Note: Descriptions are shown in the official language in which they were submitted.
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HISTONE DEACETYLASE INHIBITORS
TECHNICAL FIELD
This invention relates to enzyme inhibitors, and more particularly to histone
deacetylase inhibitors.
BACKGROUND
DNA in the nucleus of the cell exists as a hierarchy of compacted chromatin
structures. The basic repeating unit in chromatin is the nucleosome. The
nucleosome
consists of a histone octomer of proteins in the nucleus of the cell around
which DNA is
twice wrapped. The orderly packaging of DNA in the nucleus plays an important
role in the
functional aspects of gene regulation. Covalent modifications of the histones
have a key role
in altering chromatin higher order structure and function and ultimately gene
expression.
The covalent modification of histones occurs by enzymatically mediated
processes, such as
acetylation.
Regulation of gene expression through the inhibition of the nuclear enzyme
histone
deacetylase (HDAC) is one of several possible regulatory mechanisms whereby
chromatin
activity can be affected. The dynamic homeostasis of the nuclear acetylation
of histones can
be regulated by the opposing activity of the enzymes histone acetyl
transferase (HAT) and
histone deacetylase (HDAC). Transcriptionally silent chromatin can be
characterized by
nucleosomes with low levels of acetylated histones. Acetylation of histones
reduces its
positive charge, thereby expanding the structure of the nucleosome and
facilitating the
interaction of transcription factors to the DNA. Removal the acetyl group
restores the
positive charge condensing the structure of the nucleosome. Acetylation of
histone-DNA
activates transcription of DNA's message, an enhancement of gene expression.
Histone
deacetylase can reverse the process and can serve to repress gene expression.
See, for
example, Grunstein, Nature 389, 349-352 (1997); Pazin et al., Cell 89, 325-328
(1997);
Wade et al., Trends Biochem. Sci. 22, 128-132 (1997); and Wolffe, Science 272,
371-372
(1996).
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SUMMARY
Histone deacetylase is a rnetallo-enzyme with zinc at the active site.
Compounds
having a zinc-binding moiety, such as, for example, a hydroxamic acid group or
a carboxylic
acid group, can inhibit histone deacetylase. Histone deacetylase inhibition
can repress gene
expression, including expression of genes related to tumor suppression.
Accordingly,
inhibition of histone deacetylase can provide an alternate route for treating
cancer,
hematological disorders, e.g., hemoglobinopathies, and genetic related
metabolic disorders,
e.g., cystic fibrosis and adrenoleukodystrophy.
In one aspect, a method of inhibiting histone deacetylation activity in cells
includes
contacting the cells with an effective amount of a compound of formula (I),
thereby treating
one or more disorders mediated by histone deacetylase, and determining whether
the level of
acetylated histones in the treated cells is higher than in untreated cells
under the same
conditions.
The compound of formula (I) can be the following:
X1
1 11 2
A -Y L Y2 C X (I)
A is a cyclic moiety selected from the group consisting of C3_14 cycloalkyl, 3-
14
membered heterocycloalkyl, C4_14 cycloalkenyl, 4-14 membered
heterocycloalkenyl, aryl, or
heteroaryl; the cyclic moiety being optionally substituted with alkyl,
alkenyl, alkynyl,
alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, alkylcarbonyloxy,
alkyloxycarbonyl, alkylcarbonyl, alkylsulfonylamino, aminosulfonyl, or
alkylsulfonyl; or A
is a saturated branched C3_12 hydrocarbon chain or an unsaturated branched
C3_12 hydrocarbon
chain optionally interrupted by -0-, -S-, -N(Ra)-, -C(O)-, -N(Ra)-SO2-, -SO2-
N(Ra)-,
-N(Ra)-C(O)-O-, -O-C(O)-N(Ra)-, -N(Ra)-C(O)-N(Rb)-, -0-C(O)-, -C(O)-O-, -O-SO2-
,
-S02-0-, or -0-C(O)-0-, where each of Ra and Rb, independently, is hydrogen,
alkyl,
alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl; each of the
saturated and the
unsaturated branched hydrocarbon chain being optionally substituted with
alkyl, alkenyl,
alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino,
alkylcarbonyloxy,
alkyloxycarbonyl, alkylcarbonyl, alkylsulfonylamino, aminosulfonyl, or
alkylsulfonyl. Each
of Y1 and Y2, independently, is -CH2-, -0-, -S-, -N(R )-, -N(R )-C(O)-O-, -0-
C(0)-N(R )-,
-2-
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-N(R )-C(O)-N(Rd)-, -O-C(O)-O-, or a bond, and each of Rc and Rd,
independently, being
hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or
haloalkyl. L is a
straight C2-12 hydrocarbon chain optionally containing at least one double
bond, at least one
triple bond, or at least one double bond and one triple bond; said hydrocarbon
chain being
optionally substituted with C1-4 alkyl, C2_4 alkenyl, C2_4 alkynyl, C1.4
alkoxy, hydroxyl, halo,
amino, nitro, cyano, C3-5 cycloalkyl, 3-5 membered heterocycloalkyl,
monocyclic aryl, 5-6
membered heteroaryl, C1-4 alkylcarbonyloxy, C1-4 alkyloxycarbonyl, C1-4
alkylcarbonyl, or
formyl; and further being optionally interrupted by -0-, -N(Re)-, -N(Re)-C(O)-
O-,
-O-C(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, or -O-C(O)-O-; each of Re and R,
independently,
being hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or
haloalkyl. X1 is
O or S. X2 is -OR', -SRI, -NR3-ORI, -NR3-SRI, -C(O)-ORI, -CHR4-ORI,
-N N-C(O)-N(R3)2, or -O-CHR4-O-C(O)-R5, where each of RI and R2,
independently, is
hydrogen, alkyl, hydroxylalkyl, haloalkyl, or a hydroxyl protecting group, R3
is hydrogen,
alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, haloalkyl, or an
amino protecting
group, R4 is hydrogen, alkyl, hydroxylalkyl, or haloalkyl, and R5 is alkyl,
hydroxylalkyl, or
haloalkyl. When L is a C2-3 hydrocarbon containing no double bonds and X2 is -
ORI, YI is
not a bond and Y2 is not a bond.
In another aspect, a method of inhibiting histone deacetylase in cells
comprising
contacting the cells with an effective amount of a compound of formula (I),
supra, and
determining whether the level of acetylated histones in the treated cells is
higher than in
untreated cells under the same conditions. A is phenyl optionally substituted
with alkyl
alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, or amino.
Each of YI and
Y2, independently, is -CH2-, -0-, -S-, -N(R )-, or a bond; where R is
hydrogen, alkyl,
alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl. L is a
straight C2-12
hydrocarbon chain optionally containing at least one double bond, at least one
triple bond, or
at least one double bond and one triple bond, the hydrocarbon chain being
optionally
substituted with C1_4 alkyl, C24 alkenyl, C2-4 alkynyl, C1-4 alkoxy, hydroxyl,
halo, amino,
nitro, cyano, C3-5 cycloalkyl, 3-5 membered heterocycloalkyl, monocyclic aryl,
5-6
membered heteroaryl, C1-4 alkylcarbonyloxy, C1.4 alkyloxycarbonyl, CI-4
alkylcarbonyl, or
formyl; and further being optionally interrupted by -0-, -N(Re)-, -N(Re)-C(O)-
O-,
-O-C(O)-N(Re)-, -N(Re)-C(O)-N(R)-, or -O-C(O)-O-, and each of Re and Rf,
independently,
-3-
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being hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or
haloalkyl. X1 is
O or S. X2 is -OR1, -SR1, -NR3-OR', -NR3-SR1, -C(O)-OR', -CHR4-OR',
-N=N-C(O)-N(R3)2, or -O-CHR4-O-C(O)-R5, where each of R1 and R2,
independently, is
hydrogen, alkyl, hydroxylalkyl, haloalkyl, or a hydroxyl protecting group, R3
is hydrogen,
alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, haloalkyl, or an
amino protecting
group, R4 is hydrogen, alkyl, hydroxylalkyl, or haloalkyl, and R5 is alkyl,
hydroxylalkyl, or
haloalkyl. When L is a C2-3 hydrocarbon containing no double bonds and X2 is -
OR1, Y1 is
not a bond and Y2 is not a bond.
In yet another aspect, a method of treating a histone deacetylase-mediated
disorder
includes administering to a subject in need thereof a therapeutically
effective amount of
compound of formula (I), supra.
In certain embodiments, X2 can be -OR', -NR3-OR1, -C(O)-OR', -CHR4-OR1, or
-O-CHR4-O-C(O)-R5, X2 can be -OR', -NR3-OR', -C(O)OR1, or -O-CHR4-O-C(O)-R5,
each
of Y1 and Y2, independently, can be -CH2-, -0-, -N(Rc)-, or a bond, L can be a
saturated
hydrocarbon chain, such as a C3_8 hydrocarbon chain substituted with C1-2
alkyl, C1-2 alkoxy,
hydroxyl, -NH2, -NH(C1-2 alkyl), or -N(C1-2 alkyl)2.
In other embodiments, L can be an unsaturated hydrocarbon chain containing at
least
one double bond and no triple bond, such as an unsaturated C4_8 hydrocarbon
chain
substituted with C1-2 alkyl, C1-2 alkoxy, hydroxyl, -NH2, -NH(C1-2 alkyl), or -
N(C1-2 alkyl)2.
In other embodiments, L can be an unsaturated hydrocarbon chain containing at
least one
double bond and one triple bond, such as an unsaturated C4-8 hydrocarbon chain
substituted
with C1_2 alkyl, C1-2 alkoxy, hydroxyl, -NH2, -NH(C1-2 alkyl), or -N(Cl_2
alkyl)2. The double
bond can be in trans configuration.
In certain embodiments, A can be a C5-8 cycloalkenyl or 5-8 membered
heteroalkenyl
containing at least one double bond. A can be phenyl, naphthyl, indanyl, or
tetrahydronaphthyl, or A can be phenyl optionally substituted with alkyl
alkenyl, alkynyl,
alkoxy, hydroxyl,.hydroxylalkyl, halo, haloalkyl, or amino. In other
embodiments, A can
contain only double bonds.
Set forth below are some examples of a compound that can be employed in the
methods of the present invention: 5-phenyl-2,4-pentadienoic acid, 3-methyl-5-
phenyl-2,4-
pentadienoic acid, 4-methyl-5-phenyl-2,4-pentadienoic acid, 4-chloro-5-phenyl-
2,4-
-4-
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pentadienoic acid, 5-(4-dimethylaminophenyl)-2,4-pentadienoic acid, 5-(2-
furyl)-2,4-
pentadienoic acid, 5-phenyl-2-en-4-yn-pentanoic acid, 6-phenyl-3,5-hexadienoic
acid, 7-
phenyl-2,4,6-heptatrienoic acid, 8-phenyl-3,5,7-octatrienoic acid, potassium 2-
oxo-6-phenyl-
3,5-hexadienoate, potassium 2-oxo-8-phenyl-3,5,7-octatrienoate,
cinnamoylhydroxamic acid,
methyl-cinnamoylhydroxamic acid, 4-cyclohexanebutyroylhydroxamic acid,
benzylthioglycoloylhydroxamic acid, 5-phenylpentanoylhydroxamic acid, 5-phenyl-
2,4-
pentadienoylhydroxamic acid, N-methyl-5-phenyl-2,4-pentadienoylhydroxamic
acid, 3-
methyl-5-phenyl-2,4-pentadienoylhydroxamnic acid, 4-methyl-5-phenyl-2,4-
pentadienoyl
hydroxamic acid, 4-chloro-5-phenyl-2,4-pentadienoylhydroxamic acid, 5-(4-
dimethylaminophenyl)-2,4-pentadienoylhydroxamic acid, 5-phenyl-2-en-4-yn-
pentanoylhydroxamic acid, 5-(2-furyl)-2,4-pentadienoylhydroxamic acid, 6-
phenylhexanoylhydroxamic acid, 6-phenyl-3,5-hexadienoylhydroxamic acid, N-
methyl-6-
phenyl-3,5-hexadienoylhydroxamic acid, 7-phenylheptanoylhydroxamic acid, 7-
phenyl-
2,4,6-hepta-trienoylhydroxamic acid or 8-phenyloctanoylhydroxamic acid.
In another aspect, hydroxamic acid-containing compounds have a structure of
formula (I):
X1
II
A -Y1 L Y2- C N X2 R2 (I)
11
R
A is a cyclic moiety selected from the group consisting of C3_14 cycloalkyl, 3-
14 membered
heterocycloalkyl, C4.14 cycloalkenyl, 3-14 membered heterocycloalkenyl (e.g.,
C3_8
cycloalkyl, 3-8 membered heterocycloalkyl, C4_8 cycloalkenyl, 3-8 membered
heterocycloalkenyl), monocyclic aryl, or monocyclic heteroaryl. Each of these
cyclic
moieties is optionally substituted with alkyl, alkenyl, alkynyl, alkoxy,
hydroxyl,
hydroxylalkyl, halo, haloalkyl, amino, alkylcarbonyloxy, alkyloxycarbonyl,
alkylcarbonyl,
alkylcarbonylamino, aminocarbonyl, alkylsulfonylamino, aminosulfonyl, or
alkylsulfonyl.
Each of X1 and X2, independently, is 0 or S. Y1 is -CH2-, -0-, -S-, -N(Ra)-, -
N(Ra)-C(O)-O-,
-O-C(O)-N(Ra)-, -N(Ra)-C(O)-N(R)-, -0-C(O)-, -C(O)-O-, -O-C(O)-0-, or a bond
wherein
each of Ra and Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl,
alkoxy, hydroxylalkyl,
hydroxyl, or haloalkyl. Y2 is -CH2-, -0-, -5-, -N(Rc)-, -N(Rc)-C(0)-0-, -0-
C(O)-N(Rc)-,
-5-
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-N(Rc)-C(O)-N(Rd)-, -O-C(O)-, -C(O)-O-, or -O-C(O)-O- wherein each of Re and
Rd,
independently, is hydrogen, allcyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl,
hydroxyl, or
haloalkyl. L is (1) a saturated straight C1-12 hydrocarbon chain substituted
with C1_4 alkyl, C2-
4 alkenyl, C2-4 alkynyl, C14 alkoxy, halo, carboxyl, amino, nitro, cyano, C3-6
cycloalkyl, 3-6
membered heterocycloalkyl, monocyclic aryl, 5-6 membered heteroaryl, Cl_4
alkylcarbonyloxy, Cl_4 alkyloxycarbonyl, C1-4 alkylcarbonyl, formyl, C1_4
alkylcarbonylamino, or C1-4 aminocarbonyl, or at least two hydroxyl; and
further optionally
interrupted by -0-, -N(Re)-, -N(Re)-C(O)-0-, -O-C(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-
,
-O-C(O)-, -C(O)-O-, or -O-C(O)-O- wherein each of Re and RR, independently, is
hydrogen,
alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl; or L
is (2) an
unsaturated straight C4-12 hydrocarbon chain containing at least two double
bonds, at least
one triple bond, or at least one double bond and one triple bond, where the
unsaturated
hydrocarbon chain is optionally substituted with C1.4 alkyl, C2-4 alkenyl,
C2.4 alkynyl, Cl_4
alkoxy, hydroxyl, halo, carboxyl, amino, nitro, cyano, C3-6 cycloalkyl, 3-6
membered
heterocycloalkyl, monocyclic aryl, 5-6 membered heteroaryl, C1_4
alkylcarbonyloxy, C1_4
alkyloxycarbonyl, C1.4 alkylcarbonyl, formyl, C1_4 alkylcarbonylamino, or C1_4
aminocarbonyl; and further being optionally interrupted by -0-, -N(Rg)-, -
N(Rg)-C(O)-O-,
-O-C(O)-N(RR)-, -N(R9)-C(O)-N(R)-, -0-C(O)-, -C(O)-0-, or -O-C(O)-O- wherein
each of
R9 and Rh, independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy,
hydroxylalkyl,
hydroxyl, or haloalkyl. R1 is hydrogen, alkyl, alkenyl, alkynyl, alkoxy,
hydroxylalkyl,
hydroxyl, haloalkyl, or an amino protecting group; and R2 is hydrogen, alkyl,
hydroxylalkyl,
haloalkyl, or a hydroxyl protecting group.
In another aspect, hydroxamic acid-containing compounds have a structure of
formula (I), supra. A is a cyclic moiety selected from the group consisting of
monocyclic
aryl or monocyclic heteroaryl. Each of the cyclic moieties is optionally
substituted with
alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, or amino. Each of X1 and X2,
independently,
is 0 or S. Y' is -CH2-, -0-, -S-, -N(Ra)-, -N(Ra)-C(O)-O-, -O-C(O)-N(Ra)-,
-N(Ra)-C(O)-N(R)-, -0-C(O)-, -C(O)-0-, -0-C(O)-0-, or a bond, where each of Ra
and Rb,
independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylallcyl,
hydroxyl, or
haloalkyl. Y2 is -CH2-, -0-, -S-, -N(R )-, -N(Re)-C(O)-0-, -0-C(O)-N(R )-,
-N(R )-C(O)-N(Rd)-, -0-C(O)-, -C(O)-0-, or -0-C(O)-0-; each of Re and Rd,
independently,
-6-
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being hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or
haloalkyl. L is
(1) a saturated straight C3_10 hydrocarbon chain substituted with C1_4 alkyl,
C24 alkenyl, C2_4
alkynyl, C1.4 alkoxy, or amino, and further optionally interrupted by -0- or -
N(Re)-, where Re
is hydrogen, alkyl, hydroxylalkyl, or haloalkyl; or L is (2) an unsaturated
straight C4.10
hydrocarbon chain containing at least two double bonds, at least one triple
bond, or at least
one double bond and one triple bond; said unsaturated hydrocarbon chain being
optionally
substituted with C1_4 alkyl, C2-4 alkenyl, C2_4 alkynyl, C1_4 alkoxy, or
amino, and further
optionally interrupted by -0- or -NW)-, where Rf is hydrogen, alkyl,
hydroxylalkyl, or
haloalkyl. Each of R1 and R2, independently, is hydrogen, alkyl,
hydroxylalkyl, or haloalkyl.
In certain embodiments, R1 is hydrogen, R2 is hydrogen, X1 is 0, X2 is 0, or
Y1 is
-CH2-, -0-, -N(Ra)-, or a bond, and Y2 is -CH2-, -0-, or -N(Rc)-. L can be a
saturated straight
C4-10 hydrocarbon chain, or C5_8 hydrocarbon chain (e.g., a saturated straight
C5 hydrocarbon
chain, a saturated straight C6 hydrocarbon chain, or a saturated straight C7
hydrocarbon
chain), substituted with C1_4 alkyl, C2_4 alkenyl, C2_4 alkynyl, C1.4 alkoxy,
or amino, and
further optionally interrupted by -0- or -N(Re)-. In other embodiments, L is
an unsaturated
straight C4_10 hydrocarbon chain, or an unsaturated straight C4.8 hydrocarbon
chain,
containing 2-5 double bonds, or 1-2 double bonds and 1-2 triple bonds,
optionally substituted
with Cl.4 alkyl, C2_4 alkenyl, C2_4 alkynyl, or C1-4 alkoxy, and further being
optionally
interrupted by -O- or -N(R8)-. In certain embodiments, L can be -(CH=CH),,,-
where m is 2
or 3 or L can be -C -(CH=CH)ri where n is 1 or 2. A can be phenyl, furyl,
thienyl,
pyrrolyl, or pyridyl or A can be phenyl optionally substituted with alkyl,
alkenyl, alkynyl,
alkoxy, hydroxylalkyl, or amino.
In a further aspect, hydroxamic acid-containing compounds have a structure of
formula (II):
R3 R9 XI
= A C RC = CR6 C=C R7C =CR3 C=C C ICN-X2-RZ (~.
b e e
14 d R10 f R1
A is a cyclic moiety selected from the group consisting of monocyclic aryl or
monocyclic
heteroaryl. Each of the cyclic moieties is optionally substituted with alkyl,
alkenyl, alkynyl,
alkoxy, hydroxylalkyl, or amino. Each of X1 and X2, independently, is 0 or S.
Each of R1
and R2, independently, is hydrogen, alkyl, hydroxylalkyl, or haloalkyl. Each
of R3, R4, R5,
-7-
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R6, R7, R8, R9 and R10, independently, is hydrogen, C1_4 alkyl, C2.4 alkenyl,
C2_4 alkynyl, C1.4
alkoxy, hydroxyl, halo, hydroxylCl_4 alkyl, haloC1_4 alkyl, or amino, and each
of a, b, c, d, e,
and f, independently, is 0 or 1. Note that at least one of b, c, d, and e
cannot be zero. In
certain embodiments, a is 0, f is 0, or the total number of b, c, d, and e is
3 or 4. In other
embodiments, each of R3, R4, R5, R6, R7, R8, R9 and R10, independently, is
hydrogen, CI.4
alkyl, CI-4 alkoxy, hydroxyl, hydroxylCl_4 alkyl, or amino. Each of R5, R6,
R7, and R8,
independently can be hydrogen, C1_4 alkyl, CI-4 alkoxy, hydroxyl, hydroxylC1_4
alkyl, or
amino, Each of R3, R4, R9 and R10, independently, can be hydrogen.
In another aspect hydroxamic acid-containing compounds have the structure of
formula (I), supra. A is a saturated branched C3_14 hydrocarbon chain or an
unsaturated
branched C3_14 hydrocarbon chain optionally interrupted by -0-, -S-, -N(Ra)-, -
C(O)-,
-N(Ra)-C(O)-, -C(O)-N(Ra)-, -N(Ra)-S02-, -SO2-N(Ra)-, -N(Ra)-C(O)-O-, -O-C(O)-
N(Ra)-,
-N(Ra)-C(O)-N(R)-, -O-C(O)-, -C(O)-O-, or -0-C(O)-0-, where each of Ra and Rb,
independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl,
hydroxyl, or
haloalkyl. Each of the saturated and the unsaturated branched hydrocarbon
chain is
optionally substituted with alkyl, alkenyl, alkynyl, alkoxy, hydroxyl,
hydroxylalkyl, halo,
haloalkyl, amino, alkylcarbonyloxy, alkyloxycarbonyl, alkylcarbonyl,
alkylcarbonylamino,
aminocarbonyl, alkylsulfonylamino, aminosulfonyl, or alkylsulfonyl. Each of X1
and X2,
independently, is 0 or S. Each of Y1 and Y2, independently, is -CH2-, -0-, -
N(Rc)-,
-N(Re)-C(O)-0-, -0-C(O)-N(R )-, -N(R )-C(O)-N(Rd)-, -O-C(O)-, -C(O)-0-, -0-
C(O)-O-, or
a bond, where each of R and Rd, independently, is hydrogen, alkyl, alkenyl,
alkynyl, alkoxy,
hydroxylalkyl, hydroxyl, or haloalkyl. L is a saturated straight C3_12
hydrocarbon or an
unsaturated straight C4_12 hydrocarbon chain, said hydrocarbon chain being
optionally
substituted with C1_4 alkyl, C2-4 alkenyl, C2-4 alkynyl, CI-4 alkoxy,
hydroxyl, halo, carboxyl,
amino, nitro, cyano, C3_6 cycloalkyl, 3-6 membered heterocycloalkyl,
monocyclic aryl, 5-6
membered heteroaryl, CI.4 alkylcarbonyloxy, C1_4 alkyloxycarbonyl, C1.4
alkylcarbonyl,
formyl, CI-4 alkylcarbonylamino, or C1_4 aminocarbonyl; and further optionally
interrupted
by -0-, -N(Re)-, -N(Re)-C(O)-O-, -O-C(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -0-C(O)-,
-C(O)-0-,
or -0-C(O)-0-, where each of Re and R f, independently, is hydrogen, alkyl,
alkenyl, alkynyl,
alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl. R1 is hydrogen, alkyl, alkenyl,
alkynyl,
-8-
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alkoxy, hydroxylalkyl, hydroxyl, haloalkyl, or an amino protecting group; and
R2 is
hydrogen, alkyl, hydroxylalkyl, haloalkyl, or a hydroxyl protecting group.
Set forth below are some examples of a hydroxamic acid-containing compound of
the
present invention: benzylthioglycoloylhydroxamic acid, N-methyl-5-phenyl-2,4-
pentadienoylhydroxamic acid, 3-methyl-5-phenyl-2,4-pentadienoyl hydroxamic
acid, 4-
methyl-5-phenyl-2,4-pentadienoylhydroxamic acid, 4-chloro-5-phenyl-2,4-
pentadienoylhydroxamic acid, 5-(4-diinethylaminophenyl)-2,4-
pentadienoylhydroxamic acid,
5-phenyl-2-en-4-yn-pentanoylhydroxamic acid, 5-(2-furyl)-2,4-
pentadienoylhydroxamic
acid, N-methyl-6-phenyl-3,5-hexadienoylhydroxamic acid, and 7-phenyl-2,4,6-
hepta-
trienoylhydroxamic acid.
In another aspect, carboxylic acid-containing compounds have a structure of
formula
(I):
X1
A Y1 L Y2 CI X2 H I
A is a cyclic moiety selected from the group consisting of C3_14 cycloalkyl, 3-
14 membered
heterocycloalkyl, C4_14 cycloalkenyl, 3-14 membered heterocycloalkenyl (e.g.,
C3_8
cycloalkyl, 3-8 membered heterocycloalkyl, C4_8 cycloalkenyl, 3-8 membered
heterocycloalkenyl), aryl, or heteroaryl. The cyclic moiety being optionally
substituted with
alkyl, alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl,
amino,
alkylcarbonyloxy, alkyloxycarbonyl, alkylcarbonyl, alkylsulfonylamino,
aminosulfonyl, or
alkylsulfonyl. Each of X1 and X2, independently, is 0 or S, and each of Y' and
Y2,
independently, is -CH2-, -0-, -S-, -N(Ra)-, -N(Ra)-C(O)-O-, -O-C(0)-N(Ra)-,
-N(Ra)-C(0)-N(R)-, -O-C(O)-0-, or a bond; each of Ra and Rb, independently,
being
hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or
haloalkyl;
L is a straight C3_12 hydrocarbon chain optionally containing at least one
double bond,
at least one triple bond, or at least one double bond and one triple bond. The
hydrocarbon
chain is optionally substituted with C1-4 alkyl, C2_4 alkenyl, C2-4 alkynyl,
C1. alkoxy,
hydroxyl, halo, amino, nitro, cyano, C3-5 cycloalkyl, 3-5 membered
heterocycloalkyl,
monocyclic aryl, 5-6 membered heteroaryl, C1_4 alkylcarbonyloxy, C14
alkyloxycarbonyl,
C1.4 alkylcarbonyl, or formyl; and is further optionally interrupted by -0-, -
N(R )-,
-N(R )-C(O)-0-, -0-C(0)-N(R )-, -N(Rc)-Q0)-N(Rd)-, or -0-C(O)-0-. Each of R
and Rd,
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independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl,
hydroxyl, or
haloalkyl. When L contains two or more double bonds, the double bonds are not
adjacent to
each other. Further, when L contains less than 6 carbon atoms in the
hydrocarbon chain, Y1
is not a bond.
In certain embodiments, A can be a C5_8 cycloalkenyl or 5-8 membered
heteroalkenyl
containing at least two double bond, A can be phenyl, naphthyl, indanyl, or
tetrahydronaphthyl, or A can be phenyl optionally substituted with alkyl
alkenyl, alkynyl,
alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, or amino.
In another aspect, carboxylic acid-containing compounds have a structure of
formula
(I), supra. A is a cyclic moiety selected from the group consisting of aryl or
heteroaryl. The
cyclic moiety is optionally substituted with alkyl, alkenyl, alkynyl, alkoxy,
hydroxylalkyl, or
amino. Each of X1 and X2, independently, is 0 or S, and each of Y1 and Y2,
independently,
is -CH2-, -0-, -S-, -N(Ra)-, -N(Ra)-C(O)-O-, -O-C(O)-N(Ra)-, -N(Ra)-C(O)-N(Rb)-
,
-0-C(O)-O-, or a bond; each of Ra and Rb, independently, being hydrogen,
alkyl,
hydroxylalkyl, or haloalkyl. L is a straight C3_12 hydrocarbon chain
optionally containing at
least one double bond, at least one triple bond, or at least one double bond
and one triple
bond. The hydrocarbon chain is optionally substituted with C1_4 alkyl, C2_4
alkenyl, C2.4
alkynyl, C1_4 alkoxy, or amino, and further optionally interrupted by -0- or -
N(Rc)-, where R
is hydrogen, alkyl, hydroxylalkyl, or haloalkyl. When L contains two or more
double bonds,
the double bonds are not adjacent to each other. Further, when L contains less
than 6 carbon
atoms in the hydrocarbon chain, Y' is not a bond.
In another aspect, carboxylic acid-containing compounds have a structure of
formula
(I), supra. A is a heteroaryl optionally substituted with alkyl, alkenyl,
alkynyl, alkoxy,
hydroxylalkyl, or amino. Each of X1 and X2, independently, is 0 or S, and each
of Y1 and
Y2, independently, is -CH2-, -0-, -S-, -N(Ra)-, -N(Ra)-C(O)-0-, -O-C(O)-N(Ra)-
,
-N(Ra)-C(O)-N(R)-, -0-C(O)-0-, or a bond; each of Ra and Rb, independently,
being
hydrogen, alkyl, hydroxylalkyl, or haloalkyl. L is a straight C3_12
hydrocarbon chain
optionally containing at least one double bond, at least one a triple bond, or
at least one
double bond and one triple bond. The hydrocarbon chain is optionally
substituted with Cl_4
alkyl, C2_4 alkenyl, C24 alkynyl, C1_4 alkoxy, or amino, and further
optionally interrupted by
-0- or -N(R )-, where R is hydrogen, alkyl, hydroxylalkyl, or haloalkyl.
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In another aspect carboxylic acid-containing compounds have the structure of
formula
(I), supra. A is a phenyl optionally substituted with alkyl, alkenyl, alkynyl,
alkoxy,
hydroxylalkyl, or amino. Each of X1 and X2, independently, is 0 or S, and each
of Y1 and
Y2, independently, is -CH2-, -0-, -S-, -N(Ra)-, -N(Ra)-C(O)-0-, -O-C(O)-N(Ra)-
,
-N(Ra)-C(O)-N(R)-, -O-C(O)-0-, or a bond. Each of Ra and Rb, independently,
being
hydrogen, alkyl, hydroxylalkyl, or haloalkyl. L is a straight C3_12
hydrocarbon chain
containing at least one double bond and one triple bond. The hydrocarbon chain
is optionally
substituted with C1_4 alkyl, C2-4 alkenyl, C2.4 alkynyl, C1.4 alkoxy, or
amino, and further
optionally interrupted by -0- or -N(Rc)-, where Re is hydrogen, alkyl,
hydroxylalkyl, or
haloalkyl.
In another aspect, carboxylic acid-containing compounds have a structure of
formula
(I), supra. A is a saturated branched C3_12 hydrocarbon chain or an
unsaturated branched
C3_12 hydrocarbon chain optionally interrupted by -0-, -S-, -N(Ra)-, -C(O)-, -
N(Ra)-SO2-,
-SO2-N(Ra)-, -N(Ra)-C(O)-O-, -O-C(O)-N(Ra)-, -N(Ra)-C(O)-N(R)-, -0-SO2-, -S02-
0-, or
-0-C(O)-0-. Each of R a and Rb, independently, is hydrogen, alkyl, alkenyl,
alkynyl, alkoxy,
hydroxylalkyl, hydroxyl, or haloalkyl. Each of the saturated and the
unsaturated branched
hydrocarbon chain is optionally substituted with alkyl, alkenyl, alkynyl,
alkoxy, hydroxyl,
hydroxylalkyl, halo, haloalkyl, amino, alkylcarbonyloxy, alkyloxycarbonyl,
alkylcarbonyl,
alkylsulfonylamino, aminosulfonyl, or alkylsulfonyl. Each of X1 and X2,
independently, is 0
or S, and each of Y' and Y2, independently, is -CH2-, -0-, -S-, -N(R )-, -C(O)-
, -N(Rc)-S02-,
-SO2-N(Rc)-, -N(R )-C(O)-0-, -0-C(O)-N(Rc)-, -N(Rc)-C(O)-N(Rd)-, -0-SO2-, -S02-
0-,
-O-C(O)-O-, or a bond. Each of Re and Rd, independently, is hydrogen, alkyl,
alkenyl,
alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl. L is a straight C2_12
hydrocarbon
chain optionally containing at least one double bond, at least one a triple
bond, or at least one
double bond and one triple bond. The hydrocarbon chain is optionally
substituted with C1-4
alkyl, C2.4 alkenyl, C2_4 alkynyl, C1_4 alkoxy, hydroxyl, halo, amino, nitro,
cyan, C3_5
cycloalkyl, 3-5 membered heterocycloalkyl, monocyclic aryl, 5-6 membered
heteroaryl, C1_4
alkylcarbonyloxy, C1-4 alkyloxycarbonyl, C1-4 alkylcarbonyl, or formyl; and is
further
optionally interrupted by -0-, -S-, -N(Re)-, -C(O)-, -N(Re)-SO2-, -SO2-N(Re)-,
-N(Re)-C(O)-O-, -O-C(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -0-SO2-, -S02-O-, or -0-
C(O)-O-.
Each of Re and R f, independently, is hydrogen, alkyl, alkenyl, alkynyl,
alkoxy, hydroxylalkyl,
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hydroxyl, or haloalkyl. When L contains two or more double bonds, the double
bonds are
not adjacent to each other. Further, A must contain a heteroatom selected from
the group
consisting of 0, S, or N or a double or triple bond. A can be furyl, thienyl,
pyrrolyl, or
pyridyl.
In certain embodiments, X1 can be 0, X2 can be 0, or each of Y' and Y2,
independently, can be -CH2-, -0-, -N(Ra)-, or a bond. L can be a saturated
C3_8 hydrocarbon
chain optionally substituted with C1_2 alkyl, C1.2 alkoxy, hydroxyl, -NH2, -
NH(C1.2 alkyl), or
-N(C1-2 alkyl)2. In other embodiments, L can be an unsaturated C4_8
hydrocarbon chain
containing at least one double bond and no triple bond, at least one double
bond and one
triple bond, or only double bonds. The unsaturated hydrocarbon chain can be
optionally
substituted with C1_2 alkyl, C1.2 alkoxy, hydroxyl, -NH2, -NH(C1.2 alkyl), or -
N(C1_2 alkyl)2.
When present, the double bond can be in trans configuration.
Set forth below are some examples of a carboxylic acid-containing compound of
the
present invention: 4-chloro-5-phenyl-2,4-pentadienoic acid, 5-(4-
dimethylaminophenyl)-2,4-
pentadienoic acid, 5-(2-furyl)-2,4-pentadienoic acid, 5-phenyl-2-en-4-yn-
pentanoic acid, 7-
phenyl-2,4,6-heptatrienoic acid, and 8-phenyl-3,5,7-octatrienoic acid.
A salt of any of the compounds of the invention can be prepared. For example,
a
pharmaceutically acceptable salt can be formed when an amino-containing
compound of this
invention reacts with an inorganic or organic acid. Some examples of such an
acid include
hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,
phosphoric acid, p-
bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic
acid, and acetic
acid. Examples of pharmaceutically acceptable salts thus formed include
sulfate, pyrosulfate
bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,
dihydrogenphosphate,
metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate,
decanoate,
caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate,
oxalate, malonate,
succinate, suberate, sebacate, fumarate, and maleate. A compound of this
invention may also
form a pharmaceutically acceptable salt when a compound of this invention
having an acid
moiety reacts with an inorganic or organic base. Such salts include those
derived from
inorganic or organic bases, e.g., alkali metal salts such as sodium,
potassium, or lithium salts;
alkaline earth metal salts such as calcium or magnesium salts; or ammonium
salts or salts of
organic bases such as morpholine, piperidine, pyridine, dimethylamine, or
diethylamine salts.
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It should be recognized that a compound of the invention can contain chiral
carbon
atoms. In other words, it may have optical isomers or diastereoisomers.
Alkyl is a straight or branched hydrocarbon chain containing 1 to 10
(preferably, 1 to
6; more preferably 1 to 4) carbon atoms. Examples of alkyl include, but are
not limited to,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-
pentyl, 2-
methylhexyl, and 3-ethyloctyl.
The terms "alkenyl" and "alkynyl" refer to a straight or branched hydrocarbon
chain
containing 2 to 10 carbon atoms and one or more (preferably, 1-4 or more
preferably 1-2)
double or triple bonds, respectively. Some examples of alkenyl and alkynyl are
allyl, 2-
butenyl, 2-pentenyl, 2-hexenyl, 2-butynyl, 2-pentynyl, and 2-hexynyl.
Cycloalkyl is a monocyclic, bicyclic or tricyclic alkyl group containing 3 to
14 carbon
atoms. Some examples of cycloalkyl are cyclopropyl, cyclopentyl, cyclohexyl,
cycloheptyl,
adamantyl, and norbornyl. Heterocycloalkyl is a cycloalkyl group containing at
least one
heteroatom (e.g., 1-3) such as nitrogen, oxygen, or sulfur. The nitrogen or
sulfur may
optionally be oxidized and the nitrogen may optionally be quaternized.
Examples of
heterocycloalkyl include piperidinyl, piperazinyl, tetrahydropyranyl,
tetrahydrofuryl, and
morpholinyl. Cycloalkenyl is a cycloalkyl group containing at least one (e.g.,
1-3) double
bond. Examples of such a group include cyclopentenyl, 1,4-cyclohexa-di-enyl,
cycloheptenyl, and cyclooctenyl groups. By the same token, heterocycloalkenyl
is a
cycloalkenyl group containing at least one heteroatom selected from the group
of oxygen,
nitrogen or sulfur.
Aryl is an aromatic group containing a 5-14 ring and can contain fused rings,
which
may be saturated, unsaturated, or aromatic. Examples of an aryl group include
phenyl,
naphthyl, biphenyl, phenanthryl, and anthracyl. If the aryl is specified as
"monocyclic aryl,"
if refers to an aromatic group containing only a single ring, i.e., not a
fused ring.
Heteroaryl is aryl containing at least one (e.g., 1-3) heteroatom such as
nitrogen,
oxygen, or sulfur and can contain fused rings. Some examples of heteroaryl are
pyridyl,
furanyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl,
benzofuranyl, and
benzthiazolyl.
The cyclic moiety can be a fused ring formed from two or more of the just-
mentioned
groups. Examples of a cyclic moiety having fused rings include fluorenyl,
dihydro-
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dibenzoazepine, dibenzocycloheptenyl, 7H-pyrazino[2,3-c]carbazole, or 9,10-
dihydro-9,10-
[2]buteno-anthracene.
Amino protecting groups and hydroxy protecting groups are well-known to those
in
the art. In general, the species of protecting group is not critical, provided
that it is stable to
the conditions of any subsequent reaction(s) on other positions of the
compound and can be
removed without adversely affecting the remainder of the molecule. In
addition, a
protecting group maybe substituted for another after substantive synthetic
transformations
are complete. Examples of an amino protecting group include, but not limited
to, carbamates
such as 2,2,2-trichloroethylcarbamate or tertbutylcarbamate. Examples of a
hydroxyl
protecting group include, but not limited to, ethers such as methyl, t-butyl,
benzyl, p-
methoxybenzyl, p-nitrobenzyl, allyl, trityl, methoxymethyl, 2-methoxypropyl,
methoxyethoxymethyl, ethoxyethyl, tetrahydropyranyl, tetrahydrothiopyranyl,
and
trialkylsilyl ethers such as trimethylsilyl ether, triethylsilyl ether,
dimethylarylsilyl ether,
triisopropylsilyl ether and t-butyldimethylsilyl ether; esters such as
benzoyl, acetyl,
phenylacetyl, formyl, mono-, di-, and trihaloacetyl such as chloroacetyl,
dichloroacetyl,
trichloroacetyl, trifluoroacetyl; and carbonates including but not limited to
alkyl carbonates
having from one to six carbon atoms such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, t-
butyl; isobutyl, and n-pentyl; alkyl carbonates having from one to six carbon
atoms and
substituted with one or more halogen atoms such as 2,2,2-trichloroethoxymethyl
and 2,2,2-
trichloro-ethyl; alkenyl carbonates having from two to six carbon atoms such
as vinyl and
allyl; cycloalkyl carbonates having from three to six carbon atoms such as
cyclopropyl,
cyclobutyl, cyclopentyl and cyclohexyl; and phenyl or benzyl carbonates
optionally
substituted on the ring with one or more C1_6 alkoxy, or nitro. Other
protecting groups and
reaction conditions can be found in T. W. Greene, Protective Groups in Organic
Synthesis,
(3rd, 1999, John Wiley & Sons, New York, N.Y.).
Note that an amino group can be unsubstituted (i.e., -NH2), mono-substituted
(i.e.,
-NHR), or di-substituted (i.e., -NR2). It can be substituted with groups (R)
such as alkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.
Halo refers to fluoro,
chloro, bromo, or iodo.
Inhibition of a histone deacetylase in a cell is determined by measuring the
level of
acetylated histones in the treated cells and measuring the level of acetylated
histones in
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untreated cells and comparing the levels. If the level of histone acetylation
in the treated
cells increases relative to the untreated cells, histone deacetylase has been
inhibited.
Some disorders or physiological conditions may be mediated by hyperactive
histone
deacetylase activity. A disorder or physiological condition that is mediated
by histone
deacetylase refers to a disorder or condition wherein histone deacetylase
plays a role in
triggering the onset thereof. Examples of such disorders or conditions
include, but not
limited to, cancer, hemoglobinopathies (e.g., thalassemia or sickle cell
anemia), cystic
fibrosis, protozoan infection, adrenoleukodystrophy, alpha-1 anti-trypsin,
retrovirus gene
vector reactivation, wound healing, hair growth, peroxisome biogenesis
disorder, and
adrenoleukodystrophy.
Other features or advantages will be apparent from the following detailed
description
of several embodiments, and also from the appended claims.
DETAILED DESCRIPTION
A carboxylic acid-containing compound of the present invention can be prepared
by
any known methods in the art. For example, a compound of the invention having
an
unsaturated hydrocarbon chain between A and -C(=X1)- can be prepared according
to the
following scheme:
0 0 X1
II II II
A-L' -C H + EtO-P-CH2-C-OH
EtO
X1
n-BuLi/THF 11
A-L' -CH =CH -C-OH
H30+
where L' is a saturated or unsaturated hydrocarbon linker between A and -CH=CH-
in a
compound of the invention, and A and X1 has the same meaning as defined above.
See'
Coutrot et al., Syn. Comm. 133-134 (1978). Briefly, butyllithium was added to
an
appropriate amount of anhydrous tetrahydrofuran (THF) at a very low
temperature (e.g.,
-65 C). A second solution having diethylphosphonoacetic acid in anhydrous THF
was added
dropwise to the stirred butyllithium solution at the same low temperature. The
resulting
solution is stirred at the same temperature for an additional 30-45 minutes
which is followed
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by the addition of a solution containing an aromatic acrylaldehyde in
anhydrous THE over 1-
2 hours. The reaction mixture is then warmed to room temperature and stirred
overnight. It
is then acidified (e.g., with HCl) which allows the organic phase to be
separated. The
organic phase is then dried, concentrated, and purified (e.g., by
recrystallization) to form an
unsaturated carboxylic acid.
Alternatively, a carboxylic acid-containing compound can be prepared by
reacting an
acid ester of the formula A-L'-C(=O)-O-lower alkyl with a Grignard reagent
(e.g., methyl
magnesium iodide) and a phosphorus oxychloride to form a corresponding
aldehyde, which
can be further oxidized (e.g., by reacting with silver nitrate and aqueous
NaOH) to form an
unsaturated carboxylic acid.
Other types of carboxylic acid-containing compounds (e.g., those containing a
linker
with multiple double bonds or triple bonds) can be prepared according to
published
procedures such as those described, for example, in Parameswara et al.,
Synthesis, 815-818
(1980) and Denny et al., J Org. Chem., 27, 3404 (1962).
Carboxylic acid-containing compounds described above can then be converted to
hydroxamic acid-containing compounds according to the following scheme:
11X1 I CH3 1~ 0
A-L' -C OH + H3C-C-CH2 O-C-CI
Xl
H2NOH.HC1 11
11 A-L' C-NHOH
DMF/TEA
Triethylamine (TEA) is added to a cooled (e.g., 0-5 C) anhydrous THE solution
containing
the carboxylic acid. Isobutyl chloroformate is then added to the solution
having carboxylic
acid, which is followed by the addition of hydroxylamine hydrochloride and
TEA. After
acidification, the solution was filtered to collect the desired hydroxamic
acid-containing
compound.
An N-substituted hydroxamic acid can be prepared in a similar manner as
described
above. A corresponding carboxylic acid A-L'-C(=O)-OH can be converted to an
acid
chloride by reacting with oxalyl chloride (in appropriate solvents such as
methylene chloride
and dimethylformamide), which in turn, can be converted to a desired N-
substituted
hydroxamic acid by reacting the acid chloride with an N-substituted
hydroxylamine
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hydrochloride (e.g., CH3NHOH=HCl) in an alkaline medium (e.g., 40% NaOH (aq))
at a low
temperature (e.g., 0-5 C). The desired N-substituted hydroxamic acid can be
collected after
acidifying the reaction mixture after the reaction has completed (e.g., in 2-3
hours).
As to compounds of the invention wherein X1 is S, they can be prepared
according to
procedures described in Sandler, S. R. and Karo, W., Organic Functional Group
Preparations, Volume III (Academic Press, 1972) at pages 436-437. For
preparation of
compounds of the invention wherein X2 is -N(R )OH- and X1 is S, see procedures
described
in U.S. Patent Nos. 5,112,846; 5,075,330 and 4,981,865.
Compounds of the invention containing an a-keto acid moiety (e.g., when X1 is
oxygen and X2 is -C(=O)OM or A-L'-C(=O)-C(=O)-OM, where A and L' have been
defined
above and M can be hydrogen, lower alkyl or a cation such as K), these
compounds can be
prepared by procedures based on that described in Schummer et al.,
Tetrahedron, 43, 9019
(1991). Briefly, the procedure starts with a corresponding aldehyde-containing
compound
(e.g., A-L'-C(=O)-H), which is allowed to react with a pyruvic acid in a basic
condition
(KOHlmethanol) at a low temperature (e.g., 0-5 C). Desired products (in the
form of a
potassium salt) are formed upon warming of the reaction mixture to room
temperature.
The compounds described above, as well as their (thio)hydroxamic acid or a-
keto
acid counterparts, can possess histone deacetylase inhibitory properties.
Note that appropriate protecting groups may be needed to avoid forming side
products during the preparation of a compound of the invention. For example,
if the linker
L' contains an amino substituent, it can be first protected by a suitable
amino protecting
group such as trifluoroacetyl or tent-butoxycarbonyl prior to being treated
with reagents such
as butyllithium. See, e.g., T. W. Greene, supra, for other suitable protecting
groups.
A compound produced by the methods shown above can be purified by flash column
chromatography, preparative high performance liquid chromatography, or
crystallization.
A pharmaceutical composition can be used to inhibit histone deacetylase in
cells and
can be used to treat disorders associated with abnormal histone deacetylase
activity. Some
examples of these disorders are cancers (e.g., leukemia, lung cancer, colon
cancer, CNS
cancer, melanoma, ovarian cancer, cervical cancer, renal cancer, prostate
cancer, and breast
cancer), hematological disorders (e.g., hemoglobinopathies, thalassemia, and
sickle cell
anemia) and genetic related metabolic disorders (e.g., cystic fibrosis,
peroxisome biogenesis
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disorder, alpha-1 anti-trypsin, and adrenoleukodystrophy). The compounds of
this invention
can also stimulate hematopoietic cells ex vivo, ameliorating protozoal
parasitic infection,
accelerate wound healing, and protecting hair follicles.
An effective amount is defined as the amount which is required to confer a
therapeutic effect on the treated patient, and is typically determined based
on age, surface
area, weight, and condition of the patient. The interrelationship of dosages
for animals and
humans (based on milligrams per meter squared of body surface) is described by
Freireich et
al., Cancer Chemother. Rep. 50, 219 (1966). Body surface area maybe
approximately
determined from height and weight of the patient. See, e.g., Scientific
Tables, Geigy
Pharmaceuticals, Ardley, New York, 1970, 537. An effective amount of a
compound
described herein can range from about 1 mg/kg to about 300 mg/kg. Effective
doses will
also vary, as recognized by those skilled in the art, dependant on route of
administration,
excipient usage, and the possibility of co-usage, pre-treatment, or post-
treatment, with other
therapeutic treatments including use of other chemotherapeutic agents and
radiation therapy.
Other chemotherapeutic agents that can be co-administered (either
simultaneously or
sequentially) include, but not limited to, paclitaxel and its derivatives
(e.g., taxotere),
doxorubicin, L-asparaginase, dacarbazine, ainascrine, procarbazine,
hexamethylmelamine,
mitoxantrone, and gemicitabine.
The pharmaceutical composition may be administered via the parenteral route,
including orally, topically, subcutaneously, intraperitoneally,
intramuscularly, and
intravenously. Examples of parenteral dosage forms include aqueous solutions
of the active
agent, in a isotonic saline, 5% glucose or other well-known pharmaceutically
acceptable
excipient. Solubilizing agents such as cyclodextrins, or other solubilizing
agents well-known
to those familiar with the art, can be utilized as pharmaceutical excipients
for delivery of the
therapeutic compounds. Because some of the compounds described herein can have
limited
water solubility, a solubilizing agent can be included in the composition to
improve the
solubility of the compound. For example, the compounds can be solubilized in
polyethoxylated castor oil (Cremophor EL ) and may further contain other
solvents, e.g.,
ethanol. Furthermore, compounds described herein can also be entrapped in
liposomes that
may contain tumor-directing agents (e.g., monoclonal antibodies having
affinity towards
tumor cells).
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A compound described herein can be formulated into dosage forms for other
routes of
administration utilizing conventional methods. For example, it can be
formulated in a
capsule, a gel seal, or a tablet for oral administration. Capsules may contain
any standard
pharmaceutically acceptable materials such as gelatin or cellulose. Tablets
may be
formulated in accordance with conventional procedures by compressing mixtures
of a
compound described herein with a solid carrier and a lubricant. Examples of
solid carriers
include starch and sugar bentonite. Compounds of this invention can also be
administered in
a form of a hard shell tablet or a capsule containing a binder, e.g., lactose
or mannitol, a
conventional filler, and a tableting agent.
The activities of a compound described herein can be evaluated by methods
known in
the art, e.g., MTT (3-[4,5-dimehtythiazol-2-yl]-2,5-diphenyltetrazolium
bromide) assay,
clonogenic assay, ATP assay, or Extreme Drug Resistance (EDR) assay. See
Freuhauf, J.P.
and Manetta, A., Cheinosensitivity Testing in Gynecologic Malignancies and
Breast Cancer
19, 39 - 52 (1994). The EDR assay, in particular, is useful for evaluating the
antitumor and
antiproliferative activity of a compound of this invention (see Example 28
below). Cells are
treated for four days with compound of the invention. Both untreated and
treated cells are
pulsed with tritiated thymidine for 24 hours. Radioactivity of each type of
cells is then
measured and compared. The results are then plotted to generate drug response
curves,
which allow ICsp values (the concentration of a compound required to inhibit
50% of the
population of the treated cells) to be determined.
The histone acetylation activity of a compound described herein can be
evaluated in
an assay using mouse erythroleukemia cells. Studies are performed with the DS
19 mouse
erythroleukemia cells maintained in RPMI 1640 medium with 25 mM HEPES buffer
and 5%
fetal calf serum. The cells are incubated at 37 C.
Histones are isolated from cells after incubation for periods of 2 and 24
hours. The
cells are centrifuged for 5 minutes at 2000 rpm in the Sorvall SS34 rotor and
washed once
with phosphate buffered saline. The pellets are suspended in 10 ml lysis
buffer (10 mM Tris,
50 mM sodium bisulfite, 1% Triton- X-100, 10 mM magnesium chloride, 8.6%
sucrose, pH
6.5) and homogenized with six strokes of a Teflon pestle. The solution is
centrifuged and the
pellet washed once with 5 ml of the lysis buffer and once with 5 ml 10 mM
Tris, 13 mM
EDTA, pH 7.4. The pellets are extracted with 2 x 1 mL 0.25N HC1. Histories are
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precipitated from the combined extracts by the addition of 20 mL acetone and
refrigeration
overnight. The histones are pelleted by centrifuging at 5000 rpm for 20
minutes in the
Sorvall SS34 rotor. The pellets are washed once with 5 mL acetone and protein
concentration are quantitated by the Bradford procedure.
Separation of acetylated histones is usually performed with an acetic acid-
urea
polyacrylamide gel electrophoresis procedure. Resolution of acetylated H4
histones is
achieved with 6,25N urea and no detergent as originally described by Panyim
and Chalkley,
Arch. Biochem. Biophys. 130, 337-346, 1969. 25 g total histones are applied
to a slab gel
which is run at 20 ma. The run is continued for a further two hours after the
Pyronon Y
tracking dye has run off the gel. The gel is stained with Coomassie Blue R.
The most
rapidly migrating protein band is the unacetylated H4 histone followed by
bands with 1, 2, 3
and 4 acetyl groups which can be quantitated by densitometry. The procedure
for
densitometry involves digital recording using the Alpha Imager 2000,
enlargement of the
image using the PHOTOSHOP program (Adobe Corp.) on a MACINTOSH computer (Apple
Corp.), creation of a hard copy using a laser printer and densitometry by
reflectance using the
Shimadzu CS9000U densitometer. The percentage of H4 histone in the various
acetylated
states is expressed as a percentage of the total H4 histone.
The concentration of a compound of the invention required to decrease the
unacetylated H4 histone by 50% (i.e., EC50) can then be determined from data
obtained using
different concentrations of test compounds.
Histone deacetylase inhibitory activity can be measured based on procedures
described by Hoffinann et al., Nucleic Acids Res., 27,'2057-2058 (1999). See
Example 30
below. Briefly, the assay starts with incubating the isolated histone
deacetylase enzyme with
a compound of the invention, followed by the addition of a fluorescent-labeled
lysine
substrate (contains an amino group at the side chain which is available for
acetylation).
HPLC is used to monitor the labeled substrate. The range of activity of each
test compound
is preliminarily determined using results obtained from HPLC analyses. IC50
values can then
be determined from HPLC results using different concentrations of compounds of
this
invention. All assays are duplicated or triplicated for accuracy. The histone
deacetylase
inhibitory activity can be compared with the increased activity of acetylated
histone for
confirmation.
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Compounds of this invention are also evaluated for effects on treating X-
linked
adrenoleukodystrophy (X-ALD), a peroxisomal disorder with impaired very long-
chain fatty
acid (VLCFA) metabolism. In such an assay, cell lines derived from human
primary
fibroblasts and (EBV-transformed lymphocytes) derived from X-ALD patients
grown in
RPMI are employed. Tissue culture cells are grown in the presence or absence
of test
compounds. For VLCFA measurements, total lipids are extracted, converted to
methyl
esters, purified by TLC and subjected to capillary GC analysis as described in
Moser et al.,
Technique in Diagnostic Biochemical Genetics: A Laboratory Manual (ed. A.,
H.F.) 177-191
(Wiley-Liss, New York, 1991). C24:0 fl-oxidation activity of lyophoclastoid
cells are
determined by measuring their capacity to degrade [1-14C]-C24:0 fatty acid to
water-soluble
products as described in Watkins et al., Arch. Biochem. Biophys. 289, 329-336
(1991). The
statistical significance of measured biochemical differences between untreated
and treated X-
ALD cells can be determined by a two-tailed Student's t-test. See Example 31
below.
Further, compounds of the present invention are evaluated for their effects in
treating
cystic fibrosis (CF). Since the initial defect in the majority of cases of CF
is the inability of
mutant CF protein (CFTR) to fold properly and exit the ER, compounds of the
invention are
tested to evaluate their efficacy in increasing the trafficking of the CF
protein out of the ER
and its maturation through the Golgi. During its biosynthesis, CFTR is
initially synthesized
as a nascent polypeptide chain in the rough ER, with a molecular weight of
around 120 kDa
(Band A). It rapidly receives a core glycosylation in the ER, giving it a
molecular weight of
around 140 kDa (Band B). As CFTR exits the ER and matures through the Golgi
stacks, its
glycosylation is modified until it achieves a terminal mature glycosylation,
affording it a
molecular weight of around 170 kDa (Band Q. Thus, the extent to which CFTR
exits the ER
and traverses the Golgi to reach the plasma membrane may be reflected in the
ratio of Band
B to Band C protein. CFTR is immunoprecipitated from control cells, and cells
exposed to
test compounds. Both wt CFTR and AF508 CFTR expressing cells are tested.
Following
lysis, CFTR are immunoprecipitated using various CFTR antibodies.
Immunoprecipitates
are then subjected to in vitro phosphorylation using radioactive ATP and
exogenous protein
kinase A. Samples are subsequently solubilized and resolved by SDS-PAGE. Gels
are then
dried and subject to autoradiography and phosphor image analysis for
quantitation of Bands
B and C are determined on a BioRad personal fix image station. See Example 32
below.
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Furthermore, compounds of this invention can be used to treat homozygous S
thalassemia, a disease in which there is inadequate production of fl globin
leading to severe
anemia. See Collins et al., Blood, 85(1), 43-49 (1995).
Still further, compounds of the present invention are evaluated for their use
as
antiprotozoal or antiparasitic agents. The evaluation can be conducted using
parasite cultures
(e.g., Asexual P. falciparuin). See Trager, W. & Jensen, J.B. Science 193, 673-
675 (1976).
Test compounds of the invention are dissolved in dimethyl sulfoxide (DMSO) and
added to
wells of a flat-bottomed 96-well microtitre plate containing human serum.
Parasite cultures
are then added to the wells, whereas control wells only contain parasite
cultures. After at
least one invasion cycle, and addition of labeled hypoxanthine
monohydrochloride, the level
of incorporation of labeled hypoxanthine is detected. IC50 values can be
calculated from data
using a non-linear regression analysis.
The toxicity of a compound described herein is evaluated when a compound of
the
invention is administered by single intraperitoneal dose to test mice. See
Example 33 below.
After administration of a predetermined dose to three groups of test mice and
untreated
controls, mortality/morbidity checks are made daily. Body weight and gross
necropsy
findings are also monitored. For reference, see Gad, S. C. (ed.), Safety
Assessment for
Pharmaceuticals (Van Nostrand Reinhold, New York, 1995).
Without further elaboration, it is believed that one skilled in the art can,
based on the
description herein, utilize the present invention to its fullest extent. The
following specific
examples, which described syntheses, screening, and biological testing of
various compounds
of this invention, are therefore, to be construed as merely illustrative, and
not limitative of the
remainder of the disclosure in any way whatsoever.
Example 1
Synthesis of 3-methyl-5-phenyl-2,4-pentadienoic acid
To a cooled (-10 to -5 C) 165 mL of 3 M solution of methyl magnesium iodide in
ether was added dropwise a solution of ethyl trans-cinnamate (25.0 g) in 200
mL of
anhydrous ether. The reaction was warmed to room temperature and stirred
overnight. The
mixture was then heated up to 33 C under reflux for two hours and cooled to 0
C. A white
solid was formed during cooling and water (105 mL) was gradually added to
dissolve the
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white precipitate followed by an additional 245 mL of saturated aqueous
ammonium chloride
solution. The mixture was then stirred until the solids were completely
dissolved and
extracted with 100 mL of ether three times. The combined extract was washed
with 100 mL
of water, dried over anhydrous sodium sulfate and filtered. The solvent was
evaporated to
give 22.1 g of the desired 4-phenyl-2-methyl-3-buten-2-ol as an oil which was
used in the
next step without further purification. 1H NMR (CDC13, 300 MHz), b(ppm) 7.41
(m, 5H),
6.58 (d, 1H), 6.34 (d, 111), 1.41 (broad s, 6H).
Dimethylformamide (DMF, anhydrous, 25 mL) was cooled to 0-5 C and phosphorus
oxychloride (16.4 mL) was added dropwise over a period of an hour. The
resulting solution
was added dropwise to a cooled (0-5 C) solution of 4-phenyl-2-methyl-3-buten-2-
ol (0.14
mol) in 60 mL of anhydrous DMF over a period of an hour. The reaction mixture
was then
warmed to room temperature, gradually heated up to 80 C, stirred at 80 C for
three hours and
cooled to 0-5 C. To the cooled reaction solution was added dropwise a solution
of sodium
acetate (80 g) in deionized water (190 mL) over a period of two hours. The
mixture was then
reheated to 80 C, stirred at 80 C for an additional 10 minutes, cooled down to
room
temperature and extracted with ether (300 mL) twice. The combined extract was
washed
with water (200 mL), dried over anhydrous sodium sulfate, filtered and
concentrated in
vacuum to yield 16.7 g of the desired 3-methyl-5-phenyl-2,4-pentadienal as a
liquid which
was used in the next step without further purification.
To a stirred solution of 3-methyl-5-phenyl-2,4-pentadienal (16.5 g) in ethanol
(330
mL) was added dropwise a solution of silver nitrate (19.28 g) in water (160
mL) followed by
dropwise addition of an aqueous sodium hydroxide (25g, 80 mL) solution. The
resulting
mixture was allowed to stir for an additional five hours and then filtered.
The solid was
washed with ethanol. The combined filtrate was concentrated in vacuum. The
residue was
dissolved in water (200 mL). The aqueous solution was extracted with ether
(300 mL) twice
and acidified with 6 N hydrochloric acid (74 mL). The solid formed was
filtered and
recrystallized from methanol (40 mL) to yield 2.65 g of the desired 3-methyl-5-
phenyl-2,4-
pentadienoic acid. 1H NMR (acetone-d6, 300 MHz), 6(ppm) 7.60 (d, 2H), 7.35 (m,
3H), 7.06
(m, 2H), 6.02 (broad s, 111), 2.50 (s, 3H).
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Example 2
Synthesis of 4-methyl-5-phenyl-2,4-pentadienoic acid
Butyllithiumn (135 mL of 2.5 N solution) was added to 600 mL of anhydrous
tetrahydrofuran (THF) at -65 C. A solution of diethylphosphonoacetic acid
(30.5 g) in 220
mL of anhydrous THF was added dropwise to the stirred solution at -65 C over a
period of
60 minutes. The resulting solution was stirred at -65 C for an additional 30
minutes and then
a solution of a-methyl-trans-cinnamaldehyde (23.2 g) in 100 mL of anhydrous
THF was
added to the reaction at -65 C over a period of 70 minutes. The reaction was
stirred for one
hour, allowed to warm to room temperature and then stirred overnight. The
reaction was
then acidified with 5% hydrochloric acid (125 mL) to a pH of 2.8. The aqueous
layer was
extracted with 100 mL of ether twice and with 100 mL of ethyl acetate once.
The combined
organic extract was dried over anhydrous sodium sulfate, filtered and
concentrated under
vacuum. The crude material was dissolved in 100 mL of hot methanol and then
refrigerated
overnight. The crystals formed were filtered and dried under vacuum to afford
25.8 g of the
desired 4-methyl-5-phenyl-2,4-pentadienoic acid. 1H NMR (acetone-d6, 300 MHz),
6(ppm)
7.53 (d, 1H), 7.43 (m, 4H), 7.37 (dd, 111), 6.97 (broad s, 1H), 6.02 (d, 1H),
2.07 (s, 3H).
Example 3
Synthesis of 4-chloro-5-phenyl-2,4-pentadienoic acid
Butyllithium (50 mL of 2.5 N solution) was added to 250 mL of anhydrous
tetrahydrofuran (THF) at -65 C. A solution of diethylphosphonoacetic acid
(11.4 g) in 90
mL of anhydrous THF was added dropwise to the stirred solution at -65 C. The
resulting
solution was stirred at -65 C for an additional 40 minutes and then a solution
of a-chloro-
cinnamaldehyde (10.0 g) in 60 mL of anhydrous THF was added to the reaction at
-65 C
over a period of 95 minutes. The reaction was stirred for one hour, allowed to
warm to room
temperature and then stirred overnight. The reaction was then acidified with
5%
hydrochloric acid (48 mL) to a pH of 3.9. The aqueous layer was extracted with
50 mL of
ether twice and with 50 mL of ethyl acetate once. The combined organic extract
was dried
over anhydrous sodium sulfate, filtered and concentrated under vacuum. The
crude material
was dissolved in 30 mL of hot methanol and then refrigerated overnight. The
crystals formed
were filtered and dried under vacuum to afford 9.2 g of the desired 4-chloro-5-
phenyl-2,4-
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pentadienoic acid. 1H NMR (acetone-d6, 300 MHz), 8(ppm) 7.86 (d, 2H), 7.60 (d,
1H), 7.45
(m, 3H), 7.36 (broad s, 1H), 6.32 (d, 1H).
Example 4
Synthesis of 5-phenyl-2-ene-4-pentynoic acid
Butyllithium (16 mL of 2.5 N solution) was added to 75 mL of anhydrous
tetrahydrofuran (THF) at -65 C. A solution of diethylphosphonoacetic acid (3.6
g) in 25 mL
of anhydrous THF was added dropwise to the stirred solution at -65 C over a
period of 15
minutes. The resulting solution was stirred at -65 C for an additional 30
minutes and then a
solution of phenylpropargyl aldehyde (2.5 g) in 20 mL of anhydrous THF was
added to the
reaction at -65 C over a period of 20 minutes. The reaction was stirred for
one hour, allowed
to warm to room temperature and then stirred overnight. The reaction was then
acidified
with 6 N hydrochloric acid (5 mL) to a pH of 1Ø The aqueous layer was
extracted with 75
mL of ethyl acetate three times. The combined organic extract was dried over
anhydrous
sodium sulfate, filtered and concentrated under vacuum. The crude material was
recrystallized with chloroform: ether (90:10) and then refrigerated overnight.
The crystals
were filtered and dried under vacuum to afford 1.1 g of the desired 5-phenyl-2-
ene-4-
pentynoic acid. 1H NMR (acetone-d6, 300 MHz), 8(ppm) 7.50 (m, 5H), 6.98 (d,
1H), 6.35 (d,
1H).
Example 5
Synthesis of 5-(p-dimethylaminophenyl)-2,4-pentadienoic acid
Butyllithium (24 mL of 2.5 N solution) was added to 120 mL of anhydrous
tetrahydrofuran (THF) at -65 C. A solution of diethylphosphonoacetic acid (5.5
g) in 45 mL
of anhydrous THF was added dropwise to the stirred solution at -65 C over a
period of one
hour. The resulting solution was stirred at -65 C for an additional 30 minutes
and then a
solution ofp-dimethylaminocinnamaldehyde (5.0 g) in 80 mL of anhydrous THF was
added
to the reaction at -65 C over a period of 30 minutes. The reaction was stirred
for one hour,
allowed to warm to room temperature and then stirred overnight. The reaction
was then
quenched with 4001nL of water and extracted with 300 mL of ethyl acetate three
times. The
aqueous layer was acidified with 5% hydrochloric acid (11 mL) to a pH of 6.1.
The solid
formed was filtered, washed with 75 mL of water and dried to yield 3.83 g of
the desired 5-
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(p-dimethylaminophenyl)-2,4-pentadienoic acid. 1H NMR (DMSO-d6, 300 MHz),
8(ppm)
7.34 (m, 3H), 6.82 (m, 2H), 6.70 (d, 2H), 5.84 (d, 1H), 2.94 (s, 6H).
Example 6
Synthesis of 5-(2-furyl)-2,4-pentadienoic acid
Butyllithium (70 mL of 2.5 N solution) was added to 350 mL of anhydrous
tetrahydrofuran (THF) at -65 C. A solution of diethylphosphonoacetic acid
(15.9 g) in 130
mL of anhydrous THE was added dropwise to the stirred solution at -65 C over a
period of
75 minutes. The resulting solution was stirred at -65 C for an additional 30
minutes and then
a solution of trans-3-(2-furyl)acrolein (10.0 g) in 85 mL of anhydrous THE was
added to the
reaction at -65 C over a period of 2 hours. The reaction was allowed to warm
to room
temperature and stirred overnight. The reaction was then acidified with 5%
hydrochloric
acid (85 mL) to a pH of 3.5 followed by addition of 30 mL of water. The
aqueous layer was
extracted with 50 mL of ether twice and with 50 mL of ethyl acetate once. The
combined
organic extract was dried over anhydrous sodium sulfate, filtered and
concentrated under
vacuum to give an oil. The crude oil was dissolved in 45 mL of hot methanol
and then
refrigerated overnight. The crystals formed were filtered and dried under
vacuum to afford
9.2 g of the desired 5-(2-furyl)-2,4-pentadienoic acid. 1H NMR (acetone-d6,
300 MHz),
8(ppm) 7.64 (broad s, 1H), 7.42 (m, 1H), 6.86 (m, 2H), 6.58 (m, 2H), 6.05 (d,
1H).
Example 7
Synthesis of 6-phenyl-3,5-hexadienoic acid
Triphenylphosphine (178.7 g) and 3-chloropropionic acid (73.9 g) were mixed in
a 1-
liter 3-neck round bottom flask equipped with a mechanical stirrer, reflux
condenser with a
nitrogen inlet and a thermocouple. The mixture was heated to 145 C under
nitrogen and
stirred for 2 hours. The reaction was then cooled to 70 C. Ethanol (550 mL)
was added and
the mixture was refluxed at 80 C until complete dissolution. The solution was
cooled to
room temperature and ether (900 mL) was added. The mixture was placed in the
freezer
overnight. The solids were collected by filtration and dried under vacuum to
afford 217 g of
3-(triphenylphosphonium)propionic acid chloride as a white solid which was
used in the next
step without further purification.
Sodium hydride (12.97 g) in an oven dried 5-liter 3-neck round bottom flask
equipped
with a mechanical stirrer and a thermocouple was cooled to 0-5 C in an ice
bath. A solution
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of 3-(triphenylphosphonium)propionic acid chloride (100.0 g) and trans-
cinnamaldehyde (34
inL) in 400 mL each of anhydrous dimethyl sulfoxide and tetrahydrofuran was
added over a
period of 3 hours. The reaction was then allowed to warm to room temperature
and stirred
overnight. The reaction mixture was cooled to 0-5 C in an ice bath and water
(1.6 liters) was
added dropwise. The aqueous solution was acidified with 12 N hydrochloric acid
(135 mL)
to a pH of 1 and extracted with ethyl acetate (1.6 liters) twice. The combined
organic layers
was washed with water (1000 mL) three times, dried over anhydrous sodium
sulfate and
concentrated under vacuum to afford a yellow oil. The crude oil was dissolved
in 125 mL of
methylene chloride and chromatographed on a Biotage 75L silica gel column and
eluted with
methylene chloride: ether (9:1). The fractions containing the desired product
were combined
and the solvents were removed under vacuum to afford 10.38 g of 6-phenyl-3,5-
hexadienoic
acid. 1H NMR (CDC13, 300 MHz), 8(ppm) 7.33 (m, 5H), 6.80 (m, 1H), 6.53 (d,
1H), 6.34
(m, 1H), 5.89 (m, 1H), 3.25 (d, 2H).
Example 8
Synthesis of 7-phenyl-2,4,6-heptatrienoic acid
To a cooled (0-55 C) 927 mL of 1 M solution of phenyl magnesium bromide in
tetrahydofuran was added dropwise a solution of crotonaldehyde (65.0 g) in 130
mL of
anhydrous ether over a period of 2 hours and 45 minutes. The reaction was
stirred for an
additional 45 minutes and then warmed to room temperature. After four more
hours of
stirring, saturated ammonium chloride aqueous solution (750 mL) was added to
the reaction.
The mixture was extracted with 750 mL of ether twice. The combined extract was
dried over
anhydrous potassium carbonate and filtered. The solvent was evaporated to give
135.88 g
(99.9%) of the desired 1-phenyl-2-buten-l-ol as an oil which was used in the
next step
without further purification.
1-Phenyl-2-buten-l-ol (135.88 g) was dissolved in 2300 mL of dioxane and
treated
with 2750 mL of dilute hydrochloric acid (2.3 mL of concentrated hydrochloric
acid in 2750
mL of water) at room temperature. The mixture was stirred overnight and then
poured into
4333 mL of ether and neutralized with 2265 mL of saturated aqueous sodium
bicarbonate.
The aqueous phase was extracted with 1970 mL of ether. The combined extract
was dried
over anhydrous potassium carbonate. Evaporation of the solvent followed by
Kugelrohr
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distillation at 30 C for 30 minutes afforded 131.73 g (96.8%) of the desired 4-
phenyl-3-
buten-2-ol as an oil which was used in the next step without further
purification.
Dimethylformamide (DMF, anhydrous, 14 mL) was cooled to 0-5 C and phosphorus
oxychloride (8.2 mL) was added dropwise over a period of 40 minutes. The
resulting
solution was added dropwise to a cooled (0-5 C) solution of 4-phenyl-3-buten-2-
ol (10 g) in
32 mL of anhydrous DMF over a period of an hour. The reaction mixture was
warmed to
room temperature over a 35-minute period and then gradually heated up to 80 C
over a
period of 45 minutes. The reaction was stirred at 80 C for three hours and
then cooled to 0-
5 C. To the cooled reaction solution was added dropwise a solution of sodium
acetate (40 g)
in deionized water (100 mL) over a period of one hour. The mixture was then
reheated to
80 C, stirred at 80 C for an additional 10 minutes, cooled down to room
temperature and
extracted with ether (100 mL) twice. The combined extract was washed with
brine (100
mL), dried over anhydrous sodium sulfate, filtered and concentrated under
vacuum to yield
8.78 g of the desired 5-phenyl-2,4-pentadienal as a liquid which was used in
the next step
without further purification. 'H NMR (CDC13, 300 MHz), 5(ppm) 7.51 (m, 2H),
7.37 (m,
3H), 7.26 (m, 1H), 7.01 (m, 2H), 6.26 (m, 1H).
Butyllithium (12.8 mL of 2.5 N solution) was added to 65 mL of anhydrous
tetrahydrofuran (THF) at -65 C. A solution of diethylphosphonoacetic acid
(2.92 g) in 25
mL of anhydrous THE was added dropwise to the stirred solution at -65 C. The
resulting
solution was stirred at -65 C for an additional 30 minutes and then a solution
of 5-phenyl-
2,4-pentadienal (2.4 g) in 15 mL of anhydrous THE was added to the reaction at
-65 C. The
reaction was stirred for one hour, allowed to warm to room temperature and
then stirred
overnight. To the reaction was added 30 mL of water, acidified with 5%
hydrochloric acid
(14 mL) to a pH of 4.7 and then added an additional 20 mL of water. The
aqueous layer was
extracted with 10 mL of ether twice and with 10 mL of ethyl acetate once. The
combined
organic extract was dried over anhydrous sodium sulfate, filtered and
concentrated under
vacuum. The crude material was dissolved in 50 mL of hot methanol and then
refrigerated
overnight. The crystals formed were filtered and dried under vacuum to afford
2.4 g of the
desired 7-phenyl-2,4,6-heptatrienoic acid. 1H NMR (DMSO-d6, 300 MHz), 5(ppm)
7.52 (m,
2H), 7.33 (m, 4H), 7.06 (m, 1H), 6.86 (m, 2H), 6.58 (m, 1H), 5.95 (d, 1H).
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Example 9
Synthesis of 8-phenyl-3,5,7-octatrienoic acid
A solution of 5-phenyl-2,4-pentadienal (15 g) and 3-(triphenylphosphonium)-
propionic acid chloride (35.2 g) in 140 mL each of anhydrous tetrahydrofuran
and anhydrous
dimethyl sulfoxide was added dropwise to sodium hydride (4.6 g) at 0-5 C under
nitrogen
over a period of four hours. The reaction was allowed to warm to room
temperature and
stirred overnight. The reaction mixture was cooled to 0-5 C and water (280 mL)
was added
dropwise over a period of 30 minutes. The aqueous layer was extracted with
ethyl acetate
(280 mL) twice, acidified with 12 N hydrochloric acid (24 mL) to a pH of 1,
extracted again
with ethyl acetate (280 mL) twice. The combined organic layers were washed
with water
(500 mL) twice, dried over anhydrous sodium sulfate and concentrated under
vacuum to give
an oil. The oily crude product was chromatographed on a Biotage 40M silica gel
column and
eluted with methylene chloride:ethyl acetate (95:5). The fractions containing
the desired
product were combined and the solvents were removed under vacuum to afford 0.7
g of 8-
phenyl-3,5,7-octatrienoic acid. 1H NMR (acetone-d6, 300 MHz), 8(ppm) 7.46 (m,
2H), 7.26
(m, 3H), 6.95 (m, 1H), 6.60 (d, 1H), 6.34 (in, 3H), 5.87 (in, 1H), 3.17 (d,
2H).
Example 10
Synthesis of potassium 2-oxo-6-phenyl-3,5-hexadienoate
A solution of trans-cinnamaldehyde (26.43 g) and pyruvic acid (11.9 mL) in 10
mL
of methanol was stirred and chilled to 0 -5 C in an ice bath. To the chilled
solution was
added 35 mL of potassium hydroxide (16.83 g in 50 mL of methanol) over a
period of 20
minutes. The remaining methanolic potassium hydroxide was added rapidly and
the ice bath
was removed. The solution changed from a yellow to a dark orange and the
precipitate was
formed. The reaction mixture was chilled in the refrigerator overnight and the
solid was
collected by filtration, washed with 50 mL of methanol three times, 50 mL of
ether and then
air dried to afford 29.3 g of the desired 2-oxo-6-phenyl-3,5-hexadienoate as a
yellow solid
(61.0%). 1H NMR (DMSO-d6/D20, 300 MHz), 8(ppm) 7.48 (d, 2H), 7.28 (m, 4H),
7.12 (d,
2H), 6.27 (d, 1H).
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Example 11
Synthesis of potassium 2-oxo-8-phenyl-3,5,7-octatrienoate
To a cooled (0-55 C) 927 mL of 1 M solution of phenyl magnesium bromide in
tetrahydofuran was added dropwise a solution of crotonaldehyde (65.0 g) in 130
mL of
anhydrous ether over a period of 2 hours and 45 minutes. The reaction was
stirred for an
additional 45 minutes and then.warmed to room temperature. After four more
hours of
stirring, saturated ammonium chloride aqueous solution (750 mL) was added to
the reaction.
The mixture was extracted with 750 mL of ether twice. The combined extract was
dried over
anhydrous potassium carbonate and filtered. The solvent was evaporated to give
135.88 g
(99.9%) of the desired 1-phenyl-2-buten-l-ol as an oil which was used in the
next step
without further purification.
1-Phenyl-2-buten-l-ol (135.88 g) was dissolved in 2300 mL of dioxane and
treated
with 2750 mL of dilute hydrochloric acid (2.3 mL of concentrated hydrochloric
acid in 2750
mL of water) at room temperature. The mixture was stirred overnight and then
poured into
4333 mL of ether and neutralized with 2265 mL of saturated sodium bicarbonate.
The
aqueous phase was extracted with 1970 mL of ether. The combined extract was
dried over
anhydrous potassium carbonate. Evaporation of the solvent followed by
Kugelrohr
distillation at 30 C for 30 minutes afforded 131.73 g (96.8%) of the desired 4-
phenyl-3-
buten-2-ol as an oil which was used in the next step without further
purification.
Dimethylformamide (DMF, anhydrous, 14 mL) was cooled to 0-5 C and phosphorus
oxychloride (8.2 mL) was added dropwise over a period of 40 minutes. The
resulting
solution was added dropwise to a cooled (0-5 C) solution of 4-phenyl-3-buten-2-
ol (10 g) in
32 mL of anhydrous DMF over a period of an hour. The reaction mixture was
warmed to
room temperature over a 35-minute period and then gradually heated up to 80 C
over a
period of 45 minutes. The reaction was stirred at 80 C for three hours and
then cooled to 0-
5 C. To the cooled reaction solution was added dropwise a solution of sodium
acetate (40 g)
in deionized water (100 mL) over a period of one hour. The mixture was then
reheated to
80 C, stirred at 80 C for an additional 10 minutes, cooled down to room
temperature and
extracted with ether (100 mL) twice. The combined extract was washed with
brine (100
mL), dried over anhydrous sodium sulfate, filtered and concentrated under
vacuum to yield
8.78 g of the desired 5-phenyl-2,4-pentadienal as a liquid which was used in
the next step
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without further purification. 'H NMR (CDC13, 300 MHz), 8(ppm) 7.51 (m, 2H),
7.37 (m,
3H), 7.26 (m, 1H), 7.01 (m, 2H), 6.26 (m, 1H).
A solution of 5-phenyl-2,4-pentadienal (6.70 g) and pyruvic acid (3.0 mL) in 5
mL of
methanol was stirred and chilled to 0 -5 C in an ice bath. To the chilled
solution was added
a solution of 35 mL of potassium hydroxide (3.5 g) in 10 mL of methanol
dropwise over a
period of 30 minutes. The remaining methanolic potassium hydroxide was added
rapidly and
the ice bath was removed. The reaction was allowed to warm to room temperature
and
stirred for another hour. The flask was then refrigerated overnight. The solid
was collected
by filtration, washed with 15 mL of methanol three times, 15 mL of ether and
then air dried
to afford 6.69 g of potassium 2-oxo-8-phenyl-3,5,7-octatrienoate as a yellow
solid. 1H NMR
(DMSO-d6, 300 MHz), 6(ppm) 7.52 (d, 2H), 7.32 (m, 3H), 7.10 (m, 2H), 6.83 (dd,
2H), 6.57
(dd, 1H), 6.13 (d, 1H).
Example 12
Synthesis of cinnamoylhydroxamic acid
Triethylamine (TEA, 17.6 mL) was added to a cooled (0-5 C) solution of trans-
cinnamic acid (15.0 g) in 200 mL of anhydrous dimethylformamide. To this
solution was
added dropwise isobutyl chloroformate (16.4 mL). The reaction mixture was
stirred for 30
minutes and hydroxylamine hydrochloride (17.6 g) was added followed by
dropwise addition
of 35 mL of TEA at 0-5 C. The reaction was allowed to warm to room temperature
and
stirred overnight. The reaction was quenched with 250 mL of 1 % (by weight)
citric acid
solution and 50 mL of 5% (by weight) citric acid solution and then extracted
with 200 mL of
methylene chloride twice and 200 mL of ether once. The solvents were removed
under
vacuum. The residue was triturated with 125 mL of water, filtered, washed with
25 mL of
water and dried under vacuum to give a tan solid. The crude product was
chromatographed
on a Biotage 75S column and eluted with methylene chloride:acetonitrile
(80:20). The
fractions containing the desired product were combined and the solvent was
removed under
vacuum to yield 4.1 g of cinnamoylhydroxamic acid. 1H NMR (DMSO-d6, 300 MHz),
S(ppm) 7.48 (m, 6H), 6.49 (d, 1H).
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Example 13
Synthesis of N-methyl-cinnamoylhydroxamic acid
A solution of cinnamoyl chloride (5 g) in 50 mL of methylene chloride was
added
dropwise to a solution of N-methylhydroxylamine hydrochloride (5 g) and 12 mL
of 40%
sodium hydroxide in 50 mL of water cooled to 0-5 C. The reaction mixture was
stirred for
two hours. The aqueous layer was acidified with concentrated hydrochloric
acid. The
precipitate was collected by filtration and dried under vacuum to afford 2.8 g
of the desired
N-methyl-cinnamoylhydroxamic acid as a white solid. 1H NMR (DMSO-d6, 300 MHz),
b(ppm) 7.66 (d, 2H), 7.53 (d, 1H), 7.42 (m, 3H), 7.26 (d, 1H), 3.22 (s, 3H).
Example 14
Synthesis of 5-phenyl-2,4-pentadienoylhydroxamic acid
Triethylamine (TEA, 29 mL) was added to a cooled (0-5 C) solution of 5-phenyl-
2,4-
pentadienoic acid (29.0 g) in 300 mL of anhydrous dimethylformamide. To this
solution was
added dropwise isobutyl chloroformate (27.0 mL). The reaction mixture was
stirred for 15
minutes and hydroxylamine hydrochloride (28.92 g) was added followed by
dropwise
addition of 581nL of TEA over a period of 60 minutes at 0-5 C. The reaction
was allowed to
warm to room temperature and stirred overnight. The reaction was then poured
into 450 mL
of a 1 % (by weight) solution of citric acid and then extracted with 200 mL of
methylene
chloride twice and 500 mL of ether once. The solvents were removed under
vacuum to give
an oil. The crude oil was crystallized with 200 mL of hot acetonitrile to give
a tan solid. The
tan solid was recrystallized from 60 mL of hot acetonitrile to afford 12.5 g
of the desired 5-
phenyl-2,4-pentadienoylhydroxamic acid. 1H NMR (DMSO-d6, 300 MHz), 6(ppm) 7.56
(d,
2H), 7.31 (m, 4H), 7.03 (m, 2H), 6.05 (s, 1H).
Example 15
Synthesis of N-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid
5-Phenyl-2,4-pentadienoic acid (6 g) and oxalyl chloride (6.1 mL) were
dissolved in
50 mL of methylene chloride and 0.2 mL of dimethylformamide was added. The
reaction
was stirred for three hours, concentrated under vacuum and then co-evaporated
with 100 mL
of chloroform to remove oxalyl chloride. The crude 5-phenyl-2,4-pentadienoic
acid chloride
was used in the next step without further purification.
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5-Phenyl-2,4-pentadienoic acid chloride was dissolved in 50 mL of methylene
chloride and added to a solution of 13.8 mL of 40% sodium hydroxide in 50 mL
of water at
0-5 C. The resulting solution was stirred for two hours and then acidified to
a pH of 4 with
concentrated hydrochloric acid. The precipitate was collected by filtration
and dried under
vacuum to afford 4.2 g of N-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid.
1H NMR
(DMSO-d6, 300 MHz), 8(ppm) 7.57 (d, 2H), 7.35 (m, 4H), 7.19 (m, 1H), 6.99 (d,
1H), 6.82
(d, 1H), 3.21 (s, 3H).
Example 16
Synthesis of 3-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid
Triethylamine (TEA, 1.8 mL) was added to a cooled (0-5 C) solution of 3-methyl-
5-
phenyl-2,4-pentadienoic acid (2.0 g) in 20 mL of anhydrous dimethylfonnamide.
To this
solution was added dropwise isobutyl chloroformate (1.7 mL) over a period of
15 minutes.
The reaction mixture was stirred for 30 minutes and hydroxylamine
hydrochloride (1.85 g)
was added followed by dropwise addition of 3.7 mL of TEA over a period of 35
minutes at
0-5 C. The reaction was allowed to warm to room temperature and stirred
overnight. To the
stirred reaction mixture at room temperature was added 20 mL of a 1% (by
weight) solution
of citric acid followed by 75 mL of water. The mixture was stirred for 30
minutes and then
filtered. The filtered cake was washed with 30 mL of water and dried in vacuum
to afford
1.49 g of the desired 3-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid in 69%
yield. 1H
NMR (DMSO-d6, 300 MHz), 5(ppm) 7.55 (d, 2H), 7.30 (m, 3H), 6.89 (broad s, 2H),
5.83 (s,
1H), 2.38 (s, 3H).
Example 17
Synthesis of 4-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid
Triethylamine (TEA, 6.5 mL) was added to a cooled (0-5 C) solution of 4-methyl-
5-
phenyl-2,4-pentadienoic acid (7.0 g) in 75 mL of anhydrous dimethylfonnamide.
To this
solution was added dropwise isobutyl chloroformate (6.0 mL) over a period of
60 minutes.
The reaction mixture was stirred for 15 minutes and hydroxylamine
hydrochloride (6.5 g)
was added followed by dropwise addition of 13 mL of TEA over a period of 60
minutes at 0-
5 C. The reaction was allowed to warm to room temperature and stirred
overnight. To the
stirred reaction mixture at room temperature was added 130 mL of a 1% (by
weight) solution
of citric acid followed by 50 mL of water. The mixture was stirred for 30
minutes and then
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filtered. The filtered cake was recrystallized from hot, acetonitrile to
afford 4.4 g of the
desired 4-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid. 1H NMR (DMSO-d6,
300
MHz), b(ppm) 7.37 (m, 6H), 6.91 (s, 1H), 6.02 (d, 1H), 1.99 (s, 3H).
Example 18
Synthesis of 4-chloro-5-phenyl-2,4-pentadienoylhydroxamic acid
Triethylamine (TEA, 2.5 mL) was added to a cooled (0-5 C) solution of 4-chloro-
5-
phenyl-2,4-pentadienoic acid (3.0 g) in 30 mL of anhydrous dimethylformamide.
To this
solution was added dropwise isobutyl chloroformate (2.3 mL) over a period of
15 minutes.
The reaction mixture was stirred for 30 minutes and hydroxylamine
hydrochloride (2.5 g)
was added followed by dropwise addition of 5.0 mL of TEA over a period of 60
minutes at
0-5 C. The reaction was allowed to warm to room temperature and stirred
overnight. The
reaction was then quenched with 30 mL of a 1% (by weight) solution of citric
acid followed
by 115 mL of water. The mixture was stirred for 30 minutes and then filtered.
The filtered
cake was washed with 100 mL of water and dried under vacuum. The crude
material was
recrystallized from 20 mL of hot acetonitrile twice to yield 1.46 g of the
desired 4-chloro-5-
phenyl-2,4-pentadienoylhydroxamic acid as a solid. 1H NMR (DMSO-d6, 300 MHz),
6(ppm) 7.75 (d, 2H), 7.40 (m, 5H), 6.31 (d, 1H).
Example 19
Synthesis of 5-phenyl-2-ene-4-pentynoylhydroxamic acid
Triethylamine (TEA, 1.1 mL) was added to a cooled (0-5 C) solution of 5-phenyl-
2-
ene-4-pentynoic acid (1.1 g) in 13 mL of anhydrous dimethylformamide. To this
solution
was added dropwise isobutyl chloroformate (1.0 mL). The reaction mixture was
stirred for
minutes and hydroxylamine hydrochloride (1.1 g) was added followed by dropwise
addition of 2.2 mL of TEA at 0-5 C. The reaction was allowed to warm to room
temperature
25 and stirred overnight. The reaction was quenched with 15 mL of a 1 % (by
weight) solution
of citric acid and extracted with 30 mL of methylene chloride twice. The
combined organic
layer was dried over anhydrous sodium sulfate. The solvents were removed under
vacuum to
give an oil which in turn was triturated with 10 mL of chloroform. The solid
was collected
by filtration to yield 0.63 g of the desired 5-phenyl-2-ene-4-
pentynoylhydroxamic acid as a
30 white powder. 1H NMR (DMSO-d6, 300 MHz), S(ppm) 7.48 (m, 5H), 6.76 (d, 1H),
6.35 (d,
1H).
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Example 20
Synthesis of 5-(p-dimethylaminophenyl)-2,4-pentadienoylhydroxamic acid
Triethylamine (TEA, 0.8 mL) was added to a cooled (0-5 C) solution of 5-(p-
dimethylaminophenyl)-2,4-pentadienoic acid (1.0 g) in 10 mL of anhydrous
dimethylformamide. To this solution was added dropwise isobutyl chloroformate
(0.7 mL).
The reaction mixture was stirred for 60 minutes and hydroxylamine
hydrochloride (0.8 g)
was added followed by dropwise addition of 1.6 mL of TEA at 0-5 C. The
reaction was
allowed to warm to room temperature and stirred overnight. The reaction was
quenched with
mL of water. The solid was filtered and dried under vacuum to yield 0.75 g of
the desired
10 5-(p-dimethylaminophenyl)-2,4-pentadienoylhydroxamic acid. 'H NMR (DMSO-d6,
300
MHz), S(ppm) 7.33 (m, 3H), 6.86 (m, 2H), 6.70 (d, 2H), 5.84 (d, 1H), 2.99 (s,
6H).
Example 21
Synthesis of 5-(2-furyl)-2,4-pentadienoylhydroxamic acid
Triethylamine (TEA, 2.1 mL) was added to a cooled (0-5 C) solution of 5-(2-
furyl)-
15 2,4-pentadienoic acid (2.0 g) in 15 mL of anhydrous dimethylformamide. To
this solution
was added dropwise isobutyl chloroformate (2.0 mL) over a period of 30
minutes. The
reaction mixture was stirred for 30 minutes and hydroxylamine hydrochloride
(2.15 g) was
added followed by dropwise addition of 4.2 mL of TEA over a period of 60
minutes at 0-5 C.
The reaction was allowed to warm to room temperature and stirred overnight. To
the stirred
reaction mixture at room temperature was added 12 mL of a 1% (by weight)
solution of citric
acid followed by 46 rnL of water. The mixture was stirred for 30 minutes and
then filtered.
The filtered cake was washed with 30 mL of water and dried in vacuum to afford
1.3 g of the
desired 5-(2-furyl)-2,4-pentadienoylhydroxamic acid. 'H NMR (DMSO-d6, 300
MHz),
5(ppm) 7.73 (broad s, 1H), 7.22 (m, 1H), 6.71 (m, 4H), 6.01 (d, 1H).
Example 22
Synthesis of 6-phenyl-3,5-hexadienoylhydroxamic acid
Triethylamine (TEA, 1.75 mL) was added to a cooled (0-5 C) solution of 6-
phenyl-
3,5-hexadienoic acid (2.0 g) in 30 mL of anhydrous dimethylformamide. To this
solution
was added dropwise isobutyl chloroformate (1.62 mL) over a period of 15
minutes. The
reaction mixture was stirred for 15 minutes and hydroxylamine hydrochloride
(1.74 g) was
added followed by dropwise addition of 3.5 mL of TEA at 0-5 C. The reaction
was allowed
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to warm to room temperature and stirred overnight. The reaction was then
poured into 20
mL of 1 % (by weight) aqueous citric acid solution and extracted with 20 mL of
methylene
chloride twice and ether once. The combined organic layer was dried over
anhydrous
sodium sulfate and concentrated under vacuum to give a dark red oil. The crude
oil was
crystallized with 10 mL of hot acetonitrile. The solid was collected by
filtration and then
purified on a Biotage 40S silica gel column using methylene chloride:ether
(95:5) as an
eluent. The fractions containing the desired product were combined and the
solvent was
removed to give 40 mg of 6-phenyl-3,5-hexadienoylhydroxamic acid as a tan
solid (2.1 %).
1H NMR (DMSO-d6, 300 MHz), 8(ppm) 7.34 (m, 5H), 6.91 (m, 1H), 6.55 (d, 1H),
6.30 (m,
1H), 5.89 (m, 1H), 3.36 (d, 2H).
Example 23
Synthesis of N-methyl-6-phenyl-3,5-hexadienoylhydroxamic acid
6-Phenyl-3,5-hexadienoic acid (1 g) was dissolved in 10 mL of tetrahydrofuran
(THF) and treated with 0.9 g of 1,1'-carbonyldiimidazole. The reaction was
stirred for 30
minutes. N-methylhydroxylamine hydrochloride (0.44 g) was neutralized with
0.29 g of
sodium methoxide in 10 mL of THF and 5 mL of methanol and then filtered to
remove the
sodium chloride. N-methylhydroxylamine was then added to the reaction mixture
and stirred
overnight. The resulting mixture was partitioned between 25 mL of water and 50
mL of
ethyl acetate. The ethyl acetate layer was washed with 25 mL each of 5%
hydrochloric acid,
saturated sodium bicarbonate and brine, dried over sodium sulfate and
concentrated under
vacuum to afford 0.9 g of a viscous yellow oil. The crude product was
chromatographed on
a Biotage 40S silica gel column and eluted with ethyl acetate:hexane (1:1).
The fractions
containing the desired product were combined and the solvent was removed under
vacuum to
yield 0.17 g of N-methyl-6-phenyl-3,5-hexadienoylhydroxamic acid. 1H NMR
(CDC13, 300
MHz), 8(ppm) 7.38 (m, 5H), 6.80 (m, 1H), 6.60 (m, 1H), 6.35 (m, 1H), 5.89 (m,
1H), 3.24
(m, 2H), 2.92 (s, 3H).
Example 24
Synthesis of 7-phenyl-2,4,6-heptatrienoylhydroxamic acid
Triethylamine (TEA, 24.1 mL) was added to a cooled (0-5 C) solution of 7-
phenyl-
2,4,6-heptatrienoic acid (27.8 g) in 280 mL of anhydrous dimethylformamide. To
this
solution was added dropwise isobutyl chloroformate (22.5 mL) over a period of
75 minutes.
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The reaction mixture was stirred for 40 minutes and hydroxylamine
hydrochloride (24.2 g)
was added followed by dropwise addition of 48 mL of TEA over a period of 70
minutes at 0-
C. The reaction was allowed to warm to room temperature and stirred overnight.
To the
stirred reaction mixture at room temperature was added 280 mL of a 1% (by
weight) solution
5 of citric acid followed by 1050 mL of water. The mixture was stirred for 30
minutes and
then filtered. The filtered cake was washed with water (200 mL) and dried
under vacuum to
afford 20.5 g of the desired 7-phenyl-2,4,6-heptatrienoylhydroxamic acid. 1H
NMR (DMSO-
d6, 300 MHz), 6(ppm) 7.48 (m, 2H), 7.32 (m, 2H), 7.19 (m, 2H), 7.01 (m, 1H),
6.75 (m, 2H),
6.51 (m, 1H), 5.93 (d, 1H).
Example 25
Synthesis of 4-cyclohexylbutyroylhydroxamic acid
To a solution of hydroxylamine hydrochloride (7.3 g) in 50 mL of methanol was
added 24 mL of sodium methoxide (25% wt.) dropwise at room temperature over a
period of
45 minutes. To this solution was added methyl 4-cyclohexylbutyrate in 50 mL of
methanol
at room temperature followed by 12 mL of sodium methoxide (25% wt.) dropwise
over a
period of 60 minutes. The resulting mixture was stirred at room temperature
overnight. The
reaction was then poured into 120 mL of water and acidified to a pH of 4 with
45 mL of
glacial acetic acid. Methanol was removed under vacuum. The solid formed was
filtered
and dried over phosphorus pentoxide to afford 8.53 g of the desired 4-
cyclohexylbutyroyl-
hydroxamic acid. 1H NMR (DMSO-d6, 300 MHz), S(ppm) 3.38 (m, 2H), 1.91 (t, 2H),
1.68
(m, 4H), 1.50 (m, 2H), 1.16 (m, 5H), 0.84 (m, 2H).
Example 26
Synthesis of S-benzylthioglycoloylhydroxamic acid
S-benzylthioglycolic acid (12.0 g) was dissolved in 250 mL of methanol and
sparged
with hydrogen chloride gas at room temperature for 20 minutes. The solvent was
then
removed under vacuum. Methyl S-benzylthioglycolate obtained was used in the
next step
without further purification.
To a solution of hydroxylamine hydrochloride (9.2 g) in 60 mL of methanol was
added 30 mL of sodium methoxide (25% wt.) dropwise at room temperature over a
period of
30 minutes. To this solution was added methyl S-benzylthioglycolate in 50 mL
of methanol
at room temperature followed by 15 mL of sodium methoxide (25% wt.) dropwise
over a
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period of 60 minutes. The resulting mixture was stirred at room temperature
overnight. The
reaction was then poured into 150 mL of water and acidified to a pH of 4 with
55 mL of
glacial acetic acid. Methanol was removed under vacuum. The solid formed was
filtered
and dried over phosphorus pentoxide to afford 8.57 g of the desired S-
benzylthioglycoloyl-
hydroxamic acid. 1H NMR (DMSO-d6, 300 MHz), 8(ppm) 7.29 (m, 5H), 3.84 (s, 2H),
2.93
(s, 2H).
Example 27
Synthesis of 5-phenylpentanoloylhydroxamic acid
5-Phenylpentanoic acid (10.0 g) was dissolved in 250 mL of methanol and
sparged
with hydrogen chloride gas at room temperature for 15 minutes. The solvent was
then
removed under vacuum. Methyl 5-phenylpentanoate obtained was used in the next
step
without further purification.
To a solution of hydroxylamine hydrochloride (7.8 g) in 50 mL of methanol was
added 26 mL of sodium methoxide (25% wt.) dropwise at room temperature over a
period of
45 minutes. To this solution was added methyl 5-phenylpentanoate in 50 mL of
methanol at
room temperature followed by 15 mL of sodium methoxide (25% wt.) dropwise over
a
period of 60 minutes. The resulting mixture was stirred at room temperature
overnight. The
reaction was then poured into 150 mL of water and acidified to a pH of 4 with
40 mL of
glacial acetic acid. The solvents were removed under vacuum to give a yellow
oil. The
yellow oil was placed on a Biotage 40M silica gel column and eluted with
methylene
chloride: ethanol (95:5). The fractions containing the desired product as
indicated by the
NMR were combined. The solvents were removed under vacuum to afford 8.30 g of
the
desired 5-phenylpentanoylhydroxamic acid. 1H NMR (DMSO-d6, 300 MHz), 8(ppm)
7.22
(m, 5H), 3.42 (s, 3H), 2.55 (t, 2H), 1.98 (t, 2H), 1.52 (m, 4H).
Example 28
In vitro Efficacy Studies - Extreme Drug Resistance (EDR) Assay
The PC3 cell line was maintained in RPM1 supplemented with 10% fetal calf
serum
and antibiotics. Cells were suspended in 0.12% soft agar in complete medium
and plated
(2,000 cells per well) in different drug concentrations onto a 0.4% agarose
underlayer in 24-
well plates. Plating calls on agarose underlayers supports the proliferation
only of the
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transformed cells, ensuring that the growth signal stems from the malignant
component of the
tumor.
All compounds were dissolved were dissolved in DMSO to 200x stock solutions.
Stock solutions were diluted to 20x working solutions using the tissue culture
medium,
serially diluted and added to the 24-well plates. The initial range of
concentrations was 1
micromolar to 200 micromolar. This concentration range was extended in the
case of N-
methyl-5-phenyl-2,4-pentadienoylhydroxamic acid to 10 M -500 M and in the
case of
tricostatin A to 0.001 M to 0.3 M. No significant changes in pH of the
culture medium
were observed under the above conditions. Diluent control wells contained PC3
cells treated
with DMSO, at the dilutions used for appropriate drug treatment. All
experimental points
were represented by two separate wells (duplicates). Four wells containing
tumor cells that
were not treated with drugs served as negative controls in each experiment.
Cells were incubated with drugs under standard culture conditions for 5 days.
Cultures were pulsed with tritiated thymidine (3H-TdR, New Life Science
Products, Boston,
MA) at 5 Ci per well for the last 48 hours of the culture period. Cell
culture plates were
then heated to 90 C to liquefy the agarose, and cells were harvested onto
glass fiber filters,
which were then placed into counting vials containing liquid scintillation
fluid. The
radioactivity trapped on the filters was counted with a Beckman scintillation
counter. The
fraction of surviving cells was determined by comparing 3H-TdR incorporation
in treated
(experimental points) and untreated (negative control) wells. Microsoft Excel
was used to
organize the raw data on EDR experiments, and the SigmaPlot program was
utilized to
generate drug response curves. All drug response curves were as approximated
as sigmoidal
equations (characteristic for typical drug response curves) to fit the data.
IC50 values were
determined using the approximated sigmoidal curves and expressed as mM.
IC50 values of the test compounds of the invention range from approximately 1
M to
approximately 2000 M.
Example 29
Histone (Hyper)Acetylation Assay
The model used in this assay was mouse erythroleukemia cells. Specifically,
the
level of acetylation of H4 histones in these erythroleukemia cells was
monitored. H4
histones was chosen as the target due to the ease of resolution of the
variably acetylated
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histones. Inhibition of histone deacetylase leads to increased
(hyper)acetylation of histones.
Activities on histone deacetylase were examined to confirm the results of this
assay. See
Example 30 below.
Studies were performed with the DS 19 mouse erythroleukemia cells maintained
in
RPMI 1640 medium with 25 mM HEPES buffer and 5% fetal calf serum. The cells
were
incubated at 37 C. In studies on proliferation, cell density was determined at
24 hour
intervals using a hemacytometer.
Histone Isolation
Histones were isolated from cells after incubation for 2 or 24 hours. The
cells were
centrifuged for 5 minutes at 2,000 rpm in the Sorvall SS34 rotor and washed
once with
phosphate buffered saline. The pellets were suspended in 5 mL lysis buffer (10
mM Tris, 50
mM sodium bisulfite, 1%Triton X-100, 10 mM magnesium chloride, 8.6% sucrose,
pH 6.5)
and homogenized with six strokes of a teflon pestle. The homogenizing tubes
were rinsed
with 5 mL lysis buffer. The combined solutions were centrifuged and the
pellets were
washed once with 5 mL of the lysis buffer and once with 5 mL 10 mM Tris, 13 mM
EDTA,
pH 7.4. The pellets were extracted with 2 x 1 mL 0.25N HC1. Histones were
precipitated
from the combined extracts by the addition of 20 mL acetone and refrigeration
overnight.
The histones were pelleted by centrifuging at 5,000 rpm for 20 minutes in the
Sorvall SS34
rotor. The pellets were washed once with 5 mL acetone and protein
concentration was
quantitated by the Bradford procedure.
Polyacrylamide Gel Electrophoresis
Separation of acetylated histones was performed with an acetic acid-urea
polyacrylamide gel electrophoresis procedure as originally described by Panyim
and
Chalkley Arch. Biochem. Biophys. 130, 337-346, 1969. 25 jig histones were
applied to a slab
gel which was run at 20 ma. The run was continued for a further two hours
after the Pyronin
Y tracking dye had run off the gel. The gel was stained with Coomassie Blue R.
The most
rapidly migrating protein band is the unacetylated H4 histone followed by
bands with 1,2,3
and 4 acetyl groups which were quantitated by densitometry.
Densitometry
Densitometry was measured through digital recording using the Alpha Imager
2000.
Enlargement of the image was done using PHOTOSHOP (Adobe Corp.) on a MACINTOSH
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(Apple Corp.) computer. After creating a hard copy of the gel by using a laser
printer, a
Shimadzu CS9000U densitometer was used to measure densitometry by reflectance.
The
percentage of H4 histone in the various acetylated states was expressed as a
percentage of the
total H4 histone.
Results
Many of the test compounds of the invention showed EC50 values in micromolar
concentration range.
Example 30
Histone Deacetylation Assay
The determination of the inhibition of histone deacetylase by compounds of the
invention was based upon the procedure described by Hoffmann et al., Nucleic
Acids Res. 27,
2057-2058 (1999). The histone deacetylase was isolated from rat liver as
previously
described in Kolle, D. et al., Methods: A Companion to Methods in Enzymology
15, 323-331
(1998). Compounds were initially dissolved in either ethanol or in DMSO to
provide a
working stock solution. The synthetic substrate used in the assay is N-(4-
methyl-7-
coumarinyl)-N-a(tert-butyloxy-carbonyl)-N-SZ -acetyllysineamide (MAL).
The assay was performed in a final total volume of 120 L consisting of 100 pL
of 15
mM tris-HC1 buffer at pH 7.9 and 0.25 mM EDTA, 10 mM NaCl, 10% glycerol, 10 mM
mercaptoethanol and the enzyme. The assay was initiated upon the addition of
10 l of a test
compound followed by the addition of a fluorescent-labeled lysine substrate to
each assay
tube in an ice bath for 15 minutes. The tubes were transferred to a water bath
at 37 C for an
additional 90 minutes.
An initial assay was performed to determine the range of activity of each test
compound. The determination of IC50 values was made from the results of five
dilutions in
range according to the expected potency for each test compound. Each assay was
duplicated
or triplicated.
Test compounds of the invention showed potent inhibition of histone
deacetylase,
having IC50 values in the low micromolar concentration range (e.g., two test
compounds
showed IC50 values of 1.7 M and 1.8 M).
Example 31
X-ALD Screening Assay
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Cell Cultures and Drug Treatment
Cell lines derived from X-ALD human patients were grown in RPMI supplemented
with fetal calf serum (10%), penicillin (100 U/mL), streptomycin (100 U/mL)
and glutamine
(2mM). On day 0, cells were divided into two separate tissue culture flasks,
and test
compounds (2.5-250 M final concentration, diluted from a 0.5 M stock solution
in PBS, pH
7.6) was added to one flask. Cells in the second flask were grown in the
absence of test
compounds for the same length of time and served as controls. The media were
changed
every 3-4 days.
Biochemical Measurements
As described above, tissue culture cells were grown in the presence or absence
of test
compounds, collected from tissue culture flasks using trypsin, washed twice
with PBS and
subjected to biochemical analysis. VLCFA measurements was conducted by
extracting total
amount of lipids, converted the lipids to methyl ester, purified by TLC, and
subjected to
capillary CC analysis as described in Moser et al., Technique in Diagnostic
Biochemical
Genetics: A Laboratory Manual (ed. A., H.F.) 177-191 (Wiley-Liss, New York,
1991).
Duplicate assays were set up independently and were assayed on different days.
C24:0 ,6-
oxidation activity of lymphoblastoid cells was determined by measuring their
capacity to
degrade [1-14C]-C24:0 fatty acid to water-soluble products as described in
Watkins et al.,
Arch. Biochem. Biophys. 289, 329-336 (1991). The statistical significance of
measured
biochemical differences between untreated and treated X-ALD cells can be
determined by a
two-tailed Student's t-test.
Compounds of the invention were found to decrease the cellular content of the
VLCFA by approximately 60 percent in the X-ALD cells.
Example 32
Cystic Fibrosis Screening Assay
As described above, during its biosynthesis, CFTR is initially synthesized as
a
nascent polypeptide chain in the rough ER, with a molecular weight of around
120 kDa
(Band A). It rapidly receives a core glycosylation in the ER, giving it a
molecular weight of
around 140 kDa (Band B). As CFTR exits the ER and matures through the Golgi
stacks, its
glycosylation is modified until it achieves a terminal mature glycosylation,
affording it a
molecular weight of around 170 kDa (Band Q. The extent to which CFTR exits the
ER and
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traverses the Golgi to reach the plasma membrane maybe reflected in the ratio
of Band B to
Band C protein. CFTR is immunoprecipitated from control cells, and cells
exposed to test
compounds. Both wt CFTR and AF508 CFTR expressing cells are tested. Following
lysis,
CFTR are immunoprecipitated using various CFTR antibodies. Immunoprecipitates
are then
subjected to in vitro phosphorylation using radioactive ATP and exogenous
protein kinase A.
Samples are subsequently solubilized and resolved by SDS-PAGE. Gels are then
dried and
subject to autoradiography and phosphor image analysis for quantitation of
Bands B and C
are determined on a BioRad personal fix image station.
Cell Culture
Chinese hamster ovary (CHO) cells stably expressing both wt and AF508 CFTR
were
used in these assays. The cultures were grown on 100 mm plastic cell dishes in
DMEM
containing 10% foetal bovine serum (FBS) and kept at 5% CO2 / 95% 02 at 376C.
Cells
were grown to confluence and used 3-5 days post-plating. All test compounds
were added to
cells for 24 hours prior to analysis.
Immunoprecipitation
Cells were treated with test compounds and CFTR immunoprecipitated as
described
in Bradbury et al., Ana. J Physiol. 276, L659 - 668 (1999). Briefly, treated
cells were lysed
in buffer containing 1% TRITON X-100 and various protease inhibitors. Soluble
material
was immunoprecipitated using both R domain and C-terminal monoclonal
antibodies.
Immunoprecipitated CFTR was then subject to in vitro phosphorylation using
camp-
dependent PKA catalytic subunit and [7-32P]ATP, followed by resolution on SDS-
PAGE
gels. After fixation, the gels were dried and processed for autoradiography
and phosphor
image analysis. Quantitation of B and C bands on a BioRad personal fix image
analysis
station.
It was found that compounds of the invention (at 100 M) showed no significant
changes in the levels of Bands B and C in treated cells relative to untreated
cells. Based on
the results obtained from using these test compounds, there was no gross
effect of the test
compounds on the expression levels of wild type CFTR. Analysis of band C of
AF508
CFTR CHO cells showed that very little Band C was present in AF508 cells
compared to
wild-type cells. Exposure of these cells to test compounds at 100 M for 24
hours at 37 C
did not affect the level of Band C CFTR in either wild-type or AF508 CFTR
expressing cells.
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In contrast, analysis of Band B CFTR in AF508 cells showed that test compounds
at 100 gM
resulted in a significant increase (about 6-7 fold) in the level of Band B
compared to AF508
cells not exposed to the test compounds.
Example 33
Toxicity Assay
Test compounds of the invention were administered to three groups of 10 mice
at
100, 300, and 1,000 mg/kg. An additional group received vehicle (20%
hydroxypropyl-/3-
cyclodextrin aqueous solution) at 10 mg/kg. Mortality/morbidity checks were
made twice
daily. Clinical observations were recorded predose and /or postdose on Day 1,
and daily
thereafter through Day 8. Body weights were recorded on the day of dosing (Day
1) and on
Day 8. Mice were euthanized by CO2 asphyxiation and necropsied on Day 8 or
upon death.
One test compound was tested so far and based on the results obtained, the no-
observed toxicity level for this compound when administered to CD-1 mice as a
single
intraperitoneal does 100 mg/kg. Clinical signs of toxicity were noted after
dosing at 300
mg/kg with recovery within 24 hours, while dosing at 1,000 mg/kg resulted in
death (80% of
animals) by the end of Day 2.
Other Embodiments
The scope of the claims should not be limited by the preferred embodiments set
forth in the
examples, but should be given the broadest interpretation consistent with the
description as a whole.
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