U.S. patent application number 16/705375 was filed with the patent office on 2020-05-28 for heterocyclic inhibitors of glutaminase.
The applicant listed for this patent is Calithera Biosciences, Inc.. Invention is credited to Lijing Chen, Bindu Goyal, Guy Laidig, Jim Li, Eric B. Sjogren, Timothy Friend Stanton.
Application Number | 20200165238 16/705375 |
Document ID | / |
Family ID | 48610735 |
Filed Date | 2020-05-28 |
View All Diagrams
United States Patent
Application |
20200165238 |
Kind Code |
A1 |
Li; Jim ; et al. |
May 28, 2020 |
Heterocyclic Inhibitors of Glutaminase
Abstract
The invention relates to novel heterocyclic compounds and
pharmaceutical preparations thereof. The invention further relates
to methods of treatment using the novel heterocyclic compounds of
the invention.
Inventors: |
Li; Jim; (San Francisco,
CA) ; Chen; Lijing; (Cupertino, CA) ; Goyal;
Bindu; (Fremont, CA) ; Laidig; Guy; (Woodside,
CA) ; Stanton; Timothy Friend; (Pacifica, CA)
; Sjogren; Eric B.; (Mountain View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Calithera Biosciences, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
48610735 |
Appl. No.: |
16/705375 |
Filed: |
December 6, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16403142 |
May 3, 2019 |
|
|
|
16705375 |
|
|
|
|
14471476 |
Aug 28, 2014 |
|
|
|
16403142 |
|
|
|
|
14051216 |
Oct 10, 2013 |
8865718 |
|
|
14471476 |
|
|
|
|
13680582 |
Nov 19, 2012 |
8604016 |
|
|
14051216 |
|
|
|
|
61665370 |
Jun 28, 2012 |
|
|
|
61562266 |
Nov 21, 2011 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 285/125 20130101;
C07D 417/14 20130101; C07D 417/06 20130101; A61N 5/00 20130101;
A61N 5/10 20130101; A61K 31/433 20130101; A61K 45/06 20130101; A61K
31/5377 20130101; A61K 31/506 20130101; C07D 285/135 20130101; A61N
7/00 20130101; A61B 18/02 20130101; A61K 31/501 20130101 |
International
Class: |
C07D 417/14 20060101
C07D417/14; C07D 285/125 20060101 C07D285/125; A61N 5/10 20060101
A61N005/10; A61K 31/433 20060101 A61K031/433; A61N 7/00 20060101
A61N007/00; A61N 5/00 20060101 A61N005/00; A61K 45/06 20060101
A61K045/06; A61K 31/5377 20060101 A61K031/5377; A61K 31/506
20060101 A61K031/506; A61K 31/501 20060101 A61K031/501; A61B 18/02
20060101 A61B018/02; C07D 417/06 20060101 C07D417/06; C07D 285/135
20060101 C07D285/135 |
Claims
1-71. (canceled)
72. A method of making a compound having the structure of formula
D: ##STR00924## comprising combining a compound having the
structure of formula F: ##STR00925## with BrZn(CH.sub.2).sub.4CN
solution and a NiCl.sub.2(dppp) catalyst, thereby forming a
compound of formula D.
73. The method of claim 72, further comprising combining a compound
having the structure of formula G: ##STR00926## with a compound
having the structure of formula H: ##STR00927## to form a mixture,
then treating the mixture with diisopropylethylamine and
propylphosphonic anhydride, thereby forming a compound of formula
F.
74. The method of claim 73, wherein combining the compound having
the structure of formula G with the compound having the structure
of formula H occurs in the presence of dimethylformamide.
75. A method of making a compound having the structure of formula
D: ##STR00928## comprising combining a compound having the
structure of formula K: ##STR00929## with Pd(OH).sub.2/C under a
hydrogen atmosphere, thereby forming the compound of formula D.
76. The method of claim 75, further comprising combining a compound
having the structure of formula L: ##STR00930## wherein X is
selected from Cl, Br, and I; with pent-4-ynenitrile, thereby
forming the compound of formula K.
77. The method of claim 76, further comprising combining a compound
having the structure of formula M: ##STR00931## with
3-trifluoromethylphenylacetyl chloride, thereby forming the
compound of formula L.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application No. 61/562,266, filed Nov. 21, 2011,
and U.S. Provisional Patent Application No. 61/665,370, filed Jun.
28, 2012, which applications are hereby incorporated by reference
in their entirety.
BACKGROUND
[0002] Glutamine supports cell survival, growth and proliferation
through metabolic and non-metabolic mechanisms. In actively
proliferating cells, the metabolism of glutamine to lactate, also
referred to as "glutaminolysis" is a maj or source of energy in the
form of NADPH. The first step in glutaminolysis is the deamination
of glutamine to form glutamate and ammonia, which is catalyzed by
the glutaminase enzyme. Thus, deamination via glutaminase is a
control point for glutamine metabolism.
[0003] Ever since Warburg's observation that ascites tumor cells
exhibited high rates of glucose consumption and lactate secretion
in the presence of oxygen (Warburg, 1956), researchers have been
exploring how cancer cells utilize metabolic pathways to be able to
continue actively proliferating. Several reports have demonstrated
how glutamine metabolism supports macromolecular synthesis
necessary for cells to replicate (Curthoys, 1995; DeBardinis,
2008).
[0004] Thus, glutaminase has been theorized to be a potential
therapeutic target for the treatment of diseases characterized by
actively proliferating cells, such as cancer. The lack of suitable
glutaminase inhibitors has made validation of this target
impossible. Therefore, the creation of glutaminase inhibitors that
are specific and capable of being formulated for in vivo use could
lead to a new class of therapeutics.
SUMMARY OF INVENTION
[0005] The present invention provides a compound of formula I,
##STR00001## [0006] or a pharmaceutically acceptable salt thereof,
wherein: [0007] L represents CH.sub.2SCH.sub.2, CH.sub.2CH.sub.2,
CH.sub.2CH.sub.2CH.sub.2, CH.sub.2, CH.sub.2S, SCH.sub.2,
CH.sub.2NHCH.sub.2, CH.dbd.CH, or
##STR00002##
[0007] preferably CH.sub.2CH.sub.2, wherein any hydrogen atom of a
CH or CH.sub.2 unit may be replaced by alkyl or alkoxy, any
hydrogen of an NH unit may be replaced by alkyl, and any hydrogen
atom of a CH.sub.2 unit of CH.sub.2CH.sub.2,
CH.sub.2CH.sub.2CH.sub.2 or CH.sub.2 may be replaced by hydroxy;
[0008] X, independently for each occurrence, represents S, O or
CH.dbd.CH, preferably S or CH.dbd.CH, wherein any hydrogen atom of
a CH unit may be replaced by alkyl; [0009] Y, independently for
each occurrence, represents H or CH.sub.2O(CO)R.sub.7; [0010]
R.sub.7, independently for each occurrence, represents H or
substituted or unsubstituted alkyl, alkoxy, aminoalkyl,
alkylaminoalkyl, heterocyclylalkyl, arylalkyl, or
heterocyclylalkoxy; [0011] Z represents H or R.sub.3(CO); [0012]
R.sub.1 and R.sub.2 each independently represent H, alkyl, alkoxy
or hydroxy; [0013] R.sub.3, independently for each occurrence,
represents substituted or unsubstituted alkyl, hydroxyalkyl,
aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl,
arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl,
heteroaryloxy, heteroaryloxyalkyl or C(R.sub.8)(R.sub.9)(R.sub.10),
N(R.sub.4)(R.sub.5) or OR.sub.6, wherein any free hydroxyl group
may be acylated to form C(O)R.sub.7; [0014] R.sub.4 and R.sub.5
each independently represent H or substituted or unsubstituted
alkyl, hydroxyalkyl, acyl, aminoalkyl, acylaminoalkyl, alkenyl,
alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl,
heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein any
free hydroxyl group may be acylated to form C(O)R.sub.7; [0015]
R.sub.6, independently for each occurrence, represents substituted
or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl,
alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,
heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl,
wherein any free hydroxyl group may be acylated to form
C(O)R.sub.7; and [0016] R.sub.8, R.sub.9 and R.sub.10 each
independently represent H or substituted or unsubstituted alkyl,
hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl,
acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl,
alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,
heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl,
or R.sub.8 and R.sub.9 together with the carbon to which they are
attached, form a carbocyclic or heterocyclic ring system, wherein
any free hydroxyl group may be acylated to form C(O)R.sub.7, and
wherein at least two of R.sub.8, R.sub.9 and R.sub.10 are not
H.
[0017] In certain embodiments, the present invention provides a
pharmaceutical preparation suitable for use in a human patient,
comprising an effective amount of any of the compounds described
herein (e.g., a compound of the invention, such as a compound of
formula I), and one or more pharmaceutically acceptable excipients.
In certain embodiments, the pharmaceutical preparations may be for
use in treating or preventing a condition or disease as described
herein. In certain embodiments, the pharmaceutical preparations
have a low enough pyrogen activity to be suitable for intravenous
use in a human patient.
[0018] The present invention further provides methods of treating
or preventing cancer, immunological or neurological diseases as
described herein, comprising administering a compound of the
invention.
DETAILED DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows that intraperitoneal administration of compound
188 to mice results in reduced tumor size in a HCT116 colon
carcinoma xenograft model.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention provides a compound of formula I,
##STR00003## [0021] or a pharmaceutically acceptable salt thereof,
wherein: [0022] L represents CH.sub.2SCH.sub.2, CH.sub.2CH.sub.2,
CH.sub.2CH.sub.2CH.sub.2, CH.sub.2, CH.sub.2S, SCH.sub.2,
CH.sub.2NHCH.sub.2, CH.dbd.CH, or
##STR00004##
[0022] preferably CH.sub.2CH.sub.2, wherein any hydrogen atom of a
CH or CH.sub.2 unit may be replaced by alkyl or alkoxy, any
hydrogen of an NH unit may be replaced by alkyl, and any hydrogen
atom of a CH.sub.2 unit of CH.sub.2CH.sub.2,
CH.sub.2CH.sub.2CH.sub.2 or CH.sub.2 may be replaced by hydroxy;
[0023] X, independently for each occurrence, represents S, O or
CH.dbd.CH, preferably S or CH.dbd.CH, wherein any hydrogen atom of
a CH unit may be replaced by alkyl; [0024] Y, independently for
each occurrence, represents H or CH.sub.2O(CO)R.sub.7; [0025]
R.sub.7, independently for each occurrence, represents H or
substituted or unsubstituted alkyl, alkoxy, aminoalkyl,
alkylaminoalkyl, heterocyclylalkyl, arylalkyl, or
heterocyclylalkoxy; [0026] Z represents H or R.sub.3(CO); [0027]
R.sub.1 and R.sub.2 each independently represent H, alkyl, alkoxy
or hydroxy; [0028] R.sub.3, independently for each occurrence,
represents substituted or unsubstituted alkyl, hydroxyalkyl,
aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl,
arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl,
heteroaryloxy, heteroaryloxyalkyl or C(R.sub.8)(R.sub.9)(R.sub.10),
N(R.sub.4)(R.sub.5) or OR.sub.6, wherein any free hydroxyl group
may be acylated to form C(O)R.sub.7; [0029] R.sub.4 and R.sub.5
each independently represent H or substituted or unsubstituted
alkyl, hydroxyalkyl, acyl, aminoalkyl, acylaminoalkyl, alkenyl,
alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl,
heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein any
free hydroxyl group may be acylated to form C(O)R.sub.7; [0030]
R.sub.6, independently for each occurrence, represents substituted
or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl,
alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,
heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl,
wherein any free hydroxyl group may be acylated to form
C(O)R.sub.7; and [0031] R.sub.8, R.sub.9 and R.sub.10 each
independently represent H or substituted or unsubstituted alkyl,
hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl,
acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl,
alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,
heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl,
or R.sub.8 and R.sub.9 together with the carbon to which they are
attached, form a carbocyclic or heterocyclic ring system, wherein
any free hydroxyl group may be acylated to form C(O)R.sub.7, and
wherein at least two of R.sub.8, R.sub.9 and R.sub.10 are not
H.
[0032] In certain embodiments wherein alkyl, hydroxyalkyl, amino,
acylamino, aminoalkyl, acylaminoalkyl, alkenyl, alkoxy,
alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl,
heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl are
substituted, they are substituted with one or more substituents
selected from substituted or unsubstituted alkyl, such as
perfluoroalkyl (e.g., trifluoromethyl), alkenyl, alkoxy,
alkoxyalkyl, aryl, aralkyl, arylalkoxy, aryloxy, aryloxyalkyl,
hydroxyl, halo, alkoxy, such as perfluoroalkoxy (e.g.,
trifluoromethoxy), alkoxyalkoxy, hydroxyalkyl, hydroxyalkylamino,
hydroxyalkoxy, amino, aminoalkyl, alkylamino, aminoalkylalkoxy,
aminoalkoxy, acylamino, acylaminoalkyl, such as perfluoro
acylaminoalkyl (e.g., trifluoromethylacylaminoalkyl), acyloxy,
cycloalkyl, cycloalkylalkyl, cycloalkylalkoxy, heterocyclyl,
heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, heteroaryl,
heteroarylalkyl, heteroarylalkoxy, heteroaryloxy,
heteroaryloxyalkyl, heterocyclylaminoalkyl,
heterocyclylaminoalkoxy, amido, amidoalkyl, amidine, imine, oxo,
carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl,
including perfluoroacyl (e.g., C(O)CF.sub.3)), carbonylalkyl (such
as carboxyalkyl, alkoxycarbonylalkyl, formylalkyl, or acylalkyl,
including perfluoroacylalkyl (e.g., -alkylC(O)CF.sub.3)),
carbamate, carbamatealkyl, urea, ureaalkyl, sulfate, sulfonate,
sulfamoyl, sulfone, sulfonamide, sulfonamidealkyl, cyano, nitro,
azido, sulfhydryl, alkylthio, thiocarbonyl (such as thioester,
thioacetate, or thioformate), phosphoryl, phosphate, phosphonate or
phosphinate.
[0033] In certain embodiments, L represents CH.sub.2SCH.sub.2,
CH.sub.2CH.sub.2, CH.sub.2CH.sub.2CH.sub.2, CH.sub.2, CH.sub.2S,
SCH.sub.2, or CH.sub.2NHCH.sub.2, wherein any hydrogen atom of a
CH.sub.2 unit may be replaced by alkyl or alkoxy, and any hydrogen
atom of a CH.sub.2 unit of CH.sub.2CH.sub.2,
CH.sub.2CH.sub.2CH.sub.2 or CH.sub.2 may be replaced by hydroxyl.
In certain embodiments, L represents CH.sub.2SCH.sub.2,
CH.sub.2CH.sub.2, CH.sub.2S or SCH.sub.2. In certain embodiments, L
represents CH.sub.2CH.sub.2. In certain embodiments, L is not
CH.sub.2SCH.sub.2.
[0034] In certain embodiments, Y represents H.
[0035] In certain embodiments, X represents S or CH.dbd.CH. In
certain embodiments, one or both X represents CH.dbd.CH. In certain
embodiments, each X represents S. In certain embodiments, one X
represents S and the other X represents CH.dbd.CH.
[0036] In certain embodiments, Z represents R.sub.3(CO). In certain
embodiments wherein Z is R.sub.3(CO), each occurrence of R.sub.3 is
not identical (e.g., the compound of formula I is not
symmetrical).
[0037] In certain embodiments, R.sub.1 and R.sub.2 each represent
H.
[0038] In certain embodiments, R.sub.3 represents arylalkyl,
heteroarylalkyl, cycloalkyl or heterocycloalkyl. In certain
embodiments, R.sub.3 represents C(R.sub.8)(R.sub.9)(R.sub.10),
wherein R.sub.8 represents aryl, arylalkyl, heteroaryl or
heteroaralkyl, such as aryl, arylalkyl or heteroaryl, R.sub.9
represents H, and R.sub.10 represents hydroxy, hydroxyalkyl, alkoxy
or alkoxyalkyl, such as hydroxy, hydroxyalkyl or alkoxy.
[0039] In certain embodiments, L represents CH.sub.2SCH.sub.2,
CH.sub.2CH.sub.2, CH.sub.2S or SCH.sub.2, such as CH.sub.2CH.sub.2,
CH.sub.2S or SCH.sub.2, Y represents H, X represents S, Z
represents R.sub.3(CO), R.sub.1 and R.sub.2 each represent H, and
each R.sub.3 represents arylalkyl, heteroarylalkyl, cycloalkyl or
heterocycloalkyl. In certain such embodiments, each occurrence of
R.sub.3 is identical.
[0040] In certain embodiments, L represents CH.sub.2SCH.sub.2,
CH.sub.2CH.sub.2, CH.sub.2S or SCH.sub.2, Y represents H, X
represents S, Z represents R.sub.3(CO), R.sub.1 and R.sub.2 each
represent H, and each R.sub.3 represents
C(R.sub.8)(R.sub.9)(R.sub.10), wherein R.sub.8 represents aryl,
arylalkyl, heteroaryl or heteroaralkyl, such as aryl, arylalkyl or
heteroaryl, R.sub.9 represents H, and R.sub.10 represents hydroxy,
hydroxyalkyl, alkoxy or alkoxyalkyl, such as hydroxy, hydroxyalkyl
or alkoxy. In certain such embodiments, each occurrence of R.sub.3
is identical.
[0041] In certain embodiments, L represents CH.sub.2CH.sub.2, Y
represents H, X represents S or CH.dbd.CH, Z represents
R.sub.3(CO), R.sub.1 and R.sub.2 each represent H, and each R.sub.3
represents substituted or unsubstituted arylalkyl, heteroarylalkyl,
cycloalkyl or heterocycloalkyl. In certain such embodiments, each X
represents S. In other embodiments, one or both occurrences of X
represents CH.dbd.CH, such as one occurrence of X represents S and
the other occurrence of X represents CH.dbd.CH. In certain
embodiments of the foregoing, each occurrence of R.sub.3 is
identical. In other embodiments of the foregoing wherein one
occurrence of X represents S and the other occurrence of X
represents CH.dbd.CH, the two occurrences of R.sub.3 are not
identical.
[0042] In certain embodiments, L represents CH.sub.2CH.sub.2, Y
represents H, X represents S, Z represents R.sub.3(CO), R.sub.1 and
R.sub.2 each represent H, and each R.sub.3 represents
C(R.sub.8)(R.sub.9)(R.sub.10), wherein R.sub.8 represents aryl,
arylalkyl or heteroaryl, R.sub.9 represents H, and R.sub.10
represents hydroxy, hydroxyalkyl or alkoxy. In certain such
embodiments, R.sub.8 represents aryl and R.sub.10 represents
hydroxyalkyl. In certain such embodiments, each occurrence of
R.sub.3 is identical.
[0043] In certain embodiments wherein L represents CH.sub.2,
CH.sub.2CH.sub.2CH.sub.2 or CH.sub.2CH.sub.2, X represents O, and Z
represents R.sub.3(CO), both R.sub.3 groups are not alkyl, such as
methyl, or C(R.sub.8)(R.sub.9)(R.sub.10), wherein R.sub.8, R.sub.9
and R.sub.10 are each independently hydrogen or alkyl.
[0044] In certain embodiments wherein L represents
CH.sub.2CH.sub.2, X represents S, and Z represents R.sub.3(CO),
both R.sub.3 groups are not phenyl or heteroaryl, such as
2-furyl.
[0045] In certain embodiments wherein L represents
CH.sub.2CH.sub.2, X represents O, and Z represents R.sub.3(CO),
both R.sub.3 groups are not N(R.sub.4)(R.sub.5) wherein R.sub.4 is
aryl, such as phenyl, and R.sub.5 is H.
[0046] In certain embodiments wherein L represents
CH.sub.2SCH.sub.2, X represents S, and Z represents R.sub.3(CO),
both R.sub.3 groups are not aryl, such as optionally substituted
phenyl, aralkyl, such as benzyl, heteroaryl, such as 2-furyl,
2-thienyl or 1,2,4-trizole, substituted or unsubstituted alkyl,
such as methyl, chloromethyl, dichloromethyl, n-propyl, n-butyl,
t-butyl or hexyl, heterocyclyl, such as pyrimidine-2,4
(1H,3H)-dione, or alkoxy, such as methoxy, pentyloxy or ethoxy.
[0047] In certain embodiments wherein L represents
CH.sub.2SCH.sub.2, X represents S, and Z represents R.sub.3(CO),
both R.sub.3 groups are not N(R.sub.4)(R.sub.5) wherein R.sub.4 is
aryl, such as substituted or unsubstituted phenyl (e.g., phenyl,
3-tolyl, 4-tolyl, 4-bromophenyl or 4-nitrophenyl), and R.sub.5 is
H.
[0048] In certain embodiments wherein L represents
CH.sub.2CH.sub.2CH.sub.2, X represents S, and Z represents
R.sub.3(CO), both R.sub.3 groups are not alkyl, such as methyl,
ethyl, or propyl, cycloalkyl, such as cyclohexyl, or
C(R.sub.8)(R.sub.9)(R.sub.10), wherein any of R.sub.8, R.sub.9 and
R.sub.10 together with the C to which they are attached, form any
of the foregoing.
[0049] In certain embodiments, the compound is not one of the
following:
##STR00005## ##STR00006## ##STR00007##
[0050] The present invention further provides a compound of formula
Ia,
##STR00008## [0051] or a pharmaceutically acceptable salt thereof,
wherein: [0052] L represents CH.sub.2SCH.sub.2, CH.sub.2CH.sub.2,
CH.sub.2CH.sub.2CH.sub.2, CH.sub.2, CH.sub.2S, SCH.sub.2,
CH.sub.2NHCH.sub.2, CH.dbd.CH, or
##STR00009##
[0052] preferably CH.sub.2CH.sub.2, wherein any hydrogen atom of a
CH or CH.sub.2 unit may be replaced by alkyl or alkoxy, any
hydrogen of an NH unit may be replaced by alkyl, and any hydrogen
atom of a CH.sub.2 unit of CH.sub.2CH.sub.2,
CH.sub.2CH.sub.2CH.sub.2 or CH.sub.2 may be replaced by hydroxy;
[0053] X represents S, O or CH.dbd.CH, preferably S or CH.dbd.CH,
wherein any hydrogen atom of a CH unit may be replaced by alkyl;
[0054] Y, independently for each occurrence, represents H or
CH.sub.2O(CO)R.sub.7; [0055] R.sub.7, independently for each
occurrence, represents H or substituted or unsubstituted alkyl,
alkoxy, aminoalkyl, alkylaminoalkyl, heterocyclylalkyl, arylalkyl,
or heterocyclylalkoxy; [0056] Z represents H or R.sub.3(CO); [0057]
R.sub.1 and R.sub.2 each independently represent H, alkyl, alkoxy
or hydroxy, preferably H; [0058] R.sub.3 represents substituted or
unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl,
alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy,
aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,
heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy,
heteroaryloxyalkyl or C(R.sub.8)(R.sub.9)(R.sub.10),
N(R.sub.4)(R.sub.5) or OR.sub.6, wherein any free hydroxyl group
may be acylated to form C(O)R.sub.7; [0059] R.sub.4 and R.sub.5
each independently represent H or substituted or unsubstituted
alkyl, hydroxyalkyl, acyl, aminoalkyl, acylaminoalkyl, alkenyl,
alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl,
heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein any
free hydroxyl group may be acylated to form C(O)R.sub.7; [0060]
R.sub.6, independently for each occurrence, represents substituted
or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl,
alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,
heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl,
wherein any free hydroxyl group may be acylated to form
C(O)R.sub.7; and [0061] R.sub.8, R.sub.9 and R.sub.10 each
independently represent H or substituted or unsubstituted alkyl,
hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl,
acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl,
alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,
heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl,
or R.sub.8 and R.sub.9 together with the carbon to which they are
attached, form a carbocyclic or heterocyclic ring system, wherein
any free hydroxyl group may be acylated to form C(O)R.sub.7, and
wherein at least two of R.sub.8, R.sub.9 and R.sub.10 are not H;
[0062] R.sub.11 represents substituted or unsubstituted aryl,
arylalkyl, aryloxy, aryloxyalkyl, heteroaryl, heteroarylalkyl,
heteroaryloxy, or heteroaryloxyalkyl, or
C(R.sub.12)(R.sub.13)(R.sub.14), N(R.sub.4)(R.sub.14) or OR.sub.14,
wherein any free hydroxyl group may be acylated to form
C(O)R.sub.7; [0063] R.sub.12 and R.sub.13 each independently
respresent H or substituted or unsubstituted alkyl, hydroxy,
hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl,
alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl,
aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl,
heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein any
free hydroxyl group may be acylated to form C(O)R.sub.7, and
wherein both of R.sub.12 and R.sub.13 are not H; and [0064]
R.sub.14 represents substituted or unsubstituted aryl, arylalkyl,
aryloxy, aryloxyalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy,
or heteroaryloxyalkyl.
[0065] In certain embodiments wherein alkyl, hydroxyalkyl, amino,
acylamino, aminoalkyl, acylaminoalkyl, alkenyl, alkoxy,
alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl,
heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl are
substituted, they are substituted with one or more substituents
selected from substituted or unsubstituted alkyl, such as
perfluoroalkyl (e.g., trifluoromethyl), alkenyl, alkoxy,
alkoxyalkyl, aryl, aralkyl, arylalkoxy, aryloxy, aryloxyalkyl,
hydroxyl, halo, alkoxy, such as perfluoroalkoxy (e.g.,
trifluoromethylalkoxy), alkoxyalkoxy, hydroxyalkyl,
hydroxyalkylamino, hydroxyalkoxy, amino, aminoalkyl, alkylamino,
aminoalkylalkoxy, aminoalkoxy, acylamino, acylaminoalkyl, such as
perfluoro acylaminoalkyl (e.g., trifluoromethylacylaminoalkyl),
acyloxy, cycloalkyl, cycloalkylalkyl, cycloalkylalkoxy,
heterocyclyl, heterocyclylalkyl, heterocyclyloxy,
heterocyclylalkoxy, heteroaryl, heteroarylalkyl, heteroarylalkoxy,
heteroaryloxy, heteroaryloxyalkyl, heterocyclylaminoalkyl,
heterocyclylaminoalkoxy, amido, amidoalkyl, amidine, imine, oxo,
carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl,
including perfluoroacyl (e.g., C(O)CF.sub.3)), carbonylalkyl (such
as carboxyalkyl, alkoxycarbonylalkyl, formylalkyl, or acylalkyl,
including perfluoroacylalkyl (e.g., -alkylC(O)CF.sub.3)),
carbamate, carbamatealkyl, urea, ureaalkyl, sulfate, sulfonate,
sulfamoyl, sulfone, sulfonamide, sulfonamidealkyl, cyano, nitro,
azido, sulfhydryl, alkylthio, thiocarbonyl (such as thioester,
thioacetate, or thioformate), phosphoryl, phosphate, phosphonate or
phosphinate.
[0066] In certain embodiments, R.sub.11 represents substituted or
unsubstituted arylalkyl, such as substituted or unsubstituted
benzyl.
[0067] In certain embodiments, L represents CH.sub.2SCH.sub.2,
CH.sub.2CH.sub.2, CH.sub.2CH.sub.2CH.sub.2, CH.sub.2, CH.sub.2S,
SCH.sub.2, or CH.sub.2NHCH.sub.2, wherein any hydrogen atom of a
CH.sub.2 unit may be replaced by alkyl or alkoxy, and any hydrogen
atom of a CH.sub.2 unit of CH.sub.2CH.sub.2,
CH.sub.2CH.sub.2CH.sub.2 or CH.sub.2 may be replaced by hydroxyl.
In certain embodiments, L represents CH.sub.2SCH.sub.2,
CH.sub.2CH.sub.2, CH.sub.2S or SCH.sub.2, preferably
CH.sub.2CH.sub.2. In certain embodiments, L is not
CH.sub.2SCH.sub.2.
[0068] In certain embodiments, each Y represents H. In other
embodiments, at least one Y is CH.sub.2O(CO)R.sub.7.
[0069] In certain embodiments, X represents S or CH.dbd.CH. In
certain embodiments, X represents S.
[0070] In certain embodiments, R.sub.1 and R.sub.2 each represent
H.
[0071] In certain embodiments, Z represents R.sub.3(CO). In certain
embodiments wherein Z is R.sub.3(CO), R.sub.3 and R.sub.11 are not
identical (e.g., the compound of formula I is not symmetrical).
[0072] In certain embodiments, Z represents R.sub.3(CO) and R.sub.3
represents arylalkyl, heteroarylalkyl, cycloalkyl or
heterocycloalkyl. In certain embodiments, Z represents R.sub.3(CO)
and R.sub.3 represents C(R.sub.8)(R.sub.9)(R.sub.10), wherein
R.sub.8 represents aryl, arylalkyl, heteroaryl or heteroaralkyl,
such as aryl, arylalkyl or heteroaryl, R.sub.9 represents H, and
R.sub.10 represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl,
such as hydroxy, hydroxyalkyl or alkoxy. In certain embodiments, Z
represents R.sub.3(CO) and R.sub.3 represents heteroarylalkyl.
[0073] In certain embodiments, L represents CH.sub.2SCH.sub.2,
CH.sub.2CH.sub.2, CH.sub.2S or SCH.sub.2, such as CH.sub.2CH.sub.2,
Y represents H, X represents S, Z represents R.sub.3(CO), R.sub.1
and R.sub.2 each represent H, R.sub.3 represents arylalkyl,
heteroarylalkyl, cycloalkyl or heterocycloalkyl, and R.sub.11
represents arylalkyl. In certain such embodiments, R.sub.3
represents heteroarylalkyl.
[0074] In certain embodiments, L represents CH.sub.2SCH.sub.2,
CH.sub.2CH.sub.2, CH.sub.2S or SCH.sub.2, such as CH.sub.2CH.sub.2,
Y represents H, X represents S, Z represents R.sub.3(CO), R.sub.1
and R.sub.2 each represent H, and each R.sub.3 represents
C(R.sub.8)(R.sub.9)(R.sub.10), wherein R.sub.8 represents aryl,
arylalkyl, heteroaryl or heteroaralkyl, such as aryl, arylalkyl or
heteroaryl, R.sub.9 represents H, and R.sub.10 represents hydroxy,
hydroxyalkyl, alkoxy or alkoxyalkyl, such as hydroxy, hydroxyalkyl
or alkoxy, and R.sub.11 represents arylalkyl. In certain such
embodiments, R.sub.8 represents heteroaryl.
[0075] In certain embodiments, L represents CH.sub.2CH.sub.2, Y
represents H, X represents S or CH.dbd.CH, such as S, Z represents
R.sub.3(CO), R.sub.1 and R.sub.2 each represent H, R.sub.3
represents substituted or unsubstituted arylalkyl, heteroarylalkyl,
cycloalkyl or heterocycloalkyl, and R.sub.11 represents arylalkyl.
In certain such embodiments, R.sub.3 represents
heteroarylalkyl.
[0076] In certain embodiments, L represents CH.sub.2CH.sub.2, Y
represents H, X represents S, Z represents R.sub.3(CO), R.sub.1 and
R.sub.2 each represent H, R.sub.3 represents
C(R.sub.8)(R.sub.9)(R.sub.10), wherein R.sub.8 represents aryl,
arylalkyl or heteroaryl, R.sub.9 represents H, and R.sub.10
represents hydroxy, hydroxyalkyl or alkoxy, and R.sub.11 represents
arylalkyl. In certain such embodiments, R.sub.8 represents aryl and
R.sub.10 represents hydroxyalkyl. In certain other embodiments,
R.sub.8 represents heteroaryl.
[0077] In certain embodiments, the compound is selected from any
one of the compounds disclosed in Table 3. Preferably, the compound
is selected from compound 1, 2, 6, 7, 8, 11, 13, 14, 15, 16, 17,
18, 19, 20, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 38,
39, 40, 41, 43, 44, 47, 48, 50, 51, 52, 54, 55, 58, 63, 64, 65, 67,
68, 69, 70, 71, 72, 73, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 92, 93, 94, 95, 97, 99, 100, 102, 105, 107, 111, 112, 114, 115,
116, 117, 118, 120, 121, 122, 123, 126, 127, 133, 135, 136, 138,
140, 141, 143, 146, 147, 148, 152, 153, 155, 156, 157, 158, 159,
160, 161, 162, 163, 164, 165, 166, 168, 169, 170, 172, 173, 174,
175, 176, 177, 178, 179, 180, 181, 182, 185, 186, 187, 188, 189,
190, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,
205, 208, 210, 211, 213, 214, 216, 217, 219, 220, 226, 227, 228,
229, 231, 232, 234, 235, 236, 237, 239, 240, 241, 242, 243, 244,
245, 246, 247, 248, 249, 250, 251, 252, 255, 256, 257, 258, 259,
260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 273,
274, 275, 276, 278, 279, 280, 281, 282, 283, 285, 286, 287, 288,
290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 302, 304,
1038, 306, 307, 308, 309, 310, 311, 313, 314, 315, 316, 317, 318,
319, 320, 321, 322, 323, 324, 325, 327, 329, 332, 333, 334, 335,
336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 527, 347,
348, 349, 350, 351, 352, 353, 354, 355, 358, 359, 360, 361, 362,
363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375,
376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,
389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401,
402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,
415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427,
428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,
441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,
454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,
467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,
480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,
493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505,
506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518,
519, 520, 521, 522, 523, 528, 529, 530, 531, 532, 533, 534, 535,
536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548,
549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561,
562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574,
575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587,
588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600,
601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613,
614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626,
627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640,
641, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655,
656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668,
669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681,
682, 683, 684, 685, 686, 687, 688, 689, 690, 692, 693, 694, 695,
696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 707, 708, or
709.
[0078] In certain embodiments, compounds of the invention may be
prodrugs of the compounds of formula I, e.g., wherein a hydroxyl in
the parent compound is presented as an ester or a carbonate, or
carboxylic acid present in the parent compound is presented as an
ester. In certain such embodiments, the prodrug is metabolized to
the active parent compound in vivo (e.g., the ester is hydrolyzed
to the corresponding hydroxyl, or carboxylic acid).
[0079] In certain embodiments, compounds of the invention may be
racemic. In certain embodiments, compounds of the invention may be
enriched in one enantiomer. For example, a compound of the
invention may have greater than 30% ee, 40% ee, 50% ee, 60% ee, 70%
ee, 80% ee, 90% ee, or even 95% or greater ee. In certain
embodiments, compounds of the invention may have more than one
stereocenter. In certain such embodiments, compounds of the
invention may be enriched in one or more diastereomer. For example,
a compound of the invention may have greater than 30% de, 40% de,
50% de, 60% de, 70% de, 80% de, 90% de, or even 95% or greater
de.
[0080] In certain embodiments, the present invention relates to
methods of treatment with a compound of formula I, or a
pharmaceutically acceptable salt thereof. In certain embodiments,
the therapeutic preparation may be enriched to provide
predominantly one enantiomer of a compound (e.g., of formula I). An
enantiomerically enriched mixture may comprise, for example, at
least 60 mol percent of one enantiomer, or more preferably at least
75, 90, 95, or even 99 mol percent. In certain embodiments, the
compound enriched in one enantiomer is substantially free of the
other enantiomer, wherein substantially free means that the
substance in question makes up less than 10%, or less than 5%, or
less than 4%, or less than 3%, or less than 2%, or less than 1% as
compared to the amount of the other enantiomer, e.g., in the
composition or compound mixture. For example, if a composition or
compound mixture contains 98 grams of a first enantiomer and 2
grams of a second enantiomer, it would be said to contain 98 mol
percent of the first enantiomer and only 2% of the second
enantiomer.
[0081] In certain embodiments, the therapeutic preparation may be
enriched to provide predominantly one diastereomer of a compound
(e.g., of formula I). A diastereomerically enriched mixture may
comprise, for example, at least 60 mol percent of one diastereomer,
or more preferably at least 75, 90, 95, or even 99 mol percent.
[0082] In certain embodiments, the present invention relates to
methods of treatment with a compound of formula I, or a
pharmaceutically acceptable salt thereof. In certain embodiments,
the therapeutic preparation may be enriched to provide
predominantly one enantiomer of a compound (e.g., of formula I). An
enantiomerically enriched mixture may comprise, for example, at
least 60 mol percent of one enantiomer, or more preferably at least
75, 90, 95, or even 99 mol percent. In certain embodiments, the
compound enriched in one enantiomer is substantially free of the
other enantiomer, wherein substantially free means that the
substance in question makes up less than 10%, or less than 5%, or
less than 4%, or less than 3%, or less than 2%, or less than 1% as
compared to the amount of the other enantiomer, e.g., in the
composition or compound mixture. For example, if a composition or
compound mixture contains 98 grams of a first enantiomer and 2
grams of a second enantiomer, it would be said to contain 98 mol
percent of the first enantiomer and only 2% of the second
enantiomer.
[0083] In certain embodiments, the therapeutic preparation may be
enriched to provide predominantly one diastereomer of a compound
(e.g., of formula I). A diastereomerically enriched mixture may
comprise, for example, at least 60 mol percent of one diastereomer,
or more preferably at least 75, 90, 95, or even 99 mol percent.
[0084] In certain embodiments, the present invention provides a
pharmaceutical preparation suitable for use in a human patient,
comprising any of the compounds shown above (e.g., a compound of
the invention, such as a compound of formula I), and one or more
pharmaceutically acceptable excipients. In certain embodiments, the
pharmaceutical preparations may be for use in treating or
preventing a condition or disease as described herein. In certain
embodiments, the pharmaceutical preparations have a low enough
pyrogen activity to be suitable for use in a human patient.
[0085] Compounds of any of the above structures may be used in the
manufacture of medicaments for the treatment of any diseases or
conditions disclosed herein.
Uses of Enzyme Inhibitors
[0086] Glutamine plays an important role as a carrier of nitrogen,
carbon, and energy. It is used for hepatic urea synthesis, for
renal ammoniagenesis, for gluconeogenesis, and as respiratory fuel
for many cells. The conversion of glutamine into glutamate is
initated by the mitochondrial enzyme, glutaminase ("GLS"). There
are two major forms of the enzyme, K-type and L-type, which are
distinguished by their Km values for glutamine and response to
glutamate, wherein the Km value, or Michaelis constant, is the
concentration of substrate required to reach half the maximal
velocity. The L-type, also known as "liver-type" or GLS2, has a
high Km for glutamine and is glutamate resistant. The K-type, also
known as "kidney-type or GLS1, has a low Km for glutamine and is
inhibited by glutamate. An alternative splice form of GLS1,
referred to as glutmainase C or "GAC", has been identified recently
and has similar activity characteristics of GLS1. In certain
embodiments, the compounds may selectively inhibit GLS1, GLS2 and
GAC. In a preferred embodiment, the compounds selectively inhibit
GLS1 and GAC.
[0087] In addition to serving as the basic building blocks of
protein synthesis, amino acids have been shown to contribute to
many processes critical for growing and dividing cells, and this is
particularly true for cancer cells. Nearly all definitions of
cancer include reference to dysregulated proliferation. Numerous
studies on glutamine metabolism in cancer indicate that many tumors
are avid glutamine consumers (Souba, Ann. Surg., 1993; Collins et
al., J. Cell. Physiol., 1998; Medina, J. Nutr., 2001; Shanware et
al., J. Mol. Med., 2011). An embodiment of the invention is the use
of the compounds described herein for the treatment of cancer.
[0088] In certain embodiments, the cancer may be one or a variant
of Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia
(AML), Adrenocortical Carcinoma, AIDS-Related Cancers (Kaposi
Sarcoma and Lymphoma), Anal Cancer, Appendix Cancer, Atypical
Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer
(including Extrahepatic), Bladder Cancer, Bone Cancer (including
Osteosarcoma and Malignant Fibrous Histiocytoma), Brain Tumor (such
as Astrocytomas, Brain and Spinal Cord Tumors, Brain Stem Glioma,
Central Nervous System Atypical Teratoid/Rhabdoid Tumor, Central
Nervous System Embryonal Tumors, Craniopharyngioma,
Ependymoblastoma, Ependymoma, Medulloblastoma, Medulloepithelioma,
Pineal Parenchymal Tumors of Intermediate Differentiation,
Supratentorial Primitive Neuroectodermal Tumors and Pineoblastoma),
Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, Basal Cell
Carcinoma, Bile Duct Cancer (including Extrahepatic), Bladder
Cancer, Bone Cancer (including Osteosarcoma and Malignant Fibrous
Histiocytoma), Carcinoid Tumor, Carcinoma of Unknown Primary,
Central Nervous System (such as Atypical Teratoid/Rhabdoid Tumor,
Embryonal Tumors and Lymphoma), Cervical Cancer, Childhood Cancers,
Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous
Leukemia (CML), Chronic Myeloproliferative Disorders, Colon Cancer,
Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma
(Mycosis Fungoides and Sdzary Syndrome), Duct, Bile (Extrahepatic),
Ductal Carcinoma In Situ (DCIS), Embryonal Tumors (Central Nervous
System), Endometrial Cancer, Ependymoblastoma, Ependymoma,
Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma Family of
Tumors, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor,
Extrahepatic Bile Duct Cancer, Eye Cancer (like Intraocular
Melanoma, Retinoblastoma), Fibrous Histiocytoma of Bone (including
Malignant and Osteosarcoma) Gallbladder Cancer, Gastric (Stomach)
Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal
Tumors (GIST), Germ Cell Tumor (Extracranial, Extragonadal,
Ovarian), Gestational Trophoblastic Tumor, Glioma, Hairy Cell
Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular
(Liver) Cancer, Histiocytosis, Langerhans Cell, Hodgkin Lymphoma,
Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors
(Endocrine, Pancreas), Kaposi Sarcoma, Kidney (including Renal
Cell), Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia
(including Acute Lymphoblastic (ALL), Acute Myeloid (AML), Chronic
Lymphocytic (CLL), Chronic Myelogenous (CML), Hairy Cell), Lip and
Oral Cavity Cancer, Liver Cancer (Primary), Lobular Carcinoma In
Situ (LCIS), Lung Cancer (Non-Small Cell and Small Cell), Lymphoma
(AIDS-Related, Burkitt, Cutaneous T-Cell (Mycosis Fungoides and
Sdzary Syndrome), Hodgkin, Non-Hodgkin, Primary Central Nervous
System (CNS), Macroglobulinemia, Waldenstrom, Male Breast Cancer,
Malignant Fibrous Histiocytoma of Bone and Osteosarcoma,
Medulloblastoma, Medulloepithelioma, Melanoma (including
Intraocular (Eye)), Merkel Cell Carcinoma, Mesothelioma
(Malignant), Metastatic Squamous Neck Cancer with Occult Primary,
Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple
Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell
Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,
Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia,
Chronic (CML), Myeloid Leukemia, Acute (AML), Myeloma and Multiple
Myeloma, Myeloproliferative Disorders (Chronic), Nasal Cavity and
Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,
Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral
Cavity Cancer, Lip and, Oropharyngeal Cancer, Osteosarcoma and
Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer (such as
Epithelial, Germ Cell Tumor, and Low Malignant Potential Tumor),
Pancreatic Cancer (including Islet Cell Tumors), Papillomatosis,
Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid
Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pineal
Parenchymal Tumors of Intermediate Differentiation, Pineoblastoma
and Supratentorial Primitive Neuroectodermal Tumors, Pituitary
Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary
Blastoma, Pregnancy and Breast Cancer, Primary Central Nervous
System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer, Renal Cell
(Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer,
Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma
(like Ewing Sarcoma Family of Tumors, Kaposi, Soft Tissue,
Uterine), Sezary Syndrome, Skin Cancer (such as Melanoma, Merkel
Cell Carcinoma, Nonmelanoma), Small Cell Lung Cancer, Small
Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma,
Squamous Neck Cancer with Occult Primary, Metastatic, Stomach
(Gastric) Cancer, Supratentorial Primitive Neuroectodermal Tumors,
T-Cell Lymphoma(Cutaneous, Mycosis Fungoides and Sezary Syndrome),
Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma,
Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and
Ureter, Trophoblastic Tumor (Gestational), Unknown Primary, Unusual
Cancers of Childhood, Ureter and Renal Pelvis, Transitional Cell
Cancer, Urethral Cancer, Uterine Cancer, Endometrial, Uterine
Sarcoma, Waldenstrom Macroglobulinemia and Wilms Tumor.
[0089] In some instances, oncogenic mutations promote glutamine
metabolism. Cells expressing oncogenic K-Ras exhibt increased
ultilization of glutamine (Weinberg et al., Proc. Natl. Acad. Sci.
USA, 2010; Gaglio et al., Mol. Syst. Biol., 2011). In certain
embodiments, the cancer cells have a mutated K-Ras gene. In certain
embodiments, the cancer is associated with tissue of the bladder,
bone marrow, breast, colon, kidney, liver, lung, ovary, pancreas,
prostate, skin or thyroid. The c-Myc gene is known to be altered in
numerous cancers (Zeller et al., Genome biology, 2003). Increased
Myc protein expression has been correlated with increased
expression of glutaminase, leading to up-regulation of glutamine
metabolism (Dang eta 1., Clin. Cancer Res., 2009; Gao et al.,
Nature, 2009). In certain embodiments, the cancer cells have an
oncogenic c-Myc gene or elevated Myc protein expression. In some
embodiments, the cancer is associated with tissue of the bladder,
bone, bowel, breast, central nervous system (like brain), colon,
gastric system (such as stomach and intestine), liver, lung, ovary,
prostate, muscle, and skin.
[0090] While many cancer cells depend on exogenous glutamine for
survival, the degree of glutamine dependence among tumor cell
subtypes may make a population of cells more susceptible to the
reduction of glutamine. As an example, gene expression analysis of
breast cancers has identified five intrinsic subtypes (luminal A,
luminal B, basal, HER2+, and normal-like) (Sorlie et al., Proc Natl
Acad Sci USA, 2001). Although glutamine deprivation has an impact
on cell growth and viability, basal-like cells appear to be more
sensitive to the reduction of exogenous glutamine (Kung et al.,
PLoS Genetics, 2011). This supports the concept that glutamine is a
very important energy source in basal-like breast cancer cell
lines, and suggests that inhibition of the glutaminase enzyme would
be beneficial in the treatment of breast cancers comprised of
basal-like cells. Triple-negative breast cancer (TNBC) is
characterized by a lack of estrogen receptor, progesterone receptor
and human epidermal growth factor receptor 2 expression. It has a
higher rate of relapse following chemotherapy, and a poorer
prognosis than with the other breast cancer subtypes (Dent et al.,
Clin Cancer res, 2007). Interestingly, there appears to be
significant similarities in metabolic profiling between TNBC cells
and basal-like breast cancer cells (unpublished data). Therefore,
an embodiment of the invention is the use of the compounds
described herein for the treatment of TNBC and basal-type breast
cancers.
[0091] Cachexia, the massive loss of muscle mass, is often
associated with poor performance status and high mortality rate of
cancer patients. A theory behind this process is that tumors
require more glutamine than is normally supplied by diet, so
muscle, a major source of glutamine, starts to breakdown in order
to supply enough nutrient to the tumor. Thus, inhibition of
glutaminase may reduce the need to breakdown muscle. An embodiment
of the invention is the use of the present compounds to prevent,
inhibit or reduce cachexia.
[0092] The most common neurotransmitter is glutamate, derived from
the enzymatic conversion of glutamine via glutaminase. High levels
of glutamate have been shown to be neurotoxic. Following traumatic
insult to neuronal cells, there occurs a rise in neurotransmitter
release, particularly glutamate. Accordingly, inhibition of
glutaminase has been hypothesized as a means of treatment following
an ischemic insult, such as stroke (Newcomb, PCT WO 99/09825,
Kostandy, Neurol. Sci., 2011). Huntington's disease is a
progressive, fatal neurological condition. In genetic mouse models
of Huntington's disease, it was observed that the early
manifestation of the disease correlated with dysregulated glutamate
release (Raymond et al., Neuroscience, 2011). In HIV-associated
dementia, HIV infected macrophages exhibit upregulated glutaminase
activity and increased glutamate release, leading to neuronal
damage (Huang et al., J Neurosci., 2011). Similarly, in another
neurological disease, the activated microglia in Rett Syndrome
release glutamate causing neuronal damage. The release of excess
glutamate has been associated with the up-regulation of glutaminase
(Maezawa et al., J. Neurosci, 2010). In mice bred to have reduced
glutaminase levels, sensitivity to psychotic-stimulating drugs,
such as amphetamines, was dramatically reduced, thus suggesting
that glutaminase inhibition may be beneficial in the treatment of
schizophrenia (Gaisler-Salomon et al., Neuropsychopharmacology,
2009). Bipolar disorder is a devastating illness that is marked by
recurrent episodes of mania and depression. This disease is treated
with mood stabilizers such as lithium and valproate; however,
chronic use of these drugs appear to increase the abundance of
glutamate receptors (Nanavati et al., J. Neurochem., 2011), which
may lead to a decrease in the drug's effectiveness over time. Thus,
an alternative treatment may be to reduce the amount of glutamate
by inhibiting glutaminase. This may or may not be in conjunction
with the mood stabilizers. Memantine, a partial antagonist of
N-methyl-D-aspartate receptor (NMDAR), is an approved therapeutic
in the treatment of Alzheimer's disease. Currently, research is
being conducted looking at memantine as a means of treating
vascular dementia and Parkinson's disease (Oliverares et al., Curr.
Alzheimer Res., 2011). Since memantine has been shown to partially
block the NMDA glutamate receptor also, it is not unresasonable to
speculate that decreasing glutamate levels by inhibiting
glutaminase could also treat Alzheimer's disease, vascular dementia
and Parkinson's disease. Alzheimer's disease, bipolar disorder,
HIV-associated dementia, Huntington's disease, ischemic insult,
Parkinson's disease, schizophrenia, stroke, traumatic insult and
vascular dementia are but a few of the neurological diseases that
have been correlated to increased levels of glutamate. Thus,
inhibiting glutaminase with a compound described herein can reduce
or prevent neurological diseases. Therefore, in one embodiment, the
compounds may be used for the treatment or prevention of
neurological diseases.
[0093] Activation of T lymphocytes induces cell growth,
proliferation, and cytokine production, thereby placing energetic
and biosynthetic demands on the cell. Glutamine serves as an amine
group donor for nucleotide synthesis, and glutamate, the first
component in glutamine metabolism, plays a direct role in amino
acid and glutathione synthesis, as well as being able to enter the
Krebs cycle for energy production (Carr et al., J. Immunol., 2010).
Mitogen-induced T cell proliferation and cytokine production
require high levels of glutamine metabolism, thus inhibiting
glutaminase may serve as a means of immune modulation. In multiple
sclerosis, an inflammatory autoimmune disease, the activated
microglia exhibit up-regulated glutaminase and release increased
levels of extracellular glutamate. Glutamine levels are lowered by
sepsis, injury, burns, surgery and endurance exercise (Calder et
al., Amino Acids, 1999). These situations put the individual at
risk of immunosuppression. In fact, in general, glutaminase gene
expression and enzyme activity are both increased during T cell
activity. Patients given glutamine following bone marrow
transplantation resulted in a lower level of infection and reduced
graft v. host disease (Crowther, Proc. Nutr. Soc., 2009). T cell
proliferation and activiation is involved in many immunological
diseases, such as inflammatory bowel disease, Crohn's disease,
sepsis, psoriasis, arthritis (including rheumatoid arthritis),
multiple sclerosis, graft v. host disease, infections, lupus and
diabetes. In an embodiment of the invention, the compounds
described herein can be used to treat or prevent immunological
diseases.
[0094] Hepatic encephalopathy (HE) represents a series of transient
and reversible neurologic and psychiatric dysfunction in patients
with liver disease or portosystemic shunting. HE is not a single
clinical entity and may reflect reversible metabolic
encephalopathy, brain atrophy, brain edema, or a combination of
these factors; however, the current hypothesis is that the
accumulation of ammonia, mostly derived from the intestine, plays a
key role in the pathophysiology (Khunger et al., Clin Liver Dis,
2012). The deamination of glutamine in small intestine, renal and
muscle synthesis all contribute to ammonia production. Impaired
hepatic clearance caused by hepatocellular clearance or
portosystemic shunting causes increased accumulation of ammonia.
Ammonia toxicity affects astrocytes in the brain via glutamine
synthetase, which metabolizes the ammonia to produce increased
glutamine. Glutamine, in turn, attracts water into the astrocytes,
leading to swelling and oxidative dysfunction of the mitochondria.
The resulting cerebral edema is thought to contribute to neurologic
dysfunction seen in HE (Kavitt et al., Clin Gastroenterol Hepatol,
2008). In an embodiment of the invention, the compounds described
herein can be used to treat or prevent HE.
[0095] Primary sensory neurons in the dorsal root ganglion have
been shown to elevate their glutaminase enzyme activity following
inflammation (Miller et al., Pain Research and Treatment, 2012). It
is believed that the resulting increased glutamate production
contributes to both central and peripheral sensitization,
identified as pain. An aspect of the invention is the use of the
present compounds herein for the treatment or diminishment of pain.
In certain embodiments, the pain can be neuropathic pain,
chemotherapy-induced pain or inflammatory pain.
[0096] High blood glucose levels, high insulin levels, and insulin
resistance are risk factors for developing diabetes mellitus.
Similarly, high blood pressure is a risk factor for developing
cardiovascular disease. In a recent report from a large human
cohort study, these four risk factors were inversely correlated
with glutamine-to-glutamate ratios in the blood stream (Chen et al,
Circulation, 2012). Furthermore, plasma glutamine-to-glutamate
ratios were inversely correlated with the eventual incidence of
diabetes mellitus over 12 years (Cheng et al, Circulation, 2012).
Experiments with animal models were consistent with these findings.
Mice fed glutamine-rich diets exhibited lower blood glucose levels
in a glucose tolerance test after 6 hours of fasting, and
intraperitoneal injection of glutamine into mice rapidly decreased
their blood pressure (Cheng et al, Circulation, 2012). Therefore,
it is plausible that glutaminase inhibitors, which cause increased
glutamine levels and decrease glutamate levels, would decrease the
incidence of diabetes mellitus and cardiovascular disease. In
particular, the liver and small intestine are major sites of
glutamine utilization in diabetic animals, and glutaminase activity
is higher than normal in these organs in streptozotocin-induced
diabetic rats (Watford et al, Biochem J, 1984; Mithieux et al, Am J
Physiol Endrocrinol Metab, 2004). In an embodiment of the
invention, the compounds described herein can be used to treat
diabetes. In another embodiment of the invention, the present
compounds can be used to reduce high blood pressure.
[0097] In one embodiment, the method of treating or preventing
cancer, immunological and neurological diseases may comprise
administering a compound of the invention conjointly with a
chemotherapeutic agent. Chemotherapeutic agents that may be
conjointly administered with compounds of the invention include:
aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg,
bicalutamide, bleomycin, buserelin, busulfan, campothecin,
capecitabine, carboplatin, carmustine, chlorambucil, chloroquine,
cisplatin, cladribine, clodronate, colchicine, cyclophosphamide,
cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin,
demethoxyviridin, dichloroacetate, dienestrol, diethylstilbestrol,
docetaxel, doxorubicin, epirubicin, estradiol, estramustine,
etoposide, everolimus, exemestane, filgrastim, fludarabine,
fludrocortisone, fluorouracil, fluoxymesterone, flutamide,
gemcitabine, genistein, goserelin, hydroxyurea, idarubicin,
ifosfamide, imatinib, interferon, irinotecan, ironotecan,
letrozole, leucovorin, leuprolide, levamisole, lomustine,
lonidamine, mechlorethamine, medroxyprogesterone, megestrol,
melphalan, mercaptopurine, mesna, metformin, methotrexate,
mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole,
octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin,
perifosine, plicamycin, porfimer, procarbazine, raltitrexed,
rituximab, sorafenib, streptozocin, sunitinib, suramin, tamoxifen,
temozolomide, temsirolimus, teniposide, testosterone, thioguanine,
thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin,
vinblastine, vincristine, vindesine, and vinorelbine.
[0098] Many combination therapies have been developed for the
treatment of cancer. In certain embodiments, compounds of the
invention may be conjointly administered with a combination
therapy. Examples of combination therapies with which compounds of
the invention may be conjointly administered are included in Table
1.
TABLE-US-00001 TABLE 1 Exemplary combinatorial therapies for the
treatment of cancer. Name Therapeutic agents ABV Doxorubicin,
Bleomycin, Vinblastine ABVD Doxorubicin, Bleomycin, Vinblastine,
Dacarbazine AC (Breast) Doxorubicin, Cyclophosphamide AC (Sarcoma)
Doxorubicin, Cisplatin AC (Neuroblastoma) Cyclophosphamide,
Doxorubicin ACE Cyclophosphamide, Doxorubicin, Etoposide ACe
Cyclophosphamide, Doxorubicin AD Doxorubicin, Dacarbazine AP
Doxorubicin, Cisplatin ARAC-DNR Cytarabine, Daunorubicin B-CAVe
Bleomycin, Lomustine, Doxorubicin, Vinblastine BCVPP Carmustine,
Cyclophosphamide, Vinblastine, Procarbazine, Prednisone BEACOPP
Bleomycin, Etoposide, Doxorubicin, Cyclophosphamide, Vincristine,
Procarbazine, Prednisone, Filgrastim BEP Bleomycin, Etoposide,
Cisplatin BIP Bleomycin, Cisplatin, Ifosfamide, Mesna BOMP
Bleomycin, Vincristine, Cisplatin, Mitomycin CA Cytarabine,
Asparaginase CABO Cisplatin, Methotrexate, Bleomycin, Vincristine
CAF Cyclophosphamide, Doxorubicin, Fluorouracil CAL-G
Cyclophosphamide, Daunorubicin, Vincristine, Prednisone,
Asparaginase CAMP Cyclophosphamide, Doxorubicin, Methotrexate,
Procarbazine CAP Cyclophosphamide, Doxorubicin, Cisplatin CaT
Carboplatin, Paclitaxel CAV Cyclophosphamide, Doxorubicin,
Vincristine CAVE ADD CAV and Etoposide CA-VP16 Cyclophosphamide,
Doxorubicin, Etoposide CC Cyclophosphamide, Carboplatin CDDP/VP-16
Cisplatin, Etoposide CEF Cyclophosphamide, Epirubicin, Fluorouracil
CEPP(B) Cyclophosphamide, Etoposide, Prednisone, with or
without/Bleomycin CEV Cyclophosphamide, Etoposide, Vincristine CF
Cisplatin, Fluorouracil or Carboplatin Fluorouracil CHAP
Cyclophosphamide or Cyclophosphamide, Altretamine, Doxorubicin,
Cisplatin ChlVPP Chlorambucil, Vinblastine, Procarbazine,
Prednisone CHOP Cyclophosphamide, Doxorubicin, Vincristine,
Prednisone CHOP-BLEO Add Bleomycin to CHOP CISCA Cyclophosphamide,
Doxorubicin, Cisplatin CLD-BOMP Bleomycin, Cisplatin, Vincristine,
Mitomycin CMF Methotrexate, Fluorouracil, Cyclophosphamide CMFP
Cyclophosphamide, Methotrexate, Fluorouracil, Prednisone CMFVP
Cyclophosphamide, Methotrexate, Fluorouracil, Vincristine,
Prednisone CMV Cisplatin, Methotrexate, Vinblastine CNF
Cyclophosphamide, Mitoxantrone, Fluorouracil CNOP Cyclophosphamide,
Mitoxantrone, Vincristine, Prednisone COB Cisplatin, Vincristine,
Bleomycin CODE Cisplatin, Vincristine, Doxorubicin, Etoposide COMLA
Cyclophosphamide, Vincristine, Methotrexate, Leucovorin, Cytarabine
COMP Cyclophosphamide, Vincristine, Methotrexate, Prednisone Cooper
Regimen Cyclophosphamide, Methotrexate, Fluorouracil, Vincristine,
Prednisone COP Cyclophosphamide, Vincristine, Prednisone COPE
Cyclophosphamide, Vincristine, Cisplatin, Etoposide COPP
Cyclophosphamide, Vincristine, Procarbazine, Prednisone CP(Chronic
Chlorambucil, Prednisone lymphocytic leukemia) CP (Ovarian Cancer)
Cyclophosphamide, Cisplatin CT Cisplatin, Paclitaxel CVD Cisplatin,
Vinblastine, Dacarbazine CVI Carboplatin, Etoposide, Ifosfamide,
Mesna CVP Cyclophosphamide, Vincristine, Prednisome CVPP Lomustine,
Procarbazine, Prednisone CYVADIC Cyclophosphamide, Vincristine,
Doxorubicin, Dacarbazine DA Daunorubicin, Cytarabine DAT
Daunorubicin, Cytarabine, Thioguanine DAV Daunorubicin, Cytarabine,
Etoposide DCT Daunorubicin, Cytarabine, Thioguanine DHAP Cisplatin,
Cytarabine, Dexamethasone DI Doxorubicin, Ifosfamide DTIC/Tamoxifen
Dacarbazine, Tamoxifen DVP Daunorubicin, Vincristine, Prednisone
EAP Etoposide, Doxorubicin, Cisplatin EC Etoposide, Carboplatin EFP
Etoposie, Fluorouracil, Cisplatin ELF Etoposide, Leucovorin,
Fluorouracil EMA 86 Mitoxantrone, Etoposide, Cytarabine EP
Etoposide, Cisplatin EVA Etoposide, Vinblastine FAC Fluorouracil,
Doxorubicin, Cyclophosphamide FAM Fluorouracil, Doxorubicin,
Mitomycin FAMTX Methotrexate, Leucovorin, Doxorubicin FAP
Fluorouracil, Doxorubicin, Cisplatin F-CL Fluorouracil, Leucovorin
FEC Fluorouracil, Cyclophosphamide, Epirubicin FED Fluorouracil,
Etoposide, Cisplatin FL Flutamide, Leuprolide FZ Flutamide,
Goserelin acetate implant HDMTX Methotrexate, Leucovorin Hexa-CAF
Altretamine, Cyclophosphamide, Methotrexate, Fluorouracil ICE-T
Ifosfamide, Carboplatin, Etoposide, Paclitaxel, Mesna IDMTX/6-MP
Methotrexate, Mercaptopurine, Leucovorin IE Ifosfamide, Etoposie,
Mesna IfoVP Ifosfamide, Etoposide, Mesna IPA Ifosfamide, Cisplatin,
Doxorubicin M-2 Vincristine, Carmustine, Cyclophosphamide,
Prednisone, Melphalan MAC-III Methotrexate, Leucovorin,
Dactinomycin, Cyclophosphamide MACC Methotrexate, Doxorubicin,
Cyclophosphamide, Lomustine MACOP-B Methotrexate, Leucovorin,
Doxorubicin, Cyclophosphamide, Vincristine, Bleomycin, Prednisone
MAID Mesna, Doxorubicin, Ifosfamide, Dacarbazine m-BACOD Bleomycin,
Doxorubicin, Cyclophosphamide, Vincristine, Dexamethasone,
Methotrexate, Leucovorin MBC Methotrexate, Bleomycin, Cisplatin MC
Mitoxantrone, Cytarabine MF Methotrexate, Fluorouracil, Leucovorin
MICE Ifosfamide, Carboplatin, Etoposide, Mesna MINE Mesna,
Ifosfamide, Mitoxantrone, Etoposide mini-BEAM Carmustine,
Etoposide, Cytarabine, Melphalan MOBP Bleomycin, Vincristine,
Cisplatin, Mitomycin MOP Mechlorethamine, Vincristine, Procarbazine
MOPP Mechlorethamine, Vincristine, Procarbazine, Prednisone
MOPP/ABV Mechlorethamine, Vincristine, Procarbazine, Prednisone,
Doxorubicin, Bleomycin, Vinblastine MP (multiple Melphalan,
Prednisone myeloma) MP (prostate cancer) Mitoxantrone, Prednisone
MTX/6-MO Methotrexate, Mercaptopurine MTX/6-MP/VP Methotrexate,
Mercaptopurine, Vincristine, Prednisone MTX-CDDPAdr Methotrexate,
Leucovorin, Cisplatin, Doxorubicin MV (breast cancer) Mitomycin,
Vinblastine MV (acute Mitoxantrone, Etoposide myelocytic leukemia)
M-VAC Vinblastine, Doxorubicin, Cisplatin Methotrexate MVP
Mitomycin Vinblastine, Cisplatin MVPP Mechlorethamine, Vinblastine,
Procarbazine, Prednisone NFL Mitoxantrone, Fluorouracil, Leucovorin
NOVP Mitoxantrone, Vinblastine, Vincristine OPA Vincristine,
Prednisone, Doxorubicin OPPA Add Procarbazine to OPA. PAC
Cisplatin, Doxorubicin PAC-I Cisplatin, Doxorubicin,
Cyclophosphamide PA-CI Cisplatin, Doxorubicin PC Paclitaxel,
Carboplatin or Paclitaxel, Cisplatin PCV Lomustine, Procarbazine,
Vincristine PE Paclitaxel, Estramustine PFL Cisplatin,
Fluorouracil, Leucovorin POC Prednisone, Vincristine, Lomustine
ProMACE Prednisone, Methotrexate, Leucovorin, Doxorubicin,
Cyclophosphamide, Etoposide ProMACE/ Prednisone, Doxorubicin,
Cyclophosphamide, cytaBOM Etoposide, Cytarabine, Bleomycin,
Vincristine, Methotrexate, Leucovorin, Cotrimoxazole PRoMACE/MOPP
Prednisone, Doxorubicin, Cyclophosphamide, Etoposide,
Mechlorethamine, Vincristine, Procarbazine, Methotrexate,
Leucovorin Pt/VM Cisplatin, Teniposide PVA Prednisone, Vincristine,
Asparaginase PVB Cisplatin, Vinblastine, Bleomycin PVDA Prednisone,
Vincristine, Daunorubicin, Asparaginase SMF Streptozocin,
Mitomycin, Fluorouracil TAD Mechlorethamine, Doxorubicin,
Vinblastine, Vincristine, Bleomycin, Etoposide, Prednisone TCF
Paclitaxel, Cisplatin, Fluorouracil TIP Paclitaxel, Ifosfamide,
Mesna, Cisplatin TTT Methotrexate, Cytarabine, Hydrocortisone
Topo/CTX Cyclophosphamide, Topotecan, Mesna VAB-6 Cyclophosphamide,
Dactinomycin, Vinblastine, Cisplatin, Bleomycin VAC Vincristine,
Dactinomycin, Cyclophosphamide VACAdr Vincristine,
Cyclophosphamide, Doxorubicin, Dactinomycin, Vincristine VAD
Vincristine, Doxorubicin, Dexamethasone VATH Vinblastine,
Doxorubicin, Thiotepa, Flouxymesterone VBAP Vincristine,
Carmustine, Doxorubicin, Prednisone VBCMP Vincristine, Carmustine,
Melphalan, Cyclophosphamide, Prednisone VC Vinorelbine, Cisplatin
VCAP Vincristine, Cyclophosphamide, Doxorubicin, Prednisone VD
Vinorelbine, Doxorubicin VelP Vinblastine, Cisplatin, Ifosfamide,
Mesna VIP Etoposide, Cisplatin, Ifosfamide, Mesna VM Mitomycin,
Vinblastine VMCP Vincristine, Melphalan, Cyclophosphamide,
Prednisone VP Etoposide, Cisplatin V-TAD Etoposide, Thioguanine,
Daunorubicin, Cytarabine 5 + 2 Cytarabine, Daunorubicin,
Mitoxantrone 7 + 3 Cytarabine with/, Daunorubicin or Idarubicin or
Mitoxantrone ''8 in 1'' Methylprednisolone, Vincristine, Lomustine,
Procarbazine, Hydroxyurea, Cisplatin, Cytarabine, Dacarbazine
[0099] The proliferation of cancer cells requires lipid synthesis.
Normally, acetyl-coA used for lipid synthesis is formed from a
mitochondrial pool of pyruvate that is derived from glycolysis. Yet
under hypoxic conditions, such as those normally found in a tumor
environment, the conversion of pyruvate to acetyl-coA within the
mitochondria is downregulated. Recent studies from Metallo et al.
(2011) and Mullen et al. (2011) revealed that under such hypoxic
conditions, cells instead largely switch to using a pathway
involving the reductive carboxylation of alpha-ketoglutarate to
make acetyl-coA for lipid synthesis. The first step in this pathway
involves converting glutamine to glutamate via glutaminase enzymes.
Subsequently, glutamate is converting to alpha-ketoglutarate, and
the resulting alpha-ketoglutarate is converted to isocitrate in a
reductive carboxylation step mediated by the isocitrate
dehydrogenase enzymes. A switch to this reductive carboxylation
pathway also occurs in some renal carcinoma cell lines that contain
either impaired mitochondria or an impaired signal for induction of
the enzyme responsible for converting glycolytic pyruvate to
acetyl-coA (Mullen et al 2011). A similar switch occurs in cells
exposed to mitochondrial respiratory chain inhibitors such as
metformin, rotenone, and antimycin (Mullen at al. 2011). Therefore,
in some embodiments of this invention, we propose using
combinations of mitochondrial respiratory chain inhibitors and
glutaminase inhibitors to simultaneously increase cancer cells'
dependence on glutaminase-dependent pathways for lipid synthesis
while inhibiting those very pathways. The increased dependence on
glycolysis in tumor cells is likely because the hypoxic tumor
environment impairs mitochondrial respiration. Furthermore,
depletion of glucose induces apoptosis in cells transformed with
the MYC oncogene.
[0100] These findings suggest that inhibiting glycolysis would have
a therapeutic value in preventing cancer cell proliferation. There
are currently many documented glycolytic inhibitors (Pelicano et
al. 2006). However, as pointed out by Zhao et al. (2012),
"available glycolytic inhibitors are generally not very potent, and
high doses are required, which may cause high levels of systemic
toxicity." Since cancer cells typically use both glucose and
glutamine at higher levels than normal cells, impairing utilization
of each of those metabolites will likely have a synergistic effect.
Therefore, in some embodiments of this invention, we propose using
combinations of glycolytic pathway inhibitors and glutaminase
inhibitors. Such glycolytic inhibitors include 2-deoxyglucose,
lonidamine, 3-bromopyruvate, imatinib, oxythiamine, rapamycin, and
their pharmacological equivalents. Glycolysis can be inhibited
indirectly by depleting NAD+ via DNA damage induced by DNA
alkylating agents through a pathway activated by poly(ADP-ribose)
polymerase (Zong et al. 2004). Therefore, in one embodiment of this
invention, we propose using a combination of DNA alkylating agents
and glutaminase inhibitors. Cancer cells use the pentose phosphate
pathway along with the glycolytic pathway to create metabolic
intermediates derived from glucose. Therefore, in another
embodiment of this invention, we propose using a combination of
pentose phosphate inhibitors such as 6-aminonicotinamide along with
glutaminase inhibitors.
[0101] In certain embodiments, a compound of the invention may be
conjointly administered with non-chemical methods of cancer
treatment. In certain embodiments, a compound of the invention may
be conjointly administered with radiation therapy. In certain
embodiments, a compound of the invention may be conjointly
administered with surgery, with thermoablation, with focused
ultrasound therapy, with cryotherapy, or with any combination of
these.
[0102] In certain embodiments, different compounds of the invention
may be conjointly administered with one or more other compounds of
the invention. Moreover, such combinations may be conjointly
administered with other therapeutic agents, such as other agents
suitable for the treatment of cancer, immunological or neurological
diseases, such as the agents identified above.
[0103] In certain embodiments, the present invention provides a kit
comprising: a) one or more single dosage forms of a compound of the
invention; b) one or more single dosage forms of a chemotherapeutic
agent as mentioned above; and c) instructions for the
administration of the compound of the invention and the
chemotherapeutic agent.
[0104] The present invention provides a kit comprising: [0105] a) a
pharmaceutical formulation (e.g., one or more single dosage forms)
comprising a compound of the invention; and [0106] b) instructions
for the administration of the pharmaceutical formulation, e.g., for
treating or preventing any of the conditions discussed above.
[0107] In certain embodiments, the kit further comprises
instructions for the administration of the pharmaceutical
formulation comprising a compound of the invention conjointly with
a chemotherapeutic agent as mentioned above. In certain
embodiments, the kit further comprises a second pharmaceutical
formulation (e.g., as one or more single dosage forms) comprising a
chemotherapeutic agent as mentioned above.
Definitions
[0108] The term "acyl" is art-recognized and refers to a group
represented by the general formula hydrocarbylC(O)--, preferably
alkylC(O)--.
[0109] The term "acylamino" is art-recognized and refers to an
amino group substituted with an acyl group and may be represented,
for example, by the formula hydrocarbylC(O)NH--.
[0110] The term "acyloxy" is art-recognized and refers to a group
represented by the general formula hydrocarbylC(O)O--, preferably
alkylC(O)O--.
[0111] The term "alkoxy" refers to an alkyl group, preferably a
lower alkyl group, having an oxygen attached thereto.
Representative alkoxy groups include methoxy, ethoxy, propoxy,
tert-butoxy and the like.
[0112] The term "alkoxyalkyl" refers to an alkyl group substituted
with an alkoxy group and may be represented by the general formula
alkyl-O-alkyl.
[0113] The term "alkenyl", as used herein, refers to an aliphatic
group containing at least one double bond and is intended to
include both "unsubstituted alkenyls" and "substituted alkenyls",
the latter of which refers to alkenyl moieties having substituents
replacing a hydrogen on one or more carbons of the alkenyl group.
Such substituents may occur on one or more carbons that are
included or not included in one or more double bonds. Moreover,
such substituents include all those contemplated for alkyl groups,
as discussed below, except where stability is prohibitive. For
example, substitution of alkenyl groups by one or more alkyl,
carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is
contemplated.
[0114] An "alkyl" group or "alkane" is a straight chained or
branched non-aromatic hydrocarbon which is completely saturated.
Typically, a straight chained or branched alkyl group has from 1 to
about 20 carbon atoms, preferably from 1 to about 10 unless
otherwise defined. Examples of straight chained and branched alkyl
groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl,
sec-butyl, tert-butyl, pentyl, hexyl, pentyl and octyl. A
C.sub.1-C.sub.6 straight chained or branched alkyl group is also
referred to as a "lower alkyl" group.
[0115] Moreover, the term "alkyl" (or "lower alkyl") as used
throughout the specification, examples, and claims is intended to
include both "unsubstituted alkyls" and "substituted alkyls", the
latter of which refers to alkyl moieties having substituents
replacing a hydrogen on one or more carbons of the hydrocarbon
backbone. Such substituents, if not otherwise specified, can
include, for example, a halogen, a hydroxyl, a carbonyl (such as a
carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl
(such as a thioester, a thioacetate, or a thioformate), an alkoxyl,
a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino,
an amido, an amidine, an imine, a cyano, a nitro, an azido, a
sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a
sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic
or heteroaromatic moiety. It will be understood by those skilled in
the art that the moieties substituted on the hydrocarbon chain can
themselves be substituted, if appropriate. For instance, the
substituents of a substituted alkyl may include substituted and
unsubstituted forms of amino, azido, imino, amido, phosphoryl
(including phosphonate and phosphinate), sulfonyl (including
sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups,
as well as ethers, alkylthios, carbonyls (including ketones,
aldehydes, carboxylates, and esters), --CF.sub.3, --CN and the
like. Exemplary substituted alkyls are described below. Cycloalkyls
can be further substituted with alkyls, alkenyls, alkoxys,
alkylthios, aminoalkyls, carbonyl-substituted alkyls, --CF.sub.3,
--CN, and the like.
[0116] The term "C.sub.x-y" when used in conjunction with a
chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl,
or alkoxy is meant to include groups that contain from x to y
carbons in the chain. For example, the term "C.sub.x-yalkyl" refers
to substituted or unsubstituted saturated hydrocarbon groups,
including straight-chain alkyl and branched-chain alkyl groups that
contain from x to y carbons in the chain, including haloalkyl
groups such as trifluoromethyl and 2,2,2-tirfluoroethyl, etc. Co
alkyl indicates a hydrogen where the group is in a terminal
position, a bond if internal. The terms "C.sub.2-yalkenyl" and
"C.sub.2-yalkynyl" refer to substituted or unsubstituted
unsaturated aliphatic groups analogous in length and possible
substitution to the alkyls described above, but that contain at
least one double or triple bond respectively.
[0117] The term "alkylamino", as used herein, refers to an amino
group substituted with at least one alkyl group.
[0118] The term "alkylthio", as used herein, refers to a thiol
group substituted with an alkyl group and may be represented by the
general formula alkylS--.
[0119] The term "alkynyl", as used herein, refers to an aliphatic
group containing at least one triple bond and is intended to
include both "unsubstituted alkynyls" and "substituted alkynyls",
the latter of which refers to alkynyl moieties having substituents
replacing a hydrogen on one or more carbons of the alkynyl group.
Such substituents may occur on one or more carbons that are
included or not included in one or more triple bonds. Moreover,
such substituents include all those contemplated for alkyl groups,
as discussed above, except where stability is prohibitive. For
example, substitution of alkynyl groups by one or more alkyl,
carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is
contemplated.
[0120] The term "amide", as used herein, refers to a group
##STR00010##
wherein each R.sup.10 independently represent a hydrogen or
hydrocarbyl group, or two R.sup.10 are taken together with the N
atom to which they are attached complete a heterocycle having from
4 to 8 atoms in the ring structure.
[0121] The terms "amine" and "amino" are art-recognized and refer
to both unsubstituted and substituted amines and salts thereof,
e.g., a moiety that can be represented by
##STR00011##
wherein each R.sup.10 independently represents a hydrogen or a
hydrocarbyl group, or two R.sup.10 are taken together with the N
atom to which they are attached complete a heterocycle having from
4 to 8 atoms in the ring structure.
[0122] The term "aminoalkyl", as used herein, refers to an alkyl
group substituted with an amino group.
[0123] The term "aralkyl", as used herein, refers to an alkyl group
substituted with an aryl group.
[0124] The term "aryl" as used herein include substituted or
unsubstituted single-ring aromatic groups in which each atom of the
ring is carbon. Preferably the ring is a 5-to 7-membered ring, more
preferably a 6-membered ring. The term "aryl" also includes
polycyclic ring systems having two or more cyclic rings in which
two or more carbons are common to two adjoining rings wherein at
least one of the rings is aromatic, e.g., the other cyclic rings
can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,
heteroaryls, and/or heterocyclyls. Aryl groups include benzene,
naphthalene, phenanthrene, phenol, aniline, and the like.
[0125] The term "carbamate" is art-recognized and refers to a
group
##STR00012##
wherein R.sup.9 and R.sup.10 independently represent hydrogen or a
hydrocarbyl group, such as an alkyl group, or R.sup.9 and R.sup.10
taken together with the intervening atom(s) complete a heterocycle
having from 4 to 8 atoms in the ring structure.
[0126] The terms "carbocycle", and "carbocyclic", as used herein,
refers to a saturated or unsaturated ring in which each atom of the
ring is carbon. The term carbocycle includes both aromatic
carbocycles and non-aromatic carbocycles. Non-aromatic carbocycles
include both cycloalkane rings, in which all carbon atoms are
saturated, and cycloalkene rings, which contain at least one double
bond. "Carbocycle" includes 5-7 membered monocyclic and 8-12
membered bicyclic rings. Each ring of a bicyclic carbocycle may be
selected from saturated, unsaturated and aromatic rings. Carbocycle
includes bicyclic molecules in which one, two or three or more
atoms are shared between the two rings. The term "fused carbocycle"
refers to a bicyclic carbocycle in which each of the rings shares
two adjacent atoms with the other ring. Each ring of a fused
carbocycle may be selected from saturated, unsaturated and aromatic
rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl,
may be fused to a saturated or unsaturated ring, e.g., cyclohexane,
cyclopentane, or cyclohexene. Any combination of saturated,
unsaturated and aromatic bicyclic rings, as valence permits, is
included in the definition of carbocyclic. Exemplary "carbocycles"
include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane,
1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene,
bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary
fused carbocycles include decalin, naphthalene,
1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane,
4,5,6,7-tetrahydro-1H-indene and bicyclo[4.1.0]hept-3-ene.
"Carbocycles" may be susbstituted at any one or more positions
capable of bearing a hydrogen atom.
[0127] A "cycloalkyl" group is a cyclic hydrocarbon which is
completely saturated. "Cycloalkyl" includes monocyclic and bicyclic
rings. Typically, a monocyclic cycloalkyl group has from 3 to about
10 carbon atoms, more typically 3 to 8 carbon atoms unless
otherwise defined. The second ring of a bicyclic cycloalkyl may be
selected from saturated, unsaturated and aromatic rings. Cycloalkyl
includes bicyclic molecules in which one, two or three or more
atoms are shared between the two rings. The term "fused cycloalkyl"
refers to a bicyclic cycloalkyl in which each of the rings shares
two adjacent atoms with the other ring. The second ring of a fused
bicyclic cycloalkyl may be selected from saturated, unsaturated and
aromatic rings. A "cycloalkenyl" group is a cyclic hydrocarbon
containing one or more double bonds.
[0128] The term "carbocyclylalkyl", as used herein, refers to an
alkyl group substituted with a carbocycle group.
[0129] The term "carbonate" is art-recognized and refers to a group
--OCO.sub.2--R.sup.10, wherein R.sup.10 represents a hydrocarbyl
group.
[0130] The term "carboxy", as used herein, refers to a group
represented by the formula --CO.sub.2H.
[0131] The term "ester", as used herein, refers to a group
--C(O)OR.sup.10 wherein R.sup.10 represents a hydrocarbyl
group.
[0132] The term "ether", as used herein, refers to a hydrocarbyl
group linked through an oxygen to another hydrocarbyl group.
Accordingly, an ether substituent of a hydrocarbyl group may be
hydrocarbyl-O--. Ethers may be either symmetrical or unsymmetrical.
Examples of ethers include, but are not limited to,
heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include
"alkoxyalkyl" groups, which may be represented by the general
formula alkyl-O-alkyl.
[0133] The terms "halo" and "halogen" as used herein means halogen
and includes chloro, fluoro, bromo, and iodo.
[0134] The terms "hetaralkyl" and "heteroaralkyl", as used herein,
refers to an alkyl group substituted with a hetaryl group.
[0135] The term "heteroalkyl", as used herein, refers to a
saturated or unsaturated chain of carbon atoms and at least one
heteroatom, wherein no two heteroatoms are adjacent.
[0136] The terms "heteroaryl" and "hetaryl" include substituted or
unsubstituted aromatic single ring structures, preferably 5- to
7-membered rings, more preferably 5-to 6-membered rings, whose ring
structures include at least one heteroatom, preferably one to four
heteroatoms, more preferably one or two heteroatoms. The terms
"heteroaryl" and "hetaryl" also include polycyclic ring systems
having two or more cyclic rings in which two or more carbons are
common to two adjoining rings wherein at least one of the rings is
heteroaromatic, e.g., the other cyclic rings can be cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or
heterocyclyls. Heteroaryl groups include, for example, pyrrole,
furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine,
pyrazine, pyridazine, and pyrimidine, and the like.
[0137] The term "heteroatom" as used herein means an atom of any
element other than carbon or hydrogen. Preferred heteroatoms are
nitrogen, oxygen, and sulfur.
[0138] The terms "heterocyclyl", "heterocycle", and "heterocyclic"
refer to substituted or unsubstituted non-aromatic ring structures,
preferably 3- to 10-membered rings, more preferably 3- to
7-membered rings, whose ring structures include at least one
heteroatom, preferably one to four heteroatoms, more preferably one
or two heteroatoms. The terms "heterocyclyl" and "heterocyclic"
also include polycyclic ring systems having two or more cyclic
rings in which two or more carbons are common to two adjoining
rings wherein at least one of the rings is heterocyclic, e.g., the
other cyclic rings can be cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
Heterocyclyl groups include, for example, piperidine, piperazine,
pyrrolidine, morpholine, lactones, lactams, and the like.
[0139] The term "heterocyclylalkyl", as used herein, refers to an
alkyl group substituted with a heterocycle group.
[0140] The term "hydrocarbyl", as used herein, refers to a group
that is bonded through a carbon atom that does not have a .dbd.O or
.dbd.S substituent, and typically has at least one carbon-hydrogen
bond and a primarily carbon backbone, but may optionally include
heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and
trifluoromethyl are considered to be hydrocarbyl for the purposes
of this application, but substituents such as acetyl (which has a
.dbd.O substituent on the linking carbon) and ethoxy (which is
linked through oxygen, not carbon) are not. Hydrocarbyl groups
include, but are not limited to aryl, heteroaryl, carbocycle,
heterocyclyl, alkyl, alkenyl, alkynyl, and combinations
thereof.
[0141] The term "hydroxyalkyl", as used herein, refers to an alkyl
group substituted with a hydroxy group.
[0142] The term "lower" when used in conjunction with a chemical
moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy
is meant to include groups where there are ten or fewer
non-hydrogen atoms in the substituent, preferably six or fewer. A
"lower alkyl", for example, refers to an alkyl group that contains
ten or fewer carbon atoms, preferably six or fewer. In certain
embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy
substituents defined herein are respectively lower acyl, lower
acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower
alkoxy, whether they appear alone or in combination with other
substituents, such as in the recitations hydroxyalkyl and aralkyl
(in which case, for example, the atoms within the aryl group are
not counted when counting the carbon atoms in the alkyl
substituent).
[0143] The terms "polycyclyl", "polycycle", and "polycyclic" refer
to two or more rings (e.g., cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which
two or more atoms are common to two adjoining rings, e.g., the
rings are "fused rings". Each of the rings of the polycycle can be
substituted or unsubstituted. In certain embodiments, each ring of
the polycycle contains from 3 to 10 atoms in the ring, preferably
from 5 to 7.
[0144] The term "silyl" refers to a silicon moiety with three
hydrocarbyl moieties attached thereto.
[0145] The term "substituted" refers to moieties having
substituents replacing a hydrogen on one or more carbons of the
backbone. It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, etc. As used
herein, the term "substituted" is contemplated to include all
permissible substituents of organic compounds. In a broad aspect,
the permissible substituents include acyclic and cyclic, branched
and unbranched, carbocyclic and heterocyclic, aromatic and
non-aromatic substituents of organic compounds. The permissible
substituents can be one or more and the same or different for
appropriate organic compounds. For purposes of this invention, the
heteroatoms such as nitrogen may have hydrogen substituents and/or
any permissible substituents of organic compounds described herein
which satisfy the valences of the heteroatoms. Substituents can
include any substituents described herein, for example, a halogen,
a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a
formyl, or an acyl), a thiocarbonyl (such as a thioester, a
thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a
phosphate, a phosphonate, a phosphinate, an amino, an amido, an
amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an
alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a
sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or
heteroaromatic moiety. It will be understood by those skilled in
the art that substituents can themselves be substituted, if
appropriate. Unless specifically stated as "unsubstituted,"
references to chemical moieties herein are understood to include
substituted variants. For example, reference to an "aryl" group or
moiety implicitly includes both substituted and unsubstituted
variants.
[0146] The term "sulfate" is art-recognized and refers to the group
--OSO.sub.3H, or a pharmaceutically acceptable salt thereof.
[0147] The term "sulfonamide" is art-recognized and refers to the
group represented by the general formulae
##STR00013##
wherein R.sup.9 and R.sup.10 independently represents hydrogen or
hydrocarbyl, such as alkyl, or R.sup.9 and R.sup.10 taken together
with the intervening atom(s) complete a heterocycle having from 4
to 8 atoms in the ring structure.
[0148] The term "sulfoxide" is art-recognized and refers to the
group --S(O)--R.sup.10 wherein R.sup.10 represents a
hydrocarbyl.
[0149] The term "sulfonate" is art-recognized and refers to the
group SO.sub.3H, or a pharmaceutically acceptable salt thereof.
[0150] The term "sulfone" is art-recognized and refers to the group
--S(O).sub.2--R.sup.1 wherein R.sup.10 represents a
hydrocarbyl.
[0151] The term "thioalkyl", as used herein, refers to an alkyl
group substituted with a thiol group.
[0152] The term "thioester", as used herein, refers to a group
--C(O)SR.sup.10 or --SC(O)R.sup.10 wherein R.sup.10 represents a
hydrocarbyl.
[0153] The term "thioether", as used herein, is equivalent to an
ether, wherein the oxygen is replaced with a sulfur.
[0154] The term "urea" is art-recognized and may be represented by
the general formula
##STR00014##
wherein R.sup.9 and R.sup.10 independently represent hydrogen or a
hydrocarbyl, such as alkyl, or either occurrence of R.sup.9 taken
together with R.sup.10 and the intervening atom(s) complete a
heterocycle having from 4 to 8 atoms in the ring structure.
[0155] "Protecting group" refers to a group of atoms that, when
attached to a reactive functional group in a molecule, mask, reduce
or prevent the reactivity of the functional group. Typically, a
protecting group may be selectively removed as desired during the
course of a synthesis. Examples of protecting groups can be found
in Greene and Wuts, Protective Groups in Organic Chemistry,
3.sub.rd Ed., 1999, John Wiley & Sons, NY and Harrison et al.,
Compendium of Synthetic Organic Methods, Vols. 1-8, 1971-1996, John
Wiley & Sons, NY. Representative nitrogen protecting groups
include, but are not limited to, formyl, acetyl, trifluoroacetyl,
benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"),
trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("TES"),
trityl and substituted trityl groups, allyloxycarbonyl,
9-fluorenylmethyloxycarbonyl ("FMOC"), nitro-veratryloxycarbonyl
("NVOC") and the like. Representative hydroxylprotecting groups
include, but are not limited to, those where the hydroxyl group is
either acylated (esterified) or alkylated such as benzyl and trityl
ethers, as well as alkyl ethers, tetrahydropyranyl ethers,
trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers,
such as ethylene glycol and propylene glycol derivatives and allyl
ethers.
[0156] The term "healthcare providers" refers to individuals or
organizations that provide healthcare services to a person,
community, etc. Examples of "healthcare providers" include doctors,
hospitals, continuing care retirement communities, skilled nursing
facilities, subacute care facilities, clinics, multispecialty
clinics, freestanding ambulatory centers, home health agencies, and
HMO's.
[0157] As used herein, a therapeutic that "prevents" a disorder or
condition refers to a compound that, in a statistical sample,
reduces the occurrence of the disorder or condition in the treated
sample relative to an untreated control sample, or delays the onset
or reduces the severity of one or more symptoms of the disorder or
condition relative to the untreated control sample.
[0158] The term "treating" includes prophylactic and/or therapeutic
treatments. The term "prophylactic or therapeutic" treatment is
art-recognized and includes administration to the host of one or
more of the subject compositions. If it is administered prior to
clinical manifestation of the unwanted condition (e.g., disease or
other unwanted state of the host animal) then the treatment is
prophylactic (i.e., it protects the host against developing the
unwanted condition), whereas if it is administered after
manifestation of the unwanted condition, the treatment is
therapeutic, (i.e., it is intended to diminish, ameliorate, or
stabilize the existing unwanted condition or side effects
thereof).
[0159] The term "prodrug" is intended to encompass compounds which,
under physiologic conditions, are converted into the
therapeutically active agents of the present invention (e.g., a
compound of formula I). A common method for making a prodrug is to
include one or more selected moieties which are hydrolyzed under
physiologic conditions to reveal the desired molecule. In other
embodiments, the prodrug is converted by an enzymatic activity of
the host animal. For example, esters or carbonates (e.g., esters or
carbonates of alcohols or carboxylic acids) are preferred prodrugs
of the present invention. In certain embodiments, some or all of
the compounds of formula I in a formulation represented above can
be replaced with the corresponding suitable prodrug, e.g., wherein
a hydroxyl in the parent compound is presented as an ester or a
carbonate or carboxylic acid present in the parent compound is
presented as an ester.
Pharmaceutical Compositions
[0160] The compositions and methods of the present invention may be
utilized to treat an individual in need thereof. In certain
embodiments, the individual is a mammal such as a human, or a
non-human mammal. When administered to an animal, such as a human,
the composition or the compound is preferably administered as a
pharmaceutical composition comprising, for example, a compound of
the invention and a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are well known in the art and
include, for example, aqueous solutions such as water or
physiologically buffered saline or other solvents or vehicles such
as glycols, glycerol, oils such as olive oil, or injectable organic
esters. In a preferred embodiment, when such pharmaceutical
compositions are for human administration, particularly for
invasive routes of administration (i.e., routes, such as injection
or implantation, that circumvent transport or diffusion through an
epithelial barrier), the aqueous solution is pyrogen-free, or
substantially pyrogen-free. The excipients can be chosen, for
example, to effect delayed release of an agent or to selectively
target one or more cells, tissues or organs. The pharmaceutical
composition can be in dosage unit form such as tablet, capsule
(including sprinkle capsule and gelatin capsule), granule, lyophile
for reconstitution, powder, solution, syrup, suppository, injection
or the like. The composition can also be present in a transdermal
delivery system, e.g., a skin patch. The composition can also be
present in a solution suitable for topical administration, such as
an eye drop.
[0161] A pharmaceutically acceptable carrier can contain
physiologically acceptable agents that act, for example, to
stabilize, increase solubility or to increase the absorption of a
compound such as a compound of the invention. Such physiologically
acceptable agents include, for example, carbohydrates, such as
glucose, sucrose or dextrans, antioxidants, such as ascorbic acid
or glutathione, chelating agents, low molecular weight proteins or
other stabilizers or excipients. The choice of a pharmaceutically
acceptable carrier, including a physiologically acceptable agent,
depends, for example, on the route of administration of the
composition. The preparation or pharmaceutical composition can be a
selfemulsifying drug delivery system or a selfmicroemulsifying drug
delivery system. The pharmaceutical composition (preparation) also
can be a liposome or other polymer matrix, which can have
incorporated therein, for example, a compound of the invention.
Liposomes, for example, which comprise phospholipids or other
lipids, are nontoxic, physiologically acceptable and metabolizable
carriers that are relatively simple to make and administer.
[0162] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0163] The phrase "pharmaceutically acceptable carrier" as used
herein means a pharmaceutically acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material. Each carrier must be
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as pharmaceutically
acceptable carriers include: (1) sugars, such as lactose, glucose
and sucrose; (2) starches, such as corn starch and potato starch;
(3) cellulose, and its derivatives, such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; (4) powdered
tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such
as cocoa butter and suppository waxes; (9) oils, such as peanut
oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil
and soybean oil; (10) glycols, such as propylene glycol; (11)
polyols, such as glycerin, sorbitol, mannitol and polyethylene
glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13)
agar; (14) buffering agents, such as magnesium hydroxide and
aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water;
(17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol;
(20) phosphate buffer solutions; and (21) other non-toxic
compatible substances employed in pharmaceutical formulations.
[0164] A pharmaceutical composition (preparation) can be
administered to a subject by any of a number of routes of
administration including, for example, orally (for example,
drenches as in aqueous or non-aqueous solutions or suspensions,
tablets, capsules (including sprinkle capsules and gelatin
capsules), boluses, powders, granules, pastes for application to
the tongue); absorption through the oral mucosa (e.g.,
sublingually); anally, rectally or vaginally (for example, as a
pessary, cream or foam); parenterally (including intramuscularly,
intravenously, subcutaneously or intrathecally as, for example, a
sterile solution or suspension); nasally; intraperitoneally;
subcutaneously; transdermally (for example as a patch applied to
the skin); and topically (for example, as a cream, ointment or
spray applied to the skin, or as an eye drop). The compound may
also be formulated for inhalation. In certain embodiments, a
compound may be simply dissolved or suspended in sterile water.
Details of appropriate routes of administration and compositions
suitable for same can be found in, for example, U.S. Pat. Nos.
6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970
and 4,172,896, as well as in patents cited therein.
[0165] The formulations may conveniently be presented in unit
dosage form and may be prepared by any methods well known in the
art of pharmacy. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will vary depending upon the host being treated, the particular
mode of administration. The amount of active ingredient that can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the compound which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 1 percent to about ninety-nine percent
of active ingredient, preferably from about 5 percent to about 70
percent, most preferably from about 10 percent to about 30
percent.
[0166] Methods of preparing these formulations or compositions
include the step of bringing into association an active compound,
such as a compound of the invention, with the carrier and,
optionally, one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing into
association a compound of the present invention with liquid
carriers, or finely divided solid carriers, or both, and then, if
necessary, shaping the product.
[0167] Formulations of the invention suitable for oral
administration may be in the form of capsules (including sprinkle
capsules and gelatin capsules), cachets, pills, tablets, lozenges
(using a flavored basis, usually sucrose and acacia or tragacanth),
lyophile, powders, granules, or as a solution or a suspension in an
aqueous or non-aqueous liquid, or as an oil-in-water or
water-in-oil liquid emulsion, or as an elixir or syrup, or as
pastilles (using an inert base, such as gelatin and glycerin, or
sucrose and acacia) and/or as mouth washes and the like, each
containing a predetermined amount of a compound of the present
invention as an active ingredient. Compositions or compounds may
also be administered as a bolus, electuary or paste.
[0168] To prepare solid dosage forms for oral administration
(capsules (including sprinkle capsules and gelatin capsules),
tablets, pills, dragees, powders, granules and the like), the
active ingredient is mixed with one or more pharmaceutically
acceptable carriers, such as sodium citrate or dicalcium phosphate,
and/or any of the following: (1) fillers or extenders, such as
starches, lactose, sucrose, glucose, mannitol, and/or silicic acid;
(2) binders, such as, for example, carboxymethylcellulose,
alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia;
(3) humectants, such as glycerol; (4) disintegrating agents, such
as agar-agar, calcium carbonate, potato or tapioca starch, alginic
acid, certain silicates, and sodium carbonate; (5) solution
retarding agents, such as paraffin; (6) absorption accelerators,
such as quaternary ammonium compounds; (7) wetting agents, such as,
for example, cetyl alcohol and glycerol monostearate; (8)
absorbents, such as kaolin and bentonite clay; (9) lubricants, such
a talc, calcium stearate, magnesium stearate, solid polyethylene
glycols, sodium lauryl sulfate, and mixtures thereof; (10)
complexing agents, such as, modified and unmodified cyclodextrins;
and (11) coloring agents. In the case of capsules (including
sprinkle capsules and gelatin capsules), tablets and pills, the
pharmaceutical compositions may also comprise buffering agents.
Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugars, as well as high molecular
weight polyethylene glycols and the like.
[0169] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0170] The tablets, and other solid dosage forms of the
pharmaceutical compositions, such as dragees, capsules (including
sprinkle capsules and gelatin capsules), pills and granules, may
optionally be scored or prepared with coatings and shells, such as
enteric coatings and other coatings well known in the
pharmaceutical-formulating art. They may also be formulated so as
to provide slow or controlled release of the active ingredient
therein using, for example, hydroxypropylmethyl cellulose in
varying proportions to provide the desired release profile, other
polymer matrices, liposomes and/or microspheres. They may be
sterilized by, for example, filtration through a bacteria-retaining
filter, or by incorporating sterilizing agents in the form of
sterile solid compositions that can be dissolved in sterile water,
or some other sterile injectable medium immediately before use.
These compositions may also optionally contain opacifying agents
and may be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain portion of the
gastrointestinal tract, optionally, in a delayed manner. Examples
of embedding compositions that can be used include polymeric
substances and waxes. The active ingredient can also be in
micro-encapsulated form, if appropriate, with one or more of the
above-described excipients.
[0171] Liquid dosage forms useful for oral administration include
pharmaceutically acceptable emulsions, lyophiles for
reconstitution, microemulsions, solutions, suspensions, syrups and
elixirs. In addition to the active ingredient, the liquid dosage
forms may contain inert diluents commonly used in the art, such as,
for example, water or other solvents, cyclodextrins and derivatives
thereof, solubilizing agents and emulsifiers, such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
oils (in particular, cottonseed, groundnut, corn, germ, olive,
castor and sesame oils), glycerol, tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and
mixtures thereof.
[0172] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0173] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0174] Formulations of the pharmaceutical compositions for rectal,
vaginal, or urethral administration may be presented as a
suppository, which may be prepared by mixing one or more active
compounds with one or more suitable nonirritating excipients or
carriers comprising, for example, cocoa butter, polyethylene
glycol, a suppository wax or a salicylate, and which is solid at
room temperature, but liquid at body temperature and, therefore,
will melt in the rectum or vaginal cavity and release the active
compound.
[0175] Formulations of the pharmaceutical compositions for
administration to the mouth may be presented as a mouthwash, or an
oral spray, or an oral ointment.
[0176] Alternatively or additionally, compositions can be
formulated for delivery via a catheter, stent, wire, or other
intraluminal device. Delivery via such devices may be especially
useful for delivery to the bladder, urethra, ureter, rectum, or
intestine.
[0177] Formulations which are suitable for vaginal administration
also include pessaries, tampons, creams, gels, pastes, foams or
spray formulations containing such carriers as are known in the art
to be appropriate.
[0178] Dosage forms for the topical or transdermal administration
include powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches and inhalants. The active compound may be mixed
under sterile conditions with a pharmaceutically acceptable
carrier, and with any preservatives, buffers, or propellants that
may be required.
[0179] The ointments, pastes, creams and gels may contain, in
addition to an active compound, excipients, such as animal and
vegetable fats, oils, waxes, paraffins, starch, tragacanth,
cellulose derivatives, polyethylene glycols, silicones, bentonites,
silicic acid, talc and zinc oxide, or mixtures thereof.
[0180] Powders and sprays can contain, in addition to an active
compound, excipients such as lactose, talc, silicic acid, aluminum
hydroxide, calcium silicates and polyamide powder, or mixtures of
these substances. Sprays can additionally contain customary
propellants, such as chlorofluorohydrocarbons and volatile
unsubstituted hydrocarbons, such as butane and propane.
[0181] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the present invention to the
body. Such dosage forms can be made by dissolving or dispersing the
active compound in the proper medium. Absorption enhancers can also
be used to increase the flux of the compound across the skin. The
rate of such flux can be controlled by either providing a rate
controlling membrane or dispersing the compound in a polymer matrix
or gel.
[0182] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention. Exemplary ophthalmic formulations are described in
U.S. Publication Nos. 2005/0080056, 2005/0059744, 2005/0031697 and
2005/004074 and U.S. Pat. No. 6,583,124, the contents of which are
incorporated herein by reference. If desired, liquid ophthalmic
formulations have properties similar to that of lacrimal fluids,
aqueous humor or vitreous humor or are compatable with such fluids.
A preferred route of administration is local administration (e.g.,
topical administration, such as eye drops, or administration via an
implant).
[0183] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrastemal injection and
infusion.
[0184] Pharmaceutical compositions suitable for parenteral
administration comprise one or more active compounds in combination
with one or more pharmaceutically acceptable sterile isotonic
aqueous or nonaqueous solutions, dispersions, suspensions or
emulsions, or sterile powders which may be reconstituted into
sterile injectable solutions or dispersions just prior to use,
which may contain antioxidants, buffers, bacteriostats, solutes
which render the formulation isotonic with the blood of the
intended recipient or suspending or thickening agents.
[0185] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0186] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents that delay
absorption such as aluminum monostearate and gelatin.
[0187] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution, which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0188] Injectable depot forms are made by forming microencapsulated
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions that are
compatible with body tissue.
[0189] For use in the methods of this invention, active compounds
can be given per se or as a pharmaceutical composition containing,
for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active
ingredient in combination with a pharmaceutically acceptable
carrier.
[0190] Methods of introduction may also be provided by rechargeable
or biodegradable devices. Various slow release polymeric devices
have been developed and tested in vivo in recent years for the
controlled delivery of drugs, including proteinacious
biopharmaceuticals. A variety of biocompatible polymers (including
hydrogels), including both biodegradable and non-degradable
polymers, can be used to form an implant for the sustained release
of a compound at a particular target site.
[0191] Actual dosage levels of the active ingredients in the
pharmaceutical compositions may be varied so as to obtain an amount
of the active ingredient that is effective to achieve the desired
therapeutic response for a particular patient, composition, and
mode of administration, without being toxic to the patient.
[0192] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound or
combination of compounds employed, or the ester, salt or amide
thereof, the route of administration, the time of administration,
the rate of excretion of the particular compound(s) being employed,
the duration of the treatment, other drugs, compounds and/or
materials used in combination with the particular compound(s)
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0193] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the therapeutically effective
amount of the pharmaceutical composition required. For example, the
physician or veterinarian could start doses of the pharmaceutical
composition or compound at levels lower than that required in order
to achieve the desired therapeutic effect and gradually increase
the dosage until the desired effect is achieved. By
"therapeutically effective amount" is meant the concentration of a
compound that is sufficient to elicit the desired therapeutic
effect. It is generally understood that the effective amount of the
compound will vary according to the weight, sex, age, and medical
history of the subject. Other factors which influence the effective
amount may include, but are not limited to, the severity of the
patient's condition, the disorder being treated, the stability of
the compound, and, if desired, another type of therapeutic agent
being administered with the compound of the invention. A larger
total dose can be delivered by multiple administrations of the
agent. Methods to determine efficacy and dosage are known to those
skilled in the art (Isselbacher et al. (1996) Harrison's Principles
of Internal Medicine 13 ed., 1814-1882, herein incorporated by
reference).
[0194] In general, a suitable daily dose of an active compound used
in the compositions and methods of the invention will be that
amount of the compound that is the lowest dose effective to produce
a therapeutic effect. Such an effective dose will generally depend
upon the factors described above.
[0195] If desired, the effective daily dose of the active compound
may be administered as one, two, three, four, five, six or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms. In certain
embodiments of the present invention, the active compound may be
administered two or three times daily. In preferred embodiments,
the active compound will be administered once daily.
[0196] The patient receiving this treatment is any animal in need,
including primates, in particular humans, and other mammals such as
equines, cattle, swine and sheep; and poultry and pets in
general.
[0197] In certain embodiments, compounds of the invention may be
used alone or conjointly administered with another type of
therapeutic agent. As used herein, the phrase "conjoint
administration" refers to any form of administration of two or more
different therapeutic compounds such that the second compound is
administered while the previously administered therapeutic compound
is still effective in the body (e.g., the two compounds are
simultaneously effective in the patient, which may include
synergistic effects of the two compounds). For example, the
different therapeutic compounds can be administered either in the
same formulation or in a separate formulation, either concomitantly
or sequentially. In certain embodiments, the different therapeutic
compounds can be administered within one hour, 12 hours, 24 hours,
36 hours, 48 hours, 72 hours, or a week of one another. Thus, an
individual who receives such treatment can benefit from a combined
effect of different therapeutic compounds.
[0198] This invention includes the use of pharmaceutically
acceptable salts of compounds of the invention in the compositions
and methods of the present invention. In certain embodiments,
contemplated salts of the invention include, but are not limited
to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In
certain embodiments, contemplated salts of the invention include,
but are not limited to, L-arginine, benenthamine, benzathine,
betaine, calcium hydroxide, choline, deanol, diethanolamine,
diethylamine, 2-(diethylamino)ethanol, ethanolamine,
ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole,
lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine,
piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium,
triethanolamine, tromethamine, and zinc salts. In certain
embodiments, contemplated salts of the invention include, but are
not limited to, Na, Ca, K, Mg, Zn or other metal salts.
[0199] The pharmaceutically acceptable acid addition salts can also
exist as various solvates, such as with water, methanol, ethanol,
dimethylformamide, and the like. Mixtures of such solvates can also
be prepared. The source of such solvate can be from the solvent of
crystallization, inherent in the solvent of preparation or
crystallization, or adventitious to such solvent.
[0200] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0201] Examples of pharmaceutically acceptable antioxidants
include: (1) water-soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal-chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0202] In certain embodiments, the invention relates to a method
for conducting a pharmaceutical business, by manufacturing a
formulation of a compound of the invention, or a kit as described
herein, and marketing to healthcare providers the benefits of using
the formulation or kit for treating or preventing any of the
diseases or conditions as described herein.
[0203] In certain embodiments, the invention relates to a method
for conducting a pharmaceutical business, by providing a
distribution network for selling a formulation of a compound of the
invention, or kit as described herein, and providing instruction
material to patients or physicians for using the formulation for
treating or preventing any of the diseases or conditions as
described herein.
[0204] In certain embodiments, the invention comprises a method for
conducting a pharmaceutical business, by determining an appropriate
formulation and dosage of a compound of the invention for treating
or preventing any of the diseases or conditions as described
herein, conducting therapeutic profiling of identified formulations
for efficacy and toxicity in animals, and providing a distribution
network for selling an identified preparation as having an
acceptable therapeutic profile. In certain embodiments, the method
further includes providing a sales group for marketing the
preparation to healthcare providers.
[0205] In certain embodiments, the invention relates to a method
for conducting a pharmaceutical business by determining an
appropriate formulation and dosage of a compound of the invention
for treating or preventing any of the disease or conditions as
described herein, and licensing, to a third party, the rights for
further development and sale of the formulation.
EXAMPLES
Example 1: Synthetic Protocols
Synthesis of Linker Cores:
5,5'-(butane-1,4-diyl)-bis(1,3,4-thiadiazol-2-amine) (1001)
##STR00015##
[0207] A mixture of adiponitrile (8.00 g, 73.98 mmol) and
thiosemicarbazide (13.48 g, 147.96 mmol) in trifluoroacetic acid
(TFA) (75 mL) was heated at 80.degree. C. for 17 hours. The
reaction was cooled to room temperature and poured into a mixture
of ice and water. Sodium hydroxide pellets were added to the
mixture until it was basic (pH 14). The white precipitate was
collected by suction filtration, rinsed with water and dried to
provide 5,5'-(butane-1,4-diyl)-bis(1,3,4-thiadiazol-2-amine) (1001,
13.07 g). .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 7.00 (s, 4H),
2.84 (bs, 4H), 1.68 (bs, 4H).
Synthesis of
5,5'-(thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazol-2-amine)
(1002)
##STR00016##
[0209] Compound 1002 was prepared as described in US/2002/0115698
A1
5,5'-(2-methylbutane-1,4-diyl)-bis(1,3,4-thiadiazol-2-amine)
(1003)
##STR00017##
[0211] A mixture of 3-methyl adipic acid (5.00 g, 31.22 mmol) and
thiosemicarbazide (5.69 g, 62.43 mmol) in POC13 (45 mL) was heated
at 90.degree. C. for 4 h. The reaction was cooled to room
temperature and poured into a mixture of ice and water. Sodium
hydroxide pellets were added to the mixture until it was basic (pH
14). The white precipitate was collected by suction filtration,
rinsed with water and dried to provide
5,5'-(2-methylbutane-1,4-diyl)-bis(1,3,4-thiadiazol-2-amine) (1003,
8.97 g). .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 7.00 (s, 4H),
2.89-2.81 (m, 3H), 2.89-2.81 (m, 3H), 2.69 (dd, J=7.6, 7.6 Hz, 1H),
1.89-1.46 (m, 3H), 0.94 (d, J=6.6 Hz, 3H).
5,5'-(propane-1,3-diyl)-bis(1,3,4-thiadiazol-2-amine) (1004)
##STR00018##
[0213] A mixture of glutaronitrile (5.00 g, 53.13 mmol) and
thiosemicarbazide (9.68 g, 106.26 mmol) in TFA (50 mL) was heated
at 85.degree. C. for 4 h. The reaction was cooled to room
temperature and poured into a mixture of ice and water. Sodium
hydroxide pellets were added to the mixture until it was basic (pH
14). The white precipitate was collected by suction filtration,
rinsed with water and dried to provide
5,5'-(propane-1,3-diyl)-bis(1,3,4-thiadiazol-2-amine) (1004, 13.72
g). .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 7.06-7.03 (s, 4H),
2.87 (t, J=7.5 Hz, 4H), 2.02-1.95 (m, 2H).
5-(2-((2-(5-amino-1,3,4-thiadiazol-2-yl)ethyl)amino)ethyl)-1,3,4-thiadiazo-
l-2-amine (1005)
##STR00019##
[0215] A mixture of 3,3'-iminodipropionitrile (1.50 g, 12.18 mmol)
and thiosemicarbazide (2.22 g, 24.36 mmol) in TFA (10 mL) was
heated at 85 for 4.5 h. The reaction was cooled to room temperature
and poured into a mixture of ice and water. Sodium hydroxide
pellets were added to the mixture until it was basic (pH 14). The
white precipitate was collected by suction filtration, rinsed with
water and dried to provide
5-(2-((2-(5-amino-1,3,4-thiadiazol-2-yl)ethyl)amino)ethyl)-1,3,4-thiadiaz-
ol-2-amine (1005, 1.47 g). .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 6.95 (s, 4H), 2.90 (d, J=6.0 Hz, 4H), 2.83 (d, J=6.3 Hz,
4H).
##STR00020##
[0216] To a solution of methyl
3-((2-methoxy-2-oxoethyl)thio)propanoate (5.0 g, 26 mmol) in
THF/MeOH/water (60 mL, 4:1:1) was added lithium hydroxide
monohydrate (4.375 g, 101 mmol). The resulting mixture was stirred
at room temperature overnight before it was concentrated under
reduced pressure. The residue obtained was diluted with water
(.about.100 mL) and the resulting solution was acidified with 6N
HCl. The mixture was partitioned between water and ethyl acetate.
The organic extract was washed with more water, separated, dried
over sodium sulfate, filtered and evaporated to afford
3-((carboxymethyl)thio)propanoic acid (3.64 g, 85%) as a white
solid. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm 2.55-2.57
(t, 2H) 2.75-2.79 (t, 2H) 3.27 (s, 2H) 12.41 (s, 2H)
[0217] To a mixture of 3-((carboxymethyl)thio)propanoic acid (3.64
g, 22.2 mmol) and thiosemicarbazide (4.1 g, 45 mmol) was added
phosphorus oxychloride (25 mL) slowly. The resulting mixture was
stirred at 90.degree. C. for 3 hr before it was poured over crushed
ice slowly. The solid separated was filtered and the filtrate was
basified to pH-13 by solid sodium hydroxide. The solid separated
was filtered, washed with water and dried at 45.degree. C. under
vacuum overnight to afford 1006 (.about.3 g, 50%) as a tan solid.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm 2.79-2.83 (t, 2H)
3.06-3.10 (t, 2H) 3.99 (s, 2H) 7.04 (s, 2H) 7.16 (s, 2H)
##STR00021##
[0218] A mixture of 2,2'-Thiodiacetic acid (5.00 g, 33.3 mmol) and
thiosemicarbazide (6.07 g, 66.6 mmol) in POCl.sub.3 (40 mL) was
heated at 90.degree. C. for 5 h. The reaction was cooled to room
temperature and carefully poured it onto a mixture of ice and
water. Sodium hydroxide pellets were added to the mixture until it
was basic (pH 14). The white precipitate was collected by suction
filtration, rinsed with water and dried to afford 1007. .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. 7.18 (s, 4H), 3.96 (s, 4H).
##STR00022##
[0219] A mixture of 1,5-dicyanopentane (1.00 g, 8.19 mmol) and
thiosemicarbazide (1.5 g, 16.40 mmol) in TFA (3 mL) was heated at
85.degree. C. for 5 h. The reaction was cooled to room temperature
and poured into a mixture of ice and water. Sodium hydroxide
pellets were added to the mixture until it was basic (pH 14). The
white precipitate was collected by suction filtration, rinsed with
water and dried to afford 1008. .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 6.98 (s, 4H), 2.81 (t, 4H), 1.67 (m, 4H), 1.20 (m, 2H).
Acylation of Diamino Core:
Method A: Via Acid Chloride
N,N'-[5,5'-(butane-1,4-diyl)-bis(1,3,4-thiadiazole-5,2-diyl)]-bis(2-phenyl-
acetamide) (21)
##STR00023##
[0221] To a suspension of 1001 (8.00 g, 31.21 mmol) in
1-Methyl-2-pyrrolidinone (NMP) 100 mL) at 0.degree. C. was added
phenylacetyl chloride (10.25 mL, 77.54 mmol) dropwise. The
resulting mixture was stirred at 0.degree. C. for 1 h before it was
quenched by addition of water (.about.200 mL). The white
precipitate was collected by suction filtration, rinsed with water
and dried to provide
N,N-[5,5'-(butane-1,4-diyl)-bis(1,3,4-thiadiazole-5,2-diyl)]-bis(2-phenyl-
acetamide) (21, 14.02 g). .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 12.66 (s, 2H), 7.34 (m, 10H), 3.81 (s, 4H), 3.01 (bs, 4H),
1.76 (bs, 4H).
##STR00024##
[0222] Compound 43 was prepared following Method A using
phenoxyacetyl chloride. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.
12.68 (s, 2H), 7.35-7.30 (m, 4H), 6.99-6.97 (m, 6H), 4.90 (s, 4H),
3.05 (bs, 4H), 1.79 (bs, 4H).
##STR00025##
[0223] Compound 100 was prepared following Method A. .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. 12.42 (s, 2H), 3.64 (t, J=5.6 Hz,
4H), 3.24 (s, 6H), 3.01 (bs, 4H), 2.72 (t, J=6.2 Hz, 4H), 1.79 (bs,
4H).
##STR00026##
[0224] Compound 5 was prepared according to Method A: .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. 12.66 (s, 4H), 3.27 (t, J=6.99 Hz,
4H), 2.95 (t, J=7.02 Hz, 4H), 2.12 (s, 6H).
##STR00027##
[0225] To a suspension of 1001 (200 mg, 0.78 mmol) in NMP (2 mL) at
0.degree. C. was added O-acetylmandelic acid chloride (0.44 mL,
1.95 mmol) dropwise. The resulting mixture was stirred at 0.degree.
C. for 1.5 h before it was quenched by addition of water (.about.10
mL). The white precipitate was collected by suction filtration,
rinsed with more water and dried. The crude material was purified
by recrystallization with a mixture of DMSO and MeOH to afford
173.
[0226] A flask was charged with 173 and 2N ammonia in MeOH (3 ml)
and the resulting mixture was stirred at room temperature for 6 h.
The solvent was removed and the resulting material was dried in the
oven to afford 174. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.
12.42 (s, 2H), 7.53-7.31 (m, 10H), 6.35 (s, 2H), 5.34 (d, J=1.14
Hz, 2H), 3.01 (bs, 4H), 1.76 (bs, 4H).
[0227] Compound 306 was prepared according to the procedure for
compound 174 above.
##STR00028##
[0228] To a suspension of 1001 (400 mg, 1.56 mmol) in NMP (4 mL) at
0.degree. C. was added (R)-(-)--O-formylmandeloyl chloride (0.61
mL, 3.90 mmol) dropwise. The resulting mixture was stirred at
0.degree. C. for 1.5 h before it was quenched by addition of water
(.about.10 mL). The white precipitate was collected by suction
filtration, rinsed with more water and dried. The crude material
was purified by recrystallization with a mixture of DMSO and MeOH
to afford 68.
[0229] A flask was charged with 68 and 2N ammonia in MeOH (5 ml)
and the resulting mixture was stirred at room temperature for 2 h.
The solvent was removed and the resulting material was dried in the
oven to afford 80. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.
7.53-7.31 (m, 10H), 6.34 (s, 2H), 5.33 (s, 2H), 3.01 (bs, 4H), 1.75
(bs, 4H).
##STR00029##
[0230] To a suspension of 1002 (544 mg, 1.89 mmol) in NMP (13 mL)
at -15.degree. C. was added phenylacetyl chloride (0.249 mL, 1.89
mmol) dropwise. The resulting mixture was stirred at 0.degree. C.
for 1 h and quenched by the addition of water (54 mL). The white
precipitate was collected by suction filtration, rinsed with water
(27 mL) and ethyl acetate (3.times.27 mL). The filtrate was
basified to pH 11 using 2.5M NaOH. The layers were separated and
the aqueous layer extracted with dichloromethane (3.times.54 mL).
The combined organic layers were dried over magnesium sulfate and
concentrated to afford
N-(5-(2-((2-(5-amino-1,3,4-thiadiazol-2-yl)ethyl)thio)ethyl)-1,3,4-thiadi-
azol-2-yl)-2-phenylacetamide (17, 56 mg).sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.71 (s, 1H), 7.32 (s, 5H), 3.81 (s, 2H),
3.25 (t, J=7.61 Hz, 2H) 3.06 (t, J=7.25 Hz, 2H), 2.92 (t, J=6.90
Hz, 2H), 2.85 (t, J=6.86 Hz, 2H)
##STR00030##
[0231] Phenylacetyl chloride (0.134 mL, 1.01 mmol) and
acetoxyacetyl chloride (0.109 mL, 1.01 mmol) were mixed together in
NMP (0.5 mL). This mixture was slowly added to a suspension of 1002
(292 mg, 1.01 mmol) in NMP (7 mL) at RT. The resulting mixture was
stirred at RT for 1 h and quenched by the addition of water (20
mL). The white precipitate was collected by suction filtration,
rinsed with water and dried under high vacuum. The crude material
was purified by preparative HPLC. Compound 26: .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.69 (s, 2H), 7.34 (3, 5H), 4.81 (s,
2H), 3.82 (s, 2H), 2.96 (bs, 4H), 2.14 (s, 3H).
##STR00031##
[0232] Compound 44 was prepared following the procedure for
compound 21 described previously. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.66 (s, 2H), 7.34-7.28 (m, 10H), 3.81 (s,
4H), 3.05-3.00 (m, 3H), 2.87 (dd, J=7.9, 8.2 Hz, 1H), 1.95-1.77 (m,
3H), 0.94 (d, J=6.5 Hz, 3H).
##STR00032##
[0233] Compound 72 was prepared following the procedure for
compound 21 described previously. To a suspension of diamine 1004
(0.70 g, 3.07 mmol) in NMP (15 mL) at 0.degree. C. was added
phenylacetyl chloride (811 .mu.L, 6.13 mmol) dropwise. The
resulting mixture was stirred at 0.degree. C. for 1 h before it was
quenched by addition of water. The white precipitate was collected
by suction filtration, rinsed with water and dried to provide
N,N-[5,5'-(propane-1,3-diyl)-bis(1,3,4-thiadiazole-5,2-diyl)]-bis(2-pheny-
lacetamide) (72, 1.37 g). .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 12.68 (s, 2H), 7.38-7.27 (m, 10H), 3.82 (s, 4H), 3.06 (t,
J=7.2 Hz, 4H), 2.17-2.12 (m, 2H).
##STR00033##
[0234] To a suspension of compound 1005 (100 mg, 0.37 mmol) in DMF
(12 mL) at room temperature was added a solution of (t-Boc).sub.2O
(88 mg, 0.41 mmol) in DMF (2 mL). The mixture was stirred at room
temperature for 24 h. To this reaction mixture was added NMP (2 mL)
and followed by addition of phenylacetyl chloride (97 .mu.L, 0.74
mmol). The reaction was stirred for 1 h before it was poured into a
mixture of ice-water. The solid was collected by suction
filtration, rinsed with water and dried to provide 1010 (180
mg).
[0235] The above product 1010 (160 mg, 0.26 mmol) in a mixture of
TFA (1.5 mL) and CH.sub.2CH.sub.2 (10 mL) was stirred at room
temperature for 4 h before it was concentrated. The residue was
re-taken up in CH.sub.2Cl.sub.2 (3.times.) and concentrated to
provide N,
N-(5,5'-(azanediyl-bis(ethane-2,1-diyl))-bis(1,3,4-thiadiazole-5,2-diyl))-
-bis(2-phenylacetamide) trifluoroacetic acid (149, 122 mg). .sup.1H
NMR (300 MHz, DMSO-d.sub.6) .delta. 12.81 (s, 2H), 8.75 (bs, 2H),
7.38-7.27 (m, 10H), 3.84 (s, 4H), 3.45 (d, J=2.9 Hz, 4H), 3.39 (d,
J=6.0 Hz, 4H).
##STR00034##
[0236] To a suspension of 1006 (0.274 g, 1 mmol) in NMP (5 mL) was
added phenyl acetyl chloride (0.263 mL, 2 mmol) dropwise. The
mixture was stirred at room temperature for 1 hr and afterwards it
was diluted with water. Solid separated was filtered, washed with
more water and dried. The crude material was purified by prep HPLC
to afford 199 as a white solid. .sup.1H NMR (300 MHz,
Dimethylsulfoxide-d.sub.6) .delta. ppm 2.87-2.91 (t, 2H) 3.25-3.29
(t, 2H) 3.82 (s, 4H) 4.19 (s, 2H) 7.26-7.33 (m, 10H) 12.71-12.72
(br s, 2H).
Method B: Via Acid Using Peptide Coupling Reagents
##STR00035##
[0238] To a flask containing
5,5'-(thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazol-2-amine) (1002)
(0.69 mmol, 0.20 g, 1.0 equiv.) was added 2-morpholinoacetic acid
(1.52 mmol, 0.22 g, 2.2 equiv.),
O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU) (2.20 mmol, 0.83 g, 3.2 equiv.),
1-Hydroxybenzotriazole (HOBT) (2.2 mmol, 0.29 g, 3.2 equiv.) 5 mL
of DMF followed by N,N-Diisopropylethylamine (DIEA) (5.52 mmol,
0.71 g, 0.960 mL, 8.0 equiv.). The mixture was stirred overnight at
room temperature and then diluted with 15 mL water. The mixture was
extracted with EtOAc and the organic layers combined, washed with
water, brine and dried over Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4
was removed by filtration and the volatiles removed under reduced
pressure to give 0.04 g of compound 12. .sup.1HNMR (300 MHz,
CDCl.sub.3) Compound 12: .delta. 3.80 (broad multiplet, 4H), 3.34
(dd, 4H, J=7.2 Hz), 3.28 (s, 4H), 3.00 (dd, 4H, J=7.1 Hz), 2.63
(broad multiplet, 4H).
##STR00036##
[0239] To a flask containing
5,5'-(butane-1,4-diyl)bis(1,3,4-thiadiazol-2-amine) (1101) (3.9
mmol, 1.0 g, 1.0 equiv.) was added
(S)-2-((tert-butoxycarbonyl)amino)-2-phenylacetic acid (8.58 mmol,
2.15 g, 2.2 equiv.), HBTU (12.48 mmol, 4.73 g, 3.2 equiv.), HOBt
(12.48 mmol, 1.69 g, 3.2 equiv.) 25 mL of DMF followed by DIEA
(31.2 mmol, 4.03 g, 5.43 mL, 8.0 equiv.). The mixture was stirred
overnight and poured into 150 mL water. The white solids that
formed were collected by vacuum filtration, washed with water and
dried under vacuum giving 2.47 g of the bis-Boc protected
intermediate.
[0240] To a slurry of the bis-Boc protected intermediate (2.76
mmol, 2.0 g, 1.0 equiv.) in 20 mL of dichloromethane (DCM) was
added 4 M HCl in dioxane (40 mmol, 10 mL) with vigorous stirring.
The mixture briefly became clear and homogeneous then a white
precipitate formed. The mixture was stirred overnight and diluted
with 20 mL diethyl ether. The solids were collected by vacuum
filtration washed with additional diethyl ether and dried under
vacuum giving 0.9 g 187. .sup.1HNMR (300 MHz, DMSO, d.sub.6)
Compound 187: .delta. 9.13 (s, 4H), 7.61 (m, 4H), 7.48 (m, 6H), 6.2
(broad singlet, 4H), 5.32 (s, 2H), 3.04 (broad multiplet, 4H), 1.77
(broad multiplet, 4H).
##STR00037##
[0241] To a solution of 2,2-bis(hydroxymethyl)propionic acid (5.00
g, 37.28 mmol) in acetone (80 mL) at room temperature was added
2,2-dimethoxypropane (6.88 mL, 55.92 mmol) and p-TsOH--H.sub.2O
(0.36 g, 1.86 mmol). The reaction was stirred for 2 h before it was
quenched with Et.sub.3N (0.30 mL). The organic volatile was removed
under reduced pressure. The residue was partitioned between EtOAc
and water. The organic layer was washed with brine, dried
(MgSO.sub.4) and concentrated to provide the desired product 1011
(5.17 g) as a white solid.
[0242] To a suspension of diamine 1001 (500 mg, 1.95 mmol),
3-fluorophenylacetic acid (361 mg, 2.34 mmol) and acid 1011 (442
mg, 2.54 mmol) in DMF (20 mL) at 0.degree. C. was added HOBt (791
mg, 5.85 mmol) and followed by
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)
(1.12 g, 5.85 mmol). The mixture was stirred from 0.degree. C. to
room temperature over 18 h before it was diluted with water. The
precipitate was collected by suction filtration, washed with water
and dried. The crude product was purified by silica gel
chromatography eluting with 1-10% MeOH in CH.sub.2Cl.sub.2 to
provide
N-(5-(4-(5-(2-(3-fluorophenyl)acetamido)-1,3,4-thiadiazol-2-yl)butyl)-1,3-
,4-thiadiazol-2-yl)-2,2,5-trimethyl-1,3-dioxane-5-carboxamide
(1012, 208 mg).
[0243] The above product 1012 (87 mg, 0.16 mmol) and TFA (2 mL) in
a mixture of THF (8 mL) and water (2 mL) was heated at 50.degree.
C. for 5 h before it was concentrated under reduced pressure. The
crude residue was purified by HPLC to provide
N,N-(5-(4-(5-(2-(3-fluorophenyl)acetamido)-1,3,4-thiadiazol-2-yl)butyl)-1-
,3,4-thiadiazol-2-yl)-3-hydroxy-2-(hydroxymethyl)-2-methylpropanamide
(152). .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.68 (s, 1H),
11.77 (s, 1H), 7.04-7.38 (m, 1H), 7.18-7.09 (m, 4H), 4.98 (s, 2H),
3.86 (s, 2H), 3.62 (dd, J=10.7, 29.0 Hz, 4H), 3.03 (bs, 4H), 1.77
(bs, 4H), 1.14 (s, 3H).
##STR00038##
[0244] To a suspension of diamine 1001 (400 mg, 1.56 mmol),
3-fluorophenylacetic acid (313 mg, 2.03 mmol),
(R)-(-)-2,2-dimethyl-5-oxo-1,3-dioxolane-4-acetic acid (353 mg,
2.03 mmol) and Et.sub.3N (200 .mu.L) in DMF (20 mL) at 0.degree. C.
was added HOBt (633 mg, 4.68 mmol) and followed by EDC (897 mg,
4.68 mmol). The mixture was stirred from 0.degree. C. to room
temperature over 18 h before it was diluted with water. The
precipitate was collected by suction filtration and washed with
water. The solid was further rinsed with a mixture of hot MeOH-THF.
The combined filtrate was concentrated and purified by silica gel
chromatography eluting with 1-10% MeOH in CH.sub.2Cl.sub.2 to
provide
(R)--N-(5-(4-(5-(2-(3-fluorophenyl)acetamido)-1,3,4-thiadiazol-2-yl)butyl-
)-1,3,4-thiadiazol-2-yl)-3,4-dihydroxybutanamide (1013, 93 mg).
[0245] The above product 1013 (87 mg, 0.16 mmol) and TFA (2 mL) in
a mixture of THF (8 mL) and water (2 mL) was heated at 50.degree.
C. for 5 h before it was concentrated under reduced pressure. The
crude residue was purified by HPLC to provide
(R)--N-(5-(4-(5-(2-(3-fluorophenyl)acetamido)-1,3,4-thiadiazol-2-yl)butyl-
)-1,3,4-thiadiazol-2-yl)-3,4-dihydroxybutanamide (153). .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. 12.67 (s, 1H), 12.43 (s, 1H),
7.41-7.38 (m, 1H), 7.20-7.12 (m, 4H), 4.45-4.40 (m, 1H), 3.86 (s,
2H), 3.03 (bs, 4H), 2.85-2.77 (m, 2H), 1.78 (bs, 4H).
##STR00039##
[0246] To a suspension of (S)-(+)-O-acetylmandelic acid (666 mg,
3.43 mmol) and
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) (1.47 g, 3.86 mmol) in DMF (4 mL) was
added DIEA (0.672 ml, 3.86 mmol) followed by 1001 (400 mg, 1.56
mmol). The resulting mixture was stirred at room temperature
overnight before it was quenched by addition of water (.about.10
mL). The white precipitate was collected by suction filtration,
rinsed with more water and dried. The crude material was purified
by recrystallization with a mixture of DMSO and MeOH to afford
66.
[0247] A flask was charged with 66 and 2N ammonia in MeOH (5 ml)
and the resulting mixture was stirred at room temperature for 6 h.
The solvent was removed and the resulting material was dried in the
oven to afford 92. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.
12.42 (s, 2H), 7.53-7.31 (m, 10H), 6.35 (s, 2H), 5.33 (s, 2H), 3.01
(bs, 4H), 1.76 (bs, 4H).
##STR00040##
[0248] A flask was charged with 1001 (200 mg, 0.78 mmol),
DL-3-phenyllactic acid (285 mg, 1.716 mmol), and HOBT (527 mg, 3.9
mmol) in DMF (3 ml) was added EDC (897 mg, 4.68 mmol) followed by
triethylamine (0.87 ml, 6.24 mmol). The resulting mixture was
stirred at room temperature overnight before it was quenched by
addition of water (.about.5 mL). The mixture was partitioned
between water and EtOAc. The organic extract was washed with water,
dried over sodium sulfate, filtered and evaporated. The crude
material was purified by silica gel chromatography eluting with
0-6% MeOH in CH.sub.2Cl.sub.2 to afford 69. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.20 (s, 2H), 7.24 (m, 10H), 5.75 (d, J=6.87
Hz, 2H), 4.43 (m, 2H), 3.10 (m, 6H), 2.89-2.81 (m, 2H), 1.80 (bs,
4H).
##STR00041##
[0249] A flask was charged with 1001 (200 mg, 0.78 mmol),
D-(+)-3-phenyllactic acid (285 mg, 1.716 mmol), and HOBt (464 mg,
3.43 mmol) in DMF (3 ml) was added EDC (822 mg, 4.28 mmol) followed
by triethylamine (0.718 ml, 5.15 mmol). The resulting mixture was
stirred at room temperature overnight before it was quenched by
addition of water (.about.5 mL). The mixture was partitioned
between water and EtOAc. The organic extract was washed with water,
dried over sodium sulfate, filtered and evaporated. The crude
material was purified by silica gel chromatography eluting with
0-6% MeOH in CH.sub.2Cl.sub.2 to afford 169. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.20 (s, 2H), 7.24 (m, 10H), 5.75 (d, J=6.87
Hz, 2H), 4.43 (m, 2H), 3.03 (m, 6H), 2.89-2.81 (m, 2H), 1.80 (bs,
4H).
##STR00042##
[0250] A flask was charged with 1001 (200 mg, 0.78 mmol),
L-(-)-3-phenyllactic acid (285 mg, 1.716 mmol), and HOBt (464 mg,
3.43 mmol) in DMF (3 ml) was added EDC (822 mg, 4.28 mmol) followed
by triethylamine (0.718 ml, 5.15 mmol). The resulting mixture was
stirred at room temperature overnight before it was quenched by
addition of water (.about.5 mL). The mixture was partitioned
between water and EtOAc. The organic extract was washed with more
water, dried over sodium sulfate, filtered and evaporated. The
crude material was purified by silica gel chromatography eluting
with 0-6% MeOH in CH.sub.2Cl.sub.2 to afford 146. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.27 (s, 2H), 7.31 (m, 10H), 5.78 (m,
2H), 4.44 (m, 2H), 3.05 (m, 6H), 2.87 (m, 2H), 1.79 (bs, 4H).
##STR00043##
[0251] To a suspension of (R)-(+)-3-hydroxy-3-phenylpropionic acid
(285 mg, 1.72 mmol) and HATU (719 mg, 1.89 mmol) in DMF (3 mL) was
added DIEA (0.329 ml, 1.89 mmol) followed by 1001 (200 mg, 0.78
mmol). The resulting mixture was stirred at room temperature
overnight before it was quenched by addition of water (.about.10
mL). The white precipitate was collected by suction filtration,
rinsed with more water and dried. The crude material was purified
by recrystallization with DMSO and MeOH to afford 127. .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. 12.38 (s, 2H), 7.34 (m, 10H), 5.56
(m, 2H), 5.10 (m, 2H), 3.04 (bs, 4H), 2.80 (m, 4H), 1.80 (bs,
4H).
##STR00044##
[0252] To a suspension of (R)-2-hydroxy-2-phenylbutyric acid (310
mg, 1.72 mmol) and HATU (719 mg, 1.89 mmol) in DMF (3 mL) was added
DIEA (0.329 ml, 1.89 mmol) followed by 1001 (200 mg, 0.78 mmol).
The resulting mixture was stirred at room temperature overnight
before it was quenched by addition of water (.about.10 mL). The
crude material was purified by HPLC to afford 143. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 7.61 (d, J=7.65 Hz, 4H), 7.34 (m, 6H),
2.99 (bs, 4H), 2.26 (m, 2H), 2.10 (m, 2H) 1.74 (bs, 4H), 0.80 (t,
6H).
##STR00045##
[0253] To a suspension of 3-Oxo-1-indancarboxylic acid (604 mg,
3.43 mmol) and HATU (1.47 g, 3.86 mmol) in DMF (5 mL) was added
DIEA (0.672 ml, 3.86 mmol) followed by 1001 (400 mg, 1.56 mmol).
The resulting mixture was stirred at room temperature overnight
before it was quenched by addition of water (.about.10 mL). The
light brown precipitate was collected by suction filtration, rinsed
with water and dried. The crude material was purified by
recrystallization with a mixture of DMSO and MeOH to afford 64.
[0254] To a suspension of 64 (100 mg, 0.175 mmol) in EtOH (20 ml)
at 0.degree. C. was added NaBH.sub.4 (15 mg, 0.384 mmol) and the
resulting mixture was stirred for 1 h before it was quenched by 1N
HCl. The mixture was partitioned between 1N HCl and EtOAc, the
organic extract was dried over sodium sulfate, filtered and
evaporated. The crude material was purified by silica gel
chromatography eluting with 0-6% MeOH in CH.sub.2Cl.sub.2 and
further purified by recrystallization with a mixture of DMSO and
MeOH to afford 94. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.
12.81 (s, 2H), 7.34 (m, 8H), 5.56 (m, 2H), 5.11 (t, 2H), 4.15 (t,
2H), 3.05 (bs, 4H), 2.70 (m, 2H), 2.15 (m, 2H), 1.80 (bs, 4H).
##STR00046##
[0255] To a solution of DL-mandelic acid (1 g, 6.57 mmol) in DMF
(10 ml) at 0.degree. C. was added NaH (700 mg, 19.7 mmol) and
allowed the mixture to stir for 20 minutes before 2-bromoethyl
methyl ether (1.24 ml, 13.1 mmol) was added dropwise. The resulting
mixture was stirred at 0.degree. C. and slowly warmed up to room
temperature overnight before it was quenched by 1N HCl. The mixture
was partitioned between 1N HCl and EtOAc, the organic extract was
washed with water, dried over sodium sulfate, filtered and
evaporated to afford 1014.
[0256] To a suspension of 1014 (500 mg, 2.37 mmol) and HATU (995
mg, 2.62 mmol) in DMF (3 mL) was added DIEA (0.456 ml, 2.62 mmol)
followed by 1001 (277 mg, 1.08 mmol). The resulting mixture was
stirred at room temperature overnight before it was quenched by
addition of water (.about.6 mL). The mixture was partitioned
between water and EtOAc. The organic extract was washed with water,
dried over sodium sulfate, filtered and evaporated. The crude
material was purified by HPLC to afford 203. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.58 (s, 2H), 7.49-7.37 (m, 10H), 5.22 (s,
2H), 3.66-3.54 (m, 8H), 3.27 (s, 6H), 3.01 (bs, 4H), 1.75 (bs,
4H).
##STR00047##
[0257] To a suspension of 2-(4-Boc-piperazinyl)-2-phenylacetic acid
(1.1 g, 3.43 mmol) and HATU (1.47 g, 3.86 mmol) in DMF (5 mL) was
added DIEA (0.672 ml, 3.86 mmol) followed by 1001 (400 mg, 1.56
mmol). The resulting mixture was stirred at room temperature
overnight before it was quenched by addition of water (.about.10
mL). The white precipitate was collected by suction filtration,
rinsed with water and dried. The crude material was purified by
recrystallization with DMSO and MeOH to afford 63.
[0258] A flask was charged with 63 and 4N HCl in 1,4-dioxane (6 ml)
and the resulting mixture was stirred at room temperature for 3 h.
The precipitation was collected by filtration, rinse with
EtOAc/CH.sub.2Cl.sub.2 and dried to afford 77. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 9.10 (bs, 4H), 7.51-7.41 (m, 10H), 4.90
(bs, 2H), 4.62 (s, 2H), 3.15 (bs, 8H), 3.03 (bs, 4H), 2.73 (bs,
8H), 1.76 (bs, 4H).
##STR00048##
[0259] To a suspension of (R)-(+)-3-hydroxy-3-phenylpropionic acid
(254 mg, 1.53 mmol) and HATU (640 mg, 1.68 mmol) in DMF (3 mL) was
added DIEA (0.292 ml, 1.68 mmol) followed by 1002 (200 mg, 0.693
mmol). The resulting mixture was stirred at room temperature
overnight before it was quenched by addition of water (.about.10
mL). The white precipitate was collected by suction filtration,
rinsed with water and dried. The crude material was purified by
recrystallization with a mixture of DMSO and MeOH to afford 126.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.40 (s, 2H), 7.38 (m,
10H), 5.55 (m, 2H), 5.09 (m, 2H), 3.27 (t, 4H), 2.95 (t, 4H), 2.82
(m, 4H).
##STR00049##
[0260] A flask was charged with 1002 (200 mg, 0.693 mmol),
2-(4-Boc-piperazinyl)-2-phenylacetic acid (244 mg, 0.763 mmol), and
HOBt (187 mg, 1.39 mmol) in DMF (3 ml) was added EDC (332 mg, 1.73
mmol) followed by triethylamine (0.290 ml, 2.08 mmol). The
resulting mixture was stirred at room temperature overnight before
phenylacetyl chloride (0.037 ml, 0.277 mmol) was added dropwise at
0.degree. C. and stirred for 1 h before it was quenched by addition
of water (.about.10 mL). The white precipitate was collected by
suction filtration, rinsed with water and dried. The crude material
was purified by HPLC to afford 70 and 76.
##STR00050##
[0261] A flask was charged with 70 and 4N HCl in 1,4-dioxane (6 ml)
and the resulting mixture was stirred at room temperature for 3 h.
The precipitation was collected by filtration, rinse with
EtOAc/CH.sub.2Cl.sub.2 and dried to afford 78. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.70 (s, 2H), 8.97 (bs, 2H), 7.50-7.29
(m, 10H), 4.72 (bs, 1H), 4.59 (s, 1H), 3.82 (s, 2H), 3.27 (t, 4H),
3.15 (bs, 4H), 2.92 (t, 4H), 2.70 (bs, 4H).
##STR00051##
[0262] A flask was charged with 76 and 4N HCl in 1,4-dioxane (6 ml)
and the resulting mixture was stirred at room temperature for 3 h.
The precipitation was collected by filtration, rinse with
EtOAc/CH.sub.2Cl.sub.2 and dried to afford 79. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.87 (s, 2H), 9.03 (bs, 4H), 7.50-7.40
(m, 10H), 4.67 (bs, 2H), 4.59 (s, 2H), 3.28 (t, 4H), 3.14 (bs, 8H),
2.97 (t, 4H), 2.71 (bs, 8H).
[0263] Amide Coupling General Procedure (used for following
examples): To a 0.2 molar concentration suspension of carboxylic
acid (2 equivalents) in DMF was added HATU (2 equivalents) and
stirred till reaction mixture is clear followed by the addition of
an amine (1 equivalent) and DIPEA (4 equivalents). The resulting
mixture was stirred at room temperature overnight before it was
quenched by the addition of water. The solid separated was
filtered, washed with water and dried.
##STR00052##
[0264] 39: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.89-2.01 (m, 6H) 2.18-2.29 (m, 2H) 2.95-3 (m, 4H) 3.79-3.86 (m,
2H) 3.94-4.02 (m, 2H) 4.55-4.6 (m, 2H) 12.29 (brs, 2H).
##STR00053##
[0265] 41: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
2.93-2.98 (m, 4H) 3.27-3.32 (m, 4H), 4.46 (s, 4H), 5.18-5.2 (br s,
2H) 6.88-7.03 (m, 8H) 12.87-12.92 (br s, 2H).
##STR00054##
[0266] 51: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.78 (br s, 4H) 3.05-3.06 (br s, 4H), 3.38-3.40 (m, 2H) 3.54-3.63
(m, 2H) 5.44-5.50 (m, 2H) 6.92-7.26 (m, 8H) 12.78 (br s, 2H).
##STR00055##
[0267] 54: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.92-2.03 (m, 10H) 2.17-2.28 (m, 2H) 3.05 (br s, 4H) 3.79-3.85 (m,
2H) 3.94-4.01 (m, 2H) 4.55-4.59 (m, 2H) 12.27 (br s, 2H).
##STR00056##
[0268] 60: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.77 (br s, 4H) 3.04 (br s, 4H) 5.20 (s, 4H) 6.31 (br s, 2H) 7.49
(br s, 2H) 7.79 (br s, 2H) 12.80 (br s, 2H).
##STR00057##
[0269] 85: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
0.20-0.21 (br s, 4H) 0.48-0.50 (br s, 4H) 1.79 (br s, 4H) 2.35-2.38
(br s, 4H) 3.04 (br s, 4H) 12.32 (br s, 2H).
##STR00058##
[0270] 87: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.78 (br s, 4H) 3.03 (br s, 4H) 4.05 (s, 4H) 6.99 (br s, 4H)
7.42-7.44 (m, 2H) 12.68 (br s, 2H).
##STR00059##
[0271] 114: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.01-1.12 (m, 4H) 1.40 (s, 18H) 1.61-1.65 (m, 4H) 1.78 (br s, 4H)
1.95 (br s, 2H) 3.84 (m, 4H) 2.65-2.75 (m, 4H) 3.03 (br s, 4H)
3.89-3.93 (m, 4H) 12.39 (br s, 2H).
##STR00060##
[0272] 123: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.43 (s, 6H) 1.79-1.94 (m, 10H) 2.22-2.31 (m, 2H) 3.05 (br s, 4H)
3.85-4.01 (m, 4H) 11.85 (br s, 2H).
##STR00061##
[0273] 133: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
2.92-2.97 (m, 4H) 3.26-3.30 (m, 4H) 4.61-4.87 (m, 6H) 6.83-6.89 (m,
4H) 7.16-7.21 (m, 2H) 7.36-7.38 (m, 2H) 12.95 (br s, 2H).
##STR00062##
[0274] 135: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.77 (br s, 4H) 3.03 (br s, 4H) 4.60-4.87 (m, 6H) 6.83-6.89 (m, 4H)
7.16-7.22 (m, 2H) 7.36-7.38 (m, 2H) 12.92 (br s, 2H).
##STR00063##
[0275] 114: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.01-1.12 (m, 4H) 1.40 (s, 18H) 1.61-1.65 (m, 4H) 1.78 (br s, 4H)
1.95 (br s, 2H) 3.84 (m, 4H) 2.65-2.75 (m, 4H) 3.03 (br s, 4H)
3.89-3.93 (m, 4H) 12.39 (br s, 2H).
##STR00064##
[0276] 323: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.76 (brs, 4H) 3.01 (brs, 4H) 4.02 (s, 4H) 6.56 (s, 2H) 6.94-7.05
(m, 4H) 7.31-7.33 (m, 4H) 11.12 (brs, 2H) 12.69 (s, 2H).
##STR00065##
[0277] 397: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.75 (brs, 4H) 2.90 (brs, 2H) 3.02 (brs, 2H) 3.67-3.82 (m, 10H)
6.85-7.03 (m, 4H) 7.26-7.36 (m, 5H) 7.55-7.58 (d, 1H) 8.18-8.21 (d,
1H) 11.26 (s, 1H) 12.65 (brs, 1H).
##STR00066##
[0278] 398: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
ppm 1.75 (brs, 4H) 2.90 (brs, 2H) 3.02 (brs, 2H) 3.72-3.78 (m, 10H)
6.42-6.51 (m, 4H) 7.36 (m, 5H) 7.54-7.58 (d, 1H) 8.18-8.21 (d, 1H)
11.26 (s, 1H) 12.65 (brs, 1H).
##STR00067##
[0279] 399: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.48 (s, 9H) 1.75 (brs, 4H) 2.90 (brs, 2H) 3.02 (brs, 2H) 3.74-3.78
(m, 4H) 6.92-6.94 (m, 1H) 7.20-7.36 (m, 7H) 7.51-7.58 (m, 2H)
8.18-8.21 (d, 1H) 9.34 (s, 1H) 11.26 (s, 1H) 12.65 (brs, 1H).
##STR00068##
[0280] 400: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.48 (s, 9H) 1.75 (brs, 4H) 2.90 (brs, 2H) 3.02 (brs, 2H) 3.71-3.78
(m, 4H) 7.18-7.42 (m, 9H) 7.54-7.58 (m, 2H) 8.18-8.21 (d, 1H) 9.34
(s, 1H) 11.26 (s, 1H) 12.65 (brs, 1H).
##STR00069##
[0281] 324: .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.39 (s, 18H) 1.76 (brs, 4H) 3.01 (brs, 4H) 3.79 (s, 4H) 4.11-4.13
(brs, 4H) 7.13-7.38 (m, 8H) 12.65 (s, 2H).
Method C: Via Aluminum Amide Coupling with Esters/Lactones
##STR00070##
[0282] To a suspension of 1002 (288 mg, 1.00 mmol) in toluene (9
mL) was added 3-isochromanone (311 mg, 2.10 mmol) followed by
trimethyl aluminum (2M in toluene, 1.0 mL, 2.00 mmol). The
resulting mixture was stirred at 75.degree. C. for 15 h, cooled to
room temperature and diluted with ethyl acetate (50 mL). The
organic layer was washed with water (3.times.20 mL), 10% sodium
chloride solution (10 mL), dried (magnesium sulfate) and
concentrated under reduced pressure. The crude product was purified
by HPLC to afford
N,N'-(5,5'-(thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(-
2-(2-(hydroxymethyl)phenyl)acetamide) (181, 78 mg). .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. 7.42 (d, J=6.84 Hz, 2H), 7.26 (bs,
6H), 4.57 (s, 4H), 3.90 (s, 4H), 3.27 (t, J=6.62 Hz, 4H), 2.94 (t,
J=6.44 Hz, 4H)
##STR00071##
[0283] To a suspension of 1001 (256 mg, 1.00 mmol) in toluene (8
mL) was added 3-isochromanone (311 mg, 2.10 mmol) followed by
trimethyl aluminum (2M in toluene, 1.0 mL, 2.00 mmol). The
resulting mixture was stirred at 75.degree. C. 15 h, cooled to room
temperature and diluted with ethyl acetate (50 mL). The organic
layer was washed with water (3.times.20 mL), 10% sodium chloride
solution (10 mL), dried (magnsesium sulfate) and concentrated under
reduced pressure. The crude product was purified by HPLC to afford
N,N'-(5,5'-(thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(-
2-(2-(hydroxymethyl)phenyl)acetamide) (208, 62 mg). .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. 7.41 (s, 2H), 7.26 (s, 6H), 4.56
(s, 4H), 3.01 (bs, 4H), 1.76 (bs, 4H)
##STR00072##
[0284] To a solution of 1015 (3.2 g, 19.5 mmol) in carbon
tetrachloride (150 mL) was added N-bromosuccinimide (3.47 g, 19.6
mmol) and benzoyl peroxide (10 mg, catalytic). The resulting
mixture was refluxed overnight before it was filtered hot. The
filtrate was concentrated under reduced pressure and the residue
obtained was purified by silica gel chromatography eluting with 20%
ethylacetate/hexane to afford 1016 (2 g, 42% yield) as an oil.
.sup.1H NMR (300 MHz, Chloroform-d) .delta. ppm 3.66 (s, 2H) 3.74
(s, 3H) 4.51 (s, 2H) 7.35 (m, 4H).
[0285] To a solution of 1016 (0.243 g, 1 mmol) in acetone (10 mL)
was added 2-methyl imidazole (0.41 g, 5 mmol). The resulting
mixture was refluxed overnight before it was concentrated under
reduced pressure and the residue obtained was diluted with water
(.about.100 mL). The resulting solution was partitioned between
water and ethyl acetate. The organic extract was washed with more
water, separated, dried over sodium sulfate, filtered and
evaporated. The residue obtained was purified by silica gel
chromatography eluting with MeOH/dichloromethane to afford 1017
(0.17 g, 69% yield) as an oil. .sup.1H NMR (300 MHz, Chloroform-d)
.delta. ppm 2.37 (s, 3H) 3.63 (s, 2H) 3.72 (s, 3H) 5.07 (s, 2H)
6.87 (s, 1H) 6.96-7.02 9 m, 2H) 7.23-7.33 (m, 3H)
[0286] To a solution of 1017 (0.17 g, 0.69 mmol) in THF/MeOH/Water
(10 mL, 2 mL, 2 mL) was added lithium hydroxide monohydrate (0.06
g, 1.42 mmol). The resulting mixture was stirred at room
temperature overnight before it was concentrated under reduced
pressure. The residue obtained was diluted with water (.about.20
mL) and the resulting solution was acidified with acetic acid. The
aqueous layer was concentrated and the product was isolated by prep
HPLC. The residue obtained was dissolved in water (5 mL) and
concentrated hydrochloric acid (83 .mu.L) was added to it before it
was concentrated and dried to afford 1018 (0.15 gm) as a
hydrochloride salt.
[0287] To a suspension of carboxylic acid 1018 (105 mg, 0.39 mmol)
in DMF (3 mL) was added HATU (150 mg, 0.39 mmol) and stirred till
reaction mixture is clear followed by the addition of an amine 1001
(50.5 mg, 0.197 mmol) and DIPEA (0.14 mL, 0.8 mmol). The resulting
mixture was stirred at room temperature overnight before it was
quenched by the addition of water. The solid separated was
filtered, washed with water and dried to afford 296 (112 mg, 83%).
.sup.1H NMR (300 MHz, Dimethylsulfoxide-d.sub.6) .delta. ppm 1.76
(brs, 4H) 2.38 (s, 6H) 3.01 (brs, 4H) 3.82 (s, 4H) 5.25 (s, 4H)
7.09-7.38 (m, 12H) 12.64-12.67 (brs, 2H).
##STR00073##
[0288] To a suspension of 1019 (1.5 g, 6.8 mmol) in
CH.sub.2Cl.sub.2 (15 mL) at 0.degree. C. was added Et.sub.3N (1.9
ml, 13.6 mmol) dropwise followed by phenyl acetyl chloride (1.07
ml, 8.1 mmol) dropwise. The resulting mixture was stirred at
0.degree. C. and then slowly warmed up to room temperature for 2
days. The crude material was purified by silica gel chromatography
eluting with 0-25% EtOAc in hexane to afford 1020.
[0289] To a solution of 4-bromo-1-butyne (7 g, 53 mmol) in DMSO (30
ml) at 0.degree. C. was added NaI (7.94 g, 53 mmol). The mixture
was stirred at room temperature for 2 h before it was cooled to
0.degree. C. and followed by addition of NaCN (5.2 g, 106 mmol).
The resulting mixture was heated at 80.degree. C. for 2.5 h and
then stirred at room temperature ovemight. The mixture was
partitioned between water and EtOAc. The organic extract was washed
with water, dried over sodium sulfate, filtered and evaporated to
afford 1021.
[0290] To a mixture of 1020 (400 mg, 1.18 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (41 mg, 0.059 mmol) and CuI (11 mg,
0.059 mmol) in Et.sub.3N (3 mm) and THF (6 m0) under argon
atmosphere was added 1021 (187 mg, 2.36 mmol), then heated at
60.degree. C. overnight. After removal of the solvent, the residue
was purified by silica gel chromatography eluting with 0-60% EtOAc
in Hexane to afford 1022.
[0291] To a solution of 1022 (118 mg, 0.406 mmol) in the mixture of
EtOAc (60 ml) and EtOH (15 ml) was added Pd(OH).sub.2/C (50 mg,
0.356 mmol). Hydrogen was bubbled through the resulting mixture and
stirred for 1 h. The Pd catalyst was filterd off and the filtrate
was concentrated to afford 1023.
[0292] A mixture of 1023 (127 mg, 0.431 mmol) and thiosemicarbazide
(51 mg, 0.561 mmol) in TFA (3 mL) was heated at 85.degree. C. for 5
h. The reaction was cooled to room temperature and poured onto a
mixture of ice-water. The mixture was basified with NaOH pellets
(pH 10). The crude material was purified by silica gel
chromatography eluting with 0-6% MeOH in CH.sub.2Cl.sub.2 to afford
1024.
[0293] To a solution of 1024 (38.4 mg, 0.104 mmol) in NMP (1 mL) at
0.degree. C. was added phenyl acetyl chloride (0.017 mL, 0.125
mmol) dropwise. The resulting mixture was stirred at 0.degree. C.
for 1.5 h before it was quenched by addition of water (.about.10
mL). The mixture was partitioned between water and EtOAc. The
organic extract was washed with water, dried over sodium sulfate,
filtered and evaporated. The crude material was purified by silica
gel chromatography eluting with 0-6% MeOH in CH.sub.2Cl.sub.2 to
afford 295. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.65 (s,
1H), 11.26 (s, 1H), 8.22-8.19 (d, J=8.82 Hz, 1H), 7.58-7.54 (d,
J=9.72 Hz, 1H), 7.36-7.28 (m, 10H), 3.81-3.78 (d, J=8.43 Hz, 4H),
3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
[0294] Compound 1024 can also be prepared according to the
following procedure:
##STR00074##
[0295] To a solution of 3-amino-6-chloropyridazine (11.14 g, 86.0
mmol) in NMP (279 mL) at 19.degree. C. was added phenylacetyl
chloride (18.2 mL, 137.6 mmol) dropwise over 5 minutes with the
internal temperature of the solution maintained
T.sub.i.ltoreq.28.degree. C. The resulting mixture was stirred at
19.degree. C. for 90 minutes and poured into ice water (557 mL).
The white precipitate was collected by suction filtration, rinsed
with water (2.times.110 mL) and diethyl ether (110 mL). The product
was dried overnight under high vacuum to afford
N-(6-chloropyridazin-3-yl)-2-phenylacetamide (xxx, 18.8 g). .sup.1H
NMR (300 MHz, DMSO-d.sub.6) .delta. 11.57 (s, 1H), 8.40 (d, J=9.636
Hz, 1H), 7.90 (d, J=9.516 Hz, 1H), 7.36 (m, 5H) 3.82 (s, 2H)
[0296] A 1000 mL three-neck flask fitted with internal temperature
probe and addition funnel was flushed with Ar.sub.(g). Under
positive Argon pressure 4-cyanobutylzinc bromide (0.5M in THF, 500
mL, 250 mmol) was charged into the addition funnel then added to
the reaction vessel at room temperature. Solid
N-(6-chloropyridazin-3-yl)-2-phenylacetamide (20.6 g, 83.3 mmol)
was added to the stirred solution at RT under Ar.sub.(g) flow,
followed by the addition of NiCl.sub.2(dppp) (4.52 g, 8.33 mmol).
The resulting mixture was stirred at 19.degree. C. for 240 minutes
and then quenched with ethanol (120 mL). Water (380 mL) added to
the stirred red solution, giving a thick precipitate. Ethyl acetate
(760 mL) added and stirred well for 30 minutes. The solids were
removed by filtration through a pad of celite. The mother liquor
was then transferred to a separatory funnel and the organic layer
was washed with H.sub.2O (380 mL), 0.5% ethylenediaminetetraacetic
acid solution (380 mL) and again with H.sub.2O (380 mL). The
organic layer was concentrated by rotoevaporation. Resulting red
oil was redissolved in EtOAc (200 mL) and 1M HCl (380 mL) was added
to the well stirred flask. After 30 minutes the mixture was
transferred to separatory funnel and the aqueous layer collected.
The organic layer was extracted with 1M HCl (2.times.380 mL). The
aqueous layer's pH was then adjusted to .about.7 using 7.5% sodium
bicarbonate solution and the pale yellow precipitate was collected
by suction filtration, rinsed with water (200 mL) and diethyl ether
(2.times.200 mL). The solid was dried overnight under high vacuum
to afford N-(6-(4-cyanobutyl)pyridazin-3-yl)-2-phenylacetamide
(1023, 14.76 g). .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.29
(s, 1H), 8.23 (d, J=9.036 Hz, 1H), 7.59 (d, J=9.246 Hz, 1H), 7.32
(m, 5H), 3.79 (s, 2H), 2.90 (t, J=7.357 Hz, 2H), 2.56 (t, J=7.038
Hz, 2H), 1.79 (t, J=7.311 Hz, 2H), 1.63 (t, J=7.01 Hz, 2H)
[0297] N-(6-(4-cyanobutyl)pyridazin-3-yl)-2-phenylacetamide (14.7
g, 50.2 mmol) was charged into a 250 mL round bottom flask fitted
with an open top reflux condenser. To the flask was added
thiosemicarbazide (5.03 g, 55.2 mmol) and trifluoroacetic acid (88
mL). The reaction slurry was heated in a 65.degree. C. bath for 2
h. After cooling to RT, H.sub.2O (150 mL) was added and stirred for
30 minutes. The mixture was then slowly transferred to a stirred
7.5% sodium bicarbonate solution (1400 mL) cooled in a 0.degree. C.
bath. The precipitate was collected by suction filtration, rinsed
with water (2.times.200 mL), diethyl ether (2.times.200 mL) and
dried under high vacuum overnight. The off-white solid was slurried
in DMSO (200 mL) and heated in an 80.degree. C. bath until the
internal temperature reached 65.degree. C. DMSO (105 mL) was used
to rinse sides of flask. H.sub.2O (120 mL) was slowly added until
the solution became slightly cloudy and then the mixture was
removed from heat bath and allowed to cool to ambient temperature
while stirring. The pale green precipitate was collected by suction
filtration, rinsed with water (200 mL) and diethyl ether
(2.times.200 mL). The solid was dried overnight under high vacuum
to provide
N-(6-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)pyridazin-3-yl)-2-phenylacet-
amide (1024, 15.01 g). .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.
11.28 (s, 1H), 8.23 (d, J=8.916 Hz, 1H), 7.59 (d, J=8.826 Hz, 1H),
7.36 (m, 5H), 7.07 (s, 2H), 3.78 (s, 2H), 2.87 (t, J=6.799 Hz, 4H),
1.69 (bm, 4H)
##STR00075##
[0298] To a solution of dimethyl adipate (28.7 mmol, 5.0 g, 4.7 mL,
1.0 equiv.) in 20 mL of MeOH was added anhydrous hydrazine (229.6
mmol, 7.36 g, 7.51 mL, 8.0 equiv.) and the mixture heated to
50.degree. C., giving a white precipitate. The mixture was heated
for one hour and then allowed to cool to room temperature. The
white solid was collected by filtration and washed with additional
MeOH then dried under high vacuum giving 4.6 g of adipohydrizide.
.sup.1HNMR (300 MHz, DMSO-d.sub.6) .delta. 8.91 (s, 2H), 4.14 (s,
4H), 2.00 (br s, 4H), 1.46 (br s, 4H).
[0299] To a 0.degree. C. cooled slurry of adipohydrizide (12.49
mmol, 4.0 g, 1.0 equiv.), potassium bicarbonate (15.61 mmol, 1.56
g, 1.25 equiv.) in 25 mL of MeOH was added solid cyanogen bromide
(13.74 mmol, 1.44 g, 1.1 equiv.) in one portion. This mixture was
stirred at 0.degree. C. and allowed to warm to RT over one hour and
then stirred overnight. The volatiles were removed under reduced
pressure and the solids diluted with water. The pH was adjusted to
12 with 2.5 N NaOH and the solids collected by filtration. The
white solid was washed with water and dried under high vacuum to
give 1.73 g of oxadiazole 1025. .sup.1HNMR (300 MHz, DMSO-d.sub.6)
.delta. 6.85 (s, 4H), 2.68 (s, 4H), 1.68 (s, 4H).
##STR00076##
[0300] To a suspension of oxadiazole 1025 (181 mg, 0.81 mmol) in
NMP (9 mL) was added triethylamine (0.564 mL, 4.05 mmol) and the
mixture warmed to 70.degree. C. The mixture was allowed to stir for
30 minutes followed by the addition of phenylacetyl chloride (0.234
mL, 1.77 mmol). The reaction temperature was held at 70.degree. C.
for 15 hours then allowed to cool to room temperature. The crude
reaction mixture was purified by reverse phase HPLC giving 305
(0.015 g). .sup.1HNMR (300 MHz, DMSO-d.sub.6) .delta. 11.74 (s,
2H), 7.33 (s, 10H), 3.74 (s, 4H), 2.85 (s, 4H), 1.76 (s, 4H).
Functionalization of Diacylated Cores:
##STR00077##
[0302] To a suspension of 21 (2.25 g, 4.57 mmol) in a mixture of
THF (250 mL) and H.sub.2O (20 mL) at room temperature was added
NaOH (1.83 g, 45.67 mmol) and formaldehyde solution (37% in water,
14.83 mL, 182.70 mmol). The resulting mixture was heated at
60.degree. C. for 7 h before it was cooled to 0.degree. C. and
acidified to pH 7 with aq. HCl solution. The white precipitate was
collected by suction filtration, rinsed with water and dried to
provide
N,N-[5,5'-(butane-1,4-diyl)-bis(1,3,4-thiadiazole-5,2-diyl)]-bis(3-hydrox-
y-2-phenylpropanamide) (36, 624 mg). The 2.sup.nd precipitation
from the filtrate provided additional product (1.29 g). .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. 12.65 (bs, 2H), 7.35-7.30 (m, 10H),
5.09 (bs, 2H), 4.10-4.02 (m, 4H), 3.61 (d, J=8.1 Hz, 2H), 3.02 (bs,
4H), 1.76 (bs, 4H).
##STR00078##
[0303] To a suspension of 199 (300 mg, 0.572 mmol) in a mixture of
THF (50 mL) and MeOH (5 ml) was added potassium carbonate (158 mg,
1.144 mmol) and formaldehyde solution (37% in water, 2 mL). The
resulting mixture was stirred at room temperature for 48 h before
it was cooled to 0.degree. C. and acidified to pH 7 with aq. HCl
solution. The white precipitate was collected by suction
filtration, rinsed with water and dried. The crude material was
purified by HPLC to afford 29. .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 7.34-7.26 (m, 10H), 4.13-4.02 (m, 2H), 3.81 (s, 2H), 3.62
(m, 2H), 3.24 (t, 4H), 2.93 (t, 4H).
##STR00079##
[0304] To a suspension of 199 (2.0 g, 3.81 mmol) in a mixture of
THF (250 mL) and MeOH (20 ml)H.sub.2O (20 mL) at room temperature
was added 1N NaOH (20 ml) and formaldehyde solution (37% in water,
15 mL). The resulting mixture was heated at 50.degree. C. overnight
before it was cooled to 0.degree. C. and acidified to pH 7 with aq.
HCl solution. The white precipitate was collected by suction
filtration, rinsed with water and dried. The crude material was
purified by HPLC to afford 24. .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 12.67 (bs, 2H), 7.36-7.30 (m, 10H), 5.10 (bs, 2H),
4.10-4.02 (m, 4H), 3.61 (d, 2H), 3.27 (t, 4H), 2.95 (t, 4H).
Prodrugs:
##STR00080##
[0306] To a flask containing
N,N'-(5,5'-(thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(-
2-phenylacetamide) (1) (9.4 mmol, 5.0 g, 1.0 equiv.) was added 100
mL DMF, K.sub.2CO.sub.3 (20.98 mmol, 2.89 g, 2.2 equiv.), and
chloromethyl butyrate (20.98 mmol, 2.86 g, 2.62 mL, 2.2 equiv.).
The mixture stirred at room temperature for 15 hours then diluted
with 200 mL water and 200 mL EtOAc. The layers were separated and
the aqueous layer extracted with EtOAc (2.times.100 mL) and the
organic layers combined, washed with water, brine and dried over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 was removed by filtration
and the volatiles removed under reduced pressure. The compounds
were purified by reverse phase chromatography (MeCN, H.sub.2O)
giving 0.235 g of compound 8 and 0.126 g of compound 7.
[0307] .sup.1HNMR (300 MHz, DMSO, d.sub.6) Compound 8: .delta. 7.31
(m, 10H), 6.18 (s, 4H), 3.82 (s, 4H), 3.17 (dd, 2H, J=6.8 Hz), 2.92
(dd, 2H, J=6.8 Hz), 2.93 (m, 4H), 2.32 (dd, 2H, J=7.2 Hz), 1.54
(dt, 2H, J=7.2, 7.4 Hz), 0.87 (t, 3H, J=7.4 Hz).
[0308] .sup.1HNMR (300 MHz, DMSO, d.sub.6) Compound 7: .delta.
12.68 (s, 1H), 7.32 (m, 10H), 6.18 (s, 2H), 3.82 (s, 4H), 3.26 (dd,
2H, J=7.0 Hz), 3.17 (dd, 2H, J=6.8 Hz), 2.93 (m, 4H), 2.32 (dd, 2H,
J=7.2 Hz), 1.54 (dt, 2H, J=7.2, 7.4 Hz), 0.87 (t, 3H, J=7.4
Hz).
##STR00081##
[0309] To a suspension of 3-morpholin-4-yl-propionic acid
hydrochloride (500 mg, 2.56 mmol) in DMF (20 mL) at 0.degree. C.
was added N-(3-dimethylaminopropyl)-N-ethylcarbodiimide
hydrochloride (534 mg, 2.79 mmol). The resulting mixture was
stirred at 0.degree. C. for 40 min and followed by addition of diol
36 (642 mg, 1.16 mmol) and 4-DMAP (454 mg, 3.72 mmol). The
resulting mixture was stirred from 0.degree. C. to room temperature
over a period of 3.5 h before it was diluted with EtOAc and cold
water. The organic layer was separated and washed with water
(3.times.50 mL), brine, dried (MgSO.sub.4) and concentrated. The
crude product was purified by silica gel chromatography eluting
with 10-25% MeOH in EtOAc to provide
{[5,5'-(butane-1,4-diyl)-bis(1,3,4-thiadiazole-5,2-diyl)]-bis(azanediyl)}-
-bis(3-oxo-2-phenylpropane-3,1-diyl)-bis(3-morpholinopropanoate)
(188, 340 mg) and a less polar product,
3-((5-{4-[5-(3-hydroxy-2-phenylpropanamido)-1,3,4-thiadiazol-2-yl]butyl}--
1,3,4-thiadiazol-2-yl)amino)-3-oxo-2-phenylpropyl
3-morpholinopropanoate (228, 103 mg). 188: .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.80 (s, 2H), 7.39 (m, 10H), 4.62 (t, J=9.6
Hz, 2H), 4.33-4.27 (m, 4H), 3.48 (bs, 8H), 3.02 (bs, 4H), 2.45 (bs,
8H), 2.25 (bs, 8H), 1.76 (bs, 4H).
[0310] 228: .sup.1H NMR (300 MHz, MeOD-d.sub.4) .delta. 7.43-7.37
(m, 10H), 4.71 (t, J=10.5 Hz, 1H), 4.41 (m, 1H), 4.30-4.24 (m, 2H),
4.06-4.03 (m, 1H), 3.80-3.76 (m, 1H), 3.62 (bs, 4H), 3.11 (bs, 4H),
2.63-2.52 (m, 4H), 2.40 (bs, 4H), 1.90 (bs, 4H).
##STR00082##
[0311] To a solution of diethyl trans-1,2-cyclopropanedicarboxylate
(5.00 g, 26.85 mmol) in THF (20 mL) at 0.degree. C. was added a
solution of LAH (67.13 mL, 1.0 M in THF, 67.13 mmol) dropwise. The
resulting mixture was stirred at 0.degree. C. for 1.5 h before it
was quenched with H.sub.2O (20 mL), 2N aq. NaOH (20 mL) and
H.sub.2O (20 mL). The mixture was stirred vigorously for 1 h at
room temperature before it was filtered through a plug of celite.
The filtrate was dried (MgSO.sub.4) and concentrated to provide the
desired diol (2.73 g) as a colorless oil.
[0312] A mixture of the diol (2.00 g, 19.58 mmol) in
CH.sub.2Cl.sub.2 (75 mL) at 0.degree. C. was added pyridine (6.34
mL, 78.33 mmol) and followed by MsCl (3.33 mL, 43.08 mmol)
dropwise. The resulting mixture was stirred 0.degree. C. for 1 h
before it was warmed up to room temperature. The reaction was
quenched with H.sub.2O and diluted with ether. The organic layer
was washed with brine, dried (MgSO.sub.4) and concentrated to
provide 1039. This crude product was dissolved in DMSO (75 mL), and
added NaCN (2.88 g, 58.75 mmol) and NaI (294 mg, 1.96 mmol). The
resulting mixture was heated at 45.degree. C. for 8 h before it was
allowed to cool to room temperature and diluted with EtOAc and
H.sub.2O. The organic layer was separated, washed with brine, dried
(MgSO.sub.4) and concentrated to provide the crude product 1040
which was used in the following step without purification.
[0313] A mixture of 1040 and thiosemicarbazide (3.75 g, 41.12 mmol)
in trifluoroacetic acid (TFA) (20 mL) was heated at 80.degree. C.
for 5 h. The reaction was cooled to room temperature and poured
into a mixture of ice and water. Sodium hydroxide pellets were
added to the mixture until it was basic (pH 14). The white
precipitate was collected by suction filtration, rinsed with water,
ether and dried to provide 1041 (472 mg).
[0314] To a suspension of 1041 (70 mg, 0.26 mmol) in
1-Methyl-2-pyrrolidinone (NMP) (5 mL) at 0.degree. C. was added
phenylacetyl chloride (72 .mu.L, 0.55 mmol) dropwise. The resulting
mixture was stirred at 0.degree. C. for 1 h before it was quenched
by addition of water (.about.3 mL). The white precipitate was
collected by suction filtration, rinsed with water and dried to
provide 1035 (37 mg). .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.
12.65 (s, 2H), 7.34-7.27 (m, 10H), 3.82 (s, 4H), 3.04-2.75 (m, 4H),
1.14-1.12 (m, 2H), 0.63-0.59 (m, 2H).
##STR00083##
[0315] To a solution of 1020 (1.50 g, 4.42 mmol),
ethynyltrimethylsilane (813 uL, 5.75 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (310 mg, 0.44 mmol) and CuI (59 mg,
0.31 mmol) in THF (20 mL) under argon atmosphere at room
temperature was added Et.sub.3N (6.16 mL, 44.23 mmol). The
resulting mixture was heated at 50.degree. C. for 5 h before it was
allowed to cool to room temperature and filtered through a plug of
celite. The filtrate was concentrated and the crude residue was
purified by flash column chromatography over silica gel eluting
with 10-50% EtOAc in hexanes to provide the desired product (1.21
g) as a solid.
[0316] A mixture of the foregoing intermediate (1.07 g, 3.48 mmol)
and K.sub.2CO.sub.3 (0.40 g, 2.90 mmol) in MeOH (100 mL) was
stirred at room temperature for 5 h before it was concentrated
under reduced pressure. The residue was re-dissolved in a mixture
of EtOAc and H.sub.2O, and was neutralized with 1N aq. HCl solution
to pH 7. The organic layer was separated, washed with brine, dried
(MgSO.sub.4) and concentrated. The crude residue was purified by
flash column chromatography over silica gel eluting with 10-50%
EtOAc in hexanes to provide the desired alkyne 1036 (0.48 g) as a
white solid.
[0317] To a solution of alkyne 1036 (52 mg, 0.22 mmol) in pyridine
(5 mL) at room temperature was added CuCl (4.3 mg, 0.04 mmol). The
resulting mixture was stirred under a stream of air for 40 min as
all of the starting material was consumed. The reaction mixture was
diluted with saturated aq. NH.sub.4Cl solution (.about.2 mL). The
off-white precipitate was collected by suction filtration, washed
with H.sub.2O and dried. This crude bis-acetylene product 1037 (52
mg) was used in the following step without further
purification.
[0318] A mixture of 1037 (52 mg) and Pd(OH).sub.2/C (100 mg) in a
mixture of DMF (5 mL) and THF (10 mL) was stirred at room
temperature under 1 atmosphere of H.sub.2 for 3 h as all of the
starting material was consumed. The palladium catalyst was filtered
off and the filtrate was concentrated. The crude residue was
purified by column chromatography over silica gel eluting with
1-10% MeOH in CH.sub.2Cl.sub.2 to provide the desired product 1038
(18 mg) as a solid. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.
11.26 (s, 2H), 8.20 (d, J=8.97 Hz, 2H), 7.56 (d, J=8.77 Hz, 2H),
7.36-7.24 (m, 10H), 3.78 (s, 4H), 2.90 (bs, 4H), 1.73 (bs, 4H).
##STR00084##
[0319] To a solution of adiponitrile (19.02 g, 175.8 mmol) in TFA
(50 mL) was added thiosemicarbazide (16.02 g, 175.8 mmol) and the
mixture heated to 70.degree. C. for 4 hours under an atmosphere of
Argon. The mixture was allowed to cool to room temperature and the
volatiles removed under reduced pressure. The residue was diluted
with water (200 mL) and the pH adjusted to 7 with solid NaOH giving
a white precipitate that was collected by filtration and washed
with water. The solids were dried under high vacuum giving 9.22 g
of 1081. .sup.1HNMR (DMSO, d.sub.6): .delta. 7.02 (br s, 2H) 2.84
(m, 2H), 2.55 (m, 2H), 1.67 (m, 4H).
[0320] To a solution of 1081 (0.625 g, 2.87 mmol) in NMP (12.5 mL)
was added phenylacetyl chloride (0.487 g, 0.42 mL, 3.15 mmol)
dropwise and the mixture stirred at room temperature for one hour
under an atmosphere of Argon. The mixture was poured into water
(100 mL) and the solids collected by filtration. The solids were
washed with water and dried under high vacuum to give 0.805 g of
1082. .sup.1HNMR (DMSO, d.sub.6): .delta. 12.65 (s, 1H) 7.31 (m,
5H), 3.80 (s, 2H), 3.00 (t, 2H, J=7.3 Hz), 2.53 (t, 2H, J=7.1 Hz),
1.78 (dq, 2H, J=7.3, 7.1 Hz), 1.61 (dq, 2H, J=7.3, 7.1 Hz).
[0321] To a solution of 1082 (0.49 g, 1.33 mmol) in TFA (10 mL) was
added thiosemicarbazide (0.23 g, 1.46 mmol) and the mixture heated
at 70.degree. C. overnight under an atmosphere of Argon. The
mixture was allowed to cool to room temperature and the volatiles
removed under reduced pressure. The residue was diluted with water
(50 mL) and the pH adjusted to 7 with solid NaOH giving a white
precipitate that was collected by filtration and washed with water.
The solids were dried under high vacuum giving 0.367 g of 1083.
.sup.1HNMR (DMSO, d.sub.6): .delta. 12.70 (s, 1H) 7.34 (br s, 5H),
7.16 (s, 2H), 3.82 (s, 2H), 3.01 (s, 2H), 2.84 (S, 2H), 1.71 (br s,
4H).
[0322] To a solution of 1083 (0.10 g, 0.267 mmol),
2,4-difluoro-3-methoxyphenylacetic acid (0.058 g, 0.267 mmol), EDC
(0.127 g, 0.667 mmol), HOBt (0.090 g, 0.667 mmol) in DMF (4 mL) was
added DIEA (0.171 g, 0.231 mL, 1.335 mmol) and the mixture stirred
overnight under an atmosphere of Argon. The mixture was poured into
water (20 mL) and the solids formed were collected by filtration,
washed with water and dried under high vacuum. The crude 1084 was
used in the following step without purification. To a solution of
1084 (0.050 g, 0.091 mmol) in dichloromethane (1 mL) was added
BBr.sub.3 (1.0 mL, 1 mmol, 1.0 M in dichloromethane) and the
mixture stirred for 4 hours at room temperature under an atmosphere
of Argon. The volatiles were removed under reduced pressure and the
residue diluted with dichloromethane (5 mL). The volatiles were
removed under reduced pressure and the residue diluted with water
(15 mL) and the pH adjusted to 12. The aqueous layer was washed
with dichloromethane (4.times.5 mL) and the pH adjusted to 4. The
solids were collected by filtration, washed with water and dried
under high vacuum giving 0.029 g of 346. .sup.1HNMR (DMSO,
d.sub.6): .delta. 12.66 (s, 2H), 10.12 (s, 1H), 7.33 (s, 5H), 7.00
(m, 1H), 6.80 (m, 1H), 3.84 (s, 2H), 3.81 (s, 2H), 3.02 (br s, 4H),
1.76 (br s, 4H).
##STR00085##
[0323] To a solution of 1083 (0.05 g, 0.133 mmol),
Boc-3-aminomethyl-phenylacetic acid (0.035 g, 0.133 mmol), EDC
(0.064 g, 0.332 mmol), HOBt (0.045 g, 0.332 mmol) in DMF (8 mL) was
added DIEA (0.086 g, 0.115 mL, 0.665 mmol) and the mixture stirred
overnight under an atmosphere of Argon. The mixture was poured into
water (20 mL) and the solids formed were collected by filtration,
washed with water and dried under high vacuum to give 0.023 g of
375. .sup.1HNMR (DMSO, d.sub.6): .delta. 12.66 (s, 2H), 7.27 (m,
10H), 4.11 (br s, 2H), 3.81 (s, 2H), 3.79 (s, 2H), 3.01 (br s, 4H),
1.76 (br s, 4H), 1.39 (s, 9H).
##STR00086##
[0324] A flask was charged with 1024 (100 mg, 0.27 mmol), tropic
acid (54 mg, 0.326 mmol) in DMF (2 ml) at 0.degree. C. was added
HOBT (88 mg, 0.652 mmol) followed by EDCI (156 mg, 0.815 mmol). The
resulting mixture was slowly warmed up to room temperature and
stirred for 3 h before it was quenched by addition of water
(.about.10 mL). The white precipitate was collected by suction
filtration, rinsed with more water and dried to afford 314. .sup.1H
NMR (300 MHz, DMSO-d.sub.6) .delta. 12.65 (s, 1H), 11.26 (s, 1H),
8.22-8.19 (d, J=8.82 Hz, 1H), 7.58-7.54 (d, J=9.72 Hz, 1H),
7.36-7.28 (m, 10H), 4.10-4.05 (m, 2H), 3.78 (s, 3H), 3.65 (s, 1H),
3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00087##
[0325] A flask was charged with 1024 (500 mg, 1.36 mmol),
DL-mandelic acid (248 mg, 1.63 mmol) in DMF (10 ml) at 0.degree. C.
was added HOBT (441 mg, 3.26 mmol) followed by EDCI (781 mg, 4.08
mmol). The resulting mixture was stirred at 0.degree. C. for 10
minutes then warmed up to room temperature and stirred for 10
minutes before it was quenched by addition of water (.about.50 mL)
at 0.degree. C. The white precipitate was collected by suction
filtration, rinsed with more water and dried to afford 315. .sup.1H
NMR (300 MHz, DMSO-d.sub.6) .delta. 12.65 (s, 1H), 11.26 (s, 1H),
8.22-8.19 (d, J=8.82 Hz, 1H), 7.58-7.50 (m, 3H), 7.36-7.28 (m, 8H),
6.35 (s, 1H), 5.32 (s, 1H), 3.78 (s, 2H), 3.01 (bs, 2H), 2.90 (bs,
2H), 1.73 (bs, 4H).
[0326] To a suspension of 3-morpholin-4-yl-propionic acid
hydrochloride (209 mg, 1.07 mmol) in DMF (10 ml) was added EDCI
(308 mg, 1.61 mmol). The resulting mixture was stirred at 0.degree.
C. for 1 hour and followed by addition of 315 (447 mg, 0.889 mmol)
and 4-DMAP (261 mg, 2.14 mmol). The resulting mixture was stirred
from 0.degree. C. to room temperature over a period of 6 h before
it was quenched by addition of ice water (.about.50 mL). The white
precipitate was collected by suction filtration, rinsed with more
water. The crude material was purified by silica gel chromatography
eluting with 0-6% MeOH in EtOAc to afford 334. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.95 (s, 1H), 11.26 (s, 1H), 8.22-8.19
(d, J=9.45 Hz, 1H), 7.58-7.26 (m, 11H), 6.14 (s, 1H), 3.78 (s, 2H),
3.54 (bs, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 2.63 (bs, 4H), 2.38
(bs, 4H), 1.73 (bs, 4H).
##STR00088##
[0327] Compound 317 was prepared according to the procedure above
for compound 315. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.40
(s, 1H), 11.26 (s, 1H), 8.22-8.19 (d, J=9.03 Hz, 1H), 7.58-7.54 (d,
J=9.72 Hz, 1H), 7.36-6.87 (m, 9H), 6.35 (bs, 1H), 5.30 (s, 1H),
3.78 (m, 5H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00089##
[0328] Compound 318 was prepared according to the procedure above
for compound 315. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.50
(s, 1H), 11.26 (s, 1H), 8.22-8.19 (d, J=9.43 Hz, 1H), 7.60-7.27 (m,
10H), 6.51 (bs, 1H), 5.35 (s, 1H), 3.78 (s, 2H), 3.01 (bs, 2H),
2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00090##
[0329] A flask was charged with 1024 (50 mg, 0.135 mmol),
3-chlorophenylacetic acid (28 mg, 0.163 mmol) in DMF (1 ml) at
0.degree. C. was added HOBT (44 mg, 0.326 mmol) followed by EDCI
(78 mg, 0.408 mmol). The resulting mixture was slowly warmed up to
room temperature and stirred for 1 h before it was quenched by
addition of water (.about.5 mL). The white precipitate was
collected by suction filtration, rinsed with more water and ether
then dried to afford 335. .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 12.65 (s, 1H), 11.26 (s, 1H), 8.22-8.19 (d, J=8.82 Hz, 1H),
7.58-7.54 (d, J=9.72 Hz, 1H), 7.36-7.28 (m, 9H), 3.84 (s, 2H), 3.78
(s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00091##
[0330] Compound 337 was prepared according to the procedure above
for compound 335. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.65
(s, 1H), 11.26 (s, 1H), 9.38 (s, 1H), 8.22-8.19 (d, J=8.37 Hz, 1H),
7.58-7.54 (d, J=9.63 Hz, 1H), 7.36-7.09 (m, 6H), 6.75-6.65 (m, 3H),
3.78 (s, 2H), 3.70 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs,
4H).
##STR00092##
[0331] 339, 341, 382: A flask was charged with 1024 (100 mg, 0.27
mmol), Boc-3-aminomethyl-phenylacetic acid (86 mg, 0.325 mmol) in
DMF (2 ml) at 0.degree. C. was added HOBT (88 mg, 0.65 mmol)
followed by EDCI (156 mg, 0.812 mmol). The resulting mixture was
stirred at 0.degree. C. for 5 minutes then warmed up to room
temperature and stirred for 1.5 h before it was quenched by
addition of water (.about.10 mL) at 0.degree. C. The white
precipitate was collected by suction filtration, rinsed with more
water and ether then dried to afford 339. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.65 (s, 1H), 11.26 (s, 1H), 8.22-8.19 (d,
J=8.82 Hz, 1H), 7.58-7.54 (d, J=9.42 Hz, 1H), 7.36-7.13 (m, 9H),
4.13-4.11 (d, J=10.62, 2H), 3.78 (s, 4H), 3.01 (bs, 2H), 2.90 (bs,
2H), 1.73 (bs, 4H), 1.38 (s, 9H).
[0332] To a suspension of 339 (50 mg, 0.081 mmol) in
dichloromethane (2 ml) was added TFA (2 ml) at 0.degree. C. The
resulting mixture was stirred at room temperature for 20 minutes
before it was evaporated under vacuo to dryness. Ether was added
and the white precipitate was collected by suction filtration,
rinsed with more ether and dichloromethane then dried to afford
341. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.65 (s, 1H),
11.26 (s, 1H), 8.22-8.19 (d, J=8.82 Hz, 1H), 8.14-8.11 (bs, 2H),
7.58-7.54 (d, J=9.42 Hz, 1H), 7.36-7.13 (m, 9H), 4.06-4.03 (m, 2H),
3.84 (s, 2H), 3.78 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs,
4H).
[0333] To a solution of 341 (10 mg, 0.0159 mmol) in DMF (1 ml) at
0.degree. C. was added triethylamine (4.4 ul, 0.0317 mmol) drop
wise followed by ethyl chloroformate (1.8 ul, 0.0191 mmol) drop
wise. The resulting mixture was slowly warmed up to room
temperature and stirred for 30 minutes before it was quenched by
addition of water (.about.1 mL) at 0.degree. C. The mixture was
partitioned between water and EtOAc. The organic extract was washed
with water, dried over sodium sulfate, filtered and evaporated. The
crude material was purified by silica gel chromatography eluting
with 0-6% MeOH in CH.sub.2Cl.sub.2 to afford 382. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.65 (s, 1H), 11.26 (s, 1H), 8.22-8.19
(d, J=8.82 Hz, 1H), 7.67-7.58 (bs, 1H), 7.58-7.54 (d, J=9.42 Hz,
1H), 7.36-7.13 (m, 9H), 4.18-4.16 (m, 2H), 4.06-4.0 (q, 2H), 3.78
(s, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H), 1.19-1.13 (t,
3H).
##STR00093##
[0334] Compound 431 was prepared according to the procedure above
for compound 382 with the appropriate reagents. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.65 (s, 1H), 11.26 (s, 1H), 8.35 (s,
1H), 8.22-8.19 (d, J=8.88 Hz, 1H), 7.57-7.54 (d, J=9.51 Hz, 1H),
7.38-7.15 (m, 9H), 4.25-4.24 (d, J=5.64 Hz, 2H), 3.76 (s, 4H), 3.01
(bs, 2H), 2.90 (bs, 2H), 1.87 (s, 3H), 1.73 (bs, 4H).
##STR00094##
[0335] Compound 432 was prepared according to the procedure above
for compound 382 with the appropriate reagents. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.63 (s, 1H), 11.26 (s, 1H), 9.04-9.01
(m, 1H), 8.22-8.19 (d, J=8.91 Hz, 1H), 7.93-7.89 (d, J=9.51 Hz,
2H), 7.58-7.25 (m, 13H), 4.50-4.48 (d, J=5.91 Hz, 2H), 3.78 (s,
4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00095##
[0336] Compound 433 was prepared according to the procedure above
for compound 382 with the appropriate reagents. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.63 (s, 1H), 11.26 (s, 1H), 8.31-8.21
(m, 1H), 8.20-8.19 (d, J=9.57 Hz, 1H), 7.57-7.54 (d, J=8.73 Hz,
1H), 7.35-7.13 (m, 9H), 4.26-4.24 (d, J=5.52 Hz, 2H), 3.78 (s, 4H),
3.01 (bs, 2H), 2.90 (bs, 2H), 2.0 (s, 3H), 1.73 (bs, 4H), 0.86-0.85
(d, J=3.99 Hz, 6H).
##STR00096##
[0337] To a solution of 341 (70 mg, 0.11 mmol) in DMF (1 ml) at
0.degree. C. was added triethylamine (31 ul, 0.22 mmol) drop wise
followed by 5-bromovaleryl chloride (12 ul, 0.122 mmol) drop wise.
The resulting mixture was slowly warmed up to room temperature and
stirred for 1 h. Potassium tert-butoxide (50 mg, 0.445 mmol) was
then added to the reaction mixture at 0.degree. C. The resulting
mixture was slowly warmed up to room temperature and stirred for
overnight before it was quenched by addition of water (.about.2 mL)
at 0.degree. C. The mixture was partitioned between water and
EtOAc. The organic extract was washed with water, dried over sodium
sulfate, filtered and evaporated. The crude material was purified
by silica gel chromatography eluting with 0-6% MeOH in
CH.sub.2Cl.sub.2 to afford 476. .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 12.65 (s, 1H), 11.26 (s, 1H), 8.22-8.19 (d, J=8.82 Hz, 1H),
7.58-7.54 (d, J=9.42 Hz, 1H), 7.36-7.13 (m, 9H), 4.50 (s, 2H), 3.78
(s, 4H), 3.35 (bs, 2H), 3.20 (bs, 2H), 3.01 (bs, 2H), 2.90 (bs,
2H), 2.30 (bs, 2H), 1.68-1.80 (d, 6H).
##STR00097##
[0338] Compound 340 was prepared according to the procedure above
for compound 315 with the appropriate reagents. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.50 (s, 1H), 11.26 (s, 1H), 8.22-8.19
(d, J=9.24 Hz, 1H), 7.60-7.27 (m, 10H), 6.51 (bs, 1H), 5.35 (s,
1H), 3.78 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00098##
[0339] Compound 349 was prepared according to the procedure above
for compound 315 with the appropriate reagents. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.41 (s, 1H), 11.26 (s, 1H), 8.22-8.19
(d, J=8.76 Hz, 1H), 7.58-7.27 (m, 11H), 6.36 (s, 1H), 5.34 (s, 1H),
3.78 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00099##
[0340] Compound 350 was prepared according to the procedure above
for compound 315 with the appropriate reagents. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.41 (s, 1H), 11.26 (s, 1H), 8.22-8.19
(d, J=8.67 Hz, 1H), 7.58-7.27 (m, 11H), 6.34 (s, 1H), 5.34 (s, 1H),
3.78 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00100##
[0341] Compound 351 was prepared according to the procedure above
for compound 315 with the appropriate reagents. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.50 (s, 1H), 11.26 (s, 1H), 8.21-8.18
(d, J=8.67 Hz, 1H), 7.58-7.54 (d, J=9.72 Hz, 1H), 7.36-7.23 (m,
8H), 6.67 (s, 1H), 5.40 (s, 1H), 3.78 (s, 2H), 3.01 (bs, 2H), 2.90
(bs, 2H), 1.73 (bs, 4H).
##STR00101##
[0342] To a solution of 1024 (50 mg, 0.136 mmol) in DMF (1 ml) at
0.degree. C. was added triethylamine (38 ul, 0.271 mmol) drop wise
followed by benzyl isocyanate (20 ul, 0.163 mmol) drop wise. The
resulting mixture was slowly warmed up to room temperature and
stirred for 40 minutes before it was quenched by addition of water
(.about.5 mL) at 0.degree. C. The white precipitate was collected
by suction filtration, rinsed with more water. The crude material
was purified by silica gel chromatography eluting with 0-6% MeOH in
CH.sub.2Cl.sub.2 to afford 352. .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 11.26 (s, 1H), 10.82 (s, 1H), 8.22-8.19 (d, J=9.42 Hz, 1H),
7.58-7.54 (d, J=8.79 Hz, 1H), 7.36-7.31 (m, 10H), 7.06 (bs, 1H),
4.37-4.35 (d, J=5.22 Hz, 2H), 3.78 (s, 2H), 2.99-2.90 (m, 4H), 1.73
(bs, 4H).
##STR00102##
[0343] Compound 353 was prepared according to the procedure above
for the preparation of compound 335. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.57 (s, 1H), 11.26 (s, 1H), 8.22-8.19 (d,
J=9.45 Hz, 1H), 7.57-7.54 (d, J=9.48 Hz, 1H), 7.36-7.25 (m, 6H),
6.91-6.84 (m, 3H), 3.76 (m, 7H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73
(bs, 4H).
##STR00103##
[0344] A flask was charged with 1024 (50 mg, 0.135 mmol),
2-pyridine acetic acid hydrochloride (27 mg, 0.156 mmol) in DMF (1
ml) at 0.degree. C. was added propylphosphonic anhydride solution
(91 ul) followed by triethylamine (54 ul, 0.39 mmol). The resulting
mixture was slowly warmed up to room temperature and stirred for 1
h before it was quenched by addition of water (.about.5 mL). The
white precipitate was collected by suction filtration, rinsed with
more water and ether then dried to afford 354. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.65 (s, 1H), 11.26 (s, 1H), 8.51 (s,
1H), 8.22-8.19 (d, J=8.97 Hz, 1H), 7.81-7.76 (m, 1H), 7.58-7.54 (d,
J=9.06 Hz, 1H), 7.42-7.26 (m, 7H), 4.02 (s, 2H), 3.78 (s, 2H), 3.01
(bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00104##
[0345] Compound 355 was prepared according to the procedure above
for the preparation of compound 354. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.70 (s, 1H), 11.26 (s, 1H), 8.53-8.49 (m,
1H), 8.22-8.19 (d, J=9.0 Hz, 1H), 7.77-7.73 (d, J=8.46 Hz, 1H),
7.58-7.54 (d, J=9.48 Hz, 1H), 7.38-7.26 (m, 7H), 3.88 (s, 2H), 3.78
(s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
[0346] Compounds 309 and 310 were prepared according to the
procedure above for the preparation of compound 354.
##STR00105##
[0347] To a solution of 1043 (3.2 g, 19.5 mmol) in carbon
tetrachloride (150 mL) was added N-bromosuccinimide (3.47 g, 19.6
mmol) and benzoyl peroxide (10 mg, catalytic). The resulting
mixture was refluxed overnight before it was filtered hot. The
filtrate was concentrated under reduced pressure and the residue
obtained was purified by silica gel chromatography eluting with 20%
ethylacetate/hexane to afford 1044 (2 g, 42% yield) as an oil.
.sup.1H NMR (300 MHz, Chloroform-d) .delta. ppm 3.66 (s, 2H) 3.74
(s, 3H) 4.51 (s, 2H) 7.35 (m, 4H)
[0348] To a solution of 1044 (0.243 g, 1 mmol) in acetone (10 mL)
was added 2-methyl imidazole (0.41 g, 5 mmol). The resulting
mixture was refluxed overnight before it was concentrated under
reduced pressure and the residue obtained was diluted with water
(.about.100 mL). The resulting solution was partitioned between
water and ethyl acetate. The organic extract was washed with more
water, separated, dried over sodium sulfate, filtered and
evaporated. The residue obtained was purified by silica gel
chromatography eluting with MeOH/dichloromethane to afford 1045
(0.17 g, 69% yield) as an oil. .sup.1H NMR (300 MHz, Chloroform-d)
.delta. ppm 2.37 (s, 3H) 3.63 (s, 2H) 3.72 (s, 3H) 5.07 (s, 2H)
6.87 (s, 1H) 6.96-7.02 9 m, 2H) 7.23-7.33 (m, 3H)
[0349] To a solution of 1045 (0.17 g, 0.69 mmol) in THF/MeOH/Water
(10 mL, 2 mL, 2 mL) was added lithium hydroxide monohydrate (0.06
g, 1.42 mmol). The resulting mixture was stirred at room
temperature overnight before it was concentrated under reduced
pressure. The residue obtained was diluted with water (.about.20
mL) and the resulting solution was acidified with acetic acid. The
aqueous layer was concentrated and the product was isolated by prep
HPLC. The residue obtained was dissolved in water (mL) and
concentrated hydrochloric acid (mL) was added to it before it was
concentrated and dried to afford 1046 (0.15 gm) as a hydrochloride
salt.
[0350] To a suspension of carboxylic acid 1046 (41.8 mg, 0.157
mmol) in DMF (3 mL) was added HATU (61.3 mg, 0.161 mmol) and
stirred till reaction mixture is clear followed by the addition of
an amine 1024 (52.5 mg, 0.142 mmol) and DIPEA (50 ul, 0.29 mmol).
The resulting mixture was stirred at room temperature overnight
before it was quenched by the addition of water. The resulting
solution was partitioned between water and ethyl acetate. The
organic extract was washed with more water, separated, dried over
sodium sulfate, filtered and evaporated. The residue obtained was
triturated with ether. The solid separated was filtered, washed
with ether and dried to afford 380 (40 mg, 48%). .sup.1H NMR (300
MHz, Dimethylsulfoxide-d.sub.6) .delta. ppm 1.74 (brs, 4H)
2.91-3.02 (brs, 4H) 3.78-3.83 (m, 4H) 5.34 (s, 2H) 7.16-7.57 (m,
12H) 8.19-8.22 (d, 1H) 11.26 (s, 1H) 12.65 (brs, 1H)
##STR00106##
[0351] To an ice cold solution of 1048 (5 g, 0.033 mol) in methanol
(50 mL) was added thionyl chloride (0.2 mL) and the resulting
mixture was stirred at room temperature overnight before it was
concentrated under reduced pressure. The residue obtained was dried
at high vacuum overnight to afford 1049 (5 gm) as an oil and was
used as such for the next step. .sup.1H NMR (300 MHz, Chloroform-d)
.delta. ppm 3.62 (s, 2H) 3.74 (s, 3H) 6.76-6.87 (m, 3H) 7.18-7.21
(m, 1H).
[0352] To a solution of 1049 (1 g, 6 mmol) in DMF (20 mL) was added
potassium carbonate (2.08 g, 15 mmol), 1050 (1.225 g, 6.62 mmol)
and sodium iodide (10 mg). The resulting mixture was stirred at
80.degree. C. overnight before it was diluted with water
(.about.100 mL). The resulting solution was partitioned between
water and ethyl acetate. The organic extract was washed with more
water, separated, dried over sodium sulfate, filtered and
evaporated. The residue obtained was purified by silica gel
chromatography eluting with MeOH/dichloromethane to afford 1051 (1
g, 60% yield) as an oil. .sup.1H NMR (300 MHz, Chloroform-d)
.delta. ppm 2.61 (s, 4H) 2.83 (t, 2H) 3.62 (s, 2H) 3.63 (s, 3H)
3.73-3.77 (m, 4H) 4.14 (t, 2H) 6.88-6.91 (m, 3H) 7.26-7.29 (m,
1H)
[0353] To a solution of 1051 (1 g, 3.57 mmol) in THF/MeOH/Water (30
mL, 5 mL, 5 mL) was added lithium hydroxide monohydrate (0.3 g,
7.14 mmol). The resulting mixture was stirred at room temperature
overnight before it was concentrated under reduced pressure. The
residue obtained was diluted with water (.about.50 mL) and the
resulting solution was acidified with 1N hydrochloric acid. The
aqueous layer was concentrated and the product was isolated by prep
HPLC. The residue obtained was dissolved in water (mL) and
concentrated hydrochloric acid (mL) was added to it before it was
concentrated and dried to afford 1052 as a hydrochloride salt.
[0354] To a suspension of carboxylic acid 1052 (47.4 mg, 0.157
mmol) in DMF (3 mL) was added HATU (61.3 mg, 0.161 mmol) and
stirred till reaction mixture is clear followed by the addition of
an amine 1024 (52.5 mg, 0.142 mmol) and DIPEA (50 ul, 0.29 mmol).
The resulting mixture was stirred at room temperature overnight
before it was quenched by the addition of water. The resulting
solution was partitioned between water and ethyl acetate. The
organic extract was washed with more water, separated, dried over
sodium sulfate, filtered and evaporated. The residue obtained was
purified by silica gel chromatography eluting with
MeOH/dichloromethane to afford 381 (40 mg, 46% yield). .sup.1H NMR
(300 MHz, Dimethylsulfoxide-d.sub.6) .delta. ppm 1.74 (brs, 4H)
2.72 (t, 2H) 2.89-2.9 (m, 4H) 3.02 (brs, 4H) 3.336 (m, 2H)
3.76-3.78 (m, 2H) 4.09 (m, 2H) 6.88-6.93 (m, 3H) 7.24-7.36 (m, 6H)
7.54-7.58 (d, 1H) 8.18-8.21 (d, 1H) 11.26 (s, 1H) 12.65 (brs,
1H).
##STR00107##
[0355] To a solution of 1044 (2.29 g, 0.01 mol) in DMF (100 mL) was
added potassium carbonate (1.38 g, 0.01 mmol) and pyrazole (0.68 g,
0.01 mol). The resulting mixture was stirred at 70.degree. C. for 5
hr before it was diluted with water (.about.100 mL). The resulting
solution was partitioned between water and ethyl acetate. The
organic extract was washed with more water, separated, dried over
sodium sulfate, filtered and evaporated. The residue obtained was
purified by silica gel chromatography eluting with EtOAc/Hexane to
afford 1053 (1 g, 50% yield). .sup.1H NMR (300 MHz, Chloroform-d)
.delta. ppm 3.94 (s, 3H) 5.40 (s, 2H) 6.33 (s, 1H) 7.42-7.48 (m,
3H) 7.58 (s, 1H) 7.95 (s, 1H) 8.00-8.02 (m, 1H)
[0356] To an ice cold solution of 1053 (1 g, 4.62 mmol) in THF (20
mL) was added lithium aluminum hydride (2.5 mL, 2M/THF) drop wise
and the resulting reaction mixture was stirred at 0.degree. C. for
5 hr before it was quenched with saturated Rochelle salt solution.
The resulting solution was partitioned between water and ethyl
acetate. The organic extract was washed with more water, separated,
dried over sodium sulfate, filtered and evaporated to afford 1054
(0.8 g, 92% yield). .sup.1H NMR (300 MHz, Chloroform-d) .delta. ppm
4.71 (s, 2H) 5.35 (s, 2H) 6.30 (s, 1H) 7.15-7.43 (m, 5H) 7.58 (s,
1H)
[0357] To a solution of 1054 (0.8 g, 4.2 mmol) in dichloromethane
(20 mL) was added thionyl chloride and the resulting mixture was
stirred at room temperature for 5 hr before it was concentrated
under the reduced pressure. The residue obtained was dried at high
vacuum overnight to afford 1055 (1 g, 97% yield) as a HCl salt.
.sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm 4.75 (s,
2H) 5.38 (s, 2H) 6.30 (s, 1H) 7.19-7.50 (m, 5H) 7.86 (s, 1H)
11.49-11.60 (brs, 1H)
[0358] To a solution of 1055 (1 g, 4.1 mmol) in DMF (20 mL) was
added sodium cyanide (0.625 g, 12.7 mmol) and sodium iodide (20 mg)
and the resulting reaction mixture was stirred at 70.degree. C. for
2 hr before it was diluted with water. The resulting solution was
partitioned between water and ethyl acetate. The organic extract
was washed with more water, separated, dried over sodium sulfate,
filtered and evaporated. The residue obtained was purified by
silica gel chromatography eluting with EtOAc/Hexane to afford 1056
(0.664 g, 83% yield). .sup.1H NMR (300 MHz, Chloroform-d) .delta.
ppm 3.76 (s, 2H) 5.38 (s, 2H) 6.35 (s, 1H) 7.19-7.46 (m, 5H) 7.61
(s, 1H)
[0359] To a solution of 1056 (0.664 g, 3.3 mmol) in dioxane (5 mL)
was added concentrated hydrochloric acid (5 mL) and the resulting
reaction mixture was stirred at 90.degree. C. overnight before it
was concentrated under the reduced pressure. The residue obtained
was purified through prep HPLC and was converted to HCl salt to
afford 1057 (0.5 g, 40% yield). .sup.1H NMR (300 MHz,
Dimethylsulfoxide-d6) .delta. ppm 3.55 (s, 2H) 5.33 (s, 2H) 6.29
(s, 1H) 7.14-7.20 (m, 4H) 7.48 (s, 1H) 7.84 (s, 1H) 11.97-11.99
(brs, 1H)
[0360] To a suspension of carboxylic acid 1057 (19.8 mg, 0.0785
mmol) in DMF (2 mL) was added HATU (30.6 mg, 0.08 mmol) and stirred
till reaction mixture is clear followed by the addition of an amine
1024 (26.25 mg, 0.07 mmol) and DIPEA (25 ul, 0.15 mmol). The
resulting mixture was stirred at room temperature overnight before
it was quenched by the addition of water. The solid separated was
filtered, washed with water and dried to afford 395 (18 mg, 45%
yield). .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.74 (brs, 4H) 2.89-3.04 (m, 4H) 3.78 (s, 4H) 5.33 (s, 2H)
6.27-6.28 (s, 1H) 7.09-7.58 (m, 11H) 7.82 (s, 1H) 8.19-8.21 (d, 1H)
11.26 (s, 1H) 12.65 (brs, 1H)
##STR00108##
[0361] To a solution of 1044 (1 g, 4.1 mmol) in THF (5 mL) was
added 2M/THF methyl amine solution (2 mL) and the resulting
reaction mixture was stirred at room temperature overnight before
it was concentrated under the reduced pressure. The residue
obtained was partitioned between water and ethyl acetate. The
organic extract was washed with more water, separated, dried over
sodium sulfate, filtered and evaporated. The residue obtained was
purified by silica gel chromatography eluting with
MeOH/dichloromethane to afford 1058 (0.26 g, 33% yield). .sup.1H
NMR (300 MHz, Chloroform-d) .delta. ppm 2.49 (s, 3H) 3.66 (s, 2H)
3.73 (s, 3H) 3.79 (s, 2H) 7.2-7.33 (m, 4H).
[0362] To a solution of 1058 (0.26 g, 1.35 mmol) in dichloromethane
(5 mL) was added boc anhydride (0.293 g, 1.35 mmol) and the
resulting reaction mixture was stirred at room temperature for 4 hr
before it was purified by silica gel chromatography eluting with
EtOAc/Hexane to afford 1059 (0.3 g, 77% yield). .sup.1H NMR (300
MHz, Chloroform-d) .delta. ppm 1.5 (s, 9H) 2.84 (s, 3H) 3.66 (s,
2H) 3.73 (s, 3H) 4.44 (s, 2H) 7.17-7.32 (m, 4H).
[0363] To an ice cold solution of 1059 (0.3 g, 1.02 mmol) in
dioxane (3 mL) and water (2 mL) was added lithium hydroxide
monohydrate (0.086 g, 2.04 mmol) and the resulting reaction mixture
was stirred at 0.degree. C. for 3 hr before it was acidified with
1N HCl. The resulting solution was partitioned between water and
ethyl acetate. The organic extract was washed with more water,
separated, dried over sodium sulfate, filtered and evaporated. The
residue obtained was dried at high vacuum overnight to afford 1060
(0.2 g, 70% yield). .sup.1H NMR (300 MHz, Chloroform-d) .delta. ppm
1.5 (s, 9H) 2.84 (s, 3H) 3.66 (s, 2H) 4.43 (s, 2H) 7.17-7.32 (m,
4H)
[0364] To a suspension of carboxylic acid 1060 (51.1 mg, 0.183
mmol) in DMF (3 mL) was added HATU (69.7 mg, 0.183 mmol) and
stirred till reaction mixture is clear followed by the addition of
an amine 1024 (61.3 mg, 0.166 mmol) and DIPEA (58 ul, 0.33 mmol).
The resulting mixture was stirred at room temperature overnight
before it was quenched by the addition of water. The resulting
solution was partitioned between water and ethyl acetate. The
organic extract was washed with more water, separated, dried over
sodium sulfate, filtered and evaporated. The residue obtained was
purified by silica gel chromatography eluting with
MeOH/dichloromethane to afford 445 (0.06 g, 57% yield). .sup.1H NMR
(300 MHz, Dimethylsulfoxide-d6) .delta. ppm 1.37-1.38 (s, 9H) 1.74
(brs, 4H) 2.76 (s, 3H) 2.89 (brs, 2H) 3.02 (brs, 2H) 3.78-3.80 (m,
4H) 4.36 (s, 2H) 7.11-7.36 (m, 9H) 7.54-7.57 (d, 1H) 8.18-8.21 (d,
1H) 11.26 (s, 1H) 12.65 (brs, 1H).
Prep of 445 Via 396 Deprotection to 408 and Re-Acylation:
##STR00109##
[0366] To an ice cold solution of 408 (26 mg, 0.04 mmol) in DMF (1
mL) was added triethylamine (12.3 uL, 0.088 mmol) and acetyl
chloride (3.16 uL, 0.044 mmol). The resulting mixture was stirred
at room temperature for 2 hr before it was diluted with water. The
solid separated was filtered, washed with water and dried at high
vacuum overnight to afford 445 (10 mg, 48% yield). .sup.1H NMR (300
MHz, Dimethylsulfoxide-d6) .delta. ppm 1.74 (brs, 4H) 2.05 (m, 3H)
2.91-3.02 (m, 7H) 3.78-3.82 (m, 4H) 4.49-4.56 (m, 2H) 7.18-7.36 (m,
9H) 7.55-7.58 (d, 1H) 8.18-8.21 (d, 1H) 8.75-8.7 (brs, 2H) 11.26
(s, 1H) 12.65 (brs, 1H).
##STR00110##
[0367] Compound 401 was prepared according to the procedure above
for the preparation of compound 339. .sup.1H NMR (300 MHz,
Dimethylsulfoxide-d6) .delta. ppm 1.40 (s, 9H) 1.75 (brs, 4H) 2.87
(brs, 2H) 2.89 (brs, 2H) 3.78 (s, 4H) 4.09-4.11 (brs, 2H) 7.18-7.36
(m, 9H) 7.54-7.58 (d, 1H) 8.18-8.21 (d, 1H) 11.26 (s, 1H) 12.65
(brs, 1H)
##STR00111##
[0368] Compound 413 was prepared according to the procedure above
for the preparation of compound 315. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.68 (bs, 1H), 11.26 (s, 1H), 8.20 (d,
J=9.46 Hz, 1H), 7.58-7.26 (m, 10H), 3.90 (s, 2H), 3.78 (s, 2H),
3.02 (bs, 2H), 2.90 (bs, 2H), 1.74 (bs, 4H).
##STR00112##
[0369] Compound 415 was prepared according to the procedure above
for the preparation of compound 315: .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.48 (s, 1H), 11.26 (s, 1H), 8.20 (d, J=8.95
Hz, 1H), 7.75 (s, 1H), 7.58-7.26 (m, 9H), 6.52 (m, 1H), 5.35 (m,
1H), 3.78 (s, 2H), 3.02 (m, 2H), 2.90 (m, 2H), 1.74 (bs, 4H).
##STR00113##
[0370] To a solution of 1063 (6.31 g, 24.9 mmol) in ethanol was
added lithium hydroxide monohydrate (1.048 g, 24.9 mmol) and the
resulting reaction mixture was stirred at room temperature for 3 hr
before it was concentrated under the reduced pressure. The residue
obtained was diluted with water and was acidified with 6N HCl. The
solution was extracted with ethyl acetate. The organic extract was
washed with more water, separated, dried over sodium sulfate,
filtered and evaporated. The residue obtained was purified by
silica gel chromatography eluting with EtOAc/hexane to afford 1064
(3 g, 53% yield).
[0371] To a suspension of carboxylic acid 1064 (0.1 g, 0.44 mmol)
in DMF (2 mL) was added HATU (0.17 g, 0.44 mmol) and stirred till
reaction mixture is clear followed by the addition of an amine 1024
(0.15 g, 0.4 mmol) and DIPEA (0.14 mL, 0.8 mmol). The resulting
mixture was stirred at room temperature overnight before it was
quenched by the addition of water. The solid separated was
filtered, washed with water and dried to afford 456 (0.2, 86%
yield). .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.18 (t, 3H) 1.74 (brs, 4H) 2.88-2.90 (m, 2H) 3.01-3.04 (m, 2H)
3.66 (s, 2H) 3.78 (s, 4H) 4.05-4.12 (q, 2H) 7.19-7.36 (m, 9H)
7.55-7.58 (m, 1H) 8.18-8.21 (d, 1H) 11.26 (s, 1H) 12.65 (brs,
1H).
[0372] To a solution of 456 (0.205 g, 0.358 mmol) in Dioxane/Water
(20 mL/6 mL) was added lithium hydroxide monohydrate (0.06 g, 1.42
mmol). The resulting mixture was stirred at room temperature for 3
hr before it was acidified with acetic acid. The solution was
concentrated under reduced pressure and the residue obtained was
diluted with water. The solid separated was filtered, washed with
water and dried at high vacuum overnight. The residue obtained was
purified by silica gel chromatography eluting with
MeOH/dichloromethane to afford 465 (0.15 g, 77% yield). .sup.1H NMR
(300 MHz, Dimethylsulfoxide-d6) .delta. ppm 1.74 (brs, 4H) 2.90
(brs, 2H) 3.01 (brs, 2H) 3.5 (s, 2H) 3.78 (s, 4H) 7.19-7.36 (m, 9H)
7.55-7.58 (m, 1H) 8.18-8.21 (d, 1H) 11.26 (s, 1H) 12.32 (brs, 1H)
12.65 (s, 1H).
[0373] To a suspension of carboxylic acid 465 (25 mg, 0.046 mmol)
in DMF (1 mL) was added HATU (19.2 mg, 0.05 mmol) and stirred till
reaction mixture is clear followed by the addition of an
N,N-dimethylamine (2M/THF, 30 uL, 0.05 mmol) and DIPEA (16 uL,
0.092 mmol). The resulting mixture was stirred at room temperature
for 3 hr before it was quenched by the addition of water. The solid
separated was filtered, washed with water and dried to afford 472
(19 mg, 73% yield). .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6)
.delta. ppm 1.74 (brs, 4H) 2.83-2.90 (brs, 6H) 3.01 (brs, 4H) 3.68
(s, 2H) 3.78 (s, 4H) 7.14-7.36 (m, 9H) 7.55-7.58 (d, 1H) 8.18-8.21
(d, 1H) 11.26 (s, 1H) 12.65 (brs, 1H).
##STR00114##
[0374] To a solution of 1049 (1 g, 6 mmol) in DMF (20 mL) was added
potassium carbonate (1.662 g, 12 mmol) and (2.16 g, 9 mmol). The
resulting mixture was stirred at 70.degree. C. overnight before it
was diluted with water (.about.100 mL). The resulting solution was
partitioned between water and ethyl acetate. The organic extract
was washed with more water, separated, dried over sodium sulfate,
filtered and evaporated. The residue obtained was purified by
silica gel chromatography eluting with EtOAc/Hexane to afford 1065
(1.78 g, 91% yield) as an oil. .sup.1H NMR (300 MHz, Chloroform-d)
.delta. ppm 0.13 (s, 6H) 0.95 (s, 9H) 3.63 (s, 2H) 3.73 (s, 2H)
3.99-4.06 (m, 4H) 6.87 (m, 3H) 7.3 (m, 1H).
[0375] To a solution of 1065 (1.78 g, 5.5 mmol) in THF/MeOH/Water
(30 mL, 3 mL, 3 mL) was added lithium hydroxide monohydrate (0.46
g, 10.9 mmol). The resulting mixture was stirred at room
temperature overnight before it was concentrated under reduced
pressure. The residue obtained was diluted with water (.about.20
mL) and the resulting solution was acidified with 6N hydrochloric
acid. The solution was partitioned between water and ethyl acetate.
The organic extract was washed with more water, separated, dried
over sodium sulfate, filtered and evaporated. The residue obtained
was purified by silica gel chromatography eluting with EtOAc/Hexane
to afford 1065 and 1066. .sup.1H NMR (300 MHz,
Dimethylsulfoxide-d6) .delta. ppm 3.54 (s, 2H) 3.72 (brs, 2H)
3.96-3.98 (brs, 2H) 4.85 (brs, 1H) 6.82-6.85 (m, 3H) 7.0-7.22 (m,
1H) 12.3 (brs, 1H).
[0376] To a suspension of carboxylic acid 1065 (27 mg, 0.137 mmol)
in DMF (2 mL) was added HATU (52.2 mg, 0.137 mmol) and stirred till
reaction mixture is clear followed by the addition of an amine 1024
(46 mg, 0.125 mmol) and DIPEA (44 ul, 0.25 mmol). The resulting
mixture was stirred at room temperature overnight before it was
quenched by the addition of water. The solid separated was
filtered, washed with water and dried. The solid obtained was
purified by prep HPLC to afford 427 (16 mg, 23% yield). .sup.1H NMR
(300 MHz, Dimethylsulfoxide-d6) .delta. ppm 1.75 (brs, 4H) 2.90
(brs, 2H) 3.02 (brs, 2H) 3.71-3.78 (m, 6H) 3.98-3.99 (brs, 2H)
4.84-4.87 (brs, 1H) 6.83-6.92 (m, 3H) 7.21-7.36 (m, 6H) 7.54-7.58
(d, 1H) 8.2-8.23 (d, 1H) 11.26 (s, 1H) 12.65 (brs, 1H).
##STR00115##
[0377] To a solution of 1049 (1 g, 6 mmol) in acetone (50 mL) was
added cesium carbonate (2.545 g, 7.83 mmol), 2-bromoethyl methyl
ether (0.92 g, 6.62 mmol) and sodium iodide (10 mg). The resulting
mixture was stirred at 50.degree. C. overnight before it was
filtered. The filtrate was evaporated and the residue obtained was
purified by silica gel chromatography eluting with EtOAc/Hexane to
afford 1075 (0.97 g, 72% yield) as oil. .sup.1H NMR (300 MHz,
Chloroform-d) .delta. ppm 3.48 (s, 3H) 3.63 (s, 2H) 3.72 (brs, 2H)
4.14-4.15 (t, 2H) 6.86-6.9 (m, 3H) 7.26-7.29 (m, 1H).
[0378] The remainder of the preparation for compound 428 followed
the procedure above for compound 427. 428: .sup.1H NMR (300 MHz,
Dimethylsulfoxide-d6) .delta. ppm 1.75 (brs, 4H) 2.90 (brs, 2H)
3.02 (brs, 2H) 3.32 (s, 3H) 3.66 (brs, 2H) 3.78 (brs, 4H) 4.08
(brs, 2H) 6.88-6.92 (m, 3H) 7.25-7.27 (m, 6H) 7.54-7.58 (d, 1H)
8.2-8.23 (d, 1H) 11.26 (s, 1H) 12.65 (brs, 1H).
##STR00116##
[0379] To an ice cold solution of 1068 (6 g, 30.9 mmoL) in ethanol
(50 mL) was added thionyl chloride (2 mL) and the resulting
reaction mixture was stirred at room temperature overnight before
it was concentrated under the reduced pressure. The residue
obtained was partitioned between water and ethyl acetate. The
organic extract was washed with more water, separated, dried over
sodium sulfate, filtered and evaporated to afford 1063 (6 gm).
[0380] To a stirred solution of 1063 (3.35 g, 13.4 mmol) in THF (50
mL) was added CDI (2.44 g, 15 mmol) and the resulting mixture was
stirred for 2 hr followed by the addition of water (13 mL). The
reaction mixture was cooled to 0.degree. C. and sodium borohydride
(2.87 g, 76 mmol) was added portionwise. The stirring was continued
at room temperature for 3 hr before it was diluted with ethyl
acetate and acidifed with 6N HCl. The organic layer was separated,
dried over sodium sulfate, filtered and evaporated. The residue
obtained was purified by silica gel chromatography eluting with
EtOAc/Hexane to afford 1069 (0.563 g, 20% yield) as an oil. .sup.1H
NMR (300 MHz, Chloroform-d) .delta. ppm 1.27-1.31 (q, 3H) 2.87-2.92
(d, 2H) 3.63 (s, 2H) 3.87-3.92 (t, 2H) 4.18-4.2 (q, 2H) 7.19-7.31
(m, 4H).
[0381] To an ice cold solution of 1069 (0.563 g, 2.7 mmol) in
dichloromethane (40 mL) and triethylamine (0.47 mL, 3.3 mmol) was
added methane sulfonylchloride (0.23 mL, 3.3 mmol) and the
resulting mixture was stirred at 0.degree. C. for 2 hr and at room
temperature for 1 hr before it was diluted with saturated aqueous
sodium bicarbonate solution. The solution was extracted with ethyl
acetate. The organic extract was washed with more water, separated,
dried over sodium sulfate, filtered and evaporated to afford 1070
(0.78 g, 100% yield). .sup.1H NMR (300 MHz, Chloroform-d) .delta.
ppm 1.27-1.31 (q, 3H) 2.87 (s, 3H) 3.08 (t, 2H) 3.63 (s, 2H)
4.18-4.2 (t, 2H) 4.45 (q, 2H) 7.19-7.31 (m, 4H).
[0382] To a solution of 1070 (0.787 g, 2.7 mmol) in DMF (6 mL) was
added sodium azide (0.358 g, 5.5 mmol) and the resulting reaction
mixture was stirred at 60.degree. C. for 3 hr before it was
partitioned between water and ethyl acetate. The organic extract
was washed with more water, separated, dried over sodium sulfate,
filtered and evaporated. The residue obtained was purified by
silica gel chromatography eluting with EtOAc/Hexane to afford 1071
(0.5 g, 78% yield) as an oil. .sup.1H NMR (300 MHz, Chloroform-d)
.delta. ppm 1.27-1.31 (q, 3H) 2.92 (t, 2H) 3.54 (t, 2H) 3.63 (s,
2H) 4.18-4.2 (q, 2H) 7.19-7.29 (m, 4H).
[0383] To a solution of 1071 (0.5 g, 2.1 mmol) in THF (25 mL) was
added triphenylphosphine (0.787 g, 3 mmol) and the reaction mixture
was stirred at room temperature under argon for overnight before it
was diluted with 1 mL of water. The reaction was continued at
50.degree. C. for 1 hr before it was concentrated under the reduced
pressure. The residue was partitioned between saturated sodium
bicarbonate solution and dichloromethane. The organic layer was
separated, dried over sodium sulfate, filtered and evaporated. The
residue obtained was purified by silica gel chromatography eluting
with MeOH/dichloromethane to afford 1072 (0.43 g, 100% yield) as an
oil. .sup.1H NMR (300 MHz, Chloroform-d) .delta. ppm 1.27-1.31 (q,
3H) 2.75-2.79 (t, 2H) 2.98-3.02 (t, 2H) 3.63 (s, 2H) 4.18-4.2 (q,
2H) 7.13-7.29 (m, 4H).
[0384] To a solution of 1072 (0.427 g, 2 mmol) in dichloromethane
(30 mL) was added di-tert-butyl dicarbonate (0.447 g, 2 mmol) and
the reaction mixture was stirred at room temperature for 5 hr
before it was purified by silica gel chromatography eluting with
EtOAc/Hexane to afford 1073 (0.577 g, 91% yield) as an oil. .sup.1H
NMR (300 MHz, Chloroform-d) .delta. ppm 1.27-1.31 (q, 3H) 1.59 (s,
9H) 2.82 (t, 2H) 3.4 (m, 2H) 3.63 (s, 2H) 4.18 (q, 2H) 7.13-7.29
(m, 4H).
[0385] To a solution of 1073 (0.577 g, 1.8 mmol) in Dioxane/Water
(10 mL/3 mL) was added lithium hydroxide monohydrate (0.158 g, 3.6
mmol). The resulting mixture was stirred at room temperature
overnight before it was concentrated under reduced pressure. The
residue obtained was diluted with water (.about.20 mL) and the
resulting solution was acidified with 1N hydrochloric acid. The
solution was partitioned between water and ethyl acetate. The
organic extract was washed with more water, separated, dried over
sodium sulfate, filtered and evaporated to afford 1074 (0.35 g, 67%
yield). .sup.1H NMR (300 MHz, Chloroform-d) .delta. ppm 2.82 (m,
2H) 3.4 (m, 2H) 3.63 (s, 2H) 4.6 (brs, 1H) 7.13-7.29 (m, 4H).
[0386] To a suspension of carboxylic acid 1074 (43.8 mg, 0.157
mmol) in DMF (2 mL) was added HATU (61.3 mg, 0.161 mmol) and
stirred till reaction mixture is clear followed by the addition of
an amine 1024 (52.5 mg, 0.142 mmol) and DIPEA (50 ul, 0.287 mmol).
The resulting mixture was stirred at room temperature overnight
before it was quenched by the addition of water. The solid
separated was filtered, washed with water and dried to afford 429
(60 mg, 67% yield). .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6)
.delta. ppm 1.37-1.38 (s, 9H) 1.74 (brs, 4H) 2.69-2.71 (m, 2H)
2.87-2.88 (m, 2H) 2.9-3.15 (m, 4H) 3.78 (s, 4H) 7.09 (brs, 1H)
7.12-7.36 (m, 9H) 7.54-7.57 (d, 1H) 8.18-8.21 (d, 1H) 11.26 (s, 1H)
12.65 (brs, 1H).
[0387] To a suspension of 429 (50 mg, 79.5 mmol) in dichloromethane
(5 mL) was added TFA (1 mL) and the reaction mixture was stirred at
room temperature for overnight before it was concentrated under the
reduced pressure. The residue obtained was triturated with ether.
The solid separated was filtered, washed with ether and dried at
high vacuum overnight to afford 441 (45 mg, 88% yield) as a TFA
salt. .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm 1.74
(brs, 4H) 2.86-3.02 (m, 8H) 3.78-3.80 (s, 4H) 7.12-7.36 (m, 8H)
7.58 (d, 1H) 7.78 (brs, 3H) 8.18-8.21 (d, 1H) 11.26 (s, 1H) 12.65
(brs, 1H).
[0388] To an ice cold solution of 441 (23 mg, 0.035 mmol) in DMF (1
mL) was added triethylamine (11 uL, 0.079 mmol) and acetyl chloride
(2.8 uL, 0.038 mmol). The resulting mixture was stirred at room
temperature for 2 hr before it was diluted with water. The solid
separated was filtered, washed with water and dried at high vacuum
overnight to afford 454 (10 mg, 50% yield). .sup.1H NMR (300 MHz,
Dimethylsulfoxide-d6) .delta. ppm 1.75-1.79 (m, 7H) 2.67-2.70 (m,
2H) 2.9 (brs, 2H) 3.00-3.02 (m, 2H) 3.21-3.26 (m, 2H) 3.78 (s, 4H)
7.12-7.36 (m, 9H) 7.58 (d, 1H) 7.9 (brs, 1H) 8.18-8.21 (d, 1H)
11.26 (s, 1H) 12.65 (brs, 1H).
##STR00117##
[0389] Compound 409 was prepared via TFA deprotection of compound
399 according to the procedure above for the preparation of
compound 441. .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta.
ppm 1.75 (brs, 4H) 2.90 (brs, 2H) 3.02 (brs, 2H) 3.78 (brs, 4H)
6.89-6.98 (m, 4H) 7.25-7.36 (m, 7H) 7.51-7.58 (d, 1H) 8.2-8.23 (d,
1H) 9.34 (s, 1H) 11.26 (s, 1H) 12.65 (brs, 1H).
##STR00118##
[0390] Compound 457 was prepared by acylation of 409 according to
the amide coupling procedure above for the preparation of compound
39. .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm 1.74
(brs, 4H) 2.32 (s, 6H) 2.89 (m, 2H) 3.02 (m, 2H) 3.13 (s, 2H) 3.78
(s, 4H) 7.01-7.04 (m, 1H) 7.25-7.38 (m, 6H) 7.54-7.58 (m, 3H)
8.18-8.21 (d, 1H) 9.77 (s, 1H) 11.26 (s, 1H) 12.65 (brs, 1H)
##STR00119##
[0391] To a suspension of 295 (30 mg, 0.0617 mmol) in MeOH (2 ml)
at 0.degree. C. was added 2N NaOH (2 ml) solution. The resulting
mixture was stirred at room temperature overnight. The solvent was
evaporated under vacuo and the mixture was acidified with 1N HCl to
pH 6. The white precipitate was collected by suction filtration,
rinsed with more water and dried to afford 348. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 7.32-7.24 (m, 5H), 7.15-7.12 (d, J=9.57
Hz, 1H), 6.72-6.69 (d, J=9.15 Hz, 1H), 6.09 (s, 2H), 3.77 (s, 2H),
2.99-2.96 (bs, 2H), 2.76-2.70 (bs, 2H), 1.70 (bs, 4H).
##STR00120##
[0392] 366: .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.65 (s,
1H), 11.26 (s, 1H), 8.22-8.19 (d, J=8.82 Hz, 1H), 7.58-7.54 (d,
J=9.32 Hz, 1H), 7.33-7.25 (m, 6H), 6.95-6.82 (m, 3H), 3.81 (s. 3H),
3.75 (s, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00121##
[0393] 367: A flask was charged with 348 (100 mg, 0.27 mmol),
Boc-3-aminomethyl-phenylacetic acid (86 mg, 0.325 mmol) in DMF (2
ml) at 0.degree. C. was added HOBT (88 mg, 0.65 mmol) followed by
EDCI (156 mg, 0.812 mmol). The resulting mixture was stirred at
0.degree. C. for 5 minutes then warmed up to room temperature
overnight before it was quenched by addition of water (.about.10
mL) at 0.degree. C. The white precipitate was collected by suction
filtration, rinsed with more water. The crude material was purified
by silica gel chromatography eluting with 0-6% MeOH in
CH.sub.2Cl.sub.2 to afford 367.
##STR00122##
[0394] Compound 368 was prepared via the deprotection of compound
367 according to the procedure above for compound 341. .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. 12.65 (s, 1H), 11.26 (s, 1H),
8.22-8.16 (m, 3H), 7.58-7.54 (d, J=9.27 Hz, 1H), 7.40-7.28 (m, 9H),
4.04 (s, 2H), 3.81 (s. 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs,
4H).
##STR00123##
[0395] Compound 383 was prepared from compound 348 according to the
procedure above for the preparation of compound 354. .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. 12.65 (s, 1H), 11.26 (s, 1H), 8.51
(s, 1H), 8.22-8.19 (d, J=9.09 Hz, 1H), 7.81-7.76 (m, 1H), 7.58-7.54
(d, J=9.12 Hz, 1H), 7.42-7.26 (m, 7H), 4.0 (s, 2H), 3.81 (s, 2H),
3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00124##
[0396] To a solution of 348 (56.5 mg, 0.153 mmol) in DMF (1 ml) at
0.degree. C. was added triethylamine (43 ul, 0.306 mmol) drop wise
followed by benzyl isocyanate (23 ul, 0.184 mmol) drop wise. The
resulting mixture was slowly warmed up to room temperature and
stirred for 6 h before it was quenched by addition of water
(.about.5 mL) at 0.degree. C. The white precipitate was collected
by suction filtration, rinsed with more water and ether and
dichloromethane then dried to afford 405. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.65 (s, 1H), 9.57 (s, 1H), 8.25 (bs, 1H),
7.74-7.71 (d, J=8.61 Hz, 1H), 7.50-7.47 (d, J=9.42 Hz, 1H),
7.34-7.27 (m, 10H), 4.42-4.40 (d, J=5.46 Hz, 2H), 3.80 (s, 2H),
3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00125##
[0397] To a suspension of 339 (1 g, 1.62 mmol) in MeOH (10 ml) at
0.degree. C. was added 2N NaOH (10 ml) solution. The resulting
mixture was stirred at room temperature overnight. The solvent was
evaporated under vacuo and the mixture was acidified with 6N HCl to
pH 6 at 0.degree. C. The mixture was triturated with EtOAc and the
white precipitate was collected by suction filtration, rinsed with
more EtOAc and dried to afford 412. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.66 (s, 1H), 7.29-7.22 (m, 2H), 7.19-7.13
(m, 4H), 6.72 (d, J=8.86 Hz, 1H), 6.12 (bs, 2H), 4.12 (d, J=6.09
Hz, 2H), 3.79 (s, 2H), 3.01 (m, 2H), 2.71 (m, 2H), 1.70 (bs, 4H),
1.39 (s, 9H).
[0398] To a solution of 412 (60 mg, 0.121 mmol) in DMF (1 ml) at
0.degree. C. was added triethylamine (34 ul, 0.242 mmol) drop wise
followed by ethyl isocyanate (11 ul, 0.145 mmol) drop wise. The
resulting mixture was slowly warmed up to room temperature and
stirred for 6 h before it was quenched by addition of water
(.about.5 mL) at 0.degree. C. The white precipitate was collected
by suction filtration. The crude material was purified by silica
gel chromatography eluting with 0-6% MeOH in CH.sub.2Cl.sub.2 to
afford 420. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .quadrature. 12.65
(s, 1H), 11.27 (s, 1H), 9.42 (s, 1H), 8.22-8.19 (d, J=8.61 Hz, 1H),
7.77-7.13 (m, 5H), 6.56-6.53 (bs, 1H), 4.12-4.11 (d, 2H), 3.78 (s,
2H), 3.23-3.16 (m, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs,
4H), 1.38 (s, 9H), 1.10-1.07 (t, 3H).
##STR00126##
[0399] 422: .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.65 (s,
1H), 10.74 (s, 1H), 8.18-8.15 (d, J=9.51 Hz, 1H), 7.61-7.12 (m,
9H), 6.62 (s, 1H), 5.33 (s, 1H), 4.13-4.11 (d, J=5.58 Hz, 2H), 3.78
(s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H), 1.38 (s,
9H).
##STR00127##
[0400] To a solution of 412 (40 mg, 0.0804 mmol) in DMF (1 ml) at
0.degree. C. was added triethylamine (17 ul, 0.121 mmol) drop wise
followed by acetic anhydride (8 ul, 0.0844 mmol) drop wise. The
resulting mixture was slowly warmed up to room temperature and
stirred overnight before it was quenched by addition of water
(.about.5 mL) at 0.degree. C. The mixture was partitioned between
water and EtOAc. The organic extract was washed with water, dried
over sodium sulfate, filtered and evaporated. The crude material
was purified by silica gel chromatography eluting with 0-6% MeOH in
CH.sub.2Cl.sub.2 to afford 424. .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 12.65 (s, 1H), 11.01 (s, 1H), 8.23-8.20 (d, J=8.61 Hz, 1H),
7.57-7.55 (d, J=8.16 Hz, 1H), 7.38-7.12 (m, 4H), 4.13-4.11 (d,
J=5.76 Hz, 2H), 3.78 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 2.14
(s, 3H), 1.75 (bs, 4H), 1.39 (s, 9H).
##STR00128##
[0401] To a suspension of 424 (10 mg, 0.018 mmol) in
dichloromethane (1 ml) was added TFA (1 ml) at 0.degree. C. The
resulting mixture was stirred at room temperature for 1 h before it
was evaporated under vacuo to dryness. Ether was added and the
white precipitate was collected by suction filtration, rinsed with
more ether and dried to afford 425. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.70 (s, 1H), 11.0 (s, 1H), 8.22-8.19 (d,
J=8.82 Hz, 1H), 8.16-8.08 (bs, 2H), 7.58-7.54 (d, J=9.42 Hz, 1H),
7.39-7.30 (m, 4H), 4.06-4.03 (m, 2H), 3.84 (s, 2H), 3.01 (bs, 2H),
2.90 (bs, 2H), 2.14 (s, 3H), 1.75 (bs, 4H).
##STR00129##
[0402] To a solution of 1076 (1.8 g, 10 mmmol) in ethanol/water (40
mL/20 mL) was added sodium cyanide (0.98 g, 20 mmol). The resulting
mixture was stirred at 90.degree. C. for 4 hr before it was cooled
to 0.degree. C. Solid separated was filtered, washed with water and
dried at high vacuum overnight to afford 1077 (1.5 g, 85%
yield).
[0403] To an ice cold solution of 1077 (1 g, 5.68 mmmol) in ethanol
(50 mL) was added sodium borohydride (0.86 g, 22.72 mmol) followed
by the addition of bismuth chloride (2 g, 6.248 mmol) portionwise.
The resulting mixture was stirred at room temperature for 3 hr
before it was filtered through the celite pad. Filtrate was
concentrated and the residue obtained was partitioned between aq
sodium bicarbonate solution and ethyl acetate. The organic extract
was separated, dried over sodium sulfate, filtered and evaporated
to afford 1078 (0.82 g, 100% yield). .sup.1H NMR (300 MHz,
Chloroform-d) .delta. ppm 2.17 (s, 3H) 3.69-3.71 (brs, 4H)
6.71-6.74 (d, 1H) 6.80-6.83 (d, 1H) 7.04-7.09 (m, 1H).
[0404] To a solution of 1078 (0.3 g, 2 mmmol) in toluene (10 mL)
was added potassium acetate (0.2 g, 2.04 mmol) and acetic anhydride
(0.55 mL, 5.83 mmol). The resulting mixture was stirred at
80.degree. C. for 1 hr followed by the addition of isoamyl nitrite
(0.4 mL, 3 mmol). Stirring was continued at 80.degree. C. overnight
before it was cooled to room temperature. The solution was
partitioned between water and ethyl acetate. The organic extract
was washed with more water, separated, dried over sodium sulfate,
filtered and evaporated. The residue obtained was purified by
silica gel chromatography eluting with EtOAc/Hexane to afford 1079
(0.22 g, 54% yield). .sup.1H NMR (300 MHz, Chloroform-d) .delta.
ppm 2.85 (s, 3H) 4.09 (s, 2H) 7.39-7.41 (d, 1H) 7.58-7.63 (m, 1H)
8.28 (s, 1H) 8.48-8.51 (d, 1H)
[0405] To a solution of 1079 (0.44 g, 2.21 mmmol) in ethanol (5 mL)
was added 20% aqueous sodium hydroxide (5 mL). The resulting
mixture was stirred at 90.degree. overnight before it was
concentrated. The residue obtained was diluted with water,
acidified with acetic acid and extracted with ethyl acetate. The
organic extract was separated, dried over sodium sulfate, filtered
and evaporated to afford 1080 (0.1 g, 51% yield). .sup.1H NMR (300
MHz, Dimethylsulfoxide-d6) .delta. ppm 3.89 (s, 2H) 6.98-7.0 (d,
1H) 7.27-7.32 (m, 1H) 7.43-7.46 (d, 1H) 8.10 (s, 1H) 12.3-13.2
(broad doublet, 2H)
[0406] To a suspension of carboxylic acid 1080 (60 mg, 0.34 mmol)
in DMF (2 mL) was added HATU (130 mg, 0.34 mmol) and stirred till
reaction mixture is clear followed by the addition of an amine 1024
(114 mg, 0.31 mmol) and DIPEA (108 uL, 0.62 mmol). The resulting
mixture was stirred at room temperature for 3 hr before it was
quenched by the addition of water. The solid separated was
filtered, washed with water and dried. The residue obtained was
purified by silica gel chromatography eluting with
MeOH/dichloromethane to afford 512 (14 mg, 9% yield). .sup.1H NMR
(300 MHz, Dimethylsulfoxide-d6) .delta. ppm 1.74 (brs, 4H) 2.89
(brs, 2H) 2.91 (brs, 2H) 3.78 (s, 2H) 4.13 (s, 2H) 7.05-7.08 (m,
1H) 7.27-7.57 (m, 8H) 8.19 (d, 2H) 11.26 (s, 1H) 12.76-12.80 (brs,
1H) 13.11 (s, 1H).
##STR00130##
[0407] Compound 389 was prepared according to the procedure above
for the preparation of compound 334. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.95 (s, 1H), 11.26 (s, 1H), 8.22-8.19 (d,
J=8.91 Hz, 1H), 7.61-7.26 (m, 10H), 6.17 (s, 1H), 3.78 (s, 2H),
3.54 (bs, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 2.67-2.62 (m, 4H),
2.38 (bs, 4H), 1.73 (bs, 4H).
##STR00131##
[0408] Compound 404 was prepared according to the procedure above
for the preparation of compound 334. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.95 (s, 1H), 11.26 (s, 1H), 8.22-8.19 (d,
J=9.60 Hz, 1H), 7.58-7.54 (d, J=9.03 Hz, 1H), 7.39-7.26 (m, 6H),
7.12 (s, 2H), 7.01-6.98 (m, 1H), 6.10 (s, 1H), 3.78 (s, 5H), 3.54
(bs, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 2.64 (bs, 4H), 2.38 (bs,
4H), 1.74 (bs, 4H).
##STR00132##
[0409] To a flask was added K.sub.2CO.sub.3 (0.28 g, 2.06 mmol),
compound 295 (0.5 g, 1.03 mmol) followed by 25 mL of DMF. The
mixture was stirred for 15 minutes and chloromethyl butyrate (0.17
g, 1.23 mmol) was added and the reaction placed under an atmosphere
of argon. The mixture was heated to 80.degree. C. for 1.5 hours,
allowed to cool to room temperature and poured into 200 ml water.
The mixture was transferred to a separatory funnel, extracted with
EtOAc (3.times.100 mL), the organic layers separated and washed
with water (3.times.50 mL), brine (2.times.50 ml) and dried over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 was removed by filtration
and the volatiles removed under reduced pressure. The crude
material was purified by reverse-phase chromatography giving 0.15 g
of compound 402.
##STR00133##
[0410] To a solution of 318 (100 mg, 0.19 mmol) in CH.sub.2Cl.sub.2
(5 mL) at 0.degree. C. was added pyridine (300 .mu.L) and followed
by addition of a solution of butyryl chloride (43 mL, 0.41 mmol) in
CH.sub.2Cl.sub.2 (5 mL) dropwise. The resulting mixture was stirred
at 0.degree. C. for 1 h before it was partitioned between EtOAc and
H.sub.2O. The organic layer was separated, dried (MgSO.sub.4) and
concentrated. The residue was purified by flash column
chromatography over silica gel eluting with 1-10% MeOH in
CH.sub.2Cl.sub.2 to provide the desired product 439 (117 mg).
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 13.01 (bs, 1H), 10.12 (s,
1H), 8.49 (d, J=9.64 Hz, 1H), 7.77 (s, 1H), 7.57 (d, J=7.11 Hz,
1H), 7.40-7.30 (m, 8H), 6.57 (s, 1H), 3.97 (s, 2H), 3.09 (bs, 2H),
3.00 (bs, 2H), 2.48 (m, 2H), 1.91 (bs, 4H), 1.85-1.62 (m, 2H), 0.98
(t, J=7.07 Hz, 3H).
##STR00134##
[0411] To a solution of sodium thiomethoxide (0.266 g, 3.8 mmol) in
DMF (10 mL) was added a solution of 1016 (0.657 g, 2.7 mmol) in DMF
and the resulting mixture was stirred at room temperature for
overnight. The solution was partitioned between water and ethyl
acetate. The organic extract was washed with more water, separated,
dried over sodium sulfate, filtered and evaporated. The residue
obtained was purified by silica gel chromatography eluting with
EtOAc/Hexane to afford 1085 (0.41 g, 72% yield). .sup.1H NMR (300
MHz, Chloroform-d) .delta. ppm 2.03-2.04 (s, 3H) 3.66-3.73 (m, 7H)
7.21-7.32 (m, 4H).
[0412] To a solution of 1085 (0.503 g, 2.39 mmol) in
dichloromethane was added MCPBA (1.338 g, 7.78 mmol) and the
resulting mixture was stirred at room temperature for 4 hr before
it was diluted with aq. Sodium thiosulfate solution. Organic layer
was separated, washed with saturated aq. Sodium bicarbonate
solution and water, dried over sodium sulfate, filtered and
concentrated. The residue obtained was purified by silica gel
chromatography eluting with EtOAc/Hexane to afford 1086 (0.5 g, 86%
yield). .sup.1H NMR (300 MHz, Chloroform-d) .delta. ppm 2.8 (s, 3H)
3.7-3.74 (m, 5H) 4.27 (s, 2H) 7.30-7.4 (m, 4H).
[0413] To an ice cold solution of 1086 (0.5 g, 2.06 mmol) in
dioxane (10 mL) and water (10 mL) was added lithium hydroxide
monohydrate (0.26 g, 6.19 mmol) and the resulting reaction mixture
was stirred at room temperature for overnight before it was
concentrated. The residue obtained was diluted with water and was
acidified with acetic acid. The resulting solution was partitioned
between water and ethyl acetate. The organic extract was washed
with more water, separated, dried over sodium sulfate, filtered and
evaporated. The residue obtained was triturated with ether. The
solid separated was filtered, washed with ether and dried at high
vacuum overnight to afford 1087 (0.3 g, 64% yield). .sup.1H NMR
(300 MHz, Dimethylsulfoxide-d6) .delta. ppm 2.92 (s, 3H) 3.61 (s,
2H) 4.48 (s, 2H) 7.31-7.35 (m, 4H) 12.37 (s, 1H).
##STR00135##
[0414] Compound 634 was prepared using procedures analogous to
those above. .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta.
ppm 1.74 (brs, 4H) 2.91 (brs, 5H) 3.03 (brs, 2H) 3.78 (s, 2H) 3.85
(s, 2H) 4.49 (s, 2H) 7.32-7.40 (m, 9H) 7.55-7.58 (d, 1H) 8.19 (d,
1H) 11.26 (s, 1H) 12.69 (s, 1H).
##STR00136##
[0415] Compound 635 was prepared using procedures analogous to
those above. .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta.
ppm 1.75 (brs, 4H) 2.91 (brs, 5H) 3.03 (brs, 2H) 3.82 (s, 4H) 4.49
(s, 2H) 7.32-7.40 (m, 9H) 7.55-7.58 (d, 1H) 8.19 (d, 1H) 11.26 (s,
1H) 12.69 (s, 1H).
##STR00137##
[0416] To a solution of 1,3-bromo chloropropane (1.57 g, 10 mmol)
in DMF (10 mL) was added sodium thiomethoxide (0.63 g, 9 mmol) and
the resulting reaction mixture was stirred at room temperature
overnight and at 70.degree. C. for another day. The solution was
partitioned between water and ethyl acetate. The organic extract
was washed with more water, separated, dried over sodium sulfate,
filtered and evaporated to afford 1088 (1.3 gm) which is used for
the next step without purification.
[0417] To a solution of 1088 (1.3 g, 7.7 mmol) in dichloromethane
(100 mL) was added MCPBA (5.15 g, 23.34 mmol) and the resulting
mixture was stirred at room temperature for overnight before it was
diluted with aq. Sodium thiosulfate solution. Organic layer was
separated, washed with saturated aq. Sodium bicarbonate solution
and water, dried over sodium sulfate, filtered and concentrated.
The residue obtained was purified by silica gel chromatography
eluting with EtOAc/Hexane to afford 1089 (0.3 gm). .sup.1H NMR (300
MHz, Chloroform-d) .delta. ppm 2.38-2.49 (m, 2H) 2.99 (s, 3H)
3.22-3.27 (m, 2H) 3.57-3.77 (m, 2H).
[0418] To a solution of 1092 (0.525 g, 3.16 mmol) in DMF (15 mL)
was added potassium carbonate (0.873 g, 6.32 mmol), 1089 (0.74 g,
4.74 mmol) and sodium iodide (10 mg). The resulting mixture was
stirred at 70.degree. C. overnight before it was diluted with water
(.about.100 mL). The resulting solution was partitioned between
water and ethyl acetate. The organic extract was washed with more
water, separated, dried over sodium sulfate, filtered and
evaporated. The residue obtained was purified by silica gel
chromatography eluting with EtOAc/Hexane to afford 1090 (0.53 g,
59% yield). .sup.1H NMR (300 MHz, Chloroform-d) .delta. ppm
2.35-2.40 (m, 2H) 2.99 (s, 3H) 3.26-3.31 (m, 2H) 3.63 (s, 2H) 3.73
(s, 3H) 4.16 (t, 2H) 6.81-6.93 (m, 3H) 7.25 (m, 1H).
[0419] To a solution of 1090 (0.53 g, 1.85 mmol) in dioxane (8 mL)
and water (4 mL) was added lithium hydroxide monohydrate (0.156 g,
3.71 mmol) and the resulting reaction mixture was stirred at room
temperature for 5 hr before it was acidified with acetic acid. The
resulting solution was partitioned between water and ethyl acetate.
The organic extract was washed with more water, separated, dried
over sodium sulfate, filtered and evaporated. The residue obtained
was triturated with ether. The solid separated was filtered, washed
with ether and dried at high vacuum overnight to afford 1091 (0.2
g, 40% yield). .sup.1H NMR (300 MHz, Chloroform-d) .delta. ppm
2.32-2.42 (m, 2H) 2.99 (s, 3H) 3.26-3.31 (m, 2H) 3.66 (s, 2H)
4.12-4.16 (t, 2H) 6.83-6.94 (m, 3H) 7.26-7.31 (m, 1H).
##STR00138##
[0420] Compound 583 was prepared by coupling of 1091 with 1024
using procedure described for Amide Coupling General Procedure.
.sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm 1.74 (brs,
4H) 2.15-2.19 (m, 2H) 2.90-3.03 (m, 7H) 3.27-3.39 (m, 2H) 3.78 (s,
4H) 4.07-4.11 (t, 2H) 6.90-6.93 (m, 3H) 7.24-7.37 (m, 6H) 7.55-7.58
(d, 1H) 8.19 (d, 1H) 11.26 (s, 1H) 12.69 (s, 1H).
##STR00139##
[0421] Compound 623 was prepared by coupling of 11 with 348 using
procedure described for Amide Coupling General Procedure. .sup.1H
NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm 1.74 (brs, 4H)
2.15-2.19 (m, 2H) 2.90-3.03 (m, 7H) 3.27-3.39 (m, 2H) 3.75-3.78 (m,
4H) 4.07-4.11 (t, 2H) 6.90-6.97 (m, 3H) 7.26-7.34 (m, 6H) 7.58 (d,
1H) 8.19 (d, 1H) 11.26 (s, 1H) 12.69 (s, 1H).
##STR00140##
[0422] To a solution of 3-hydroxyphenylacetic acid (1 g, 0.00657
mol) in MeOH (10 ml) at 0.degree. C. was added (Trimethylsilyl)
diazomethane solution (2 M in hexanes, 20 ml) dropwise. The
resulting mixture was stirred at room temperature for 30 minutes
before it was evaporated to dryness. The crude material was
purified by silica gel chromatography eluting with 0-25% EtOAc in
Hexanes to afford 1093.
[0423] 1094 was made using procedure described for compound
1119.
[0424] 1095 was made using procedure described for compound
1102.
##STR00141##
[0425] 646 was made using procedure described for compound 666.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.32 (s, 1H), 8.50-8.47
(d, J=8.52 Hz, 1H), 7.90-7.70 (m, 1H), 7.40-7.36 (m, 6H), 7.03-6.86
(m, 3H), 4.72 (s, 2H), 4.02 (s, 2H), 3.90 (s, 2H), 3.44-3.39 (m,
4H), 3.09-2.96 (d, 4H), 1.87 (bs, 4H), 1.24-1.16 (m, 6H).
##STR00142##
[0426] 647 was made using procedure described for compound 666.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.61 (s, 1H), 11.22
(s, 1H), 8.22-8.19 (d, J=9.18 Hz, 1H), 8.02-8.10 (t, 1H), 7.58-7.55
(d, J=9.12 Hz, 1H), 7.36-7.24 (m, 5H), 6.99-6.84 (m, 3H), 4.48 (s,
2H), 3.82 (s, 2H), 3.75 (s, 2H), 3.50 (s, 2H), 3.01-2.90 (m, 5H),
1.73 (bs, 4H), 0.82-0.80 (d, J=6.69 Hz, 6H).
##STR00143##
[0427] A solution of hydroxylamine (50% in water, 7.4 mL) was added
to acetonitrile (60 mL) and the mixture heated to 90.degree. C. for
16 hours. The mixture was cooled to room temperature then cooled in
a wet-ice bath giving a precipitate. The solids were collected by
filtration and rinsed with cold acetonitrile (10 mL) and dried
under high vacuum giving 4.47 g of N'-hydroxyacetimidamide 1096.
See Zemolka, S. et al PCT Int Appl 2009118174. .sup.1H NMR 300 MHz
CDCl.sub.3: .delta. 4.57 (br s, 2H), 1.89 (s, 3H).
##STR00144##
[0428] A flask was charged with N'-hydroxyacetimidamide 1096 (0.45
g, 6.17 mmol) followed by THF (25 mL), NaH (60% in oil, 0.246 g,
6.17 mmol), 4 A molecular sieves (4.5 g) and the mixture heated to
60.degree. C. under an atmosphere of argon for 1 hour. A solution
of ethyl 2-(3-bromophenyl)acetate 1097 (1.5 g, 6.17 mmol) in THF
(12.5 mL) was added to the N'-hydroxyacetimidamide mixture and
heated at 60.degree. C. for 16 hours. The mixture was diluted with
water (100 mL) and extracted with EtOAc (2.times.25 mL). The
organic layers were combined, washed with water (25 mL), brine
(2.times.25 mL) and dried over Na.sub.2SO.sub.4. The
Na.sub.2SO.sub.4 was removed by filtration and the volatiles
removed under reduced pressure. The crude material was purified by
normal phase chromatography 0-30% EtOAc/hexanes giving 0.56 g of
5-(3-bromobenzyl)-3-methyl-1,2,4-oxadiazole 1098. .sup.1H NMR 300
MHz CDCl.sub.3: .delta. 7.48-7.42 (m, 2H), 7.26-7.24 (m, 2H), 4.15
(s, 2H), 2.38 (s, 3H).
##STR00145##
[0429] To a solution of 5-(3-bromobenzyl)-3-methyl-1,2,4-oxadiazole
1098 (0.50 g, 1.97 mmol) in dioxane (1 mL), under an atmosphere of
Argon, was added Bis(tri-t-butylphosphine)palladium(0) (0.15 g,
0.295 mmol) followed by the addition of
2-tert-butoxy-2-oxoethylzinc chloride (0.5 M in diethyl ether, 4.92
mmol, 9.84 mL). The mixture was allowed to stir under argon for 20
hours and the volatiles were removed under reduced pressure. The
residue was taken up in EtOAc (10 mL) and washed with water
(2.times.5 mL), brine (2.times.5 mL) and dried over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 was removed by filtration
and the volatiles removed under reduced pressure. The crude
material was purified by normal phase chromatography 0-50%
EtOAc/Hexanes to give 0.300 g tert-butyl
2-(3-((3-methyl-1,2,4-oxadiazol-5-yl)methyl)phenyl)acetate 1099.
.sup.1H NMR 300 MHz CDCl.sub.3: .delta. 7.40-7.18 (m, 4H), 4.17 (s,
2H), 3.51 (s, 2H), 2.36 (s, 3H), 1.43 (s, 9H).
##STR00146##
[0430] To a mixture of tert-butyl
2-(3-((3-methyl-1,2,4-oxadiazol-5-yl)methyl)phenyl)acetate 1099
(0.127 g, 0.44 mmol) in dioxane (3 mL) was added 4N HCl in dioxane
(1 mL) and stirred under an atmosphere of argon for 2 hours. The
volatiles were removed under reduced pressure and the residue
diluted with water (5 mL) and the pH adjusted to 12 with 2.5 N
NaOH. The mixture was washed with dichloromethane (4.times.2 mL)
and the pH adjusted to 6 with 1 N HCl. The mixture was extracted
with EtOAc (3.times.2 mL) and the organic layers combined, washed
with brine and dried over Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4
was removed by filtration and the volatiles removed under reduced
pressure to give 0.041 g of
2-(3-((3-methyl-1,2,4-oxadiazol-5-yl)methyl)phenyl)acetic acid
1100. .sup.1H NMR 300 MHz CDCl.sub.3: .delta. 7.40-7.18 (m, 4H),
4.18 (s, 2H), 3.63 (s, 2H), 2.36 (s, 3H).
##STR00147##
[0431] To a solution of
N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-phenylacet-
amide 348 (0.061 g, 0.0165 mmol),
2-(3-((3-methyl-1,2,4-oxadiazol-5-yl)methyl)phenyl)acetic acid 1100
(0.040 g, 0.18 mmol), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (0.078 g, 0.41 mmol), 1-hydroxybenzotriazole (0.055 g,
0.41 mmol) in DMF (3 mL) was added DIEA (0.085 g, 0.115 mL, 0.66
mmol) and the mixture stirred for 16 hours. The mixture was diluted
with water (20 mL) and extracted with EtOAc (3.times.20 mL). The
organic layers were combined, washed with water (3.times.20 mL),
brine (2.times.20 mL) and dried over Na.sub.2SO.sub.4. The
Na.sub.2SO.sub.4 was removed by filtration and the volatiles
removed under reduced pressure. The crude material was purified by
normal phase chromatography 0-5% MeOH/dichloromethane giving 0.003
g of
2-(3-((3-methyl-1,2,4-oxadiazol-5-yl)methyl)phenyl)-N-(6-(4-(5-(2-phen-
ylacetamido)-1,3,4-thiadiazol-2-yl)butyl)pyridazin-3-yl)acetamide
648. .sup.1H NMR 300 MHz CDCl.sub.3: .delta. 12.59 (s, 1H), 10.53
(s, 1H), 8.45 (d, 1H, J=12.2 Hz), 7.4-7.1 (m, 10H), 4.15 (s, 2H),
4.03 (s, 2H), 3.94 (s, 2H), 3.02 (m, 2H), 2.94 (m, 2H), 2.33 (s,
3H), 1.85 (m, 4H).
##STR00148##
[0432] 1101 was made using procedure described for compound
1119.
[0433] To a solution of 1101 (470 mg, 1.41 mmol) in MeOH (5 ml) and
H.sub.2O (5 ml) at 0.degree. C. was added lithium hydroxide
monohydrate (296 mg, 7.05 mmol). The resulting mixture was stirred
at room temperature for 3 days before it was evaporated to dryness.
The mixture was then acidified with 1N HCl (pH 4), and it was
partitioned between water and EtOAc. The organic extract was washed
with water, dried over sodium sulfate, filtered and evaporated to
afford 1102.
##STR00149##
[0434] 608 was made using procedure described for compound 664.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.71 (s, 1H), 11.32
(s, 1H), 8.22-8.19 (d, J=9.15 Hz, 1H), 7.58-7.54 (d, J=9.27 Hz,
1H), 7.38-7.28 (m, 8H), 4.63 (bs, 4H), 3.82 (s, 2H), 3.78 (s, 2H),
3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H), 1.48-1.44 (d, J=5.93
Hz, 9H).
##STR00150##
[0435] 612 was made using procedure described for compound 666.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.32 (s, 1H),
8.22-8.19 (d, J=9.78 Hz, 1H), 7.58-7.54 (d, J=9.72 Hz, 1H),
7.48-7.28 (m, 7H), 4.67-4.61 (m, 4H), 3.88 (s, 2H), 3.80 (s, 2H),
3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H), 1.48-1.44 (d, J=9.93
Hz, 9H).
##STR00151##
[0436] 649 was made using procedure described for compound 695.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.36 (s, 1H),
8.20-8.17 (d, J=9.78 Hz, 1H), 7.60-7.57 (d, J=8.92 Hz, 1H),
7.52-7.32 (m, 7H), 4.61-4.56 (d, J=16.99 Hz, 4H), 3.91 (s, 2H),
3.87 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00152##
[0437] 650 was made using procedure described for compound 695.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.71 (s, 1H), 11.32
(s, 1H), 9.40 (bs, 1H), 8.22-8.19 (d, J=9.09 Hz, 1H), 7.58-7.54 (d,
J=9.36 Hz, 1H), 7.38-7.28 (m, 8H), 4.63 (bs, 4H), 3.82 (s, 2H),
3.78 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00153##
[0438] To a solution of 650 (30 mg, 0.0468 mmol) in DMF (1 ml) at
0.degree. C. was added triethylamine (13 ul, 0.0936 mmol) dropwise
followed by acetic anhydride (4.64 ul, 0.0491 mmol) dropwise. The
resulting mixture was stirred at 0.degree. C. for 20 minutes before
it was quenched by addition of ice water (.about.5 mL). The white
precipitate was collected by suction filtration, rinsed with more
water. The crude material was purified by silica gel chromatography
eluting with 0-6% MeOH in CH.sub.2Cl.sub.2 to afford 651. .sup.1H
NMR (300 MHz, DMSO-d.sub.6) .delta. 12.71 (s, 1H), 11.32 (s, 1H),
8.22-8.19 (d, J=9.27 Hz, 1H), 7.58-7.54 (d, J=9.00 Hz, 1H),
7.38-7.28 (m, 8H), 4.88 (bs, 2H), 4.67 (bs, 2H), 3.82 (s, 2H), 3.78
(s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 2.11 (s, 3H), 1.73 (bs,
4H).
##STR00154##
[0439] To a solution of 2-(3-bromophenyl)acetic acid 1103 (10.0 g,
46.5 mmol) in 100 mL EtOH was added conc. H.sub.2SO.sub.4 (10
drops) and the mixture heated to relux temperature for 3 hours. The
mixture was allowed to cool to room temperature and the volatiles
were removed under reduced pressure. The residue was taken up in
EtOAc (100 mL) and washed with water (2.times.50 mL), saturated
NaHCO.sub.3 (1.times.25 mL), brine (2.times.25 mL) and dried over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 was removed by filtration
and the volatiles removed under reduced pressure to give ethyl
2-(3-bromophenyl)acetate 1097 (11.1 grams) as a liquid). .sup.1H
NMR 300 MHz CDCl.sub.3: .delta. 7.41 (m, 2H), 7.20 (m, 2H), 4.14
(q, 2H, J=9.5 Hz), 3.57 (s, 2H), 1.25 (t, 3H, J=9.5 Hz).
##STR00155##
[0440] To a solution of ethyl 2-(3-bromophenyl)acetate 1097 (1.5 g,
6.17 mmol) in MeOH (20 mL) was added hydrazine (0.79 g, 24.7 mmol)
and the mixture heated to reflux temperature for 4 hours. The
mixture was allowed to cool to room temperature giving rise to a
white precipitate which was collected by filtration and rinsed with
MeOH (10 mL). After drying under reduced pressure 1.4 grams of
2-(3-bromophenyl)acetohydrazide 1104 was isolated. .sup.1H NMR 300
MHz CDCl.sub.3: .delta. 7.42 (s, 2H), 7.20 (s, 2H), 6.73 (br s,
1H), 3.51 (s, 2H), 1.81 (br s, 2H).
##STR00156##
[0441] To a solution of 2-(3-bromophenyl)acetohydrazide 1104 (1.0
g, 4.37 mmol) in AcOH (10 mL) was added trimethylorthoacetate (2.62
g, 21.83 mmol) and the mixture heated to 115.degree. C. for 18
hours. The volatiles were removed under reduced pressure and the
residue purified by reverse phase chromatography to give 0.59 g of
2-(3-bromobenzyl)-5-methyl-1,3,4-oxadiazole 1105. .sup.1H NMR 300
MHz CDCl.sub.3: .delta. 7.45 (m, 2H), 7.23 (m, 2H), 4.12 (s, 2H),
2.49 (s, 3H).
##STR00157##
[0442] To a solution of 2-(3-bromobenzyl)-5-methyl-1,3,4-oxadiazole
1105 (0.50 g, 1.97 mmol) in dioxane (1 mL), under an atmosphere of
Argon, was added Bis(tri-t-butylphosphine)palladium(0) (0.15 g,
0.295 mmol) followed by the addition of
2-tert-butoxy-2-oxoethylzinc chloride (0.5 M in diethyl ether, 4.92
mmol, 9.84 mL). The mixture was allowed to stir under Argon for 20
hours and the volatiles were removed under reduced pressure. The
residue was taken up in EtOAc (10 mL) and washed with water
(2.times.5 mL), brine (2.times.5 mL) and dried over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 was removed by filtration
and the volatiles removed under reduced pressure. The crude
material was purified by normal phase chromatography 0-50%
EtOAc/Hexanes to give 0.338 g of tert-butyl
2-(3-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)phenyl)acetate 1106.
.sup.1H NMR 300 MHz CDCl.sub.3: .delta. 7.24 (m, 4H), 4.12 (s, 2H),
3.51 (s, 2H), 2.46 (s, 3H), 1.43 (s, 9H).
##STR00158##
[0443] To a mixture of tert-butyl
2-(3-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)phenyl)acetate 1106
(0.127 g, 0.44 mmol) in dioxane (3 mL) was added 4N HCl in dioxane
(1 mL) and stirred under an atmosphere of Argon for 2 hours. The
volatiles were removed under reduced pressure and the residue
diluted with water (5 mL) and the pH adjusted to 12 with 2.5 N
NaOH. The mixture was washed with dichloromethane (4.times.2 mL)
and the pH adjusted to 6 with 1 N HCl. The mixture was extracted
with EtOAc (3.times.2 mL) and the organic layers combined, washed
with brine and dried over Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4
was removed by filtration and the volatiles removed under reduced
pressure to give 0.023 g of
2-(3-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)phenyl)acetic acid
1107.
##STR00159##
[0444] A solution of
N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-phenylacet-
amide 348 (0.035 g, 0.094 mmol),
2-(3-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)phenyl)acetic acid 1107
(0.023 g, 0.094 mmol), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (0.045 g, 0.235 mmol), 1-hydroxybenzotriazole (0.032
g, 0.235 mmol) in DMF (1.75 mL) was stirred for 16 hours and
diluted with water (20 mL). The mixture was extracted with EtOAc
(3.times.20 mL) the organic layers combined, washed with water
(3.times.20 mL), brine (2.times.20 mL) and dried over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 was removed by filtration
and the volatiles removed under reduced pressure. The crude
material was purified by reverse phase chromatography giving 0.004
g of
2-(3-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)phenyl)-N-(6-(4-(5-(2-phenyla-
cetamido)-1,3,4-thiadiazol-2-yl)butyl)pyridazin-3-yl)acetamide 652.
.sup.1H NMR 300 MHz DMSO-d.sub.6: .delta. 12.62 (s, 1H), 11.24 (s,
1H), 8.16 (d, 1H, J=12.2 Hz), 7.54 (d, 1H, J=12.2 Hz), 7.3-7.1 (m,
9H), 4.20 (s, 2H), 3.78 (s, 2H), 3.74 (s, 2H), 2.99 (m, 2H), 2.87
(m, 2H), 2.41 (s, 3H), 1.72 (m, 4H).
##STR00160##
[0445] A mixture of 3-bromoacetophenone (5 g, 25.1 mmol) in formic
acid (6 gm) and formamide (25 mL) was heated to 170.degree. C. for
overnight before it was extracted with toluene. Organic layer was
separated and concentrated. The residue obtained was diluted with
3N HCl and the resulting mixture was refluxed overnight before it
was cooled to room temperature. The solution was extracted with
ether. Aqueous layer was separated, basified with aq. Sodium
hydroxide solution and extracted with ether. Organic layer was
separated, dried over sodium sulfate, filtered and concentrated to
afford 1108 (3 g, 60% yield). .sup.1H NMR (300 MHz, Chloroform-d)
.delta. ppm 1.22-1.25 (d, 3H) 3.97-3.99 (q, 1H) 7.23-7.4 (m, 3H)
7.6 (s, 1H).
[0446] To a solution of 1108 (2.945 g, 14.7 mmol) in
dichloromethane (100 mL) was added boc anhydride (3.21 g, 14.7
mmol) and the reaction mixture was stirred at room temperature
overnight before it was concentrated and purified by silica gel
chromatography eluting with EtOAc/Hexane to afford 1109 (3 g, 68%
yield). .sup.1H NMR (300 MHz, Dimethylsulfoxide-d.sub.6) .delta.
ppm 1.29-1.31 (d, 3H) 1.38 (s, 9H) 4.61-4.63 (q, 1H) 7.3 (brs, 2H)
7.41-7.5 (m, 3H).
[0447] To a degassed solution of 1109 (0.5 g, 1.66 mmol) and
bis(tri-tert-butylphosphine)palladium(0) (0.085 g, 0.166 mmol) in
dioxane (3 mL) was added 2-tert-Butoxy-2-oxoethylzinc chloride (8.5
mL, 4.15 mmol) under Argon and the resulting reaction mixture was
stirred at room temperature for 4 hr before it was quenched with
saturated aqueous ammonium chloride solution. The resulting
solution was partitioned between water and ethyl acetate. The
organic extract was washed with more water, separated, dried over
sodium sulfate, filtered and evaporated. The residue obtained was
purified by silica gel chromatography eluting with EtOAc/Hexane to
afford 1110 (0.35 g, 62% yield). .sup.1H NMR (300 MHz,
Dimethylsulfoxide-d6) .delta. ppm 1.29-1.31 (d, 3H) 1.388-1.42
(brs, 18H) 3.53 (s, 2H) 4.59-4.63 (q, 1H) 7.09 (brs, 1H) 7.12-7.20
(brs, 2H) 7.25-7.27 (m, 1H) 7.27-7.30 (m, 1H).
[0448] To a solution of 1110 (0.44 g, 1.3 mmol) in methanol (40 mL)
and water (10 mL) was added lithium hydroxide monohydrate (0.4 gm)
and the resulting reaction mixture was stirred at room temperature
for 2 days before it was concentrated. The residue obtained was
diluted with ice cold water and acidified with acetic acid. The
resulting solution was partitioned between water and ethyl acetate.
The organic extract was washed with more water, separated, dried
over sodium sulfate, filtered and evaporated. The residue obtained
was purified by silica gel chromatography eluting with EtOAc/Hexane
to afford 1111 (0.316 g, 86% yield). .sup.1H NMR (300 MHz,
Dimethylsulfoxide-d6) .delta. ppm 1.22-1.39 (m, 12H) 3.55 (s, 2H)
4.58-4.63 (q, 1H) 7.11-7.38 (m, 5H) 12.29 (s, 1H).
##STR00161##
[0449] .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm 1.43
(m, 12H) 1.89 (brs, 4H) 2.97-3.08 (m, 4H) 3.95-4.03 (m, 4H)
4.71-4.77 (q, 1H) 7.24-7.43 (m, 11H) 8.45-8.48 (d, 1H) 10.99 (s,
1H) 12.4 (brs, 1H).
##STR00162##
[0450] .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm 1.43
(m, 12H) 1.89 (brs, 4H) 2.97-3.08 (m, 4H) 3.95-4.03 (m, 4H)
4.71-4.77 (q, 1H) 7.24-7.43 (m, 11H) 8.45-8.48 (d, 1H) 10.22 (brs,
1H) 12.4 (brs, 1H).
##STR00163##
[0451] .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.5-1.52 (d, 3H) 1.75 (brs, 4H) 2.88-2.93 (m, 2H) 3.03-3.05 (m, 2H)
3.79 (s, 2H) 3.86 (s, 2H) 4.38-4.44 (q, 1H) 7.27-7.59 (m, 10H)
8.20-8.23 (m, 4H) 11.27 (s, 1H) 12.71 (s, 1H).
##STR00164##
[0452] .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.5-1.52 (d, 3H) 1.75 (brs, 4H) 2.88-2.93 (m, 2H) 3.03-3.05 (m, 2H)
3.86 (s, 4H) 4.38-4.44 (q, 1H) 7.27-7.59 (m, 10H) 8.20-8.23 (m, 4H)
11.27 (s, 1H) 12.71 (s, 1H).
##STR00165##
[0453] .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.5-1.52 (d, 3H) 1.75 (brs, 4H) 2.88-2.93 (m, 2H) 3.03-3.05 (m, 2H)
3.78 (s, 2H) 3.82 (s, 2H) 4.91-4.96 (q, 1H) 7.20-7.35 (m, 9H)
7.55-7.58 (d, 1H) 8.20-8.23 (d, 1H) 8.68-8.71 (m, 1H) 11.27 (s, 1H)
12.71 (s, 1H).
##STR00166##
[0454] To an ice cold solution of
1-(5-bromo-2-fluorophenyl)ethanone (4.5 g, 20.7 mmol) in methanol
(100 mL) was added ammonium acetate (32 g, 414.7 mmol) and sodium
cyanoborohydride (6.15 g, 28.98 mmol). The reaction mixture was
stirred at room temperate over the weekend before it was
concentrated. The residue obtained was diluted with water, basified
to pH.about.13 with 1N NaOH and extracted with dichloromethane. The
organic extract was separated, dried over sodium sulfate, filtered
and evaporated. The residue obtained was purified by silica gel
chromatography eluting with EtOAc/Hexane to afford 1112 (1.8 g, 40%
yield). .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.24-1.26 (d, 3H) 4.22-4.24 (q, 1H) 7.1-7.16 (t, 1H) 7.41-7.46 (m,
1H) 7.76 (m, 1H).
[0455] To a solution of 1112 (1.97 g, 9 mmol) in dichloromethane
(100 mL) was added boc anhydride (1.97 g, 9 mmol) and the reaction
mixture was stirred at room temperature overnight before it was
concentrated and purified by silica gel chromatography eluting with
EtOAc/Hexane to afford 1113 (2.4 g, 83% yield). .sup.1H NMR (300
MHz, Dimethylsulfoxide-d6) .delta. ppm 1.29-1.32 (d, 3H) 1.39 (s,
9H) 4.87 (q, 1H) 7.14-7.21 (t, 1H) 7.46-7.58 (m, 3H).
[0456] To a degassed solution of 1113 (2.4 g, 7.54 mmol) and
bis(tri-tert-butylphosphine)palladium(0) (0.77 g, 1.508 mmol) in
dioxane (l2 mL) was added 2-tert-Butoxy-2-oxoethylzinc chloride (38
mL, 18.85 mmol) under Argon and the resulting reaction mixture was
stirred at room temperature for 4 hr before it was quenched with
saturated aqueous ammonium chloride solution. The resulting
solution was partitioned between water and ethyl acetate. The
organic extract was washed with more water, separated, dried over
sodium sulfate, filtered and evaporated. The residue obtained was
purified by silica gel chromatography eluting with EtOAc/Hexane to
afford 1114 (2 g, 75% yield). .sup.1H NMR (300 MHz,
Dimethylsulfoxide-d6) .delta. ppm 1.29-1.32 (d, 3H) 1.38-1.41 (m,
18H) 3.53 (s, 2H) 4.87 (q, 1H) 7.05-7.16 (m, 2H) 7.26-7.29 (m, 1H)
7.48 (m, 1H).
[0457] To a solution of 1114 (2 g, 5.66 mmol) in methanol (100 mL)
and water (25 mL) was added lithium hydroxide monohydrate (2 gm)
and the resulting reaction mixture was stirred at room temperature
for 2 days before it was concentrated. The residue obtained was
diluted with ice cold water and acidified with acetic acid. The
resulting solution was partitioned between water and ethyl acetate.
The organic extract was washed with more water, separated, dried
over sodium sulfate, filtered and evaporated. The residue obtained
was purified by silica gel chromatography eluting with EtOAc/Hexane
to afford 1115 (1.5 g, 89% yield). .sup.1H NMR (300 MHz,
Dimethylsulfoxide-d6) .delta. ppm 1.29-1.31 (d, 3H) 1.38 (s, 9H)
3.53 (s, 2H) 4.87 (q, 1H) 7.05-7.19 (m, 2H) 7.26-7.29 (m, 1H)
7.45-7.48 (m, 1H) 12.32 (s, 1H).
##STR00167##
[0458] .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.30-1.33 (m, 12H) 1.74 (brs, 4H) 2.89 (m, 2H) 3.02 (m, 2H) 3.78
(s, 4H) 4.85 (q, 1H) 7.10-7.57 (m, 11H) 8.19-8.22 (d, 1H) 11.26 (s,
1H) 12.64 (s, 1H).
##STR00168##
[0459] .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.28-1.32 (m, 12H) 1.73-1.75 (brs, 4H) 2.87 (m, 2H) 2.89 (m, 2H)
3.75 (s, 2H) 3.81 (s, 2H) 4.85 (q, 1H) 7.06-7.57 (m, 11H) 8.18-8.21
(d, 1H) 11.26 (s, 1H) 12.64 (s, 1H).
##STR00169##
[0460] .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm
1.51-1.53 (m, 3H) 1.75 (brs, 4H) 2.90 (m, 2H) 3.02 (m, 2H) 3.78 (s,
2H) 3.85 (s, 2H) 4.65 (q, 1H) 7.25-7.61 (m, 10H) 8.21-8.25 (d, 1H)
8.33-8.35 (brs, 3H) 11.29 (s, 1H) 12.68 (s, 1H).
##STR00170##
[0461] .sup.1H NMR (300 MHz, Dimethylsulfoxide-d6) .delta. ppm 1.54
(d, 3H) 1.75-1.76 (brs, 4H) 2.91 (m, 2H) 3.02 (m, 2H) 3.81-3.83 (m,
4H) 4.65 (q, 1H) 7.24-7.63 (m, 10H) 8.22-8.25 (d, 1H) 8.36 (brs,
3H) 11.35 (s, 1H) 12.66 (s, 1H).
##STR00171##
[0462] To a mixture of 413 (1.62 g) in MeOH (25 mL), THF (10 mL)
and H.sub.2O (10 mL) at room temperature was added 1N aq. NaOH (8
mL). This mixture was stirred for 24 h before the organic volatile
was removed under reduced pressure. The residue was neutralized to
pH 7 with 1N aq. HCl solution and extracted with EtOAc (2.times.20
mL). The combined extract was dried (MgSO.sub.4) and concentrated.
The crude was purified by silica gel chromatography eluting with
1-15% MeOH in dichloromethane to afford amine 1116. The resulting
amine 1116 was converted to 660 as described for 335. .sup.1H NMR
(300 MHz, DMSO-d.sub.6) 12.68 (bs, 1H), 11.31 (s, 1H), 8.20 (d,
J=9.2 Hz, 1H), 7.57 (d, J=8.8 Hz, 1H), 7.52-7.21 (m, 8H), 3.90 (s,
2H), 3.87 (s, 2H), 3.06-2.86 (m, 4H), 1.77-1.72 (m, 4H).
##STR00172##
3-Amino-6-chloropyridazine (55.5 g, 0.428 mol) and
3-(Trifluoromethoxy)phenylacetic acid (1.1 equiv., 0.471 mol, 104
g) were dissolved in DMF (30.0 vol., 1.66 L) in a 3000 mL three
neck round-bottom flask. Addition of DIEA (1.1 equiv., 0.471 mol,
82 mL) via addition funnel was done over 5 minutes.
Propylphosphonic anhydride solution (300 mL of a 50% solution in
DMF, 1.1 equiv., 0.471 mol,) was charged into a 500 mL addition
funnel and added dropwise to reaction solution (keeping reaction
temperature .ltoreq.+30.degree. C.). The reaction usually goes to
completion after 3 hours (TLC: 6:4 hexanes-ethyl acetate). Reaction
mixture was then poured into 7.5% sodium bicarbonate (80.0 vol.,
4.4 L) which was chilled in an ice bath. Off-white crystalline
powder was filtered through a Buchner funnel, rinsed with water
(20.0 vol., 1.1 L). Dried in a 50.degree. C. vacuum to a constant
weight to afford
N-(6-chloropyridazin-3-yl)-2-(3-(trifluoromethoxy)phenyl)acetamide
1117: yield of 119.6 g (77%). .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 11.63 (s, 1H), 8.38 (d, J=9.4 Hz, 1H), 7.88 (d, J=9.4 Hz,
1H), 7.52-7.27 (m, 4H), 3.90 (s, 2H).
##STR00173##
[0463] 4-Cyanobutylzinc bromide solution (3.0 equiv., 0.50 mol, 1.0
L) was charged into an argon gas purged 5000 mL 3 neck round bottom
flask. Argon.sub.(g) purge for 5 minutes followed by the addition
of 1117 (1.0 equiv., 0.167 mol, 55.3 g) and NiCl.sub.2(dppp) (0.15
equiv., 0.0251 mol, 13.6 g) under a blanket of argon.sub.(g). The
reaction usually goes to completion after 4 hours (TLC: 1:1
hexanes-ethyl acetate). EtOAc (15 vol., 832 mL) added to deep red
solution. Water (15 vol., 832 mL) was added, thick slurry formed.
1N HCl added until slurry breaks to pale blue layer (.about.6 vol.,
333 mL). Transferred to separatory funnel and organic layer was
washed with 1N HCl (2.times.500 mL), dried (MgSO.sub.4) and
concentrated by rotary evaporation (bath .ltoreq.30.degree. C.) to
a solid reddish oil. Oil dissolved in dichloromethane (15 vol., 832
mL), silica gel (100 g) was slurried into red solution, this was
concentrated by rotary evaporation (bath .ltoreq.30.degree. C.) to
a solid reddish powder. Loaded onto a bed of silica gel (5
cm.times.11 cm), flushed with 25% hexanes in ethyl acetate (3 L),
combined organics concentrated by rotary evaporation (bath
.ltoreq.30.degree. C.). Dried under high vacuum to a constant
weight to afford
N-(6-(4-cyanobutyl)pyridazin-3-yl)-2-(3-(trifluoromethoxy)phenyl)acetamid-
e 1118: yield of 58.2 g (92%). .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 11.41 (s, 1H), 8.28 (d, J=9.2 Hz, 1H), 7.65 (d, J=9.2 Hz,
1H), 7.52-7.27 (m, 4H), 3.89 (s, 2H), 2.92 (t, J=7.5 Hz, 2H), 2.56
(t, J=7.0 Hz, 2H), 1.80 (m, 2H), 1.61 (m, 2H).
##STR00174##
[0464] 1118 (1.0 equiv., 0.154 mol, 58.2 g) was charged into a 500
mL round bottom flask along with thiosemicarbazide (1.2 equiv.,
0.184 mol, 16.8 g). TFA (5 vol., 291 mL) slowly added to reaction
vessel while stirring. The reaction slurry was heated in a
65.degree. C. bath with an open top reflux condenser. The reaction
usually goes to completion after 5 hours (determined by LC/MS).
Toluene (10 vol., 582 mL) added to deep red solution, azeotroped by
rotary evaporation (bath .ltoreq.30.degree. C.) to a red oil.
Slowly transferred oil to a well stirred 6000 mL Erlenmeyer flask
containing 7.5% sodium bicarbonate solution (69 vol., 4.0 L) cooled
in a 0.degree. C. bath. The crystals were filtered through a
Buchner funnel and rinsed twice with diethyl ether (5 vol.,
2.times.250 mL). Dried under high vacuum to a constant weight to
afford
N-(6-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)pyridazin-3-yl)-2-(3-(triflu-
oromethoxy)phenyl)acetamide 657; yield of 55.7 g (80%). .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. 11.33 (s, 1H), 8.21 (d, J=9.2 Hz,
1H), 7.58 (d, J=9.2 Hz, 1H), 7.51-7.26 (m, 4H), 6.99 (s, 2H), 3.88
(s, 2H), 2.87 (m, 4H), 1.71 (n, 4H).
##STR00175##
[0465] To a solution of 657 (50 mg, 0.11 mmol) in DMF (3 mL) at
0.degree. C. was added 4-fluorophenyl acetic acid (22 mg, 0.14
mmol), HOBt (30 mg, 0.22 mmol) and EDCI (42 mg, 0.22 mmol). The
resulting mixture was stirred at room temperature for 1.5 h before
it was cooled to 0.degree. C. and quenched with H.sub.2O. The
precipitate was collected by suction filtration and further
purified by silica gel chromatography eluting with 1-10% MeOH in
dichloromethane to afford 661. .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 12.65 (bs, 1H), 11.31 (s, 1H), 8.20 (d, J=9.1 Hz, 1H), 7.57
(d, J=9.4 Hz, 1H), 7.49-7.14 (m, 8H), 3.87 (s, 2H), 3.81 (s, 2H),
3.06-2.86 (m, 4H), 1.77-1.72 (m, 4H).
##STR00176##
[0466] 662 was prepared by the procedure as described for compound
661. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.67 (bs, 1H),
11.31 (s, 1H), 8.20 (d, J=9.1 Hz, 1H), 7.57 (d, J=9.1 Hz, 1H),
7.51-7.07 (m, 7H), 3.89 (s, 2H), 3.87 (s, 2H), 3.06-2.86 (m, 4H),
1.77-1.72 (m, 4H).
##STR00177##
[0467] 663 was prepared by the procedure as described for compound
661. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.74 (bs, 1H),
11.31 (s, 1H), 8.20 (d, J=9.2 Hz, 1H), 7.57 (d, J=9.2 Hz, 1H),
7.51-7.19 (m, 7H), 3.97 (s, 2H), 3.87 (s, 2H), 3.06-2.86 (m, 4H),
1.77-1.72 (m, 4H).
##STR00178##
[0468] To a mixture of 1-bromo-3-(difluoromethoxy) benzene (1 g,
4.5 mmol), bis(tri-tert-butylphosphine) palladium(0) (460 mg, 0.9
mmol) in 1,4-dioxane (30 ml) under argon atmosphere was added 0.5 M
of 2-tert-butoxy-2-oxoethyl zinc chloride in ether (22.5 ml). The
resulting mixture was stirred at room temperature overnight. The
mixture was partitioned between saturated NH.sub.4Cl and EtOAc. The
organic extract was washed with brine, dried over sodium sulfate,
filtered and evaporated. The crude material was purified by silica
gel chromatography eluting with 0-10% EtOAc in Hexane to afford
1119.
[0469] To a solution of 1119 (300 mg, 1.16 mmol) in dichloromethane
(5 ml) at 0.degree. C. was added TFA (3 ml) dropwise. The resulting
mixture was stirred at room temperature overnight before it was
evaporated to dryness then triturated the residue with ether to
afford 1120.
##STR00179##
[0470] 1121 was made using procedure described for compound 1120
from 1-Bromo-3-(2,2,2-trifluoroethoxy)benzene.
##STR00180##
[0471] A flask was charged with 1024 (50 mg, 0.135 mmol), 1120 (28
mg, 0.142 mmol) in DMF (1 ml) at 0.degree. C. was added HOBT (39
mg, 0.285 mmol) followed by EDCI (68 mg, 0.356 mmol). The resulting
mixture was slowly warmed up to room temperature and stirred for 2
h before it was quenched by addition of ice water (.about.5 mL).
The white precipitate was collected by suction filtration, rinsed
with more water to afford 664. .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 12.71 (s, 1H), 11.32 (s, 1H), 8.22-8.19 (d, J=9.12 Hz, 1H),
7.58-7.54 (d, J=9.03 Hz, 1H), 7.48-6.99 (m, 10H), 3.85 (s, 2H),
3.78 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00181##
[0472] 665 was made using procedure described for compound 664.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.71 (s, 1H), 11.32
(s, 1H), 8.22-8.19 (d, J=9.12 Hz, 1H), 7.58-7.54 (d, J=9.03 Hz,
1H), 7.38-7.28 (m, 6H), 7.03-6.97 (m, 3H), 4.77-4.74 (q, 2H),
3.80-3.78 (d, J=5.82 Hz, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73
(bs, 4H).
##STR00182##
[0473] A flask was charged with 348 (50 mg, 0.135 mmol), 1120 (28
mg, 0.142 mmol) in DMF (1 ml) at 0.degree. C. was added HOBT (39
mg, 0.285 mmol) followed by EDCI (68 mg, 0.356 mmol). The resulting
mixture was slowly warmed up to room temperature and stirred
overnight before it was quenched by addition of ice water (.about.5
mL). The white precipitate was collected by suction filtration,
rinsed with more water. The crude material was purified by silica
gel chromatography eluting with 0-6% MeOH in dichloromethane to
afford 666. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.71 (s,
1H), 11.32 (s, 1H), 8.22-8.19 (d, J=9.12 Hz, 1H), 7.58-7.54 (d,
J=9.03 Hz, 1H), 7.48-6.98 (m, 10H), 3.81 (bs, 4H), 3.01 (bs, 2H),
2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00183##
[0474] 667 was made using procedure described for compound 666.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.71 (s, 1H), 11.32
(s, 1H), 8.22-8.19 (d, J=9.12 Hz, 1H), 7.58-7.54 (d, J=8.97 Hz,
1H), 7.35-7.28 (m, 6H), 7.03-6.97 (m, 3H), 4.77-4.74 (q, 2H), 3.87
(s, 2H), 3.78 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs,
4H).
##STR00184##
[0475] 668 was made using procedure described for compound 675.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.71 (s, 1H), 11.32
(s, 1H), 8.22-8.19 (d, J=9.15 Hz, 1H), 7.58-6.99 (m, 10H),
3.87-3.84 (d, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00185##
[0476] 669 was made using procedure described for compound 675.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.71 (s, 1H), 11.32
(s, 1H), 8.22-8.19 (d, J=9.09 Hz, 1H), 7.58-7.54 (d, J=9.37 Hz,
1H), 7.48-7.28 (m, 6H), 7.03-6.97 (m, 2H), 4.77-4.74 (q, 2H), 3.87
(s, 2H), 3.78 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs,
4H).
##STR00186##
[0477] A flask was charged with 657 (50 mg, 0.111 mmol), 2-pyridine
acetic acid hydrochloride (20 mg, 0.116 mmol) in DMF (1 ml) at
0.degree. C. was treated with propylphosphonic anhydride solution
(91 ul) followed by triethylamine (40 ul, 0.29 mmol). The resulting
mixture was slowly warmed up to room temperature and stirred for 1
h before it was quenched by addition of ice water (.about.5 mL).
The yellow precipitate was collected by suction filtration, rinsed
with more water. The crude material was purified by silica gel
chromatography eluting with 0-6% MeOH in dichloromethane to afford
670. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.67 (s, 1H),
11.32 (s, 1H), 8.53-8.49 (m, 1H), 8.22-8.19 (d, J=9.12 Hz, 1H),
7.78-7.76 (t, 1H), 7.58-7.26 (m, 7H), 4.01 (s, 2H), 3.87 (s, 2H),
3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00187##
[0478] 671 was made using procedure described for compound 670.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.70 (s, 1H), 11.32
(s, 1H), 8.53-8.48 (m, 2H), 8.22-8.19 (d, J=9.12 Hz, 1H), 7.76-7.26
(m, 7H), 3.87 (s, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs,
4H).
##STR00188##
[0479] 672 was made using procedure described for compound 670.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.32 (s, 1H),
8.53-8.52 (bs, 2H), 8.22-8.19 (d, J=9.12 Hz, 1H), 7.58-7.26 (m,
7H), 3.87 (s, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00189##
[0480] 673 was prepared by the procedure as described for compound
661. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.69 (bs, 1H),
11.31 (s, 1H), 8.20 (d, J=9.1 Hz, 1H), 7.57 (d, J=9.1 Hz, 1H),
7.51-7.21 (m, 8H), 3.90 (s, 2H), 3.87 (s, 2H), 3.06-2.86 (m, 4H),
1.77-1.72 (m, 4H).
##STR00190##
[0481] 674 was prepared by the procedure as described for compound
661. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.63 (bs, 1H),
11.32 (s, 1H), 8.20 (d, J=9.2 Hz, 1H), 7.57 (d, J=9.2 Hz, 1H),
7.51-7.38 (m, 3H), 7.33-7.09 (m, 5H), 3.87 (s, 2H), 3.79 (s, 2H),
3.06-2.86 (m, 4H), 2.48 (s, 3H), 1.77-1.72 (m, 4H).
##STR00191##
[0482] A flask was charged with 657 (70 mg, 0.155 mmol),
5-pyrimidineacetic acid (22 mg, 0.162 mmol) in DMF (1 ml) at
0.degree. C. was added HOBT (44 mg, 0.326 mmol) followed by EDCI
(78 mg, 0.408 mmol). The resulting mixture was slowly warmed up to
room temperature and stirred for overnight before it was quenched
by addition of ice water (.about.5 mL). The white precipitate was
collected by suction filtration, rinsed with more water. The crude
material was purified by silica gel chromatography eluting with
0-6% MeOH in dichloromethane to afford 675. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.75 (s, 1H), 11.32 (s, 1H), 9.11 (s, 1H),
8.76 (s, 1H), 8.22-8.19 (d, J=9.12 Hz, 1H), 7.59-7.26 (m, 6H), 3.94
(s, 2H), 3.87 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs,
4H).
##STR00192##
[0483] 676 was made using procedure described for compound 675.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.75 (s, 1H), 11.32
(s, 1H), 8.70 (s, 1H), 8.61-8.57 (m, 2H), 8.22-8.19 (d, J=9.36 Hz,
1H), 7.59-7.26 (m, 5H), 4.11 (s, 2H), 3.87 (s, 2H), 3.01 (bs, 2H),
2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00193##
[0484] 677 was made using procedure described for compound 675.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.75 (s, 1H), 11.32
(s, 1H), 8.89 (s, 1H), 8.22-8.19 (d, J=9.15 Hz, 1H), 7.59-7.26 (m,
5H), 6.62 (s, 1H), 3.99 (s, 2H), 3.87 (s, 2H), 3.01 (bs, 2H), 2.90
(bs, 2H), 1.73 (bs, 4H).
##STR00194##
[0485] 678 was made using procedure described for compound 675.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.75 (s, 1H), 11.32
(s, 1H), 9.06 (s, 1H), 8.22-8.19 (d, J=9.21 Hz, 1H), 7.59-7.26 (m,
6H), 4.03 (s, 2H), 3.87 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73
(bs, 4H).
##STR00195##
[0486] 679 was prepared by the procedure as described for compound
661. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.67 (bs, 1H),
11.31 (s, 1H), 8.20 (d, J=9.2 Hz, 1H), 7.57 (d, J=9.2 Hz, 1H),
7.51-7.36 (m, 4H), 7.29-7.12 (m, 4H), 3.87 (s, 2H), 3.85 (s, 2H),
3.06-2.86 (m, 4H), 1.77-1.72 (m, 4H).
##STR00196##
[0487] 680 was prepared by the procedure as described for compound
661. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.67 (bs, 1H),
11.31 (s, 1H), 8.20 (d, J=9.3 Hz, 1H), 7.57 (d, J=9.0 Hz, 1H),
7.51-7.28 (m, 8H), 3.87 (s, 2H), 3.84 (s, 2H), 3.06-2.86 (m, 4H),
1.77-1.72 (m, 4H).
##STR00197##
[0488] To a solution of 674 (100 mg, 0.16 mmol) in dichloromethane
at -78.degree. C. was added m-CPBA (60 mg, 0.24 mmol) in 4
portions. The resulting mixture was stirred at that temperature for
1 h before it was slowly warmed up to -10.degree. C. and quenched
with 25% aq. Na.sub.2S.sub.2O.sub.3 solution. The reaction was
diluted with EtOAc, washed with saturated aq. NaHCO.sub.3
(3.times.10 mL). The combined organic layer was separated, washed
with brine, dried (MgSO.sub.4) and concentrated. The crude was
purified by HPLC to afford 682. .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 12.72 (bs, 1H), 11.31 (s, 1H), 8.20 (d, J=9.0 Hz, 1H), 7.68
(m, 1H), 7.60-7.26 (m, 8H), 3.91 (s, 2H), 3.87 (s, 2H), 3.06-2.86
(m, 4H), 2.76 (s, 3H), 1.77-1.72 (m, 4H).
##STR00198##
[0489] 681 was prepared from 657 and 3-methylsulphonylphenyl acetic
acid by the procedure as described for compound 661. .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. 12.72 (bs, 1H), 11.31 (s, 1H), 8.20
(d, J=9.0 Hz, 1H), 7.92-7.83 (m, 2H), 7.70-7.26 (m, 7H), 3.93 (s,
2H), 3.87 (s, 2H), 3.23 (s, 3H), 3.06-2.86 (m, 4H), 1.77-1.72 (m,
4H).
##STR00199##
[0490] 683 was made using procedure described for compound 675.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.75 (s, 1H), 11.32
(s, 1H), 8.36 (s, 1H), 8.21-8.18 (d, J=9.18 Hz, 1H), 7.84-7.80 (d,
J=9.36 Hz, 1H), 7.59-7.26 (m, 6H), 3.90-3.87 (d, 4H), 3.01 (bs,
2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00200##
[0491] 684 was made using procedure described for compound 675.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.75 (s, 1H), 11.32
(s, 1H), 8.57 (s, 1H), 8.51-8.49 (d, J=9.18 Hz, 1H), 8.21-8.18 (d,
J=9.06 Hz, 1H), 7.79-7.75 (d, J=9.36 Hz, 1H), 7.59-7.26 (m, 6H),
4.07 (t, 2H), 3.87 (s, 2H), 3.30-3.28 (m, 1H), 3.19 (s, 3H), 3.01
(bs, 2H), 2.90 (bs, 2H), 2.3-2.5 (m, 1H), 1.99-1.96 (m, 1H), 1.73
(bs, 4H).
##STR00201##
[0492] 685 was prepared by the procedure as described for compound
661. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.52 (bs, 1H),
11.31 (s, 1H), 8.20 (d, J=9.1 Hz, 1H), 7.61-7.25 (m, 7H), 3.87 (s,
2H), 3.80 (s, 3H), 3.62 (s, 2H), 3.06-2.86 (m, 4H), 1.77-1.72 (m,
4H).
##STR00202##
[0493] 686 was prepared by the procedure as described for compound
661. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.53 (bs, 1H),
11.32 (s, 1H), 8.20 (d, J=9.1 Hz, 1H), 7.58 (d, J=9.2 Hz, 1H),
7.52-7.26 (m, 4H), 5.96 (s, 1H), 3.87 (s, 2H), 3.67 (s, 2H), 3.64
(s, 3H), 3.06-2.86 (m, 4H), 2.21 (s, 3H), 1.77-1.72 (m, 4H).
##STR00203##
[0494] 687 was prepared by the procedure as described for compound
661. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.56 (bs, 1H),
11.32 (s, 1H), 8.20 (d, J=9.3 Hz, 1H), 7.61-7.38 (m, 6H), 6.17 (d,
J=2.2 Hz, 1H), 3.87 (s, 2H), 3.79 (s, 3H), 3.75 (s, 2H), 3.03-2.90
(m, 4H), 1.7-1.72 (m, 4H).
##STR00204##
[0495] 688 was prepared by the procedure as described for compound
661. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.61 (bs, 1H),
11.32 (s, 1H), 8.20 (d, J=9.3 Hz, 1H), 7.58 (d, J=9.3 Hz, 1H),
7.51-7.26 (m, 4H), 3.87 (s, 2H), 3.84 (s, 2H), 3.07-2.86 (m, 4H),
1.77-1.72 (m, 4H).
##STR00205##
[0496] To a solution of 657 (200 mg, 0.44 mmol) in DMF (4 mL) at
0.degree. C. was added mandelic acid (124 mg, 0.66 mmol), HOBt (119
mg, 0.88 mmol) and EDCI (170 mg, 0.88 mmol). The resulting mixture
was stirred at room temperature for 1.5 h before it was cooled to
0.degree. C. and quenched with H.sub.2O. The precipitate was
collected by suction filtration and further purified by silica gel
chromatography eluting with 1-10% MeOH in dichloromethane to afford
690 and a more polar 689. 689: .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 12.42 (bs, 1H), 11.31 (s, 1H), 8.20 (d, J=9.2 Hz, 1H),
7.58-7.27 (m, 10H), 6.35 (d, J=4.4 Hz, 1H), 5.34 (d, J=4.3 Hz, 1H),
3.87 (s, 2H), 3.03-2.89 (m, 4H), 1.77-1.73 (m, 4H). 690: .sup.1H
NMR (300 MHz, DMSO-d.sub.6) .delta. 13.05 (bs, 1H), 11.31 (s, 1H),
8.20 (d, J=9.0 Hz, 1H), 7.59-7.26 (m, 15H), 6.26 (d, J=5.5 Hz, 1H),
6.11 (s, 1H), 5.38 (d, J=5.3 Hz, 1H), 3.87 (s, 2H), 3.03-2.88 (m,
4H), 1.76-1.73 (m, 4H).
##STR00206##
[0497] 447 was prepared from 657 and 3-chloromandelic acid by the
procedure as described for compound 689. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 12.48 (bs, 1H), 11.31 (s, 1H), 8.20 (d, J=9.2
Hz, 1H), 7.59-7.26 (m, 9H), 6.53 (m, 1H), 5.36 (t, J=0.7 Hz, 1H),
3.87 (s, 2H), 3.03-2.90 (m, 4H), 1.75-1.71 (m, 4H).
##STR00207##
[0498] 692 was made using procedure described for compound 675.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.75 (s, 1H), 11.32
(s, 1H), 8.21-8.18 (d, J=9.18 Hz, 1H), 7.80-7.26 (m, 9H), 3.92 (s,
2H), 3.87 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).
##STR00208##
[0499] 693 was made using procedure described for compound 675.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.75 (s, 1H), 11.32
(s, 1H), 8.22-8.19 (d, J=9.06 Hz, 1H), 7.79 (s, 1H), 7.59-7.26 (m,
6H), 6.31 (s, 1H), 5.20 (s, 2H), 3.87 (s, 2H), 3.01 (bs, 2H), 2.90
(bs, 2H), 1.73 (bs, 4H).
##STR00209##
[0500] 694 was made using procedure described for compound 675.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.71 (s, 1H), 11.32
(s, 1H), 8.22-8.18 (d, J=9.15 Hz, 1H), 7.58-7.54 (d, J=9.18 Hz,
1H), 7.48-7.26 (m, 4H), 3.87 (s, 2H), 3.63 (s, 2H), 3.01 (bs, 2H),
2.90 (bs, 2H), 2.39 (s, 3H), 2.13 (s, 3H), 1.73 (bs, 4H), 1.57 (s,
9H).
##STR00210##
[0501] To a solution of 694 (50 mg, 0.081 mmol) in dichloromethane
(2 ml) was added TFA (2 ml) at 0.degree. C. The resulting mixture
was stirred at room temperature for 1 h before it was evaporated
under vacuo to dryness. Ether was added and the white precipitate
was collected by suction filtration, rinsed with more ether to
afford 695. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.71 (s,
1H), 11.32 (s, 1H), 8.22-8.19 (d, J=9.36 Hz, 1H), 7.60-7.57 (d,
J=9.27 Hz, 1H), 7.51-7.28 (m, 4H), 3.88 (s, 2H), 3.57 (s, 2H), 3.01
(bs, 2H), 2.90 (bs, 2H), 2.45 (s, 3H), 2.15 (s, 3H), 1.73 (bs,
4H).
##STR00211##
[0502] 696 was made using procedure described for compound 695.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 12.71 (s, 1H), 11.32
(s, 1H), 8.22-8.19 (d, J=9.30 Hz, 1H), 8.15 (s, 1H), 7.58-7.54 (d,
J=9.30 Hz, 1H), 7.48-7.28 (m, 5H), 3.87 (s, 2H), 3.76 (s, 2H), 3.01
(bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H), 1.59 (s, 9H).
##STR00212##
[0503] 697 was made using procedure described for compound 695.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 14.22 (s, 1H), 12.71
(s, 1H), 11.32 (s, 1H), 9.01 (s, 1H), 8.22-8.19 (d, J=9.15 Hz, 1H),
7.59-7.26 (m, 6H), 4.04 (s, 2H), 3.87 (s, 2H), 3.01 (bs, 2H), 2.90
(bs, 2H), 1.73 (bs, 4H).
Preparative HPLC Purification
[0504] All reverse phase preparative HPLC purifications were
performed using a Shimadzu Prominence Preparative Liquid
Chromatograph with the column at ambient temperature. Mobile phases
A and B consisted of 0.1% formic acid in water and 0.1% formic acid
in acetonitrile, respectively. Crude product mixtures were
dissolved in DMF, DMSO or mixtures thereof at concentrations of
approximately 100 mg/mL and chromatographed according to the
methods described in Table 2. Appropriate chromatographic fractions
were then evaporated under high vacuum at 45.degree. C. using a
Savant Speed Vac Plus Model SC210A to yield purified products.
TABLE-US-00002 TABLE 2 Preparative HPLC Method Descriptions Product
Flow Retention Compound Time % Rate Time ID Column (min) MPB
(mL/min) (min) 7 1 0 20 2 7.4 1 20 2 2 20 5 3 70 5 14 100 5 8 1 0
20 2 11.5 1 20 2 2 20 5 3 70 5 14 100 5 26 1 0 40 1 6 1 40 2 3.5 40
4 4 40 4.73 10 90 4.73 29 2 0 40 2 7.7 1 40 3 2 40 18.9 13 50 18.9
36 2 0 32 3 12.1 0.5 32 5 1 32 18.9 13 35 18.9 143 2 0 50 3 9.1 1
50 3 2 50 18.9 5 50 18.9 15 80 18.9 153 2 0 35 3 6.2 1 35 3 2 35
18.9 4 35 18.9 14 75 18.9 199 2 0 45 3 8.3 1 45 3 2 45 18.9 3 45
18.9 13 65 18.9 203 2 0 50 3 9.6 1 50 3 2 50 18.9 5 50 18.9 15 60
18.9 208 2 0 35 3 7.6 1 35 3 2 35 18.9 4 35 18.9 14 50 18.9
[0505] The following representative synthetic protocols may also be
used for producing compounds of the invention.
##STR00213##
[0506] 3,6-Dichloropyridazine is treated with di-tertbutyl malonate
and sodium hydride in THF or DMF to give 1026. Intermediate 1026 is
then treated with sodium hydride in THF or DMF followed by
bis-(chloromethyl)sulfide to give 1027. Intermediate 1027 is
treated with TFA in dichloromethane to give 1028. Intermediate 1028
is treated with ammonia to give 1029. Intermediate 1028 is also
converted to 1029 by sequential treatment with 2,4-dimethoxybenzyl
amine and TFA. The bis-amino intermediate 1029 may be converted to
acylated products analogous to those described in Table 3 using the
methods described in Synthetic Protocols section above for
acylation of 1001-1008.
##STR00214##
[0507] Both trans- and cis-cyclopropane-1,2-diyldimethanols are
converted into the corresponding bis-nitrile 1031 via bis-mesylated
intermediate 1030. The bismesylate intermediate 1030 is prepared by
treating the diol with methanesulfonyl chloride in the presence of
pyridine or triethylamine in dichloromethane. The bisnitrile 1031
is prepared by treating 1030 with sodium cyanide in DMSO or
ethanol/water. Using a procedure similar to that described for the
preparation 1001, bis-nitrile 1031 undergoes cyclization with
thiosemicarbazide in TFA to provide bis-amino intermediate 1032.
The bis-amino intermediate 1032 may be converted to acylated
products analogous to those described in Table 3 using the methods
described in Synthetic Protocols section above for acylation of
1001-1008.
##STR00215##
[0508] The alkene analog 1033 is prepared from
trans-3-hexenedinitrile using a procedure similar to that described
for the preparation 1001. The bis-amino intermediate 1033 may be
converted to acylated products analogous to those described in
Table 3 (for example, 1034) using the methods described in
Synthetic Protocols section above for acylation of 1001-1008. The
products may be further converted to cyclopropyl analogs
(exemplified by 1035) under the Simmons-Smith conditions
(Et.sub.2Zn, CH.sub.2I.sub.2,1,2-dimethoxyethane).
Example 2: Compound Assays
[0509] Compounds were assayed in both an in vitro biochemical assay
and a cell proliferation assay as follows. The IC50 results are
provided in Table 3.
Recombinant Enzyme Assay
[0510] Compounds were assessed for their ability to inhibit the
enzymatic activity of a recombinant form of Glutaminase 1 (GAC)
using a biochemical assay that couples the production of glutamate
(liberated by GAC) to glutamate dehydrogenase (GDH) and measuring
the change in absorbance for the reduction of NAD.sup.+ to NADH.
Substrate solution was prepared (50 mM Tris-HCl pH 8.0, 0.2 mM
EDTA, 150 mM K.sub.2HPO.sub.4, 0.1 mg/ml BSA, 1 mM DTT, 20 mM
L-glutamine, 2 mM NAD.sup.+, and 10 ppm antifoam) and 50 .mu.L
added to a 96-well half area clear plate (Corning #3695). Compound
(2 .mu.L) was added to give a final DMSO concentration of 2% at
2.times. the desired concentration of compound. Enzymatic reaction
was started with the addition of 50 .mu.L of enzyme solution (50 mM
Tris-HCl pH 8.0, 0.2 mM EDTA, 150 mM K.sub.2HPO.sub.4, 0.1 mg/ml
BSA, 1 mM DTT, 10 ppm antifoam, 4 units/ml GDH, 4 mM adenosine
diphosphate, and 4 nM GAC) and read in a Molecular Devices M5 plate
reader at 20.degree. C. The plate reader was configured to read
absorbance (.lamda.=340 nm) in kinetic mode for 15 minutes. Data
was recorded as milli-absorbance units per minute and slopes were
compared to a control compound and a DMSO-only control on the same
plate. Compounds with slopes less than the DMSO control were
considered inhibitors and plate variability was assessed using the
control compound.
[0511] Results from this assay for several compounds of the
invention are shown in Table 3, expressed as IC50, or half maximal
inhibitory concentration, wherein IC50 is a quantitative measure
indicating how much compound is needed to inhibit a given
biological activity by half.
Recombinant Enzyme Assay--Time Dependence
[0512] Compounds were assessed for their ability to inhibit the
enzymatic activity of a recombinant form of Glutaminase 1 (GAC)
using a biochemical assay that couples the production of glutamate
(liberated by GAC) to glutamate dehydrogenase (GDH) and measuring
the change in absorbance for the reduction of NAD.sup.+ to NADH.
Enzyme solution was prepared (50 mM Tris-HCl pH 8.0, 0.2 mM EDTA,
150 mM K.sub.2HPO.sub.4, 0.1 mg/ml BSA, 1 mM DTT, 10 ppm antifoam,
4 units/ml GDH, 4 mM adenosine diphosphate, and 4 nM GAC) and 50
.mu.L added to a 96-well half area clear plate (Corning #3695).
Compound (2 .mu.L) was added to give a final DMSO concentration of
2% at 2.times. the desired concentration of compound. The
enzyme/compound mix was sealed with sealing foil (USA Scientific)
and allowed to incubate, with mild agitation, for 60 minutes at
20.degree. C. Enzymatic reaction was started with the addition of
50 .mu.L of substrate solution (50 mM Tris-HCl pH 8.0, 0.2 mM EDTA,
150 mM K.sub.2HPO.sub.4, 0.1 mg/ml BSA, 1 mM DTT, 20 mM
L-glutamine, 2 mM NAD.sup.+, and 10 ppm antifoam) and read in a
Molecular Devices M5 plate reader at 20.degree. C. The plate reader
was configured to read absorbance (X=340 nm) in kinetic mode for 15
minutes. Data was recorded as milli-absorbance units per minute and
slopes were compared to a control compound and a DMSO-only control
on the same plate. Compounds with slopes less than the DMSO control
were considered inhibitors and plate variability was assessed using
the control compound.
[0513] Results from this assay for several compounds of the
invention are shown in Table 3, expressed as IC50, or half maximal
inhibitory concentration, wherein IC50 is a quantitative measure
indicating how much compound is needed to inhibit a given
biological activity by half.
Cell Proliferation Assay
[0514] P493-6 (myc "on") cells were maintained in growth media
(RPMI-1640, 10% FBS, 2 mM glutamine, 100 units/ml Penicillin and
100 g/ml streptomycin) at 37.degree. C. with 5% CO.sub.2. For
compound assay, P493-6 cells were plated in 96-well V-bottom plates
on the day of compound addition in 50 .mu.l of growth media at a
cell density of 200,000 cells/ml (10,000 cells/well). Compounds
were serially diluted in 100% DMSO at 200-times the final
concentration. Compounds were diluted 100-fold into growth media
and then 50 l of this mixture was added to cell plates making the
final concentration of DMSO 0.5%. Cells were incubated with
compound for 72 hrs at 37.degree. C. with 5% CO.sub.2 and analyzed
for antiproliferative effects either by Cell Titer Glo (Promega) or
FACS analysis using the Viacount (Millipore) kit on the Guava
instrument.
[0515] Results from this assay for several compounds of the
invention are shown in Table 3, expressed as IC50, or half maximal
inhibitory concentration, wherein IC50 is a quantitative measure
indicating how much compound is needed to inhibit a given
biological activity by half.
TABLE-US-00003 TABLE 3 GAC Delta GAC N2 Delta Cell IC50 N2 prolif
60 IC50 P493 min no 72 h Cmpd preinc preinc IC50 ID Structure
(.mu.M) (.mu.M) (.mu.M) 1 ##STR00216## 0.10 0.20 0.47 2
##STR00217## 4.1 0.63 3 ##STR00218## >50 >50 4 ##STR00219##
13 >50 5 ##STR00220## >50 >50 6 ##STR00221## >50 2.7 7
##STR00222## >50 1.0 8 ##STR00223## >50 1.6 9 ##STR00224##
>50 >50 10 ##STR00225## >50 >50 11 ##STR00226## 1.4
0.89 12 ##STR00227## >50 36 13 ##STR00228## 7.7 12 14
##STR00229## 2.8 1.8 15 ##STR00230## >50 1.2 16 ##STR00231##
>50 0.80 17 ##STR00232## 15 4.2 18 ##STR00233## 4.5 8.2 19
##STR00234## 11 1.7 20 ##STR00235## 6.6 2.6 21 ##STR00236## 0.16
0.02 22 ##STR00237## >50 >50 23 ##STR00238## >50 >50 24
##STR00239## 0.51 2.3 25 ##STR00240## 1.2 1.5 26 ##STR00241## 5.6
0.70 27 ##STR00242## >50 0.47 28 ##STR00243## >50 1.0 29
##STR00244## 0.56 4.1 30 ##STR00245## 1.2 2.5 31 ##STR00246##
>50 4.3 32 ##STR00247## 7.0 11 33 ##STR00248## 13 5.3 34
##STR00249## >50 >50 35 ##STR00250## 18 3.8 36 ##STR00251##
0.04 0.22 0.16 37 ##STR00252## >50 >50 38 ##STR00253## >50
3.2 39 ##STR00254## 26 4.5 40 ##STR00255## 3.7 0.56 41 ##STR00256##
7.9 33 42 ##STR00257## >50 >50 43 ##STR00258## 2.3 >50 44
##STR00259## 4.9 2.6 45 ##STR00260## >50 >50 46 ##STR00261##
>50 16 47 ##STR00262## 8.3 35 48 ##STR00263## >50 0.42 49
##STR00264## 36 17 50 ##STR00265## 2.5 8.2 51 ##STR00266## 1.2 1.3
52 ##STR00267## 8.3 30 53 ##STR00268## >50 34 54 ##STR00269##
9.2 1.6 55 ##STR00270## >50 3.9 56 ##STR00271## >50 57
##STR00272## 40 58 ##STR00273## >50 3.7 59 ##STR00274## >50
60 ##STR00275## 24 14 61 ##STR00276## >50 62 ##STR00277## >50
19 63 ##STR00278## 25 2.6 64 ##STR00279## 1.3 0.23 65 ##STR00280##
1.3 0.52 66 ##STR00281## 20 67 ##STR00282## 3.0 1.8 68 ##STR00283##
4.9 0.34 69 ##STR00284## 0.69 0.33 70 ##STR00285## 3.4 3.4 71
##STR00286## >50 6.9 72 ##STR00287## 0.59 0.47 73 ##STR00288##
>50 74 ##STR00289## >50 75 ##STR00290## >50 76
##STR00291## >50 77 ##STR00292## 6.1 34 78 ##STR00293## 0.84 10
79 ##STR00294## 2.0 20 80 ##STR00295## 1.8 1.3 81 ##STR00296## 10
7.6 82 ##STR00297## 0.80 1.3 83 ##STR00298## 3.9 1.4 84
##STR00299## 0.23 0.89 85 ##STR00300## 1.5 1.8 86 ##STR00301## 0.32
0.52 87 ##STR00302## 0.18 0.06 88 ##STR00303## 0.20 0.12 89
##STR00304## >20 90 ##STR00305## >20 91 ##STR00306## >20
92 ##STR00307## 0.14 0.38 0.47 93 ##STR00308## 0.90 2.0 94
##STR00309## 0.28 0.47 95 ##STR00310## 2.9 45 96 ##STR00311##
>20 97 ##STR00312## 0.56 17 98 ##STR00313## >20 3.9 99
##STR00314## 2.7 1.0 100 ##STR00315## 8.1 9.0 101 ##STR00316## 24
17 102 ##STR00317## 0.24 1.4 103 ##STR00318## 19 >50 104
##STR00319## >20 105 ##STR00320## 9.9 119 106 ##STR00321##
>20 107 ##STR00322## 4.3 1.2 108 ##STR00323## >20 109
##STR00324## >20 110 ##STR00325## >20 111 ##STR00326## 0.95
0.88 112 ##STR00327## 0.51 0.89 113 ##STR00328## >20 114
##STR00329## 0.60 0.56 115 ##STR00330## 0.62 1.1 116 ##STR00331##
0.24 0.72 117 ##STR00332## 2.4 6.2 118 ##STR00333## 5.0 36 119
##STR00334## >20 13
120 ##STR00335## 1.8 38 121 ##STR00336## 1.7 3.5 122 ##STR00337##
3.5 43 123 ##STR00338## 12 6.6 124 ##STR00339## >20 125
##STR00340## >20 126 ##STR00341## 5.8 12 127 ##STR00342## 1.8
0.45 128 ##STR00343## 32 >50 129 ##STR00344## >20 >50 130
##STR00345## >20 131 ##STR00346## 19 132 ##STR00347## >20 133
##STR00348## 0.51 0.15 134 ##STR00349## 14 28 135 ##STR00350## 0.30
0.49 136 ##STR00351## 7.0 4.7 137 ##STR00352## >20 138
##STR00353## 0.75 2.7 139 ##STR00354## >20 140 ##STR00355## 3.4
>50 141 ##STR00356## 1.7 4.3 142 ##STR00357## >20 143
##STR00358## 0.57 2.2 144 ##STR00359## >20 145 ##STR00360##
>20 146 ##STR00361## 0.43 0.46 147 ##STR00362## 0.62 0.37 148
##STR00363## 0.59 0.39 149 ##STR00364## 15 150 ##STR00365## >20
151 ##STR00366## 14 >50 152 ##STR00367## 0.73 1.1 153
##STR00368## 1.0 >50 154 ##STR00369## 19 >50 155 ##STR00370##
0.27 1.9 156 ##STR00371## 0.12 0.63 157 ##STR00372## 0.34 0.18 158
##STR00373## 0.22 8.1 159 ##STR00374## 0.11 0.05 160 ##STR00375##
0.16 >50 161 ##STR00376## 0.15 1.4 162 ##STR00377## 0.23 0.15
163 ##STR00378## 0.13 >50 164 ##STR00379## 0.24 0.13 165
##STR00380## 0.51 33 166 ##STR00381## 7.4 6.8 167 ##STR00382## 11
34 168 ##STR00383## 1.3 >50 169 ##STR00384## 0.71 3.4 170
##STR00385## 7.4 9.3 171 ##STR00386## >20 172 ##STR00387## 1.7
3.7 173 ##STR00388## 24 0.76 174 ##STR00389## 0.29 0.44 175
##STR00390## 6.3 23 176 ##STR00391## 0.57 1.5 177 ##STR00392## 1.1
>50 178 ##STR00393## 1.5 >50 179 ##STR00394## 3.1 >50 180
##STR00395## 8.8 >50 181 ##STR00396## 0.33 30 182 ##STR00397##
0.58 >50 183 ##STR00398## >20 184 ##STR00399## >20 185
##STR00400## >20 0.09 186 ##STR00401## 3.1 13 187 ##STR00402##
2.8 21 188 ##STR00403## 2.0 0.46 189 ##STR00404## 4.4 190
##STR00405## 0.25 0.49 191 ##STR00406## >20 192 ##STR00407##
>20 0.03 193 ##STR00408## 3.4 194 ##STR00409## 10 195
##STR00410## 0.30 1.3 196 ##STR00411## 0.19 0.61 197 ##STR00412##
6.9 198 ##STR00413## 0.18 >50 199 ##STR00414## 0.12 0.17 200
##STR00415## 0.61 201 ##STR00416## 2.7 202 ##STR00417## 0.18 0.14
203 ##STR00418## 1.7 1.7 204 ##STR00419## 0.92 2.4 205 ##STR00420##
0.38 4.1 206 ##STR00421## >20 207 ##STR00422## 13 208
##STR00423## 0.17 9.0 209 ##STR00424## >20 22 210 ##STR00425##
0.38 0.42 211 ##STR00426## 1.2 1.0 212 ##STR00427## >20 213
##STR00428## 2.5 4.4 214 ##STR00429## 0.82 1.2 215 ##STR00430## 16
216 ##STR00431## 0.89 >50 217 ##STR00432## 0.24 >50 218
##STR00433## >20 219 ##STR00434## 0.17 0.57 220 ##STR00435## 1.6
0.31 221 ##STR00436## >20 222 ##STR00437## >20 223
##STR00438## >20 224 ##STR00439## >20 225 ##STR00440## >20
226 ##STR00441## 2.3 >50 227 ##STR00442## 9.9 3.3 228
##STR00443## 0.57 0.13 229 ##STR00444## 3.9 230 ##STR00445## 12 231
##STR00446## 7.4 232 ##STR00447## 9.8 233 ##STR00448## 15 234
##STR00449## 2.0 2.5 235 ##STR00450## 0.11 0.21 236 ##STR00451##
0.20 1.4 237 ##STR00452## 0.20 0.25 238 ##STR00453## 13 239
##STR00454## 0.30 0.30 240 ##STR00455## 0.54 1.3 241 ##STR00456##
0.38 0.87 242 ##STR00457## 0.36 0.22 243 ##STR00458## 2.7 33 244
##STR00459## 0.84 1.7 245 ##STR00460## 0.52 2.5
246 ##STR00461## 0.40 1.6 247 ##STR00462## 0.19 0.83 248
##STR00463## 2.3 249 ##STR00464## 0.12 0.16 250 ##STR00465## 0.12
0.14 251 ##STR00466## 2.8 2.8 252 ##STR00467## 1.2 6.3 253
##STR00468## 21 254 ##STR00469## >20 255 ##STR00470## 0.38 256
##STR00471## 0.11 257 ##STR00472## 0.12 0.073 258 ##STR00473## 0.19
0.18 259 ##STR00474## 0.23 0.57 260 ##STR00475## 0.15 0.084 261
##STR00476## 0.70 2.6 262 ##STR00477## 0.36 3.1 263 ##STR00478##
0.32 3.9 264 ##STR00479## 0.072 0.01 265 ##STR00480## 0.27 0.31 266
##STR00481## 2.2 >50 267 ##STR00482## 0.61 0.64 268 ##STR00483##
0.60 5.4 269 ##STR00484## 0.26 0.52 270 ##STR00485## >5 7.4 0.85
271 ##STR00486## 0.10 0.63 272 ##STR00487## >20 273 ##STR00488##
0.14 0.07 274 ##STR00489## 0.75 0.68 275 ##STR00490## 0.15 2.2 0.34
276 ##STR00491## 1.5 56 277 ##STR00492## >20 278 ##STR00493##
0.38 0.16 279 ##STR00494## 0.68 7.0 280 ##STR00495## 0.29 0.23 281
##STR00496## 0.74 0.66 282 ##STR00497## 0.082 0.37 283 ##STR00498##
0.66 0.74 284 ##STR00499## 0.05 >20 285 ##STR00500## 0.19 0.14
286 ##STR00501## 0.54 6.4 287 ##STR00502## 0.52 1.3 288
##STR00503## 0.04 0.67 0.028 289 ##STR00504## 32 290 ##STR00505##
0.80 0.79 291 ##STR00506## 1.5 1.8 292 ##STR00507## 0.12 0.012 293
##STR00508## 0.24 0.04 294 ##STR00509## 0.20 1.1 295 ##STR00510##
0.01 0.057 0.039 296 ##STR00511## 0.10 0.17 297 ##STR00512## 6.4
298 ##STR00513## 0.73 5.1 299 ##STR00514## 0.33 300 ##STR00515##
0.16 0.16 301 ##STR00516## >20 0.23 302 ##STR00517## 7.0 0.87
303 ##STR00518## >20 304 ##STR00519## 1.2 4.9 305 ##STR00520##
>20 102 1038 ##STR00521## 0.080 1.5 306 ##STR00522## 0.031 0.52
0.066 307 ##STR00523## 6.4 9.3 308 ##STR00524## 0.60 1.2 309
##STR00525## 0.11 0.18 310 ##STR00526## 0.083 0.12 311 ##STR00527##
0.20 22. 312 ##STR00528## >20 N/D 313 ##STR00529## 0.27 94 314
##STR00530## 0.14 0.048 315 ##STR00531## 0.017 0.12 0.035 316
##STR00532## 0.19 0.075 317 ##STR00533## 0.007 0.18 0.010 318
##STR00534## 0.006 0.18 0.017 0 319 ##STR00535## 0.64 10 320
##STR00536## 0.40 0.19 321 ##STR00537## 2.5 2.6 322 ##STR00538##
2.8 3.0 323 ##STR00539## 0.056 0.20 324 ##STR00540## 0.011 4.6 0.10
325 ##STR00541## 0.17 0.66 0.030 326 ##STR00542## >20 N/D 327
##STR00543## >20 0.15 328 ##STR00544## >20 N/D 329
##STR00545## 0.17 0.45 330 ##STR00546## >20 N/D 331 ##STR00547##
>20 N/D 332 ##STR00548## 3.3 0.087 333 ##STR00549## 0.10 1.6 334
##STR00550## 0.64 0.030 335 ##STR00551## 0.062 0.050 336
##STR00552## 0.068 0.052 337 ##STR00553## 0.073 0.021 338
##STR00554## 0.15 0.043 339 ##STR00555## 0.005 0.16 0.009 340
##STR00556## 0.096 0.038 341 ##STR00557## 0.013 0.13 0.039 342
##STR00558## 1.4 2.7 343 ##STR00559## 0.16 0.25 344 ##STR00560##
0.088 345 ##STR00561## 0.16 0.24 346 ##STR00562## 0.12 0.087 527
##STR00563## 0.024 0.13 0.098 347 ##STR00564## 0.22 0.71 348
##STR00565## 1.0 1.7 349 ##STR00566## 0.12 0.12 350 ##STR00567##
0.079 0.029 351 ##STR00568## 0.11 0.049 352 ##STR00569## 0.069 0.13
353 ##STR00570## 0.049 0.021 354 ##STR00571## 0.10 0.047 355
##STR00572## 0.10 0.039 356 ##STR00573## >20 N/D 357
##STR00574## >20 N/D 358 ##STR00575## 1.4 0.11 359 ##STR00576##
0.38 0.91 360 ##STR00577## 0.28 0.67 361 ##STR00578## 1.8 >20
1035 ##STR00579## >20 N/D 362 ##STR00580## 0.35 0.054 363
##STR00581## 0.065 >20 364 ##STR00582## 0.030 0.15 0.26 265
##STR00583## 0.009 0.092 0.089 366 ##STR00584## 0.074 0.024 367
##STR00585## 0.002 0.12 0.006
368 ##STR00586## 0.009 0.11 0.017 369 ##STR00587## 0.81 1.9 370
##STR00588## 0.28 0.70 371 ##STR00589## 0.43 5.2 372 ##STR00590##
0.16 0.15 373 ##STR00591## 0.17 0.28 374 ##STR00592## 0.26 0.47 375
##STR00593## 0.005 0.38 0.041 376 ##STR00594## 0.35 0.091 377
##STR00595## 0.28 0.10 378 ##STR00596## 0.22 0.090 379 ##STR00597##
0.097 0.038 380 ##STR00598## 0.12 0.019 381 ##STR00599## 0.16 0.018
382 ##STR00600## 0.003 0.099 0.007 383 ##STR00601## 0.086 0.022 384
##STR00602## 0.003 0.081 0.005 385 ##STR00603## 0.26 0.72 386
##STR00604## 0.085 0.15 387 ##STR00605## 1.2 2.3 388 ##STR00606##
0.21 0.75 389 ##STR00607## 0.084 0.032 390 ##STR00608## 0.042 0.16
391 ##STR00609## 0.007 0.027 392 ##STR00610## 0.014 0.072 393
##STR00611## 0.10 0.90 394 ##STR00612## 0.088 1.2 395 ##STR00613##
0.004 0.015 396 ##STR00614## 0.004 0.005 397 ##STR00615## 0.008
0.041 398 ##STR00616## 0.004 0.023 399 ##STR00617## 0.005 0.026 400
##STR00618## 0.015 0.053 401 ##STR00619## 0.005 0.011 402
##STR00620## 1.1 0.054 403 ##STR00621## 0.018 0.12 404 ##STR00622##
0.060 0.022 405 ##STR00623## 0.081 0.67 406 ##STR00624## 0.016 0.27
407 ##STR00625## 0.012 0.044 408 ##STR00626## 0.018 0.19 409
##STR00627## 0.008 0.037 410 ##STR00628## 0.009 0.057 411
##STR00629## 0.22 0.74 412 ##STR00630## 0.028 0.11 413 ##STR00631##
0.007 0.045 414 ##STR00632## 0.010 0.058 415 ##STR00633## 0.006
0.018 416 ##STR00634## 0.055 0.35 417 ##STR00635## 0.056 0.32 418
##STR00636## 0.14 0.32 419 ##STR00637## 0.024 0.064 420
##STR00638## 0.013 0.070 421 ##STR00639## 0.29 0.16 422
##STR00640## 0.007 0.006 423 ##STR00641## 0.022 0.042 424
##STR00642## 0.006 0.008 425 ##STR00643## 0.086 0.015 426
##STR00644## 0.011 0.033 427 ##STR00645## 0.007 0.027 428
##STR00646## 0.007 0.019 429 ##STR00647## 0.004 0.007 430
##STR00648## 0.009 0.027 431 ##STR00649## 0.007 0.026 432
##STR00650## 0.002 0.004 433 ##STR00651## 0.002 0.007 434
##STR00652## 0.005 0.017 435 ##STR00653## 0.002 0.006 436
##STR00654## 0.006 0.010 437 ##STR00655## 0.070 0.072 438
##STR00656## 0.74 0.88 439 ##STR00657## 0.25 0.056 440 ##STR00658##
0.008 0.031 441 ##STR00659## 0.011 0.18 442 ##STR00660## 0.007
0.025 443 ##STR00661## 0.011 0.10 444 ##STR00662## 0.003 0.008 445
##STR00663## 0.004 0.022 446 ##STR00664## 0.011 0.15 447
##STR00665## 0.005 0.016 448 ##STR00666## 0.005 0.051 449
##STR00667## 0.11 0.12 450 ##STR00668## 0.006 0.042 451
##STR00669## 0.003 0.056 452 ##STR00670## 0.004 0.049 453
##STR00671## 0.003 0.015 454 ##STR00672## 0.006 0.13 455
##STR00673## 0.003 0.012 456 ##STR00674## 0.003 0.024 457
##STR00675## 0.009 0.11 458 ##STR00676## 0.003 0.013 459
##STR00677## 0.048 0.57 460 ##STR00678## 0.005 0.031 461
##STR00679## 0.011 0.062 462 ##STR00680## 0.006 0.053 463
##STR00681## 0.052 0.96 464 ##STR00682## 0.005 0.059 465
##STR00683## 0.006 0.92 466 ##STR00684## 0.051 1.3 467 ##STR00685##
0.005 0.047 468 ##STR00686## 0.016 0.27 469 ##STR00687## 0.007
0.049 470 ##STR00688## 0.003 0.009 471 ##STR00689## 0.003 0.006 472
##STR00690## 0.006 0.024 473 ##STR00691## 0.002 0.006 474
##STR00692## 0.003 0.004 475 ##STR00693## 0.002 0.003 476
##STR00694## 0.004 0.012 477 ##STR00695## 0.005 0.015 478
##STR00696## 0.018 0.046 479 ##STR00697## 0.005 0.030 480
##STR00698## >20 6.3 481 ##STR00699## 0.004 0.012 482
##STR00700## 0.007 0.038 483 ##STR00701## 0.004 0.009 484
##STR00702## 0.003 0.011 485 ##STR00703## 0.004 0.012 486
##STR00704## 0.004 0.024 487 ##STR00705## 0.005 0.042 488
##STR00706## 0.32 1.9 489 ##STR00707## 0.008 0.023 490 ##STR00708##
0.011 0.25 491 ##STR00709## 0.008 0.023 492 ##STR00710## 0.006
0.014 493 ##STR00711## 0.019 0.057
494 ##STR00712## 0.019 0.58 495 ##STR00713## 0.005 0.014 496
##STR00714## 0.003 0.017 497 ##STR00715## 0.004 0.032 498
##STR00716## 0.003 0.017 499 ##STR00717## 0.010 0.19 500
##STR00718## 0.004 0.029 501 ##STR00719## 0.004 0.069 502
##STR00720## 0.007 0.075 503 ##STR00721## 0.008 0.15 504
##STR00722## 0.007 0.12 505 ##STR00723## 0.008 0.24 506
##STR00724## 0.010 0.17 507 ##STR00725## 0.013 0.041 508
##STR00726## 0.011 0.020 509 ##STR00727## 0.010 0.009 510
##STR00728## 0.022 0.094 511 ##STR00729## 0.58 1.1 512 ##STR00730##
0.005 0.046 513 ##STR00731## 0.007 0.022 514 ##STR00732## 0.009
0.063 515 ##STR00733## 0.007 0.059 516 ##STR00734## 0.003 0.028 517
##STR00735## 0.003 0.046 518 ##STR00736## 0.004 0.063 519
##STR00737## 0.009 0.059 520 ##STR00738## 0.007 0.056 521
##STR00739## 0.006 0.052 522 ##STR00740## 0.023 0.060 523
##STR00741## 0.021 0.055 524 ##STR00742## 525 ##STR00743## 526
##STR00744## 528 ##STR00745## 0.007 0.044 529 ##STR00746## 0.032
0.16 530 ##STR00747## 0.055 0.28 531 ##STR00748## 0.006 0.042 532
##STR00749## 0.006 0.059 533 ##STR00750## 0.007 0.041 534
##STR00751## 0.008 0.044 535 ##STR00752## 0.007 0.090 536
##STR00753## 0.006 0.071 537 ##STR00754## 0.007 0.076 538
##STR00755## 0.004 0.030 539 ##STR00756## 0.009 0.045 540
##STR00757## 0.007 0.050 541 ##STR00758## 0.004 0.006 542
##STR00759## 0.004 0.043 543 ##STR00760## 0.004 0.005 544
##STR00761## 0.006 0.044 545 ##STR00762## 0.006 0.046 546
##STR00763## 0.005 0.027 547 ##STR00764## 0.006 0.031 548
##STR00765## 0.010 0.085 549 ##STR00766## 0.006 0.045 550
##STR00767## 0.005 0.036 551 ##STR00768## 0.010 0.127 552
##STR00769## >20 0.005 553 ##STR00770## 0.005 0.019 554
##STR00771## 0.008 0.172 555 ##STR00772## 0.004 0.010 556
##STR00773## 0.005 0.12 557 ##STR00774## 0.025 0.12 558
##STR00775## 0.006 0.028 559 ##STR00776## 0.012 0.066 560
##STR00777## 0.010 0.037 561 ##STR00778## 0.004 0.004 562
##STR00779## 0.003 0.002 563 ##STR00780## 0.003 0.003 564
##STR00781## 0.004 0.002 565 ##STR00782## 0.005 0.013 566
##STR00783## 0.006 0.015 567 ##STR00784## 0.43 0.021 568
##STR00785## 0.009 0.028 569 ##STR00786## 0.006 0.011 570
##STR00787## 0.43 0.009 571 ##STR00788## 0.011 0.010 572
##STR00789## 0.003 0.004 573 ##STR00790## 0.004 0.015 574
##STR00791## 0.006 0.028 575 ##STR00792## 0.007 0.040 576
##STR00793## 0.003 0.013 577 ##STR00794## 0.004 0.034 578
##STR00795## 0.004 0.022 579 ##STR00796## 0.004 0.009 580
##STR00797## 0.005 0.013 581 ##STR00798## 0.011 0.24 582
##STR00799## 0.005 0.046 583 ##STR00800## 0.005 0.042 584
##STR00801## 0.22 1.4 585 ##STR00802## 0.006 0.070 586 ##STR00803##
0.013 0.031 587 ##STR00804## 0.007 0.057 588 ##STR00805## 0.008
0.27 589 ##STR00806## 0.004 0.025 590 ##STR00807## 0.007 0.087 591
##STR00808## 0.004 0.033 592 ##STR00809## 0.004 0.011 593
##STR00810## 0.005 0.033 594 ##STR00811## 0.007 0.050 595
##STR00812## 0.007 0.059 596 ##STR00813## 0.015 0.33 597
##STR00814## 0.005 0.017 598 ##STR00815## 0.005 0.004 599
##STR00816## 0.010 0.039 600 ##STR00817## 0.005 0.008 601
##STR00818## 0.006 0.036 602 ##STR00819## 0.006 0.036 603
##STR00820## 0.009 0.023 604 ##STR00821## 0.015 0.042 605
##STR00822## 0.013 0.018 606 ##STR00823## 0.007 0.045 607
##STR00824## 0.007 0.047 608 ##STR00825## 0.007 0.037 609
##STR00826## 0.009 0.014 610 ##STR00827## 0.005 0.011 611
##STR00828## 0.006 0.040 612 ##STR00829## 0.065 0.10 613
##STR00830## 0.019 0.45 614 ##STR00831## 0.008 0.082 615
##STR00832## 0.009 0.12 616 ##STR00833## 0.008 0.13 617
##STR00834## 0.005 0.040 618 ##STR00835## 0.008 0.035 619
##STR00836## 0.013 0.15
620 ##STR00837## 0.005 0.011 621 ##STR00838## 0.005 0.020 622
##STR00839## 0.004 0.010 623 ##STR00840## 0.003 0.026 624
##STR00841## 0.004 0.009 625 ##STR00842## 0.004 0.006 626
##STR00843## 0.004 0.017 627 ##STR00844## 0.028 0.85 628
##STR00845## 0.027 0.17 629 ##STR00846## >20 0.065 630
##STR00847## 0.004 0.009 631 ##STR00848## 0.005 0.006 632
##STR00849## 0.010 0.20 633 ##STR00850## 0.007 0.13 634
##STR00851## 0.006 0.048 635 ##STR00852## 0.005 0.030 636
##STR00853## 0.008 0.059 637 ##STR00854## >20 >50 638
##STR00855## 0.48 5.7 639 ##STR00856## 0.17 23 640 ##STR00857##
0.12 0.070 641 ##STR00858## 0.14 0.50 644 ##STR00859## 0.003 0.013
645 ##STR00860## 0.002 0.015 646 ##STR00861## 0.007 0.037 647
##STR00862## 0.004 0.018 648 ##STR00863## 0.004 0.011 649
##STR00864## 0.004 0.034 650 ##STR00865## 0.013 0.14 651
##STR00866## 0.006 0.037 652 ##STR00867## 0.004 0.039 653
##STR00868## 0.005 0.010 654 ##STR00869## 0.005 0.007 655
##STR00870## 0.019 0.35 656 ##STR00871## 0.018 0.40 657
##STR00872## 0.24 1.5 658 ##STR00873## 0.005 0.040 659 ##STR00874##
0.010 0.058 660 ##STR00875## 0.025 0.037 661 ##STR00876## 0.007
0.12 662 ##STR00877## 0.007 0.055 663 ##STR00878## 0.007 0.089 664
##STR00879## 0.005 0.060 665 ##STR00880## 0.005 0.10 666
##STR00881## 0.004 0.058 667 ##STR00882## 0.004 0.11 668
##STR00883## 0.009 0.026 669 ##STR00884## 0.021 0.026 670
##STR00885## 0.005 0.030 671 ##STR00886## 0.004 0.035 672
##STR00887## 0.010 0.045 673 ##STR00888## 0.006 0.033 674
##STR00889## 0.008 0.024 675 ##STR00890## 0.040 676 ##STR00891##
0.030 677 ##STR00892## 0.056 678 ##STR00893## 0.026 679
##STR00894## 0.036 680 ##STR00895## 0.033 681 ##STR00896## 0.019
682 ##STR00897## 0.017 683 ##STR00898## 0.024 684 ##STR00899##
0.042 685 ##STR00900## 0.022 686 ##STR00901## 0.010 687
##STR00902## 0.011 688 ##STR00903## 0.012 689 ##STR00904## 0.013
690 ##STR00905## 0.017 692 ##STR00906## 0.020 693 ##STR00907##
0.070 694 ##STR00908## 0.029 695 ##STR00909## 0.030 696
##STR00910## 0.034 697 ##STR00911## 0.050 698 ##STR00912## 0.098
699 ##STR00913## 0.12 700 ##STR00914## 0.17 701 ##STR00915## 0.11
702 ##STR00916## 0.31 703 ##STR00917## 0.012 704 ##STR00918## 0.88
705 ##STR00919## 0.032 706 ##STR00920## 14 707 ##STR00921## 0.085
708 ##STR00922## 2.8 709 ##STR00923## 0.14
Example 3: Xenograft Efficacy Studies
[0516] Certain compounds were assayed for in vivo efficacy in
xenograft models as follows.
[0517] Female scid/bg mice, approximately 6 weeks of age, were
implanted subcutaneously on the right flank with 5.times.10.sup.6
HCT116 cells per mouse in a volume of 100 uL of sterile PBS. When
tumors reached a volume of 50-100 mm.sup.3, mice were randomized to
groups of n=10 to receive either vehicle or test compound delivered
twice daily by intraperitoneal injection. Tumors were measured
three times per week using Vernier calipers and tumor volume
calculated using the formula: Volume=(Length.times.Width.sup.2/2),
where length and width are the longest perpendicular sides of the
tumor. Dosing continued twice daily until control tumors reached a
size of 2000 mm.sup.3. Statistical comparisons were made using a
2-way ANOVA with Bonferroni post-test.
[0518] FIG. 1 shows that intraperitoneal administration of compound
188 to mice results in reduced tumor size in this HCT116 colon
carcinoma xenograft model.
Example 4: Caco-2 Permeability Assay
[0519] Caco-2 cells are commonly used in a confluent monolayer on a
cell culture insert filter. When cultured in this format and under
specific conditions, the cells become differentiated and polarized
such that their phenotype, morphologically and functionally
resembles the enterocytes lining the small intestine. The cell
monolayer provides a physical and biochemical barrier to the
passage of small molecules, and is widely used across the
pharmaceutical industry as an in vitro model of the human small
intestinal mucosa to predict the absorption of orally administered
drugs (Hidalgo et al., Gastroenterology, 1989; Artursson, J. Pharm.
Sci., 1990). The correlation between the in vitro apparent
permeability (P-app) across Caco-2 monolayers and the in vivo
absorption is well established (Artursson et al., Biochem. Biophys.
Res. Comm., 1991).
[0520] The present assay was used to determine the bidirectional
permeability of the compounds of the invention through Caco-2 cell
monolayers. Caco-2 cells were grown in confluent monolayers where
the media of both the apical (A) and basolateral (B) sides were at
pH 7.4. Compounds were dosed at 1 M in the presence of 200 M
Lucifer Yellow, on the apical side (A.fwdarw.B) or the basolateral
side (B.fwdarw.A) for assessment, in duplicate. Samples from both A
and B sides were taken after 120 minutes exposure, and compound
concentration (reported as percent recovery) was determined using a
generic LC-MS/MS method with a minimum four-point calibration
curve.
[0521] The absorption potential of compounds were classified as
either Low (P-app <1.times.10.sup.-6 cm/s) or High (P-app
>1.times.10.sup.-6 cm/s). The efflux ratio was calculated as
(Papp B.fwdarw.A)/(Papp A.fwdarw.B), with efflux ratios being
significant when greater than or equal to 3 when the Papp
(B.fwdarw.A) was greater than or equal to 1.times.10.sup.-6 cm/s.
Results for certain compounds of the invention are shown in Table
4.
TABLE-US-00004 TABLE 4 Caco-2 Permeability Results Recovery Papp
Efflux Permeability Significant Cmpd Direction (%) (avg.) Ratio
Classification Efflux 533 A.fwdarw.B 41 4.94 7.6 High Yes
B.fwdarw.A 52 37.5 585 A.fwdarw.B 42 7.52 3.1 High Yes B.fwdarw.A
53 23.4 616 A.fwdarw.B 65 8.23 6.0 High Yes B.fwdarw.A 76 49.5 295
A.fwdarw.B 89 8.17 7.3 High Yes B.fwdarw.A 96 59.8 318 A.fwdarw.B
73 2.45 18 High Yes B.fwdarw.A 82 44.5 339 A.fwdarw.B 73 2.39 17
High Yes B.fwdarw.A 80 41.6 354 A.fwdarw.B 117 1.38 33 High Yes
B.fwdarw.A 101 45.0 436 A.fwdarw.B 44 3.75 6.6 High Yes B.fwdarw.A
57 24.7 660 A.fwdarw.B 56 0.61 3.9 Low Yes B.fwdarw.A 68 2.37 670
A.fwdarw.B 70 9.64 6.2 High Yes B.fwdarw.A 72 59.6 679 A.fwdarw.B
34 7.59 2.6 High No B.fwdarw.A 42 19.6 447 A.fwdarw.B 71 7.76 3.5
High Yes B.fwdarw.A 56 27.2 703 A.fwdarw.B 51 6.26 6.6 High Yes
B.fwdarw.A 66 41.0 705 A.fwdarw.B 60 8.52 6.0 High Yes B.fwdarw.A
67 51.0
Example 5: Solubility
[0522] Ca. 1 mg portions of test article were combined with 120
.mu.L solvent in wells of a 96.times.2 mL polypropylene plate. The
plate was vigorously vortex mixed at room temperature (ca. 20 C)
for 18 hr and each well checked visually for undissolved solid;
wells containing no visible solid were charged with additional
solid test article and vortex mixed another 6 hr at room
temperature after which all wells showed visible solid. The
contents of all wells were then filtered through a 0.45 m GHP
filter plate to yield clear filtrates. 5 .mu.L of each filtrate was
diluted into 100 .mu.L DMF and vortex mixed to yield HPLC samples.
Duplicate quantitation standards for each test article were
prepared by diluting weighed portions of solid test article in
measured volumes of DMF. 2 .mu.L of each HPLC sample and
quantitation standard were analyzed by HPLC using the method
outlined in Table 5. Dissolved test article concentrations were
calculated by peak area ratio against the appropriate quantitation
standards. Solubility results are presented in Table 6.
TABLE-US-00005 TABLE 5 Outline of HPLC Method Instrument Shimadzu
Prominence UFLC with Diode Array UV/Vis Detector Column VWR Sonoma
C8(2), 3.5 .mu.m, 2.1 .times. 50 mm Column 40.degree. C. Temp
Mobile 0.1% (v/v) formic acid in water Phase A Mobile 0.1% (v/v)
formic acid in acetonilrile Phase B Flow Rate 0.4 mL/min Gradient
Time (min) % Mobile Phase B 0 20 8 100 8.5 100 8.6 20 9.6 END
TABLE-US-00006 TABLE 6 Measured Solubilities Solvent Solubility
(mg/mL) 1 295 402 585 water <0.002 <0.002 <0.004 <0.002
0.9% NaCl <0.002 <0.002 <0.004 <0.002 0.1M HCl
<0.002 0.003 <0.004 <0.002 50 mM Cit <0.002 <0.002
<0.004 <0.002 pH 2.3 50 mM Cit <0.002 <0.002 <0.004
<0.002 pH 3.3 50 mM Cit <0.002 <0.002 <0.004 <0.002
pH 4.4 50 mM Cit <0.002 <0.002 <0.004 <0.002 pH 5.4 PBS
<0.002 <0.002 <0.004 <0.002 0.1M NaOH 14.420 0.268
<0.004 0.192 10% PS80/ 0.050 0.027 0.153 0.261 50 mM cit 10%
CrEL/ 0.076 0.055 0.157 0.228 50 mM cit 20% SBECD/ 0.046 0.090
0.019 0.125 50 mM cit 20% HPBCD/ 0.042 0.167 0.056 0.327 50 mM cit
Labrasol 0.258 0.918 31.032 5.004 Caprvol 0.042 1.540 11.210 1.780
PGMC Caprvol 90 0.081 0.215 13.676 1.744 canola oil <0.002
<0.002 0.529 0.072 PEG400 0.451 1.644 30.179 3.944 PG 0.048
0.234 1.365 1.422 EtOH 0.040 0.083 2.958 1.991 670 447 703 water
0.007 <0.004 <0.004 0.9% NaCl <0.002 0.005 <0.004 0.1M
HCl 0.005 <0.004 <0.004 50 mM Cit 0.066 <0.004 <0.004
pH 2.3 50 mM Cit 0.003 <0.004 <0.004 pH 3.3 50 mM Cit
<0.002 <0.004 <0.004 pH 4.4 50 mM Cit <0.002 <0.004
<0.004 pH 5.4 PBS <0.002 <0.004 <0.004 0.1M NaOH 0.227
0.192 0.656 10% PS80/ 1.204 0.851 0.378 50 mM cit 10% CrEL/ 0.458
0.732 0.309 50 mM cit 20% SBECD/ 5.256 2.718 0.476 50 mM cit 20%
HPBCD/ 9.685 2.177 0.651 50 mM cit Labrasol 5.042 77.164 20.727
Caprvol 1.519 7.916 3.683 PGMC Caprvol 90 1.974 11.114 7.409 canola
oil 0.012 0.071 0.014 PEG400 9.901 57.334 22.419 PG 2.569 8.265
4.698 EtOH 0.964 3.921 2.645
INCORPORATION BY REFERENCE
[0523] All publications and patents mentioned herein are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference. In case of conflict, the present
application, including any definitions herein, will control.
EQUIVALENTS
[0524] While specific embodiments of the subject invention have
been discussed, the above specification is illustrative and not
restrictive. Many variations of the invention will become apparent
to those skilled in the art upon review of this specification and
the claims below. The full scope of the invention should be
determined by reference to the claims, along with their full scope
of equivalents, and the specification, along with such
variations.
* * * * *