2005, 12(3): 211-5).
Briefly, the pool is 1.2 m in diameter and made of white metal. The
water is maintained at 25.+-.1.degree. C. and is made opaque with
nontoxic white paint to hide the square, white escape platform (10
cm.times.10 cm). During training, the platform is submerged (1 cm)
below the water surface and remains in the same position to avoid
quandrant biases. The mice receive six trials per day for 4 days (3
blocks of two trials; 1 min intertrial intervals, 1-hour interblock
intervals). The mouse is placed into the water facing the wall of
the pool and is allowed to search for the platform. The starting
position varies among four locations in a pseudorandom manner for
each trial. The trial ends when an animal climbs onto the platform
or when a maximum of 60 sec has elapsed. The mouse is placed on the
platform for 60 sec before and after each trial. At the end of the
training, all mice are given a probe test with the platform removed
from the pool. The behaviour of the mouse is recorded by a video
camera and analyzed computationally for several parameters such as
latency to finding the platform, total distance traveled, and
percent of time spent in the target quadrant.
[0353] At post-operative day 60 mice will be anesthetized and
perfused with a Hepes buffer containing a protease inhibitor
cocktail. The brains are then removed and longitudinally bisected.
The right half of the brain is fixed in a
paraformaldehyde/phosphate buffer solution and embedded in paraffin
for histological examination, while the hippocampus is isolated
from the left hemisphere and snap frozen for biochemical evaluation
of endpoints.
Example 3
Efficacy in the Tg6799 5X FAD Mouse Model
[0354] MW01-2-151SRM will be tested in the Tg6799 mouse at 5, 10
and 25 mg/kg. As above, neuroinflammation and synaptic dysfunction
biochemical endpoints and Y-maze behavioral endpoint will be
determined. A higher dose is proposed based on the start of
administration to animals that are already showing signs of
pathology based on characterization of strain. More animals needed
for significance are compared to the infusion model and longer time
due to required expansion of colony via breeding.
Example 4
Selection of Lead Drug Compound
[0355] The following eight compounds were synthesized:
MW01-4-179LKM; MW01-2-151SRM; MW01-7-107WH; MW01-6-189WH;
MW01-7-084WH; MW01-7-085WH7) MW01-7-133WH; and MW01-7-057WH (See
FIGS. 1 to 15 and Example 1).
A. The compounds were tested in glial cell-based assays for
concentration-dependent suppression of neuroinflammation endpoints
(nitric oxide, IL-1.beta.). All eight compounds inhibited
LPS-induced IL-1.beta. production in BV-2 microglia cells in a
concentration-dependent manner. Most compounds were also selective,
in that they did not inhibit production of nitric oxide (NO). The
lack of an effect on NO production was further validated by showing
no effect on up-regulated levels of iNOS. No effect over the same
concentration range was seen on up-regulation of COX-2. The
following were selective compounds: MW01-2-151SRM; MW01-4-179LKM;
MW01-6-189WH; MW01-7-084WH; MW01-7-085WH; MW01-7-133WH; and
MW01-7-057WH. One compound, MW01-7-107WH, was non-selective in that
it also inhibited production of NO, iNOS and COX-2 over the same
concentration ranges. (See FIGS. 16 to 23 showing the results of
the cell-based activity of MW01-2-151SRM; MW01-6-189WH;
MW01-4-107WH; MW01-4-179LKM; MW01-7-084WH; MW01-7-085WH;
MW01-7-133WH; and MW01-7-057WH in BV-2 microglial cells.) B.
Testing of Compounds in the human A.beta. infusion mouse model for
suppression of neuroinflammation and neuronal dysfunction
biochemical endpoints (IL-1.beta., S100B, synaptophysin).
Specifically, the following five active compounds were tested in
vivo: MW01-2-151SRM; MW01-6-189WH; MW01-7-084WH MW01-7-085WH; and
MW01-7-057WH. The best compounds in vivo were MW01-2-151SRM and
MW01-6-189WH. These two compounds blocked the up-regulation of
IL-1.beta. and S100B, and prevented the loss of PSD-95.
MW01-2-151SRM also prevented the loss of synaptophysin.
MW01-6-189WH showed a trend toward preventing the synaptophysin
loss; however, statistical significance was not reached due to
limitations in sample size. MW01-7-084WH and MW01-7-085WH blocked
the upregulation of IL-1.beta. and S100B, and prevented loss of
PSD-95. They were not as effective as MW01-2-151SRM in preventing
the synaptophysin loss. MW01-7-057WH blocked S100B upregulation and
synaptophysin loss, but did not block IL-1.beta. upregulation or
PSD-95 loss. (See FIGS. 24 to 28 showing the results of in vivo
activity of MW01-2-151SRM; MW01-6-189WH; MW01-7-084WH;
MW01-7-085WH; and MW01-7-057WH in the A.beta. infusion mouse
model.) C. The lead compounds were tested in the human A.beta.
infusion mouse model using the Y-maze behavioral assay at 1.25,
2.5, 5, and 10 mg/kg. Neuroinflammation biochemical endpoints
(hippocampus levels of IL-1.alpha., TNF.alpha.) are based on
proposed mechanism of action, and a synaptic dysfunction
biochemical endpoint (hippocampus levels of synaptophysin) is used,
as well as a Y-maze behavioral endpoint. MW01-2-151SRM,
MW01-6-189WH, and MW01-7-057WH were significantly effective in
preventing the Y-maze behavioral deficit brought about by human
A.beta. infusion. MW01-7-084WH and MW01-7-085WH showed a trend
toward preventing the Y maze behavioral deficit.
Example 5
hERG Channel Inhibition Assays and Cardiac QT Interval Assays
[0356] Compounds have been screened for hERG (human ether-a-go-go)
potassium ion channel binding and inhibition in order to eliminate
early in the process any compounds with high potential to induce
prolongation of cardiac QT interval in later studies due to
off-target toxicities. The hERG channel conducts rapidly activating
delayed rectifier potassium currents that critically contribute to
cardiac repolarization. Mutations in the hERG channel gene and
drug-induced blockade of the currents have been linked to delayed
repolarization of action potentials resulting in prolonged QT
interval (Finlayson et al., 2004; Recanatini et al., 2005; Roden,
2004). QT prolongation is considered a significant risk factor
against cardiac safety of new drugs. Therefore, consideration of
cardiac safety early in the development process by testing for hERG
channel inhibition provides an efficient and predictive means to
assess potential compound cardiac safety liabilities. In addition,
the FDA (USA) is considering this as an approval criteria in the
future and has specific recommendations. The assays done to date
have been by a commercial service (MDS PharmaService).
[0357] The initial assay is a radioligand binding assay that tests
the ability of the test compound to compete with .sup.3H-astemizole
(a reference standard that binds to hERG channels with nM affinity)
for binding to recombinant hERG channels stably expressed on human
HEK-293 cells. This cell line was chosen because it is of human
origin, has been fully characterized with regard to hERG
electrophysiology and pharmacology and displays the expected
characteristics of I.sub.Kr current as well as expected
pharmacological sensitivities, and is easy to maintain in culture
(Zhou et al., J. Gen Physiol. 1998, 111(6): 781-94). A single
concentration (10 .mu.M) of test compound is assayed, and %
inhibition of .sup.3H-astemizole binding is calculated. Generally,
any compounds that show >50% inhibition are tested further in
the hERG channel activity assay. This is usual for medium
throughout screens but is not recommended in the FDA document and
tends to give false positives, as evidenced by the results reported
below.
[0358] The hERG channel activity inhibition assay provides whole
cell electrophysiological data about compound effects on the hERG
K.sup.+ channel function. Whole cell patch clamp methodology is
generally considered to be the gold-standard determination of ion
channel activity, rather than simply measuring channel binding. The
standard testing procedure is to use 3 to 5 concentrations of
compound at log dilutions with each concentration tested in
triplicate (three cells). This allows a balance between achieving a
reasonably accurate IC.sub.50 measurement against a broad
concentration range, and reducing cell attrition that would occur
during more protracted experiment durations. After completion of
compound dose-response procedures, a known hERG channel inhibitor,
such as astemizole, is applied as a positive control.
[0359] Compounds which exhibit inhibition of hERG channel activity
are verified as positives (the hERG channel activity assay can give
false positives and false negatives) by testing in vivo for
prolongation of cardiac QT interval. The QT interval studies are
performed by evaluating compounds for effects on QT interval in
Lead II electrocardiograms measured in anesthetized guinea pigs
(Hirohashi et al., 1991, Arzneim.-Forsch./Drug Res 41:9-18), one of
the species recommended in the FDA white paper. Vehicle or compound
is administered orally at 15 mg/kg (dosing volume of 10 ml/kg) to
groups of male guinea pigs (weighing 330-350 g), with 5 animals per
group. This dose corresponds approximately to 20-fold the
therapeutic dose by taking into account the body surface area of
the animals. Heart rate, arterial blood pressure, and QT intervals
are measured at baseline, and at 15, 30, 45, and 60 min after
compound administration. Sotalol administered iv at 0.3 mg/kg
serves as the positive control compound. The QT intervals are
corrected for changes in heart rate using both Bazett's and
Fridericia's formulae. Any increase in QT interval values over
baseline values exceeding the upper 95% confidence limit of the
mean changes at the corresponding time point in the vehicle-treated
control group for two consecutive observation times indicates
significant QT interval prolongation in the individually treated
animals. This functional testing in early discovery provides a
rapid and cost-effective method to better anticipate and eliminate
compounds that may have adverse QT prolongation potential in
humans.
Calculations of Amount of Compound Needed:
[0360] Competition binding assay: 1-2 mg Patch clamp assay: 1-2 mg
QT interval assay: 5 mg/animal/dose=25 mg per assay at 15 mg/kg
dose
[0361] Because the ex vivo activity assays are subject to false
positives and negatives, it is considered better to complete
studies of in vivo QT interval assay following the guidelines of
the FDA position paper.
Results:
Competition Inhibition Assay:
[0362] MW01-5-188WH, MW01-2-151SRM, and MW01-6-127WH were tested at
10 .mu.M concentration.
[0363] MW01-5-188WH showed 91% inhibition at 10 .mu.M.
MW01-2-151SRM and MW01-6-127WH were negative, showing only 8% and
19% inhibition, respectively.
Patch Clamp Inhibition Assay:
[0364] MW01-2-151SRM and MW01-6-189WH were tested at three
concentrations (0.1, 1, 10 .mu.M). These compounds showed minimal
inhibition, with IC.sub.50 values of 4.81 .mu.M for MW01-6-189WH
and 9.21 .mu.M for MW01-2-151SRM.
Cardiac QT Interval Prolongation Assay
[0365] A summary of the results as well as the materials and
methods are set out below.
Summary
[0366] A test substance (e.g., MW01-2-151SRM) was evaluated for
possible effects on QT interval in Lead II electrocardiogram
measure in anesthetized guinea pigs. The QT intervals (QTc) were
corrected for changes in heart rate using both Bazett's and
Fridericia's formulae. Any increase in QTc values over baseline
values exceeding the upper 95% confidence limit of the change at
corresponding time point in the vehicle-treated control group for 2
consecutive observation times indicates significant QTc
prolongation in the individually treated animals. The test
substance at 15 mg/kg PO did not cause any significant prolongation
in QTc interval in all of the 5 treated animals during the
60-minute period post-dosing (FIGS. 29 and 31). On the other hand,
intravenous administration of sotalol at 0.3 mg/kg caused
significant prolongation in QTc interval in all (5.5) animals
(FIGS. 30 and 32). The results reached similar conclusion by using
either Bazett's or Fridericia's formula for QT correction.
[0367] MW01-5-188WH and MW01-2-151SRM were administered PO at 15
mg/kg to 5 guinea pigs (330-350 g weight). QT intervals were
obtained at baseline and at 15 min, 30 min, 45 min, and 60 min
after compound administration. Neither compound increased cardiac
QT interval above the mean+2SD of corresponding values in the
vehicle control group. There were also no significant effects on
mean blood pressure or heart rate after compound
administration.
[0368] Example data for MW01-5-188WH are shown in FIG. 33. The
positive control compound, sotalol, induces a significant increase
in cardiac QTc interval.
Materials and Methods
[0369] The test substance was dissolved in 2% Tween 80 and
administered by oral administration. The substance was treated at
15 mg/kg with a dosing volume of 10 ml/kg with a dosing volume of
10 ml/kg. Duncan Hartley derived guinea pigs provided by MDS Pharma
Services--Taiwan Ltd were used. Sotalol was obtained from Sigma,
USA.
[0370] Groups of guinea pigs (weighing 330-350 g) with 5 animals
each were employed. The animals were anesthetized with urethane
(1500 mg/kg, IV bolus injection in a volume of 5 ml/kg) and
breathed spontaneously. Lead II ECG was obtained with subdermal
needle electrodes and ECG signal conditioner. Heart rate was
measured with a pulse rate tachometer. The carotid artery was
cannulated with a catheter that was connected to a pressure
transducer and a pressure processor for measurements of arterial
blood pressure (BP). Five parameters [HR, Q-T Interval,
QTc(Bazett's), QTc(Fredericia's), BP] were recorded and displayed
on a Digital Acquisition Analysis and Archive System (PO-NE-MAH,
Inc. USA). QTC intervals were obtained by correction for heart rate
changes using Bazett's and Fridericia's formulae. Increase in QTc
interval in individual treated guinea pigs that lies outside the
upper limit of 95% confidence limits (mean.+-.SD) of the changes
for the vehicle-treated control at corresponding time points for
two consecutive times is considered significant.
Example 6
Acute and Chronic Toxicity Assays
[0371] Liver toxicity is an especially important initial
consideration for orally administered compounds, as the liver is
the major site of initial drug metabolism and is critical to
overall metabolism and homeostasis of an animal. Liver injury is
also a component of idiopathic tissue injury seen in certain
chronically administered drugs. Therefore, it is important to do
initial assessments of liver toxicity after oral administration of
compounds to mice.
Methods:
[0372] A standard approach is to test compounds in two initial in
vivo toxicity assays: an acute, escalating-dose paradigm and a
chronic, therapeutic dose regimen. For the escalating-dose, acute
toxicity assays, mice (5 per experimental group) are administered
either compound or vehicle in 0.5% carboxymethylcellulose
(alternatively, castor oil or sesame oil can be used) by oral
gavage once daily for 3 days. Standard compound doses are 3.1,
12.5, and 50 mg/kg; the highest dose is 20.times. a therapeutic
dose. On the 4.sup.th day, mice are sacrificed and the liver
harvested and fixed for histology. Paraffin-embedded, hematoxylin
& eosin (H&E)-stained sections of liver tissue are analyzed
microscopically for injury by two individuals blinded to the
treatment groups. A semi-quantitative histological scoring system
from 0 (best) to 9 (worst) is applied that considers architecture
features (normal to extensive fibrosis), cellular features (normal
to extensive edema and widespread necrosis), and degree of
inflammatory infiltrate (normal to extensive infiltrate). For each
acute toxicity assay, 15 mg of compound is required.
[0373] For the therapeutic dose, chronic toxicity assays, mice (5
per experimental group) are administered either compound or vehicle
in 0.5% carboxymethylcellulose by oral gavage once daily for 2
weeks at a therapeutic dose of 2.5 mg/kg/day. After two weeks of
treatment, mice are sacrificed and liver toxicity analyzed as
described above. For each chronic toxicity assay, 5 mg of compound
is required.
Results:
[0374] The results of the toxicity study are shown in FIG. 34.
[0375] MW01-5-188WH has been tested in the acute, escalating-dose
assay and the chronic, therapeutic dose assay. There was no
histological evidence of tissue toxicity at the lower doses but
some vacuolisation was observed at the 50 mg/kg dose.
[0376] MW01-2-151SRM has been tested in the chronic, therapeutic
dose assay. There was no histological evidence of tissue toxicity;
no differences were seen by histology in livers from mice treated
with vehicle or with compound.
[0377] MW01-6-189WH has been tested in the chronic, therapeutic
dose assay. There was no histological evidence of tissue toxicity;
no differences were seen by histology in livers from mice treated
with vehicle or with compound.
[0378] MW01-5-188WH was tested in the chronic, therapeutic dose
assay. In particular, mice were administered by oral gavage either
MW01-5-188WH (2.5 mg/kg) or diluent (10% DMSO) in a 0.5% (w/v)
carboxymethylcellulose suspension once daily for 2 weeks. Mice were
anesthetized and killed as described above. Livers were removed,
fixed in 4% (v/v) paraformaldehyde, and paraffin-embedded for
histology. To assess histological toxicity, 41m liver sections were
stained with hematoxylin and eosin. Two independent observers
blinded to the treatment groups performed microscopic assessment of
the tissue for injury. Histological assessment of liver tissue
showed that oral administration of MW01-5-188WH at 2.5 mg/kg daily
for 2 weeks did not induce any indices of hepatotoxic tissue injury
compared with mice treated with the diluent.
Example 7
[0379] In vitro stability, oral bioavailability, and brain uptake.
The stability of MW01-5-188WH (1 .mu.M) in a standard incubation
with rat liver microsomes (BD Biosciences, Bedford, Mass.) and an
NADPH-regenerating system was done at 37.degree. C. for 30 and 120
min. Reactions were stopped by acetonitrile, and the reaction
mixture was centrifuged at 16,000 .mu.g for 10 min. Ten microliters
of the supernatant were analyzed by calibrated HPLC to quantify the
percentage of the initial amount of MW01-5-188WH remaining after
the incubation. The HPLC system (Dionex, Sunnyvale, Calif.)
includes a Dionex P680 pump, a Phenomenex (Torrance, Calif.) Luna
C18 column (250.times.2.0 mm; 5 .mu.m) with a guard column, and a
Dionex UVD340U ultraviolet detector. The mobile phase consisted of
0.1% formic acid as reagent A and 0.08% formic acid/water in 80%
acetonitrile as reagent B at a flow rate of 0.2 ml per minute. The
gradient consisted of the following linear and isocratic gradient
elution changes in reagent B: isocratic at 60% from 0 to 5 min,
60-90% from 5 to 39 min, isocratic at 90% until 44 min. Peak
quantification was done based on absorption measured at 260 nm
relative to a standard curve obtained by using serial dilutions of
MW01-5-188WH. To estimate oral bioavailability (concentration of
compound in the blood as a function of time after oral
administration) and to gain insight into potential brain uptake,
MW01-5-188WH (2.5 mg/kg) was administered to mice by oral gavage in
a 0.5% (w/v) carboxymethylcellulose suspension. At 5, 15, 60, and
120 min after compound administration, the animals were
anesthetized with pentobarbital (50 mg/kg). Blood was harvested by
intracardiac puncture, collected in heparinized tubes, and plasma
obtained by centrifugation. Mice were perfused with PBS. Brain
homogenates were centrifuged at 12,000 .mu.g for 10 min and the
supernatant acidified by diluting 1:3 with 0.1% formic acid (Fluka,
Sigma-Aldrich, St. Louis, Mo.). Solid phase extraction followed by
HPLC analysis was used to quantify the amount of compound in brain
supernatants. Briefly, cartridges (Sep-Pak C18; Waters Associates,
Milford, Mass.) were conditioned with 1 ml of acetonitrile (HPLC
grade; EMD Biosciences, San Diego, Calif.) and equilibrated with 1
ml of water. A structural analog of MW01-5-188WH was used as an
internal standard. The acidified brain supernatant was added to the
cartridge followed by a 1 ml wash with 30% acetonitrile.
MW01-5-188WH was eluted from the cartridge using 80% acetonitrile.
The eluate was evaporated to dryness, reconstituted in 0.08% formic
acid/water in 80% acetonitrile, and analyzed by HPLC using the
following gradient in reagent B: 0-60% from 2 to 5 min, isocratic
at 65% until 7 min, 65-80% from 7 to 12 min, isocratic at 80% until
15 min, 89-100% from 15 to 18 min, and isocratic at 100% until 23
min. Plasma samples were deproteinized in 0.1 M perchloric acid and
centrifuged at 12,000 .mu.g for 10 min. The supernatant was
neutralized with 1 M NaOH, then extracted with dichloromethane, and
the layers separated at 3000 .mu.g for 5 min. The organic phases
from three successive extractions were pooled and then evaporated
to dryness under reduced pressure. The dried residue was
reconstituted in 50 .mu.l of reagent B, and 10 .mu.l of the
reconstituted material was analyzed by HPLC using the gradient
described above for brain supernatants.
Results:
Oral Bioavailability and Brain Uptake of MW01-5-188WH
[0380] Integrative chemical biology tools for neurosciences and CNS
targeted drugs must exhibit appropriate bioavailability and brain
uptake or penetration of the blood-brain barrier. Daily oral
administration is the preferred method of administration for
longer-term and time-delimited in vivo studies using animal models
and is the preferred mode in drug development for a variety of
reasons, including better patient compliance. In this regard, it is
critical to demonstrate bioavailability and appropriate rates of
initial brain uptake for an inhibitor, to fully interpret the
outcomes from in vivo studies. Therefore, the rate of MW01-5-188WH
concentration change in the blood after oral administration (oral
bioavailability) and its rate of change in the brain were
determined. Using the protocols described above for the
quantitative analysis of MW01-5-188WH extracted from biological
samples, the rates of appearance in blood and brain after a
low-dose oral administration (2.5 mg/kg) to mice were examined. The
appearance of MW01-5-188WH in the blood (FIG. 35 A) is readily
detected within the earliest possible time point, 5 min, with a
peak concentration being reached within 15 min and bulk clearance
happening within 120 min after oral administration. This
demonstrates that MW01-5-188WH has good oral bioavailability
properties. A similar pattern of time-dependent change in
concentration is seen for the brain (FIG. 35B), indicative of
MW01-5-188WH initial brain uptake reflecting that of the blood.
However, the MW01-5-188WH peak brain/blood concentration ratio is
>3.3, comparable with those of CNS drugs in clinical use. For
example, the brain/blood ratio for minaprine, a
6-phenylaminopyridazine CNS drug, is about 2 (Caccia et al., 1985
Xenobiotica, 15(12): 1111-9). These results demonstrate that
MW01-5-188WH fulfills criteria that typically exclude many
compounds that are active in cell culture from being used for in
vivo investigations and indicate its potential to work in vivo
after oral administration and within the experimental constraints
imposed by the human A.beta. ICV infusion model.
MW01-5-188WH Dosing is Selective for CNS Inflammation
[0381] The de novo focus on suppression of selected glia activation
pathways and the excellent brain uptake properties of orally
administered MW01-5-188WH raised the possibility that the compound
might exhibit selectivity for CNS proinflammatory cytokine
suppression versus suppression of proinflammatory cytokine
production by peripheral tissues. To examine this possibility,
MW01-5-188WH was administered daily at a standard therapeutic dose
(2.5 mg/kg) by oral gavage for 2 weeks, and then mice were
challenged with an intraperitoneal injection of bacterial LPS. Six
hours after the LPS challenge, the serum and brain levels of
IL-1.beta. and TNF-.alpha. were measured. As anticipated, the LPS
challenge induced an increase in the levels of IL-1.beta. and
TNF-.alpha. in the serum (FIG. 35C,D) and brain (FIG. 35E,F),
compared with control mice injected with saline. The interesting
finding was that treatment with MW01-5-188WH for 2 weeks suppressed
the LPS-induced upregulation of IL-1.beta. and TNF-.alpha.
production in the brain (FIG. 35E,F) but did not suppress the serum
response (FIG. 35 C,D). The suppression of brain cytokine responses
by MW01-5-188WH is consistent with its ability to suppress
proinflammatory cytokine production by activated glia and its oral
bioavailability and brain uptake properties shown above.
Example 8
Pharmacokinetics Studies
[0382] Plasma Pharmacokinetics and Absolute Bioavailability in Dog
and/or Rat
[0383] Two groups (3 animals per group; male animals) will be dosed
PO and IV. There will be one dose level (2.5 mg/kg), and a
crossover design will be used with 1 week washout between dose
periods. Plasma drug concentrations will be measured at not less
than eight time points not exceeding 24 hrs post-dose (e.g. 15, 30,
60, 90, 120, 240, and 480 minutes and 24 hr after administration of
single dose). PK parameters that will be derived include C.sub.max,
T.sub.max, t.sub.1/2, AUC, CI/F, V.sub.d and MRT. Dosing
formulation: oral gavage/CMC solution.
Mass Balance Study in Dog and/or Rat
[0384] A study may be performed utilizing .sup.14C-labelled minozac
(MW01-2-151SRM) to analyze excretion (urine, feces) and plasma
distribution.
Dose Range Finding Study in Rat
[0385] Phase A of the study will be a single dose MTD (3M/3 F for
each dose level, n=up to MTD or MFD found). Dose levels to be
designed based on available data if any; doses provided below may
be utilized for example purposes only. Dosing will be by oral
gavage with CMC solution.
Dose level 1: 10 mg/kg; Dose level 2: 100 mg/kg; Dose level 3: 500
mg/kg; Dose level 4: 1000 mg/kg; Dose level 5: 3000 mg/kg; Result:
An estimated single-dose MTD/MFD (sdMTD)
[0386] Phase B of the study may be performed. The phase comprises a
7-day dose range finding study (3M/3 F in each group, n=24). There
will be a control plus one dose level (a fraction of sdMTD);
additional dose level(s) will be incorporated as required by the
outcome of the initial 7-day dose range finding study.
Result: An estimated repeat-dose MTD in rats
Dose Range Finding Study in Dog
[0387] This study will utilize a single dose MTD (SM crossover
study, n=up to MTD or MFD found). Dose levels will be designed
based on available data. Examples of doses are provided below. The
dosing will be by oral gavage with a CMC solution preferred;
alternatively, filled gelatin capsules will be utilized.
[0388] Oral dose level 1: 30 mg/kg; Oral dose level 2: 100 mg/kg;
Oral dose level 3: 300 mg/kg
[0389] IV dose 1: 100 mg/kg; IV dose 2: 300 mg/kg; Oral dose level
4: 1000 mg/kg; Oral dose level 5: 3000 mg/kg.
[0390] Each subsequent dosing will be followed by an appropriate
washout period (2 days or 5 days after IV exposure).
Pharmacokinetics and absolute bioavailability will be determined
for dose levels 1 and 2. Plasma drug concentrations will be
measured at eight time points not exceeding 24 hrs post-dose (e.g.
15, 30, 60, 120, 240 and 480 minutes after administration of single
oral doses). PK parameters to be derived include C.sub.max,
T.sub.max, t.sub.1/2, AUC, CI/F, V.sub.d and MRT.
28-Day Repeat Dose Toxicology Study in Rats
[0391] The Main Study will involve 10M/10 F in each treatment
group, n=80. The dosing will be by oral gavage with CMC solution.
There will be a Control, Low dose, Mid dose, and High dose.
Results: PK (plasma and CSF drug levels) will be determined at Day
1 and Day 28. Necropsy will be determined after the completion of
treatment. Mortality, clinical observations, body weights, food
consumption, clinical pathology, opthalmoscopy, gross pathology,
and organ weights will be determined. Histopathology will be
determined on control and high dose groups.
[0392] A Recovery Study with 5M/5 F in each treatment group, n=20
will be conducted. There will be a Control and High dose. Results:
Necropsy will be determined after 28 days additional follow-up
period. Mortality, clinical observations, body weights, food
consumption, clinical pathology, opthalmoscopy, gross pathology,
and organ weights will also be determined. Histopathology will be
determined if required by observation of treatment effects.
28-Day Repeat Dose Toxicology Study in Dogs
[0393] A Main Study utilizing 3M/3 F in each treatment group, n=24
will be conducted. Dosing will be by oral gavage with CMC solution
preferred; alternatively, filled gelatin capsules will be used if
required. There will be a Control, Low dose, Mid dose, and High
dose. Results: PK (plasma and CSF drug levels) will be determined
at Day 1 and Day 28 Necropsy will be determine after the completion
of treatment. Mortality, clinical observations, body weights, food
consumption, clinical pathology, gross pathology, and organ weights
will be determined. Histopathology will be done on all dose
groups
[0394] A Recovery Study using 3M/3 F in each treatment group, n=12
will also be conducted. The study will use Control and High dose.
Results: Necropsy will be determined after 28 days additional
follow-up period. Mortality, clinical observations, body weights,
food consumption, clinical pathology, opthalmoscopy, gross
pathology, and organ weights will be determined. Histopathology if
required will be determined by observation of treatment
effects.
Example 9
General Methods
[0395] Chemicals were generally purchased from Aldrich (Milwaukee,
Wis.) or through VWR International and used as received. All
solvents were used as received unless stated otherwise in the text.
All organic solutions were dried with magnesium sulfate before
final evaporation. Microwave irradiation was carried out using the
CEM-Discover microwave synthesis system (Matthews, N.C.).
[0396] All intermediates were characterized by MS (ESI) and HPLC
and in some cases by .sup.1H-NMR. Final compounds were
characterized by HRMS, HPLC and .sup.1H-NMR, and in some cases, by
elemental analysis. NMR spectra were acquired on a Varian Inova 500
MHz spectrometer at room temperature. Electrospray mass spectra
(EI-MS) were collected on a Micromass Quattro II Triple Quadrupole
HPLC/MS/MS Mass Spectrometer. High resolution mass spectra (HR-MS)
were obtained on a VG70-250SE mass spectrometer.
[0397] All syntheses were monitored by analytical HPLC. HPLC traces
were obtained on a Rainin Instruments HPLC on commercially
available SUPELCO C18 reverse phase column (25.times.4.6 mm, 5
.mu.m). The mobile phase consisted of 0.1% formic acid in Milli-Q
water as reagent A and 0.08% formic acid/Milli-Q water in 80%
acetonitrile as reagent B. The flow rate of 1.5 ml/min was used in
a gradient of 0 to 100% of reagent B over 22 minutes. The HPLC
traces were tracked by UV absorption at 260 nm.
[0398] A separate HPLC system was used to obtain final compound
purity. The HPLC system (Dionex, Sunnyvale, Calif.) consisted of
the following components: a Dionex P680 pump, a Dionex ASI-100
autosampler, a Phenomenex (Torrance, Calif.) Luna C18 column
(250.times.2.0 mm; 5 .mu.m) with a guard column, and a Dionex
UVD1700 ultraviolet detector. The mobile phase consisted of 0.1%
formic acid in Milli-Q water as reagent A and 0.08% formic
acid/Milli-Q water in 80% acetonitrile as reagent B. The flow rate
of 0.2 mL/min was used, unless stated otherwise in the text. For
determination of compound purity, the gradient consisted of a
linear change from 0 to 100% of reagent B over 30 minutes. UV
absorption was monitored at four wavelengths (215, 230, 260 and 300
nm) with the 260 nm trace being reported. Compounds were injected
at concentrations 100-times greater than the lower detection limit
of the instrument (500 ng injected).
[0399] Elemental analysis was carried out by Quantitative
Technologies Inc. (QTI, Whitehouse, N.J.). Melting point data for
the dichloro monohydrate salt 26 (234.1-234.7.degree. C.) and of
compound 16 (>215.degree. C., decomposes to black solid) were
acquired on a Buchi Melting Point B-540 (Flawil, Switzerland).
Synthesis of R4Analogs (FIG. 37))
2-benzyl-6-phenyl-4,5-dihydropyridazin-3(2H)-one (18)
[0400] 3-benzoylpropionic acid 17 (17.8 g, 0.1 mol),
benzylhydrazine dihydrochloride (19.5 g, 0.1 mol) and sodium
acetate (74.9 g, 0.55 mol) were suspended in 500 mL ethanol (95%).
The white suspension was heated under reflux for 29 hours. Ethanol
was removed under reduced pressure and the residue was treated with
water (300 mL). The pH of the aqueous layer was adjusted with
concentrated solution of sodium carbonate to pH=8 and extracted
with ethylacetate (1.times.200 mL). The organic layer was washed
with brine and concentrated to dryness under reduced pressure. The
product 18 was obtained as yellow oil in 78% yield and was used in
the following step without further purification. HPLC
(t.sub.r/purity): 23.4 min, 80%.
3,4-dichloro-6-phenylpyridazine (19)
[0401] Compound 18 (26 g, 0.079 mol--estimated on 80% purity),
phosphorus oxychloride (59 mL, 0.64 mol, 6.5 equiv) and phosphorus
pentachloride (133.2 g, 0.64 mol, 6.5 equiv) were heated at
120.degree. C. for 12 hrs. To control the HCl gas forming during
the course of reaction, a NaOH solution was used to absorb the
acid. Most of the phosphoryl chloride was distilled under reduced
pressure, ice water was added to the residue and stirred for 30
min. The yellow crystalline solid which separated upon cooling was
filtered, washed with water (3.times.100 mL) and recrystallized
from anhydrous ethanol to give desired product 19 as yellow needles
in 44% yield. .sup.1H NMR (CDCl.sub.3): .delta. 8.06 (dd,
.sup.3J=6.5 Hz, .sup.4J=2.5 Hz, 2H), 7.98 (s, 1H), 7.56 (t,
.sup.3J=6.5 Hz, 3H). HPLC (t/purity): 23.6 min, >95%.
3-chloro-6-phenylpyridazin-4-ol (20)
[0402] A mixture of 19 (158 g, 0.7 mol) and acetic acid (700 mL)
was heated under reflux for 5 hrs. The reaction mixture was cooled
to room temperature, the precipitate filtered and the bright yellow
filter cake washed with water (5.times.500 mL). The filter cake was
recrystallized from ethyl acetate (200 mL), filtered and dried over
a medium frit sintered glass funnel in vacuo to give the desired
product 20 in 32% yields. HPLC (t.sub.r/purity): 15.37 min,
>95%. ESI m/z (MeOH): 207.3 (MH.sup.+).
6-phenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazin-4-ol
(21)
[0403] Compound 20 (14 g, 0.068 mol) was placed in a reaction tube
with 1-butanol (30 mL) and 4 equiv of 1-(2-pyrimidyl)piperazine (45
g, 0.27 mol, 4 equiv). The flask was capped and heated at
130.degree. C. for 41 h. The reaction mixture was cooled to ambient
temperature, and the 1-butanol removed under reduced pressure to
give a dark oil residue. The oil was treated with water to give a
suspension which is then filtered and washed with water. The filter
cake was dried over a medium frit sintered glass funnel in vacuo to
give the desired product 21 in 97% yields. HPLC (t.sub.r/purity):
17.30 min, >99%. ESI m/z (MeOH): 334.38 (MH.sup.+).
4-chloro-6-phenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazine
(6)
[0404] Compound 21 (22 g, 0.066 mol) was suspended in phosphorus
oxychloride (80 mL). The reaction mixture was heated at 100.degree.
C. for 3 h, cooled to room temperature and poured on crushed ice (2
kg). The aqueous mixture was neutralized with NaOH solution to give
white suspension. The precipitate was filtered and dried over a
medium frit sintered glass funnel in vacuo to give the desired
product 6 in 91% yields (21 g). .sup.1H NMR (CDCl.sub.3): .delta.
8.35 (d, J=4.6 Hz, 2H), 8.01 (d, J=7.5 Hz, 2H), 7.81 (s, 1H), 7.50
(t, J=7.0 Hz, 2H), 7.48 (t, J=7.0 Hz, 1H), 6.54 (t, J=4.4 Hz, 1H),
4.05 (t, J=4.4 Hz, 4H), 3.65 (t, J=4.4 Hz, 4H). HPLC
(t.sub.r/purity): 22.4 min, >99%; HRMS calcd for
C.sub.18H.sub.17ClN.sub.6 352.1198, found 352.1201.
4-Benzyl-6-phenyl-3-(4-pyrimidin-2-yl)piperazin-1-yl)pyridazine
(2)
[0405] Following the procedure of Zou et al (Tet Lett. 2001 42:
7213-7215), compound 6 (100 mg, 0.28 mmol) was suspended in THF
with the 1.37 equiv of benzyl boronic acid (42 mg, 0.31 mmol), 0.2
equiv of Pd(dppf)Cl.sub.2CH.sub.2Cl.sub.2 (23 mg, 0.02 mmol), 2.5
equiv of silver oxide (164 mg, 0.71 mmol) and 3 equiv of potassium
carbonate (117 mg, 0.85 mmol). The mixture was purged with argon
and was heated at 120.degree. C. for 16 h in a sealed tube. The
reaction mixture was then cooled to ambient temperature and
quenched with either 33% hydrogen peroxide or 10% sodium hydroxide.
The aqueous layer was extracted with ether (3.times.30 mL) and the
ethereal layers are combined and evaporated under reduced pressure.
The crude mixture is run on a silica gel column and eluted with
hexanes:ethyl acetate (1:1 v/v). The product 2 is obtained as a
pale pink solid in 45% yield. .sup.1H NMR (CDCl.sub.3): .delta.
8.36 (d, J=4.4 Hz, 2H), 7.94 (d, J=7.1 Hz, 2H), 7.46-7.42 (m, 3H),
7.41 (s, 1H), 7.36 (t, J=7.3 Hz, 2H), 7.30 (t, J=7.1 Hz, 1H), 7.22
(d, J=7.3 Hz, 2H), 6.55 (t, J=4.4 Hz, 1H), 4.10 (s, 2H), 4.01 (s,
4H), 3.44 (s, 4H). HPLC (t.sub.r/purity): 30.32 min, >95%; HRMS
calcd for C.sub.25H.sub.24N.sub.6 408.2057, found 408.2066.
6-Phenyl-4-(pyridin-4-yl)-3-(4-pyrimidin-2-yl)piperazin-1-yl)pyridazine
(3)
[0406] Compound 6 (700 mg, 2.0 mmol) was placed in a reaction
vessel with 3.1 equiv potassium carbonate (851 mg, 6.2 mmol), 1.37
equiv (330 mg, 2.7 mmol) 4-pyridinylboronic acid and 0.05 equiv
Pd(PPh.sub.3).sub.4 (120 mg, 0.1 mmol). DME (10 mL) was added and
the mixture was purged with argon. The reaction mixture was sealed
and heated at 110.degree. C. for 20 h. The solution was cooled to
ambient temperature and filtered through celite. The filtrate was
concentrated under reduced pressure, dissolved in ethyl acetate (30
mL) and washed with 2N HCl (50 mL). The organic layer was
concentrated under reduced pressure and recrystallized with ethyl
acetate/petroleum ether mixture to give the product 3 as light
yellow needles in 41% yield. .sup.1H NMR (CDCl.sub.3): .delta. 8.79
(d, J=5.5 Hz, 2H), 8.32 (d, J=5.0 Hz, 2H), 8.07 (d, J=7.5 Hz, 2H),
7.68 (d, J=5.5 Hz, 2H), 7.63 (s, 1H), 7.51 (t, J=7.0 Hz, 2H), 7.48
(t, J=7.0 Hz, 1H), 6.53 (t, J=4.5 Hz, 1H), 3.85 (d, J=4.5 Hz, 4H),
3.39 (t, J=5.0 Hz, 4H). HPLC (t.sub.r/purity): 21.61 min, >95%;
HRMS calcd for C.sub.23H.sub.21N.sub.7 395.1853, found
395.1852.
4-Isobutyl-6-phenyl-3-(4-pyrimidin-2-yl)piperazin-1-yl)pyridazine
(4)
[0407] Following the procedure of Zou et al (supra), compound 6
(200 mg, 0.56 mmol) was suspended in THF with the 1.37 equiv of
(2-methylpropyl)boronic acid (79 mg, 0.77 mmol), 0.2 equiv of
Pd(dppf)Cl.sub.2CH.sub.2Cl.sub.2 (92.5 mg, 0.11 mmol), 2.5 equiv
silver oxide (328 mg, 1.41 mmol) and 3 equiv of potassium carbonate
(234 mg, 1.7 mmol). The mixture was purged with argon and heated at
120.degree. C. for 42 hours in a sealed tube. The reaction was
cooled to ambient temperature and the reaction was quenched with
aqueous solution of sodium hydroxide (10%) and extracted with ether
(3.times.50 ml). The ethereal layers were combined, dried with
magnesium sulfate and evaporated under reduced pressure leaving a
sticky solid. The crude mixture was purified with column
chromatography and eluted with 40% ethyl acetate in hexanes to give
4 as a white powder in 52.5% yield. .sup.1H NMR (CDCl.sub.3):
.delta. 8.36 (d, J=4.2 Hz, 2H), 8.06 (d, J=7.1 Hz, 2H), 7.60 (s,
1H), 7.51 (t, J=7.0 Hz, 2H), 7.47 (t, J=7.0 Hz, 1H), 6.55 (t, J=4.2
Hz, 1H), 4.03 (s, 4H), 3.42 (s, 4H), 2.62 (d, J=6.7 Hz, 2H), 2.18
(sp, J=6.4 Hz, 1H), 0.97 (d, J=6.2 Hz, 6H). HPLC (t.sub.r/purity):
29.5 min, >95%; HRMS calcd for C.sub.22H.sub.26N.sub.6 374.2213,
found 374.2208.
4-Methyl-6-phenyl-3-(4-pyrimidin-2-yl)piperazin-1-yl)pyridazine
(5)
[0408] Following the procedure of Zou et al (supra), compound 6
(250 mg, 0.71 mmol) was suspended in THF with the 1.37 equiv of
methylboronic acid (59 mg, 0.97 mmol), 0.25 equiv of
Pd(dppf)Cl.sub.2CH.sub.2Cl.sub.2 (144 mg, 0.18 mmol), 2.5 equiv of
silver oxide (410 mg, 1.78 mmol) and 3 equiv of potassium carbonate
(294 mg, 2.1 mmol). The mixture was purged with argon and was
heated at 120.degree. C. for 18.5 h in a sealed tube. After cooling
to ambient temperature the reaction was quenched with aqueous
sodium hydroxide (10%) and extracted with ether (3.times.75 ml).
The compound was purified by column chromatography and eluted with
a mixture of ethyl acetate:hexanes (1:3 v/v). The compound 5 was a
white crystallize solid obtained in 45.8% yield. .sup.1H NMR
(CDCl.sub.3): .delta. 8.36 (d, J=4.5 Hz, 2H), 8.05 (d, J=7.5 Hz,
2H), 7.61 (s, 1H), 7.50 (t, J=7.1 Hz, 2H), 7.44 (t, J=7.1 Hz, 1H),
6.55 (t, J=4.5 Hz, 1H), 4.04 (t, J=4.5 Hz, 4H), 3.46 (t, J=4.5 Hz,
4H), 2.45 (s, 3H). HPLC (t.sub.r/purity): 24.91 min, >95%; HRMS
calcd for C.sub.19H.sub.20N.sub.6 332.1744, found 332.1740. Anal.
Calcd for C.sub.19H.sub.20N.sub.6C, 68.65; H, 6.06; N, 25.28. Found
C, 68.73; H, 5.97; N, 25.22.
Synthesis of R3Analogs (FIG. 38)
4-Methyl-6-phenyl-3-(4-pyrazin-2-yl)piperazin-1-yl)pyridazine
(7)
[0409] Compound 15 (500 mg, 2.4 mmol) was placed in a capped flask
and suspended in 20 mL water. 2.5 equiv (1 g, 6 mmol) of
1-(2-pyrazinyl)piperazine and 5 equiv (1.69 mL, 12 mmol) of
triethylamine were added and the flask was capped and heated to
130.degree. C. for 160 h. The reaction was cooled to ambient
temperature to give a dark brown oil at the bottom of the flask.
The water was decanted off of the oil, the oil was dissolved in
minimal isopropanol and heated to 70.degree. C. Upon cooling, a
brown solid formed and was filtered on a sintered glass funnel and
rinsed with hexanes to afford product 7 as a brown powder in 28.8%
yield. .sup.1H NMR (CDCl.sub.3): .delta. 8.25 (bs, 1H), 8.16 (bs,
1H), 8.08 (d, J=7.0 Hz, 2H), 7.93 (bs, 1H), 7.69 (s, 1H), 7.54-7.48
(m, 3H), 3.83 (t, J=5.0 Hz, 4H), 3.57 (bs, 4H), 2.48 (s, 3H). HPLC
(t.sub.r/purity): HRMS calcd for C.sub.19H.sub.20N.sub.6, found.
Given in Apr. 21, 2006
4-Methyl-6-phenyl-3-(4-pyridin-2-yl)piperazin-1-yl)pyridazine
(8)
[0410] Compound 15 (190 mg, 0.93 mmol) was placed in a reaction
tube with 1-butanol and 4 equiv of 1-(pyridin-2-yl)piperazine (605
mg, 3.7 mmol), capped and heated at 140.degree. C. for 48 h. The
reaction mixture was cooled to ambient temperature and the
1-butanol removed under reduced pressure to give a dark oil
residue. The oil was treated with water to give a suspension which
is filtered and washed first with water, then with a mixture of
ethyl acetate:hexanes (1:6 v/v) to afford the product 8 as a brown
yellow powder in 54.5% yield. .sup.1H NMR (CDCl.sub.3): .delta.
8.23 (d, J=3.7 Hz, 1H), 8.05 (d, J=7.5 Hz, 2H), 7.60 (s, 1H), 7.54
(t, J=6.8 Hz, 1H), 7.49 (t, J=7.1 Hz, 2H), 7.44 (t, J=7.3 Hz, 1H),
6.75 (d, J=8.2 Hz, 1H), 6.68 (t, J=5.5 Hz, 1H), 3.76 (s, 4H), 3.51
(t, J=4.8 Hz, 4H), 2.43 (s, 3H). HPLC (t.sub.r/purity): 15.66 min,
>95%; HRMS calcd for C.sub.20H.sub.21N.sub.5 331.1791, found
331.1800.
4-Methyl-6-phenyl-3-(4-pyridin-4-yl)piperazin-1-yl)pyridazine
(9)
[0411] Compound 15 (190 mg, 0.93 mmol) was placed in a reaction
tube with 1-butanol and 4 equiv of 4-piperazino-pyridazine (605 mg,
3.7 mmol). The flask was capped and heated at 140.degree. C. for 72
h. The reaction mixture was cooled to ambient temperature and the
1-butanol removed under reduced pressure to give a dark red oil
residue. The oil was treated with 20 mL of water, and then
extracted with 10 mL of ethyl acetate. A brown suspension was
formed in the organic layer. The precipitate was collected by
filtration and washed with 10 mL of water and then 10 mL of ethyl
acetate to afford the product 9 as a brown yellow powder in 34.1%
yield. 1H NMR (CDCl.sub.3): .delta. 8.33 (d, J=4.9 Hz, 2H), 8.06
(d, J=7.1 Hz, 2H), 7.64 (s, 1H), 7.52 (t, J=7.6 Hz, 2H), 7.48 (t,
J=7.1 Hz, 1H), 6.79 (d, J=5.8 Hz, 2H), 3.58 (s, 4H), 3.56 (s, 4H),
2.45 (s, 3H). HPLC (t.sub.r/purity): 14.95 min, >95%; HRMS calcd
for C.sub.20H.sub.21N.sub.5 331.1791, found 331.1799.
3-(4-cyclohexylpiperazin-1-yl)-4-methyl-6-phenylpyridazine (10)
[0412] Compound 15 (200 mg, 0.96 mmol) was suspended in 5 mL water
with 4 equiv cyclohexyl piperazine (651.5 mg, 3.87 mmol) in a 10-mL
microwave glass vessel and capped with a septum. Microwave
irradiation of 75W was used, the temperature being ramped from room
temperature to 175.degree. C. Once 175.degree. C. was reached, the
reaction mixture was held at this temperature for 3 h. The reaction
mixture was allowed to cool to room temperature, the dark brown
solution was poured over water to give a suspension, which was
filtered to afford a beige solid. The solid was washed with 20 mL
saturated sodium bicarbonate to give 10 in 95% yield. .sup.1H NMR
(CDCl.sub.3): .delta. 7.56 (s, 1H), 7.49 (t, J=7.5 Hz, 3H), 7.44
(m, 2H), 3.41 (s, 4H), 2.79 (s, 4H), 2.38 (s, 3H), 2.34 (m, 1H),
1.62 (m, 2H), 1.26 (m, 8H). HPLC (t.sub.r/purity): PENDING min,
>95%; HRMS calcd for C.sub.21H.sub.28N.sub.4, found GIVEN Apr.
21, 2006.
4-Methyl-3-(4-methylpiperazin-1-yl)-6-phenylpyridazine (11)
[0413] Compound 15 (500 mg, 2.4 mmol) was suspended in 20 mL water
in a capped flask with 4 equiv 1-methyl-piperazine (961 mg, 9.6
mmol). The vessel was capped and heated at 120.degree. C. for 120 h
until complete. The mixture was cooled to ambient temperature to
afford a pale yellow solution with a white solid precipitate. The
reaction was filtered, and the aqueous filtrate washed with ether
to remove trace starting materials and then extracted with ethyl
acetate (5.times.10 mL). The organic washes are combined, dried
with magnesium sulfate and the ethyl acetate removed under reduced
pressure. The remaining oil was treated with ether and cooled,
resulting in the product 11 as yellow needles in 38.7% yield.
.sup.1H NMR (CDCl.sub.3): .delta. 8.02 (d, J=7.0 Hz, 2H), 7.55 (s,
1H), 7.47 (t, J=7.0 Hz, 2H), 7.43 (m, 1H), 3.41 (t, J=4.5 Hz, 4H),
2.63 (bs, 4H), 2.38 (s, 3H), 2.36 (s, 3H). HPLC (t.sub.r/purity):
PENDING 95%; HRMS calcd for C.sub.16H.sub.20N.sub.4 Given Apr. 21,
2006
Synthesis of a Pyrazine analog (FIG. 39)
3-methyl-5-phenylpyrazin-2(1H)-one (24)
[0414] This compound was prepared following the procedure of Jones
(J. Amer. Chem. Soc. 1949, 71, 78-81). Briefly, commercially
available phenylglyoxal 22 (1.02 g, 7.62 mmol) was dissolved in
methanol and cooled to -41.degree. C. Commercially available
alanine amide 23 (672 mg, 7.62 mmol) was dissolved in 25 ml
methanol and added to the reaction mixture. A 12.5 N NaOH (0.760
mL, 9.53 mmol) solution was added dropwise while stirring,
maintaining the temperature of the reaction below -10.degree. C.
When the addition was complete, the reaction was placed at
-5.degree. C. for 2 h. The reaction was then warmed to room
temperature and quenched with 12 N HCl solution (0.76 mL), followed
by sodium bicarbonate to neutralize the solution. The methanol was
removed under reduced pressure, and the residue was extracted with
chloroform and precipitated with ethyl acetate. The compound was
isolated as a white powder to give 24 in 18% yield. HPLC
(t.sub.r/purity): 15.91 min, >97%. ESI m/z (MeOH): 187.35
(MH.sup.+).
2-(4-(3-methyl-5-phenylpyrazin-2-yl)piperazin-1-yl)pyrimidine
(25)
[0415] This compound was prepared via the pyrazine triflate with
1-(2-pyrimidyl)piperazine as the amine following the procedure of
Adams et al (Synlett 2004, 11, 2031-2033). Pyridine was used as an
anhydrous reagent kept under argon in a sure-seal bottle (Aldrich).
The compound 24 (100 mg, 0.52 mmol) and DMAP (65.7, 0.52 mmol) were
dissolved in pyridine and methylene chloride (0.5: 4 ml v/v), and
cooled to 0.degree. C. The trifluoromethane sulfonic acid (0.8
mmol, 135.5 .mu.L) was added dropwise and stirred for 15 min at
0.degree. C. and then 3 h at RT. The triflate was confirmed by ESI
(363.7 (MH.sup.+)) and HPLC (t.sub.R=25.33 min). The reaction
mixture was diluted with dichloromethane and washed one time each
with 20 ml of water, sodium bicarbonate and brine. The
dichloromethane was removed under reduced pressure, and the
remaining residue was dissolved directly in DMSO.
1-(2-pyrimidyl)piperazine (5.3 mmol, 750 .mu.L) was added and the
reaction heated to 60.degree. C. and stirred for 2 h. When
complete, the reaction was diluted with ethyl acetate and washed
with 1N HCl, after washes with brine and water to remove remaining
pyridine. The organics were then dried and evaporated in vacuo to
give 25 as a yellow solid (63% yield). HPLC (t.sub.r/purity): 24.74
min, >98%. ESI m/z (CH.sub.2Cl.sub.2) 333.29 (MH.sup.+). .sup.1H
NMR (500 MHz, CDCl.sub.3): .delta. 8.56 (s, 1H); 8.36 (d, J=4 Hz,
2H); 8.01 (d, J=7.5 Hz, 2H); 7.48-7.40 (m, 3H); 6.54 (bs, 1H); 4.03
(bs, 4H), 3.42 (bs, 4H); 2.62 (s, 6H). HRMS calculated GIVEN Apr.
21, 2006
Synthesis of 26
4,6-diphenyl-3-(4-pyrimidin-2-yl)piperazine-1-yl)pyridazine
dichloro monohydrate salt (26)
[0416] 700 mg (1.77 mmol) of 1 was suspended in 10 mL of anhydrous
isopropanol and heated to 70.degree. C. 2.5 eq (0.375 mL, 4.4 mmol)
of concentrated HCl was added at once to the solution. The
suspension was stirred at 70.degree. C. for 10 min, cooled to
ambient temperature and cooled on ice for 1 h. The precipitate was
collected by filtration and washed once with cold isopropanol (5
mL) to provide the product 26 as bright yellow powder in 55% yield.
.sup.1H NMR (DMSO-d.sub.6): .delta. 8.55 (s, 2H), 8.16 (s, 2H),
7.86 (s, 1H), 7.7 (s, 2H), 7.58 (s, 6H), 6.84 (s, 1H), 4.14 (s,
4H), 3.57 (s, 4H). HPLC (t.sub.r/purity): PENDING min, >98%. EA
calculated for C.sub.24H.sub.26Cl.sub.2N.sub.6OC 59.38, H 5.40, N,
17.31. Found C, 59.38; H, 5.40; N, 17.31.
Production Scheme for Pyrazine Analogs
[0417] 4,5-dihydro-4-methyl-6-phenylpyridazin-3(2H)-one (13)
(Hansen, K B et al. Org. Process Res. Dev., 2005, 9, 634-639,
Nelson, D A. US 20050137397A1). A 250 ml three-neck round bottom
flask fit with a temperature probe and condenser was charged with
7.7 g (40 mmol) of 2-methyl-4-oxo-4-phenylbutanoic acid 12 and 20
ml of ethanol (95%). The suspension was cooled to below 10.degree.
C. and 2.2 ml (42 mmol, 1.05 equiv) of hydrazine monohydrate in 10
ml of ethanol was added dropwise. After addition, the reaction
mixture was heated to reflux and stirred for 2 h. The reaction
mixture was cooled to ambient temperature and forming white
crystals were collected by filtration. The solid was then washed
with 2N NaHCO.sub.3 (1.times.30 mL), Milli-Q water (3.times.60 mL)
and dried over a medium frit sintered glass funnel in vacuo to give
the desired product 13 in 96.1% yield. .sup.1H NMR (DMSO-d.sub.6):
.delta. 10.84 (s, 1H), 7.75 (m, 2H), 7.41 (m, 3H), 3.12 (m, 1H),
2.60 (m, 1H), 2.50 (m, 1H), 1.13 (d, J=7 Hz, 3H). HPLC (t/purity):
PENDING min, >95%; ESI m/z (MeOH) 189.08 (MH.sup.+)
4-methyl-6-phenylpyridazin-3(2H)-one (14) (Csende, F et al.
Synthesis, 1995, 1240-1242) 7.0 g (35 mmol) of 13 was dissolved in
30 ml of acetonitrile in a 250 ml single-necked round bottom flask.
11.3 g (84 mmol, 2.4 equiv) of anhydrous copper (II) chloride was
added to the solution and the reaction mixture was heated to reflux
for 2 hours. To control the HCl gas that formed during the course
of the reaction, a NaOH solution was used to absorb the HCl that
escapes from dry tube. The reaction mixture was cooled to ambient
temperature, and placed into an ice-water bath. 150 mL of ice-water
was added to quench the reaction. The mixture was stirred
vigorously for 10 minutes to give a gray precipitate and blue
liquid containing copper (I) chloride. The precipitate was then
collected by filtration (pH of the filtrate is 0-1) and washed
first with 1N HCl (100 mL), then with Milli-Q water (5.times.100
mL). To remove remaining copper by-products, the filter cake was
stirred in 1N HCl (150 mL) for 0.5 h and then filtered. The filter
cake was washed with Milli-Q water until the filtrate is at pH 7
(approximately 7 washes). The solid was dried over a medium frit
sintered glass funnel in vacuo to give 14 as a light gray powder in
93.8% yield. .sup.1H NMR (DMSO-d.sub.6): .delta. 7.95 (s, 1H), 7.85
(d, J=7.5 Hz, 2H), 7.47 (m, 2H), 7.43 (m, 1H), 2.13 (s, 3H). HPLC
(t.sub.r/purity): 21.48 min, >97%; ESI m/z (MeOH) 187.36
(MH.sup.+).
3-chloro-4-methyl-6-phenylpyridazine (15)
[0418] 6.0 g (32 mmol) of 14 was placed in a 250 mL single neck
round bottom flask and 30 ml of acetonitrile was added to create a
pale yellow slurry. 6.0 ml (64 mmol, 2 equiv) of phosphorus
oxychloride was added and the reaction mixture was heated at reflux
for 2.5 h. After the reaction was completed, the mixture was cooled
to ambient temperature and placed in an ice water bath. Ice water
(150 mL) was slowly poured into the reaction mixture with stirring
to decompose the phosphorus oxychloride into HCl and
H.sub.3PO.sub.4. The solid was then collected by filtration and
washed with Milli-Q water (3.times.50 mL). The solid was suspended
in 100 mL of water and 1N NaOH was added until the aqueous
suspension was at pH=8. The mixture was stirred for 5 minutes to
remove all trace starting material contaminants. The solid was
filtered and washed with Milli-Q water (3.times.100 mL). The
product was dried over a medium frit sintered glass funnel in vacuo
to provide 15 as a light pink powder in 96% yield. .sup.1H NMR
(DMSO-d.sub.6): .delta. 8.29 (s, 1H), 8.10 (m, 2H), 7.53 (m, 3H),
2.41 (s, 3H), HPLC (t.sub.r/purity): 2888.98 min, >94%.ESI m/z
(MeOH) 205.49 (MH.sup.+).
2-(4-(4-methyl-6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine
(5)
[0419] 7.5 g (36.6 mmol) of 15 was suspended in 125 mL of Milli-Q
water. 60.17 g (366.0 mmol, 10 equiv.) of 1-(2-pyrimidyl)piperazine
was added and the reaction mixture was heated at reflux with rapid
stirring for 60 h. When complete, the reaction mixture was cooled
to ambient temperature and two layers were observed in the flask
consisting of an orange aqueous layer and a brown oil that settled
to the bottom of the flask. The water was decanted off, the oil was
dissolved in minimal volume of isopropanol and heated to reflux.
After 10 minutes of reflux, the solution was slowly cooled to
0.degree. C. to induce crystallization. Pale yellow crystals were
filtered from isopropanol and rinsed with minimal cold ether to
provide 5 in 54% yield. .sup.1H NMR (CDCl.sub.3): .delta. 8.36 (d,
J=4.5 Hz, 2H), 8.05 (d, J=7.5 Hz, 2H), 7.61 (s, 1H), 7.50 (t, J=7.1
Hz, 2H), 7.44 (t, J=7.1 Hz, 1H), 6.55 (t, J=4.5 Hz, 1H), 4.04 (t,
J=4.5 Hz, 4H), 3.46 (t, J=4.5 Hz, 4H), 2.45 (s, 3H). HPLC
(t.sub.r/purity): 24.91 min, >95%; HRMS calcd for
C.sub.19H.sub.20N.sub.6 332.1744, found 332.1740. Anal. Calcd for
C.sub.19H.sub.20N.sub.6: C, 68.65; H, 6.06; N, 25.28; found C,
68.73; H, 5.97; N, 25.22.
[0420]
2-(4-(4-methyl-6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine
dihydrochloride monohydrate salt (16) (Wermuth C G, Stahl P H.
Selected Procedures for the Preparation of Pharmaceutically
Acceptable Salts, in Stahl PH., Wermuth CG. (Ed.) Handbook of
Pharmaceutical Salts, Wiley-VCH, p 249-264). 6.3 g (19.0 mmol) of 5
was suspended in 50 mL of anhydrous isopropanol and heated to
70.degree. C. 2.5 eq (4.0 mL) of concentrated HCl was added at once
to the solution. The suspension was stirred at 70.degree. C. for 10
min, cooled to ambient temperature and cooled on ice 0.5 h. The
precipitate is collected by filtration and washed once with cold
isopropanol (30 mL) to provide the product 16 as a yellow powder in
93.3% yield. .sup.1H NMR (DMSO-d.sub.6): .delta. 8.47 (s, 3H), 8.07
(d, J=4.0 Hz, 2H), 7.61 (s, 3H), 6.76 (d, J=2.7 Hz, 2H), 3.99 (s,
4H), 3.60 (s, 4H), 2.59 (s, 3H). HPLC (t.sub.r/purity): 25.06 min,
99%. HRMS calcd for C.sub.19H.sub.20N.sub.6 332.1744, found
332.1744. EA calculated for C.sub.19H.sub.22Cl.sub.2N.sub.6: C,
53.91; H, 5.71; N, 19.85; Cl, 16.75; 0, 3.78. Found C, 53.66; H,
5.52; N, 19.67; Cl, 16.86; 0, 4.12. Copper found to be 2 ppm.
Example 10
Physicochemical Properties
Materials/Methods:
[0421] The HPLC system (Dionex Corp., Sunnyvale, Calif.) consisted
of the following components: a Dionex P680 Pump, a Dionex ASI-100
autosampler, a Phenomenex (Torrance, Calif.) Luna C18 column
(250.times.2.0 mm; 5 .mu.M) with a guard column, and a Dionex
UVD170U detector. The mobile phase consisted of 0.1% formic acid
(Fluka) in Milli-Q water as solvent A and 80% acetonitrile (Burdick
& Jackson), with 0.08% formic acid in Milli-Q water as solvent
B. Peak quantification was performed based upon absorption at 254
nm relative to a standard curve obtained by serial dilutions of the
compound.
[0422] Capillary tubes used in the micro scale aqueous solubility
determination were purchased from Buchi, Switzerland. The weighting
of the compounds was performed on SartoriusAG (Germany) analytical
balance. Milli-Q water was obtained using Millipore System
(Bedford, Mass.). The orbital shaker/incubator was purchased from
Barnstead International (Melrose Park, Ill.).
Micro Scale Aqueous Solubility Determination
[0423] Dry, clean borosilicate capillary tubes were weighed using
an analytical balance. Between 17-30 mg of 16 was weighed and added
to the tubes. Distilled, purified Milli-Q water was added to the
tubes to create solutions with concentrations ranging from 1-2
g/ml. Sample tubes were mixed manually to ensure sufficient wetting
and were placed in an incubator set at 37.degree. C. overnight. A
sample was collected from each tube, centrifuged at 10,000 rpm for
10 min, and injected onto a reversed-phase HPLC.
Macro Scale Aqueous Solubility Determination
[0424] Dry, clean glass Erlenmeyer flasks were weighed using an
analytical balance. Up to 30 mg of 26 was added to the flasks.
Distilled, purified water was added to the flask to create a
saturated solution. The flasks were placed in an orbital
shaker/incubator at 37.degree. C., 175 rpm for 72 hours. Samples
were removed at 24 hour intervals, centrifuged at 10,000 rpm for 10
min to remove particulate and injected onto a reversed-phase HPLC
system.
Partition Coefficient Determination
[0425] The partition coefficients of 16 and 26 were determined
using 1-octanol (Sigma) and water. Between 0.5-1 mg/ml of each
compound was dissolved in Milli-Q water and allowed to partition
into presaturated octanol. The samples were placed horizontally in
an orbital shaker/incubator at 37.degree. C. for 1 hour. After 1 h,
the samples were centrifuged for 5 min at 1500 rpm and the aqueous
phase separated. The concentration of compound in both the aqueous
and octanol phases was determined.
Activity Assays
[0426] Cell culture assays. Glia cell-based assays of the
concentration-dependent activity of the compounds were done as
previously described (Hu W, Ralay Ranaivo et al., Current
Alzheimer's Research 2005, 2:197-205; Mirzoeva S, et al., J Med
Chem 2002, 45:563-566; Ralay Ranaivo H, et al., J Neurosci 2006,
26:662-670). BV-2 mouse microglial cells were cultured for one day
in multiwell plates and then treated in serum-free media for 16 hrs
with either control buffer or the standard glial activating
stimulus lipopolysaccharide (LPS, from Salmonella typhimurium; 100
ng/ml) in the presence of diluent or different concentrations of
compounds. The accumulation of nitrite, the stable metabolite of
nitric oxide (NO), was measured in BV-2 conditioned media by the
Griess assay as previously described (Hu W, Ralay Ranaivo et al.,
Current Alzheimer's Research 2005, 2:197-205; Mirzoeva S, et al., J
Med Chem 2002, 45:563-566; Mirzoeva S, et al., Brain Res 1999,
844:126-134). Levels of IL-1.beta., TNF.alpha., MCP-1 and
IL-1.beta. in cell lysates were measured by the Mesoscale Discovery
system as per the manufacturer's instructions. Cell lysates were
analyzed by Western blots as described (Mirzoeva S, et al., J Med
Chem 2002, 45:563-566; Ralay Ranaivo H, et al., J Neurosci 2006,
26:662-670) to determine the levels of inducible nitric oxide
synthase (iNOS) and cyclooxygenase-2 (COX-2). Results for compounds
of the invention are shown in Table 1.
Oral Bioavailability and Brain Uptake
[0427] To estimate oral bioavailability (concentration of compound
in the blood as a function of time after oral administration) and
to gain insight into potential brain uptake, compound 5 (2.5 mg/kg)
was administered to mice by oral gavage in a 0.5% (w/v)
carboxymethylcellulose suspension (Ralay Ranaivo H, et al., J
Neurosci 2006, 26:662-670). At 5, 15, 30, 60 and 120 min after oral
administration, mice were sacrificed, perfused and their blood and
brain were harvested. Brains were homogenized in acetonitrile and
then centrifuged at 12000.times.g for 10 minutes. Next, the plasma
and the brain supernatant were acidified by diluting with 0.1%
formic acid (Fluka) 1:1 and 1:3, respectively. Solid phase
extraction followed by HPLC analysis was used to quantify the
amount of compound in the plasma brain supernatants. Briefly,
cartridges (Sep-Pak.RTM. C18, Waters) were conditioned with 1 ml of
acetonitrile (HPLC grade, EMD Biosciences) and equilibrated with 1
ml of water. A structural analog,
6-methyl-4-phenyl-3-(4(pyrimidin-2-yl)piperazin-1-yl)pyridazine
(MW01-7-057WH), was used as an internal recovery standard.
Acidified samples were loaded to the cartridge followed by a 1 ml
wash with 10% acetonitrile. Compound 5 was eluted from the
cartridge using 80% acetonitrile. The eluate was evaporated to
dryness, reconstituted in 0.08% formic acid/water in 80%
acetonitrile and analyzed by HPLC with 0.1% formic acid in water as
reagent A and 0.1% formic acid in acetonitrile as reagent B using
the following gradient in reagent B: 0% to 50% to 3 min, isocratic
at 50% until 6 min, 50% to 70% from 6 to 10 min, isocratic at 70%
until 13 min, 70% to 80% from 13 to 18 min, isocratic at 80% until
21 min, 80% to 70% from 21 to 23 min, and finally returning from
70% to 0% from 23 to 28 min.
[0428] In vivo efficacy studies in mice. The study design and
treatment paradigm for intracerebroventricular (ICV) infusion of
human oligomeric A.beta..sub.1-42 into the mouse were as described
previously (Craft J M, et al., Neurobiol Aging 2004, 25,:
1283-1292) except that compound administration was by mouth (Ralay
Ranaivo H, et al., J Neurosci 2006, 26:662-670). It was previously
shown that A.beta.-induced neuroinflammation is an early event
associated with the onset and progression of pathophysiology, and
can be suppressed by an inhibitor of glial activation. Female
C57Bl/6 mice (Harlan) weighing 20-25 g (3-4 months old) were housed
in a pathogen free facility under an approximate 12 h/12 h dark and
light cycle and had access ad libitum to food and water. All animal
procedures were approved by the Northwestern Animal Care and Use
Committee.
[0429] Mice were administered by oral gavage either compound 5 (2.5
mg/kg/day) or solvent control (10% DMSO) in a 0.5% (w/v)
carboxymethylcellulose suspension, once per day treatment began at
day 21 after start of A.beta. ICV infusion and continued for 14
days (Ralay Ranaivo H, et al., J Neurosci 2006, 26:662-670).
Beginning at day 50 after start of A.beta. ICV infusion, the Y maze
test of spontaneous alternation was used to evaluate
hippocampus-dependent spatial learning as described previously
(Ralay Ranaivo H, et al., J Neurosci 2006, 26:662-670). At day 60
after start of A.beta. ICV infusion, mice were sacrificed, perfused
with a HEPES buffer (10 mM, pH 7.2) containing a protease inhibitor
cocktail and brain was harvested and dissected as described
previously (Ralay Ranaivo H, et al., J Neurosci 2006, 26:662-670).
Levels of IL-1.beta. and TNF.alpha., S100B, synaptophysin, PSD-95,
levels in hippocampal supernatants were measured as previously
described (Ralay Ranaivo H, et al., J Neurosci 2006, 26:662-670;
Craft J M, et al., Neurobiol Aging 2004, 25: 1283-1292; Eldik, L J,
1994).
[0430] Immunohistochemical detection of GFAP-positive activated
astrocytes and F4/80 positive microglia was performed on 10 .mu.m
sections as described previously (Ralay Ranaivo H, et al., J
Neurosci 2006, 26:662-670; Craft J M, et al., Neurobiol Aging 2004,
25: 1283-1292).
[0431] Statistical analyses. Experimental and control groups were
compared using one-way ANOVA with Newman-Keuls post-hoc analysis
using a statistical software package (GraphPad Prism version 4.00,
GraphPad Software, San Diego Calif.). Statistical significance was
assumed when p<0.05.
[0432] The present invention is not to be limited in scope by the
specific embodiments described herein, since such embodiments are
intended as but single illustrations of one aspect of the invention
and any functionally equivalent embodiments are within the scope of
this invention. Indeed, various modifications of the invention in
addition to those shown and described herein will become apparent
to those skilled in the art from the foregoing description and
accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
[0433] All publications, patents and patent applications referred
to herein are incorporated by reference in their entirety to the
same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference in its entirety. All publications,
patents and patent applications mentioned herein are incorporated
herein by reference for the purpose of describing and disclosing
the methods etc. which are reported therein which might be used in
connection with the invention. Nothing herein is to be construed as
an admission that the invention is not entitled to antedate such
disclosure by virtue of prior invention.
TABLE-US-00001 TABLE 1 Medicinal chemistry refinement ##STR00011##
log log IL- R4 R3 MW* S* P* 1.beta..sup..perp. NO.sup..perp. 1
##STR00012## ##STR00013## 394.47 -5.40 3.88 2.5 .+-. 1.6 >25 2
##STR00014## ##STR00015## 408.50 -5.33 3.82 5.3 .+-. 0.6 >25 3
##STR00016## ##STR00017## 395.46 -4.41 2.40 25.8 .+-. 7.0 >25 4
##STR00018## ##STR00019## 374.48 -4.89 3.71 6.1 .+-. 2.5 22 .+-. 3
5 CH.sub.3 ##STR00020## 332.40 -4.08 2.29 8.3 .+-. 5.8 >25 6 Cl
##STR00021## 352.82 -4.64 2.76 9.5 .+-. 4.0 19 .+-. 8 7 CH.sub.3
##STR00022## 332.40 -1.48 2.01 46.1 .+-. 23.3 8 CH.sub.3
##STR00023## 331.41 -2.11 2.45 7.6 .+-. 2.9 >25 9 CH.sub.3
##STR00024## 331.41 -2.09 2.38 17.7 .+-. 7.2 >25 10 CH.sub.3
##STR00025## 336.47 -2.80 3.88 31.4 .+-. 4.9 11 CH.sub.3 CH.sub.3
268.36 -1.22 1.83 48.9 .+-. 22.2 *Calculated using ACD/Solubility
DB 9.03. logS is intrinsic solubility of neutral form of compounds.
PSA: 1,2,4-7 = 58.04; 3 = 70.93; 8,9 = 45.15; 10,11 = 32.26.
.sup..perp.Concentration (.mu.M) required for 50% inhibition.sup.8.
IL-1.beta. = interleukin-1.beta.; NO = nitric oxide.
TABLE-US-00002 TABLE 2 Compounds of the formula II Compound Final
Code ##STR00026## MWol-2-069A-SRM ##STR00027## MW01-6-127WH
##STR00028## MW01-6-189WH ##STR00029## WH 151SRM ##STR00030##
MW01-2-069A- SRM ##STR00031## MW01-1-030A- LKM ##STR00032##
MW01-2-127LKM ##STR00033## MW01-2-134LKM ##STR00034## MW01-2-023SRM
##STR00035## MW01-2-141SRM ##STR00036## MW01-2-163MAS ##STR00037##
MW01-3-024SRM ##STR00038## MW01-3-027SRM ##STR00039## MW01-7-100WH
##STR00040## MW01-2103LPI
* * * * *