Note: Descriptions are shown in the official language in which they were submitted.
CA 02646383 2013-08-19
. .
s
INHIBITORS OF LETHAL FACTOR PROTEASE OF ANTHRAX TOXIN
GOVERNMENT SUPPORT
This invention was made with government support under Grant Numbers
AI070494 and AI055789 awarded by the National Institutes of Health. The United
States
Government has certain rights in the invention.
BACKGROUND OF THE INVENTION
The development of new biodefense therapeutics against anthrax and botulinum
neurotoxins (BoNT) has heightened with the recent threat of these agents being
used as
biological weapons. Lethal factor (LF), a component of the anthrax tripartite
exotoxin,
cleaves mitogen-activated protein kinase kinases (MAPKK; 1-3) thus disrupting
signal
transduction leading to macrophage lysis (4). Following inhalation of anthrax
spores,
spores can adhere to alveolar macrophages and subsequently germinate. Bacteria
migrate
to lymph nodes where they rapidly multiply and excrete the anthrax exotoxin
composed
of protective antigen (PA; 83 kDa), LF, and calmodulin-activated edema factor
adenylate
cyclase (EF; 89 kDa). To exert its lethal effect, LF must enter inside the
cell
compartment. This is mediated by PA that binds to cellular receptors and,
following its
proteolytic activation by the furin-like proprotein convertases and the
release of the N-
terminal 20-kDa fragment, generates the mature PA protein (PA63). Finally,
PA63
heptamerizes and binds both LF and EF. Following endocytosis of the resulting
complexes, the engulfed molecules of LF and EF are liberated and exert their
toxic
action.
Botulinium neurotoxins, represent even more dreadful bioterrorism agents,
because BoNTs may be delivered by aerosol (7,8). With the increased use of
BoNTs in
physical/cosmetic ailments (8-13), the potential misuse of these toxins is
worrisome (14).
Currently, the only available treatment once BoNT has invaded the nervous
system is
critical care mechanical ventilation (14). However, the effects of
internalized BoNTs can
last for months (15) in which mechanical ventilation is an intolerable burden
for those
affected (14). Botulinum neurotoxins are composed of a heavy chain (HC) and a
light
1
CA 02646383 2008-12-12
chain (LC) which is connected by a disulfide bridge (16). The HC binds to
neurons
which transports the LC into the cytosol (17). The LC is a zinc
metalloprotease, similar
to anthrax that cleaves neuronal proteins involved in the neurotransmitter
release. There
are seven serotypes (BoNT A¨G) involved in the cleavage of a component of the
soluble
NSF-ethylmaleimide-sensitive factor attachment protein receptor proteins (18),
which
mediates the exocytosis of acetylcholine into neuromuscular junctions (14).
Both BoNT
serotypes A and E cleave SNAP-25 [synaptosomal-associated protein (25 KDa)]
(19),
while serotypes B, D, F, and G cleave vesicle- associated membrane protein (20-
23) and
serotype C cleaves both SNAP25 and syntaxin 1 (14,24).
The lethal action of anthrax toxin can be neutralized at several stages during
its
entry into the cell. In fact, it would be possible to inhibit PA63 processing,
pore
assembly or binding to receptor; moreover a successful therapeutic treatment
could
prevent LF or EF binding or their translocation into the cytosol (Selman,
B.R., Mourez,
M., Collier, R.J. Science 292, 695-697 (2001)). Nevertheless inhibition of LF
protease
activity is still the most promising avenue for this harmful disease
(Schwarze, S.R.,
Hruska, K.A., Dowdy, S.F. Trends Cell BioL 10, 290-295 (2000)).
Inhibition of LF protease activity is believed to be a promising avenue for
this
harmful disease (Schwarze, S.R., Hruska, K.A., Dowdy, S.F. Trends Cell Biol.
10, 290-
295 (2000)). Thus, a continuing need exists for compounds that inhibit lethal
factor (LF)
protease activity of anthrax toxin.
SUMMARY
The present invention provides compounds that can efficiently and specifically
inhibit lethal factor (LF) protease activity of anthrax toxin. Accordingly,
the invention
provides comounds of formula I and a therapeutic method for inhibiting lethal
factor
protease activity comprising administering an effective inhibitory amount of a
compound
of formula I:
R1
N
N-R2
S
R" 'S (I)
2
CA 02646383 2008-12-12
=
=
wherein
RI is phenyl, pyridyl, or thiophenyl optionally substituted with 1, 2, or 3
substituents independently selected from halo, Ci.3alkyl, C1.3alkoxy,
(aryl)C1.3alkoxy
optionally substituted with halo, ¨CF3, ¨NO2, ¨CO2H, -SO2NH2, -SO2NleR3'
wherein Rx
and RY taken together with the nitrogen to which they are attached form a
morpholino or
piperidino group, or an ortho fused tetrahydrofuran optionally substituted
with Ci_3alkyl;
R2 is ¨(CH2)1_3CO2H, --(CH2)1-3 SO3H, or heterocycle;
wherein any CH2 or heterocycle of R2 is optionally substituted with 1 or 2
substituents independently selected from halo, ¨0Ra, ¨NO2, ¨NH2, ¨S02NH2,
¨CO2H,
-CONH2, ¨0O2CH3,10CF3, or ¨CF3; wherein Ra is hydrogen, or C1_4a1ky1; and
R3 is H or phenyl optionally substituted with 1-5 halo groups;
or a pharmaceutical acceptable salt thereof.
Additionally, the invention provides comounds of formula II and a therapeutic
method for inhibiting lethal factor protease activity comprising administering
an effective
inhibitory amount of a compound a compound of formula II:
r
R1-
Al
A
(II)
wherein
Rl is hydrogen, or phenyl optionally substituted with 1, 2, or 3 substituents
independently selected from alkyl, halo, ¨NO2, ¨0O2H, or ¨S02N112;
----- optionally forms a double bond;
Al and A2 are each independently CH, C when substituted, or N;
A3 is S or NH;
R2 is hydrogen, ¨Ci4alkyl, ¨0O2H, ¨(CH2)1.3CO2H,
Ci_6alkoxycarbonyl, furyl, fury1C1.3alkylene-, phenyl, (phenyl)C1_3alkylene-,
or
(pyridyl)C1-3 alkylene¨;
wherein the alkyl or phenyl groups of R2 are optionally substituted with 1, 2,
or 3
substituents independently selected from halo, ¨0Ra, ¨NO2, ¨NH2, ¨S02NH2,
¨0O2H, ¨
3
CA 02646383 2008-12-12
CONH2,
¨CO2CH3, ¨0CF3, or ¨CF3; wherein Ra is hydrogen, or Ci4alkyl;
or a pharmaceutical acceptable salt thereof.
The invention also provides comotmds of formula III and a therapeutic method
for
inhibiting lethal factor protease activity comprising administering an
effective inhibitory
amount of a compound a compound of formula III:
0
N¨R2
R1-111
C A
R10 Q
= (111)
wherein
R1 and R1 are each independently hydrogen, halo, heterocycle, phenyl, or a
heterocycle
substituted N-H or N-alkyl atninoalkoxy group; or
R1 and R1 together form an ortho-fused aryl, heteroaryl, or heterocyclic
ring;
wherein any heterocycle, heteroaryl, or aryl of R1 and R1 is optionally
substituted
with 1, 2, or 3 substituents independently selected from alkyl, halo, ¨NO2,
¨CO2H, or ¨
SO2NH2;
A is CH, C when substituted, or N;
Q is 0 or S; and .
------------- optionally forms a double bond;
R2 is hydrogen, ¨Ci4alkyl, ¨Cmalkenyl, ¨0O2H, ¨(CH2)1-3CO211, --(CH2)1-
3S03H, -(CH2)1_3S02NH2, C1_6alkoxycarbonyl, furyl, fury1C1.3alkylene-, phenyl,
(phenyl)Ci_3alkylene-, or (pyridyl)Ci_3alkylene¨;
wherein any alkyl or phenyl group of R2 is optionally substituted with 1, 2,
or 3
substituents independently selected from halo, ¨NO2, ¨NH2, ¨SO2NH2, ¨0O2H, ¨
CONH2,
¨0O2CH3, ¨CF3, ¨0CF3, or ¨ORb, wherein Rb is hydrogen, or Ci4alkyl;
or a pharmaceutical acceptable salt thereof.
Additionally, the invention provides comounds of formula IV and a therapeutic
method for inhibiting lethal factor protease activity comprising administering
an effective
inhibitory amount of a compound a compound of formula IV:
4
_
CA 02646383 2008-12-12
0
W
N ¨R2
Z¨Y
(IV)
wherein
R1 is hydrogen, or phenyl optionally substituted with 1, 2, or 3 substituents
independently selected from alkyl, halo, ¨NO2, ¨CO2H, or ¨SO2NH2;
X, Y and Z are each independently 0, S, ¨NH¨, ¨CH2¨, =CH¨, or =N¨; and
R2 is ¨(CH2)1_3S03H, ¨(CH2)1.3heterocycle, or heterocycle;
= wherein any CH2 or heterocycle group of R2 is optionally substituted with
1 or 2
substituents independently selected from halo,¨NO2, ¨NH2, ¨S02NH2, ¨CO2H,
¨CONH2,
¨CO2CH3, ¨0CF3, ¨CF3, or ¨0Ra, wherein Ra is hydrogen, or C14alkyl;
or a pharmaceutical acceptable salt thereof.
The invention further provides comounds of formula V and a therapeutic method
for inhibiting lethal factor protease activity comprising administering an
effective
inhibitory amount of a compound a compound of formula V:
(R1 ____________________________________________ 0
). 11.
1.1
N N
0
N¨R2
0
S
(V)
wherein
each RI is independently hydrogen, alkyl, alkoxy, halo, haloalkyl, hydroxy,
hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl,
alkoxycarbonyl, amino,
alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy,
carboxyalkyl,
alkylthio, alkylsulfinyl, alkylsulfonyl, or cyano;
n is 1, 2, 3, 4, or 5;
Ra is hydrogen or Ci_aalkyl;
R2 is hydrogen, ¨CI _4 alkyl, alkenyl, ¨C 02 H, ¨(CH2)1-3C 02H, ¨(CH2)1-
3S03H, -(CH2)1-3S02NH2, Ci..6alkoxycarbonyl, furyl, furylC1_3alkylene-,
phenyl,
= (phenyl)C1.3alkylene-, or (pyridyl)C1.3alkylene¨; and
5
CA 02646383 2008-12-12
wherein any alkyl or phenyl group of R2 is optionally substituted with 1, 2,
or 3
substituents independently selected from halo, ¨NO2, ¨NH2, ¨S02N112, ¨CO2H, -
CONH2,
¨CO2CH3, ¨CF3, ¨0CF3, or ¨01e, wherein Rb is hydrogen, or Ci_aalkyl;
or a pharmaceutical acceptable salt thereof.
Additionally, the invention provides comounds of formula VI and a therapeutic
method for inhibiting lethal factor protease activity comprising administering
an effective
inhibitory amount of a compound a compound of formula VI:
R1
s
N¨R2
R1 R12 (W)
wherein
o
ICO2H
-CH .'N==\)N'N---/
101 i
R s halo, heterocycle, heteroaryl, or s
=
R2 is hydrogen, ¨CI-Alkyl, ¨CO2H, ¨(CH2)1_3CO2H,
C1_6alkoxycarbonyl, furyl, furylCi_3alkylene-, phenyl, (phenyl)C1_3alkylene-,
or
(pyridyl)Ci _3 alkylene¨;
wherein the alkyl or phenyl groups of R2 are optionally substituted with 1, 2,
or 3
substituents independently selected from halo,¨NO2, ¨NH2, ¨SO2NH2, ¨CO2H,
¨00NH2,
¨CO2CH3, ¨CF3, ¨0CF3, or ¨0Ra, wherein Ra is hydrogen or Ci_aallcyl;
¨11
x and R12 are each H or R11 and R12 taken together form an ethylenedioxy
group;
or a pharmaceutical acceptable salt thereof.
Additionally, the invention provides comounds of formula VII and a therapeutic
method for inhibiting lethal factor protease activity comprising administering
an effective
inhibitory amount of a compound a compound of formula VII:
o
N ¨R2
\z S
R1 S (VII)
6
CA 02646383 2008-12-12
wherein
Ri is hydrogen, ¨CiAalkyl, or phenyl;
wherein X, Y and Z are a combination of 0, S, ¨NH¨, ¨CH2¨, =CH¨, or =N¨;
X is -S- or =N-;
Y is -0- or -C- substituted by Ci4alkyl or heterocycle;
Z is -0-, -S-, =N-, or -C- substituted by heterocycle;
the ring containing X, Y, and Z is aromatic and includes two double bonds, and
one of X, Y, or Z comprises a carbon atom;
R2 is hydrogen, ¨Ci4allcyl, ¨0O2H, ¨(CH2)1.3CO2H,
Ci_6alkoxycarbonyl, furyl, fury1C1_3alkylene-, phenyl, (phenyl)Ci_3alkylene-,
or
(pyridyl)C1.3alkylene¨;
wherein the alkyl or phenyl groups of R2 are optionally substituted with 1, 2,
or 3
substituents independently selected from halo,¨NO2, ¨NH2, ¨SO2NH2, ¨0O2H,
¨CONH2,
¨CO2CH3, ¨CF3, ¨0CF3, or ¨01e, wherein Ra is hydrogen or Ci4a1kyl;
or a pharmaceutical acceptable salt thereof.
Accordingly, the invention provides compounds of formulas I-VII and methods of
treatment using compounds of formulas I-VII. One method of treatment includes
inhibiting bacterial toxins. The bacterial toxin can be, for example, anthrax
lethal factor
and/or BoNT/A. Another method of treatment includes inhibiting a
metalloprotease. The
metalloprotease can be a human matrix metalloprotease, such as MMP-2 and/or
MMP-9.
The invention further provides a method for preventing or inhibiting lethal
factor-induced
cell death of macrophages.= Additionally, the invention provides methods for
treating
and/or inhibiting anthrax or botulinium infections. The treating or inhibiting
can include
administering an effective inhibitory amount of a compound of any one of
formulas I-
VII, and/or contacting a patient, cell, or group of cells with an effective
amount of a
compound of any one of formulas I-VII.
In some embodiments of the invention, the compound of any one of formulas I-
VII can be used to prepare a composition that includes a phamaceutiaclly
acceptable
= diluent or carrier, optionally in combination with an antibacterial
agent. The antibacterial
agent can be ciprofloxacin (commonly referred to as cipro). Accordingly, the
invention
7
-
CA 02646383 2008-12-12
also provides methods of treatment that include the use of a compound of any
one of
formulas I-VII in combination with an antibacterial agent, such as
ciprofloxacin.
The invention further provides compounds that inhibit LF protease activity in
in
vitro assays. Accordingly, a therapeutic method is provided for treating a
mammal in
need of inhibition on LF protease activity, by administering an effective
inhibitory
amount of a compound of any one of formulas I-VII. In one embodiment the
mammal is
human. The invention also provides a therapeutic method to inhibit lethal
factor (LF)
protease activity of anthrax toxin comprising contacting the cell, in vitro or
in vivo, with
. an effective amount of a compound of a formula described herein.
The invention also provides a compound of a formula described herein for use
in
medical therapy, in some embodiments for use in treating lethal factor (LF)
protease
activity of anthrax toxin, as well as the use of a compound of a formula
described herein
for the manufacture of a medicament for the treatment of a pathological
condition or
symptom in a mammal, such as a human, which is associated with lethal factor
(LF)
protease activity from anthrax.
The invention also provides a method of identifying an agent that inhibits the
lethal factor (LF) protease activity of anthrax toxin, comprising: a)
identifying detecting a
selective lethal factor (LF) protease inhibitor; b) contacting a bound lethal
factor (LF)
protease inhibitor with a test compound, said test compound suspected of being
able to
inhibit lethal factor (LF) protease; and c) detecting dissociation of said
lethal factor (LF)
protease inhibitor from said labeled Bc1-XL, whereby said candidate agent is
identified as
an agent that inhibits Bc1-XL. The invention provides novel compounds as
described
herein, such as compounds included in any one of formulas I-VII.
The invention also provides novel intermediates for the synthesis of compounds
of formulas I-VII, as well as methods of preparing compounds formulas I-VII.
The
invention also provides compounds of formulas I-VII that are useful as
intermediates for
the synthesis of other useful compounds. The compounds and compositions can
also be
used to prepare a medicament to treat a diseases in a mammal, for example,
anthrax
disease in a human.
8
CA 02646383 2013-08-19
In accordance with an aspect of the present invention there is provided a
compound of
formula I:
R1 9
N\ V /
N-R2
N
S ______________________________________
R3 \ S (I)
wherein
RI is phenyl, pyridyl, or thiophenyl optionally substituted with 1, 2, or 3
substituents
independently selected from the group consisting of halo, C1_3alkyl,
Ci_3alkoxy, (aryl)C1..
3alkoxy optionally substituted with halo, ¨CF3, ¨NO2, ¨CO2H, -SO2NH2, and
SO2NleRY
wherein Rx and RY taken together with the nitrogen to which they are attached
form a
morpholino or piperidino group, or an ortho fused tetrahydrofuran optionally
substituted with
Ci_3alkyl;
R2 is -CH2CO2H, ¨(CH2)1-3S03H, or heterocycle;
wherein any CH2 or heterocycle of R2 is optionally substituted with 1 or 2
substituents
independently selected from the group consisting of halo, ¨0Ra, ¨NO2, ¨NH2,
¨SO2NH2, ¨
CO2H, -CONH2, ¨CO2CH3, ¨0CF3, and ¨CF3; wherein Ra is hydrogen, or C1_4alkyl;
and
R3 is phenyl optionally substituted with 1-5 halo groups;
or a pharmaceutical acceptable salt thereof.
In accordance with a further aspect of the present invention there is provided
use of an
effective inhibitory amount of a compound of formula I for inhibiting lethal
factor protease in
a mammal in need of such therapy,
R1 0
N\
N-R2
N
S ______________________________________
R3 \ S (I)
wherein
RI is phenyl, pyridyl, or thiophenyl optionally substituted with 1, 2, or 3
substituents
independently selected from the group consisting of halo, Ci_3alkyl,
Ci_3alkoxy, (aryl)C1_
3alkoxy optionally substituted with halo, ¨CF3, ¨CO2H, -SO2NH2, and -
SO2NWRY
wherein Rx and RY taken together with the nitrogen to which they are attached
form a
morpholino or piperidino group, or an ortho fused tetrahydrofuran optionally
substituted with
Ci_3alkyl;
R2 is -CH2CO2H, ¨(CH2)1_3S03H, or heterocycle;
8a
CA 02646383 2013-08-19
wherein any CH2 or heterocycle of R2 is optionally substituted with 1 or 2
substituents
independently selected from the group consisting of halo, ¨0Ra, ¨NO2, ¨NH2,
¨SO2NH2, ¨
CO2H, ¨CONH2, ¨CO2CH3, ¨0CF3, or ¨CF3; wherein Ra is hydrogen, or C1_4a1ky1;
and
R3 is phenyl optionally substituted with 1-5 halo groups;
or a pharmaceutical acceptable salt thereof
In accordance with a further aspect of the present invention there is provided
use of an
effective inhibitory amount of a compound of formula II for inhibiting lethal
factor protease
in a mammal in need of such therapy,
o
Al
R1 I I
A2 A3 S __
(II)
wherein
RI is hydrogen, or phenyl optionally substituted with 1, 2, or 3 substituents
independently selected from the group consisting of alkyl, halo, ¨NO2, ¨CO2H,
and ¨
SO2NH2;
------- optionally forms a double bond;
AI and A2 are each independently CH, C when substituted, or N;
A3 is S or NH;
R2 is hydrogen, ¨C ¨C _4 alkenyl, ¨CO2H, ¨(CH2)1-3CO2H,
C _6 alkoxycarbonyl, furyl, fury1C t_3alkylene-, phenyl, (phenyl)Ci _3
alkylene-, or (pyridyl)Ci _
3 alkylene¨;
wherein the alkyl or phenyl groups of R2 are optionally substituted with 1, 2,
or 3
substituents independently selected from the group consisting of halo, ¨01e,
¨NO2, ¨NH2, ¨
SO2NH2, ¨CO2H, ¨CONH2, ¨CO2CH3, ¨0CF3, and ¨CF3; wherein Ra is hydrogen, or CI-
4alkyl;
or a phaimaceutical acceptable salt thereof
In accordance with a further aspect of the present invention there is provided
use of an
effective inhibitory amount of a compound of formula III for inhibiting lethal
factor protease
in a mammal in need of such therapy,
8b
CA 02646383 2013-08-19
0
N¨R2
R1
S _______________________________________
R10 Q (M)
wherein
RI and RI are each independently hydrogen, halo, heterocycle, phenyl, or a
heterocycle substituted N-H or N-alkyl aminoalkoxy group; or
RI and RI together form an ortho-fused aryl, heteroaryl, or heterocyclic
ring;
wherein any heterocycle, heteroaryl, or aryl of RI and RI is optionally
substituted
with 1, 2, or 3 substituents independently selected from the group consisting
of alkyl, halo, ¨
NO2, ¨CO2H, and ¨SO2NH2;
A is CH, C when substituted, or N;
Q is 0 or S; and
------- optionally forms a double bond;
R2 is hydrogen, ¨Ci_4alkyl, ¨Ci_aalkenyl, ¨CO2H, ¨(CH2)1-3CO2H, ¨(CH2)1 -
3 S 03H, -(CH2)1_3 SO2NH2, C1_6alkoxycarbonyl, furyl, fury1C1_3alkylene-,
phenyl, (phenyl)C
3alkylene-, or (pyridyl)C1.3alkylene¨;
wherein any alkyl or phenyl group of R2 is optionally substituted with 1, 2,
or 3
substituents independently selected from the group consisting of halo, ¨NO2,
¨NH2, ¨
SO2NH2, ¨CO2H, ¨CONH2,
¨CO2CH3, ¨CF3, ¨0CF3, and ¨ORb, wherein Rb is hydrogen, or Ci_aalkyl;
or a pharmaceutical acceptable salt thereof
In accordance with a further aspect of the present invention there is provided
use of an
effective inhibitory amount of a compound of formula IV for inhibiting lethal
factor protease
in a mammal in need of such therapy,
o
RlIX
N _________________________________________ R2
Z¨Y S
\S (IV)
wherein
RI is hydrogen, or phenyl optionally substituted with 1, 2, or 3 substituents
independently selected from the group consisting of alkyl, halo, ¨NO2, ¨CO2H,
and ¨
SO2NH2;
8c
CA 02646383 2013-08-19
X, Y and Z are each independently 0, S, -NH-, -CH2-, =CH-, or =1\1-; and
R2 is -(CH2)1_3S03H, 4CH2)1_3heterocycle, or heterocycle;
wherein any CH2 or heterocycle group of R2 is optionally substituted with 1 or
2
substituents independently selected from the group consisting of halo,-NO2, -
NH2, -
SO2NH2, -CO2H, -CONH2,
-CO2CH3, -0CF3, -CF3, and -01V, wherein Ra is hydrogen, or Ci_aalkyl;
or a pharmaceutical acceptable salt thereof.
In accordance with a further aspect of the present invention there is provided
use of an
effective inhibitory amount of a compound of formula V for inhibiting lethal
factor protease
in a mammal in need of such therapy,
Ra
i(`=NN 0
(Ri)n _______________
0
0 N-R2
S
(V)
wherein
each R1 is independently hydrogen, alkyl, alkoxy, halo, haloalkyl, hydroxy,
hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl,
alkoxycarbonyl, amino,
alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy,
carboxyalkyl,
alkylthio, alkylsulfinyl, alkylsulfonyl, or cyano;
n is 1, 2, 3, 4, or 5;
Ra is hydrogen or Ci_4alkyl;
R2 is hydrogen, -Ci_4alkyl, -C1_4alkenyl, -CO2H, -(CH2)1-3CO2H, -(CH2)1-
3S03H, -(CH2)1_3S02NH2, C1_6alkoxycarbonyl, fury', furylCi_3alkylene-, phenyl,
(phenyl)Ci_
3alkylene-, or (pyridyl)C1_3alkylene-; and
wherein any alkyl or phenyl group of R2 is optionally substituted with 1, 2,
or 3
substituents independently selected from the group consisting of halo, -NO2, -
NH2, -
SO2NH2, -CONH2,
-CO2CH3, -CF3, -0CF3, and -ORb, wherein Rb is hydrogen, or Ci_4alkyl;
or a pharmaceutical acceptable salt thereof
In accordance with a further aspect of the present invention there is provided
use of an
effective inhibitory amount of a compound of foimula VI for inhibiting lethal
factor protease
in a mammal in need of such therapy,
8d
CA 02646383 2013-08-19
,
0
R1S _______________________________ /
S _________________________________________________ cR2
R11 R12 S (VI)
wherein
O
-CH (NN¨ICO2H
S __
R1 is halo, heterocycle, heteroaryl, or s =
,
R2 is hydrogen, ¨Ci_4alkyl, ¨Ci_4alkenyl, ¨CO2H, ¨(CH2)1_3CO2H,
C1_6alkoxycarbonyl, furyl, furylCi_3alkylene-, phenyl, (phenyl)Ci_3alkylene-,
or (pyridyl)Ci_
3alkylene-;
wherein the alkyl or phenyl groups of R2 are optionally substituted with 1, 2,
or 3
substituents independently selected from the group consisting of halo,¨NO2,
¨NH2, ¨
SO2NH2, ¨CO2H, ¨CONH2,
¨CO2CH3, ¨CF3, ¨0CF3, and ¨01V, wherein Ra is hydrogen or Ci_4alkyl;
R11 and R12 are each H or R11 and R12 taken together form an ethylenedioxy
group;
or a pharmaceutical acceptable salt thereof
In accordance with a further aspect of the present invention there is provided
use of an
effective inhibitory amount of a compound of formula VII for inhibiting lethal
factor protease
in a mammal in need of such therapy,
O
x
7
Y N __ R2
\z ______________________________________ S __
R1 S (VII)
wherein
RI is hydrogen, ¨C1_4alkyl, or phenyl;
wherein X, Y and Z are a combination of 0, S, ¨NH¨, ¨CH=)--, =CH¨, or =N¨;
X is -S- or =N-;
Y is -0- or -C- substituted by C1_4alkyl or heterocycle;
Z is -0-, -S-, =N-, or -C- substituted by heterocycle;
Se
CA 02646383 2013-08-19
=
the ring containing X, Y, and Z is aromatic and includes two double bonds, and
one of
X, Y, or Z comprises a carbon atom;
R2 is hydrogen, ¨CiAalkyl, ¨C1.4alkenyl, ¨CO2H, ¨(CH2)1_3CO2H,
Ci_6alkoxycarbonyl, furyl, fury1C1_3alkylene-, phenyl, (phenyl)Ci_3alkylene-,
or (pyridyl)Ci_
3alkylene¨;
wherein the alkyl or phenyl groups of R2 are optionally substituted with 1, 2,
or 3
substituents independently selected from the group consisting of halo,¨NO2,
¨NH2, ¨
SO2NH2, ¨CO2H, ¨CONH2,
¨CO2CH3, ¨CF3, ¨0CF3, and ¨01V, wherein Ra is hydrogen or Ci_4a1ky1;
or a pharmaceutical acceptable salt thereof
8f
CA 02646383 2008-12-12
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the specification and are included to
further
demonstrate certain embodiments or various aspects of the invention. In some
instances,
embodiments of the invention can be best understood by referring to the
accompanying
drawings in combination with the detailed description. The description and
accompanying drawings may highlight a certain specific example, or a certain
aspect of
the invention, however, one skilled in the art will understand that portions
of the example
or aspect may be used in combination with other examples or aspects of the
invention,
and that that portions of the example or aspect may be excluded from other
examples or
aspects of the invention.
= Figure 1: Kinetics of inhibition of lethal factor (LF) for compounds 9
and 13.
IC50 evaluation of (A) compound 9 and (B) compound 13 against LF in the
absence
(squares) and presence (triangles) of PA63; Ki evaluation of compound 9 (C)
measured at
various concentration of inhibitor (top line, 5 M; middle line, 3 M; bottom
line, no
inhibitor) and compound 13 (D) measured at various concentrations of inhibitor
(top line,
1 M; middle line, 0.5 M; bottom line, no inhibitor).
Figure 2: Molecular docking studies. Stereo views of the molecular model of
compound 34 docked into the catalytic pockets of BoNT A (PDB-ID 2G7N; A and B)
or
lethal factor (PDB-1D 1YQY; C and D). For both targets, the protein surface
was
generated with MOLCAD and the Zn2+ ion is shown as a sphere.
Figure 3: Compound 34 and ciprofloxacin (cipro) protect A/J mice from anthrax.
Mice (eight animals per group) were infected intranasally with 4 H 105
Bacillus anthracis
Sterne spores. Treatment with compound 34 (25 mg/kg in DMSO administered via
intraperitoneal injection) was started 24 h postexposure and continued for the
next 5 days.
On the fourth day following infection, mice were given daily injections of
cipro (25
mg/kg subcutaneously). Mice given DMSO alone were used as the control, and all
died
on day 6 (dashed line; triangles). The group treated with compound 34 and
cipro
(squares) had the best survival (p < 0.01 compared to control group), while
the group
receiving compound 34 alone (dashed line; circles) also survived significantly
better than
control (p < 0.05) and the group treated with cipro alone (diamonds).
9
CA 02646383 2008-12-12
õ1.
DETAILED DESCRIPTION
New componds and methods have been developed that inhibit LF and provide
significant protection against Bacillus anthracis. The compounds and methods
can be
used in combination with an antibiotic such as ciprofloxacin to treat or
prevent conditions
associated with bacterial infections, such as a Bacillus anthracis infection.
There has been relatively limited progress in identifying effective small
molecule
inhibitors of BoNTs with only a few exceptions. Disclosed herein is a series
of rhodanine
derivatives, which have been discovered to be potent and/or selective BoNT A
inhibitors.
Whereas previously reported rhodanine-based LF protease inhibitors were
inactive
against human metalloproteases MMP-2 and MMP-9 (5), several rhodanine
derivatives
of the invention can selectively inhibit MMP-2 and/or MMP-9.
The compounds disclosed herein can also be inhibitors of targets such as HCV
NS3 protease (30), aldose reductase (31), fl-lactamase (32), UDP-N-
acetylmuramate/L-
alanine ligase (33), cathepsin D (34), histidine decarboxylase, and Bc1-XL
(35). The
compounds can display a wide range of pharmacological activities, including
antimicrobial (36-41), antiviral (42), anticonvulsant (42,43), and
antidiabetic (44,45).
For example, the compounds can be used for the treatment of the type II
diabetes by
contributing to improved glycemic control by increasing insulin sensitivity.
Accordingly, the rhodanine derivatives of the invention can be antitoxin
protease
inhibitors, such as anthrax LF inhibitors or BoNT/A inhibitors. The rhodanine
derivatives of the invention were analyzed for potency and selectivity against
LF and =
BoNT A as well as for solubility and in vitro ADME-Tox properties, cell-based
assays
and preliminary in vivo efficacy.
As used herein, the following terms and expressions have the indicated
meanings.
It will be appreciated that the methods of the present invention can employ
and/or
provide compounds that can contain asymmetrically substituted carbon atoms,
and can be
isolated in optically active or racemic forms. It is well known in the art how
to prepare
optically active forms, such as by resolution of racemic forms or by
synthesis, from
optically active starting materials.
All chiral, diastereomeric, racemic forms and all geometric isomeric forms of
a
structure are intended, unless the specific stereochemistry or isomeric form
is specifically
CA 02646383 2008-12-12
indicated. The processes to prepare or manufacture compounds useful in the
present
invention are contemplated to be practiced on at least a multigram scale,
kilogram scale,
multikilogram scale, or industrial scale. Multigram scale, as used herein, is
preferably
the scale wherein at least one starting material is present in 10 grams or
more, more
preferably at least 50 grams or more, even more preferably at least 100 grams
or more.
Multi-kilogram scale, as used herein, is intended to mean the scale wherein
more than
one kilogram of at least one starting material is used. Industrial scale as
used herein is
intended to mean a scale which is other than a laboratory scale and which is
sufficient to
supply product sufficient for either clinical tests or distribution to
consumers.
One diastereomer of a compound disclosed herein may display superior activity
compared with the other. When required, separation of the racemic material can
be
achieved by HPLC using a chiral column or by a resolution using a resolving
agent such
as camphonic chloride as in Tucker, et al., J. Med. Chem., 37:2437 (1994). A
chiral
compound described herein may also be directly synthesized using a chiral
catalyst or a
chiral ligand, e.g. Huffman, et al., J. Org. Chem., 60:1590 (1995).
The present invention is intended to include all isotopes of atoms occurring
on the
compounds useful in the present invention. Isotopes include those atoms having
the same
atomic number but different mass numbers. By way of general example and
without
limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of
carbon
include C-13 (13C) and C-14 (14C).
General Definitions
As used herein, certain terms have the following meanings. All other terms and
phrases used in this specification have their ordinary meanings as one of
skill in the art
would understand. Such ordinary meanings may be obtained by reference to
technical
dictionaries, such as Hawley 's Condensed Chemical Dictionary 14th Edition, by
R.J.
Lewis, John Wiley & Sons, New York, N.Y., 2001.
The term "and/or" means any one of the items, any combination of the items, or
all of the items with which this term is associated.
The singular forms "a," "an," and "the" include plural reference unless the
context
clearly dictates otherwise. Thus, for example, a reference to "a compound"
includes a
plurality of such compounds, so that a compound X includes a plurality of
compounds X.
11
CA 02646383 2008-12-12
The term "about" can refer to a variation of 5%, 10%, or 20% of the value
specified. For example, "about 50" percent can in some embodiments cam/ a
variation
from 45 to 55 percent. For integer ranges, the term "about" can include one or
two
integers greater than and less than a recited integer.
It should be noted that references in the specification to "one embodiment",
"an
embodiment", "an example embodiment", etc., indicate that the embodiment
described
may include a particular feature, structure, or characteristic, but every
embodiment may
not necessarily include that particular feature, structure, or characteristic.
Moreover, such
phrases are not necessarily referring to the same embodiment. Further, when a
particular
feature, structure, or characteristic is described in connection with an
embodiment, it is
submitted that it is within the knowledge of one skilled in the art to affect
such feature,
structure, or characteristic in connection with other embodiments whether or
not
explicitly described.
As used herein, "contacting" refers to the act of touching, making contact, or
of
bringing to immediate or close proximity, including at the molecular level,
such as in
vivo, in vitro, or in an aqueous solution.
Compound and Composition Definitions
As used herein, "pharmaceutically acceptable salts" refer to derivatives of
the
disclosed compounds wherein the parent compound is modified by making acid or
base
salts thereof. Examples of pharmaceutically acceptable salts include, but are
not limited
to, mineral or organic acid salts of basic residues such as amines; alkali or
organic salts of
acidic residues such as carboxylic acids; and the like. The pharmaceutically
acceptable
salts include the conventional non-toxic salts or the quaternary ammonium
salts of the
parent compound formed, for example, from non-toxic inorganic or organic
acids. For
example, such conventional non-toxic salts include those derived from
inorganic acids
such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and
the like; and
the salts prepared from organic acids such as acetic, propionic, succinic,
glycolic, stearic,
lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,
phenylacetic,
glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fiimaric,
toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.
12
CA 02646383 2013-08-19
The pharmaceutically acceptable salts of the compounds useful in the present
invention can be synthesized from the parent compound, which contains a basic
or acidic
moiety, by conventional chemical methods. Generally, such salts can be
prepared by
reacting the free acid or base forms of these compounds with a stoichiometric
amount of
the appropriate base or acid in water or in an organic solvent, or in a
mixture of the two;
generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol,
or acetonitrile
are preferred. Lists of suitable salts are found in Remington's Pharmaceutical
Sciences,
17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985).
The phrase "pharmaceutically acceptable" is employed herein to refer to those
compounds, materials, compositions, and/or dosage foims which are, within the
scope of
sound medical judgment, suitable for use in contact with the tissues of human
beings and
animals without excessive toxicity, irritation, allergic response, or other
problem or
complication commensurate with a reasonable benefit/risk ratio.
"Stable compound" and "stable structure" are meant to indicate a compound that
is sufficiently robust to survive isolation to a useful degree of purity from
a reaction
mixture, and foimulation into an efficacious therapeutic agent. Only stable
compounds
are contemplated by the present invention.
The teini "substituted" means that a specified group or moiety can bear one or
more (e.g., 1, 2, 3, 4, 5, or 6) substituents. The teini "unsubstituted" means
that the
specified group bears no substituents. The teini "optionally substituted"
means that the
specified group is unsubstituted or substituted by one or more substituents.
Where the
term "substituted" is used to describe a structural system, the substitution
is meant to
occur at any valency-allowed position on the system. In cases where a
specified moiety
or group is not expressly noted as being optionally substituted or substituted
with any
specified substituent, it is understood that such a moiety or group is
intended to be
unsubstituted in some embodiments but can be substituted in other embodiments.
The
telin substituted is intended to indicate that one or more hydrogens on the
substituted
atom or group is replaced with a selection from the indicated group(s),
provided that the
indicated atom's normal valency is not exceeded, and that the substitution
results in a
stable compound. Suitable substituent groups include, e.g., alkyl, alkenyl,
alkynyl,
13
CA 02646383 2008-12-12
alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, aroyl, heteroaryl,
heterocycle,
cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino,
trifluoromethylthio, difluoromethyl, acylamino, nitro, trifluoromethyl,
trifluoromethoxy,
carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl,
arylsulfmyl,
arylsulfonyl, heteroarylsulfinyl, heteroarylsulfonyl, heterocyclesulfinyl,
heterocyclesulfonyl, phosphate, sulfate, hydroxyl amine, hydroxyl
(alkyl)amine, and/or
cyano.
The term "alkyl" refers to a monoradical branched or unbranched saturated
hydrocarbon chain preferably having from 1 to 8 carbon atoms. This term is
exemplified
by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-
butyl, n-
hexyl, and the like.
The alkyl can optionally be substituted with one or more alkoxy, halo,
haloalkyl,
hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl,
alkoxycarbonyl, amino, alkylamino, acylamino, nitro, trifluoromethyl,
trifluoromethoxy,
carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl,
cyano, NR.Rx
or COORõ, wherein each R.õ is independently H or alkyl.
The alkyl can optionally be interrupted with one or more non-peroxide oxy (-OA
thio (-S-), sulfonyl (SO) or sulfoxide (S02).
The alkyl can optionally be at least partially unsaturated, thereby providing
an
alkenyl or alkynyl.
The term "alkoxy" refers to the groups alkyl-O-, where alkyl is defined
herein.
Preferred alkoxy groups include, e.g., methoxy, ethoxy, n-propoxy, iso-
propoxy, n-
butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and
the like.
The alkoxy can optionally be substituted with one or more alkyl, halo,
haloalkyl,
hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl,
alkoxycarbonyl, amino, alkylamino, acylamino, nitro, trifluoromethyl,
trifluoromethoxy,
carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl
and cyano.
The term "aryl" refers to an unsaturated aromatic carbocyclic group of from 6
to
20 carbon atoms having a single ring (e.g., phenyl) or multiple condensed
(fused) rings,
wherein at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl,
fluorenyl, or
anthryl). Preferred aryls include phenyl, naphthyl and the like.
14
CA 02646383 2008-12-12
The aryl can optionally be substituted with one or more alkyl, alkoxy, halo,
haloalkyl, hydroxy, hydroxyalkyl, heteroaryl, heterocycle, cycloalkyl,
alkanoyl,
alkoxycarbonyl, amino, alkylamino, acylamino, nitro, trifluoromethyl,
trifluoromethoxy, =
carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl
and cyano.
The term "cycloalkyl" refers to cyclic alkyl groups of from 3 to 20 carbon
atoms
having a single cyclic ring or multiple condensed rings. Such cycloalkyl
groups include,
by way of example, single ring structures such as cyclopropyl, cyclobutyl,
cyclopentyl,
cyclooctyl, and the like, or multiple ring structures such as adatnantanyl,
and the like.
The cycloalkyl can optionally be substituted with one or more alkyl, alkoxy,
halo,
alkoxycarbonyl, amino, alkylamino, acylamino, nitro, trifluoromethyl,
trifluoromethoxy,
carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl
and cyano.
The cycloalkyl can optionally be at least partially unsaturated, thereby
providing a
cycloalkenyl.
The term "halo" refers to fluoro, chloro, bromo, and iodo. Similarly, the term
"halogen" refers to fluorine, chlorine, bromine, and iodine.
"Haloalkyl" refers to alkyl as defined herein substituted by 1-4 halo groups
as
defined herein, which may be the same or different. Representative haloalkyl
groups
include, by way of example, trifluoromethyl, 3-fluorododecyl, 12,12,12-
trifluorododecyl,
The term "heteroaryl" is defined herein as a monocyclic, bicyclic, or
tricyclic ring
system containing one, two, or three aromatic rings'and containing at least
one nitrogen,
oxygen, or sulfur atom in an aromatic ring, and which can be unsubstituted or
substituted,
for example, with one or more, and in particular one to three, substituents,
like halo,
alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino,
alkylamino,
acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl. Examples of heteroaryl
groups
include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, 4nH-
carbazolyl,
acridinyl, benzo[b]thienyl, benzothiazoly1,13-carbolinyl, carbazolyl,
chromenyl,
cinnaolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl,
indazolyl,
indolisinyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl,
isoxazolyl,
naphthyridinyl, naptho[2,3-b], oxazolyl, perimidinyl, phenanthridinyl,
phenanthrolinyl,
CA 02646383 2008-12-12
phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl,
phthalazinyl,
pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl,
pyrimidinyl,
pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl,
thianthrenyl,
thiazolyl, thienyl, triazolyl, and xanthenyl. In one embodiment the term
"heteroaryl"
denotes a monocyclic aromatic ring containing five or six ring atoms
containing carbon
and 1, 2, 3, or 4 heteroatoms independently selected from the group non-
peroxide
oxygen, sulfur, and N(Z) wherein Z is absent or is H, 0, alkyl, phenyl or
benzyl. In
another embodiment heteroaryl denotes an ortho-fused bicyclic heterocycle of
about eight
to ten ring atoms derived therefrom, particularly a benz-derivative or one
derived by
fusing a propylene, or tetramethylene diradical thereto.
The heteroaryl can optionally be substituted with one or more alkyl, alkoxy,
halo,
haloalkyl, hydroxy, hydroxyalkyl, aryl, heterocycle, cycloalkyl, alkanoyl,
alkoxycarbonyl, amino, alkylamino, acylamino, nitro, trifluoromethyl,
trifluoromethoxy,
carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfmyl, alkylsulfonyl
and cyano.
The term "heterocycle" refers to a saturated or partially unsaturated ring
system,
containing at least one heteroatom selected from the group oxygen, nitrogen,
and sulfur,
and optionally substituted with alkyl or C(=0)0Rb, wherein Rb is hydrogen or
alkyl.
Typically heterocycle is a monocyclic, bicyclic, or tricyclic group containing
one or more
heteroatoms selected from the group oxygen, nitrogen, and sulfur. A
heterocycle group
also can contain an oxo group (=0) attached to the ring. Non-limiting examples
of
heterocycle groups include 1,3-dihydrobenzofuran, 1,3-dioxolane, 1,4-dioxane,
1,4-
dithiane, 2H-pyran, 2-pyrazoline, 4H-pyran, chromanyl, imidazolidinyl,
irnidazolinyl,
indolinyl, isochromanyl, isoindolinyl, morpholine, piperazinyl, piperidine,
piperidyl,
pyrazolidine, pyrazolidinyl, pyrazolinyl, pyrrolidine, pyrroline,
quinuclidine, and
thiomorpholine.
The heterocycle can optionally be substituted with one or more alkyl, alkoxy,
halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, cycloalkyl,
alkanoyl,
alkoxycarbonyl, amino, alkylamino, acylamino, nitro, trifluoromethyl,
trifluoromethoxy,
carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl
and cyano.
Examples of nitrogen heterocycles and heteroaryls include, but are not limited
to,
pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine,
indolizine,
16
CA 02646383 2008-12-12
isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline,
phthalazine,
naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole,
carboline,
phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole,
phenoxazine,
phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline,
morpholino,
piperidinyl, tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen
containing
heterocycles.
Another class of heterocyclics is known as "crown compounds" which refers to a
specific class of heterocyclic compounds having one or more repeating units of
the
formula [.-(CH2-)aA-] where a is equal to or greater than 2, and A at each
separate
occurrence can be 0, N, S or P. Examples of crown compounds include, by way of
example only, [-(CH2)3-NH-]3, [4(CH2)2-0)44(CH2)2-NH)2] and the like.
Typically such
crown compounds can have from 4 to 10 heteroatoms and 8 to 40 carbon atoms.
The term. "alkanoyl" refers to C(=0)R, wherein R is an alkyl group as
previously
defined.
The term "acyloxy" refers to ¨0-C(=0)R, wherein R is an alkyl group as
previously defined. Examples of acyloxy groups include, but are not limited
to, acetoxy,
propanoyloxy, butanoyloxy, and pentanoyloxy. Any alkyl group as defined above
can be
used to form an acyloxy group.
The term "alkoxycarbonyl" refers to C(=0)0R, wherein R is an alkyl group as
previously defined.
The term "amino" refers to -NH2, and the term "alkylamino" refers to -NR2,
wherein at least one R is alkyl and the second R is alkyl or hydrogen. The
term
"acylamino" refers to RC(=0)N, wherein R is alkyl or aryl.
As to any of the above groups, which contain one or more substituents, it is
understood, of course, that such groups do not contain any substitution or
substitution
patterns which are sterically impractical and/or synthetically non-feasible.
In addition,
the compounds of this invention include all stereochemical isomers arising
from the
substitution of these compounds.
Selected substituents within the compounds described herein are present to a
recursive degree. In this context, "recursive substituent" means that a
substituent may
recite another instance of itself. Because of the recursive nature of such
substituents,
17
CA 02646383 2008-12-12
. =
theoretically, a large number may be present in any given claim. One of
ordinary skill in
the art of medicinal chemistry understands that the total number of such
substituents is
reasonably limited by the desired properties of the compound intended. Such
properties
include, by of example and not limitation, physical properties such as
molecular weight,
solubility or log P, application properties such as activity against the
intended target, and
practical properties such as ease of synthesis.
Recursive substituents are an intended aspect of the invention. One of
ordinary
skill in the art of medicinal and organic chemistry understands the
versatility of such
substituents. To the degree that recursive substituents are present in an
claim of the
invention, the total number will be determined as set forth above.
Obviously, numerous modifications and variations of the present invention are
possible in light of the above teachings. It is therefore to be understood
that within the
scope of the appended claims, the invention may be practiced otherwise than as
specifically described herein.
Certain compounds and methods of the invention include lethal factor inhibitor
compound 1 (BI-11B1) and compound 2 (rosiglitazone), an inhibitor of
peroxisome
proliferators-activated receptor-gamma.
H3 CO
=
Cl.=
I / \ 9113 NH
\ S.
CT 0
SrNjOH 0
1 2
These compounds may be used in combination with the therapeutic compounds,
compositions, and methods described herein.
Compounds of the Invention
The invention provides compounds of formula I wherein R1 can be phenyl,
substituted phenyl, heterocycle, or substituted heterocycle. Specific
substitutents include
halo (e.g., F, Cl, Br, or I), trifluoromethyl, morpholinosulfonyl,
phenylsulfonyl, alkoxy
such as methoxy or ethoxy, benzyloxy, or halobenzyloxy.
Specific values for R2 include -(CH2)CO2H, C2114S03H, or -(1,1-dioxo-
tetrahydro-thiophen-3-y1).
18
CA 02646383 2008-12-12
=
Specific values for R3 include H, phenyl, or substituted phenyl. Specific
substitutions for phenyl include 1-5 halo groups, each of which may be located
at the 2,
3, 4, 5, and/or 6 position of the phenyl group. Similar positional
substitutions can be
made for other aryl, heteroaryl, or heterocyclic groups of other formulas of
the invention.
In some embodiments, RI is not phenyl substituted with chloro. In other
embodiments, Rl is not phenyl substituted with halo. In yet other embodiments,
RI is
not phenyl substituted with halo, nitro, carboxy, or sulfonamide.
The invention provides compounds of formula II wherein R1 can be H, halo, for
example, chloro or bromo.
1 0 A specific values for R2 is -(CH2)CO2H.
The bond represented by ---- can be absent or present. The alkene group of the
thiazole ring of formula II can be attached to the bicyclic ring of formula II
at a position
either alpha or beta to A3. Specific values for A" include CH, S, N, and/or
NH.
The invention provides compounds of formula III wherein R1 can be H,
1 5 heterocycle, or heteroaryl. Rl can also be linked to RIO to form a
group that includes an
aryl, a heterocyclic, or a heteroaryl group, which can be optionally
substituted, for
example, with one or more halo or alkyl groups.
Specific values for R2 include H or -(CH2)CO2H and the bond represented by ----
- can be absent or present.
20 Specific values for A include C, CH, and/or N. Specific values for Q
are S or O.
Other specific values for the formulas described herein include the variables
lised
in Table A. Table A below illustrates various compounds of formulas I-VII and
certain
specific definitions of their corresponding variables. In some embodiments,
the one or
more groups from the variables of one formula can be included in the
definition of one or
25 more variables of another formula, and/or the one or more groups from
the variables of
one formula can be excluded from the definition of one or more variables of
another
formula.
19
_
,
Table A: Specific Compounds of the Invention.
Compound
Chemical Name Compound of Formula:
Substituent Values
No.
R1 0
(Z)-2-(4-oxo-2-thioxo-5-((3-(4-
N V/
RI = 4-(trifluoromethyl)phenyl
27 (trifluoromethyl)pheny1)-1H-pyrazol-4- \ N¨R2
W
s---
R2 = H00O21.1
/N /
yl)methylene)thiazolidin-3-yl)acetic acid
R3 = H
R3 s
(I)
R1 - 0
0 :
(Z)-2-(4-oxo-5-((1-pheny1-3-(thiophen-2-y1)-1H-
N V
RI = -(thiophene-2-y1)
1..)
29 pyrazol-4-yOmethylene)-2-thioxothiazolidin-3- \ / N¨R2
R2 = (CH2)CO2H 0,
0.
0,
yl)acetic acid N S---
R3 = Ph w
co
R3/S (I)
w
F')
.
o
R1 0
co
1
(Z)-2-(541,3-dipheny1-1H-pyrazol-4-
N V
R
2
RI = Ph 1
30 yOmethylene)-4-oxo-2-thioxothiazolidin-3-yp s¨N¨R
RI R1
acetic
(CH2)CO2il
"
acid
R3 = = Ph
= S
(17)
i
,
,
0
,
,
Ri =
31
4-
(Z)-2-(543-(4-(morpholinosulfonyl)pheny1)-1-
N
(morpholinosulfonyl)phenyl
/N
1
pheny1-1H-pyrazol-4-yl)methylene)-4-oxo-2- \ /
R2 = (CH2)CO2H
thioxothiazolidin-3-yl)acetic acid s4,N¨R2
R3 = Ph
R3 s
(I)
=
'
1
Ri o
(Z)-2-(54(1-(2,4-difluoropheny1)-3-(pyridin-3-y1)-
N 7
R1= N-pyridinium
32 1H-pyrazol-4-yl)methylene)-4-oxo-2- \ / N¨R2
S k
R2 = (CH2)CO2H
thioxothiazolidin-3-yl)acetic acid N
R3 = (2,4-difluorophenyl)
Ri S (I)
R1 0
(Z)-2-(54(1,3-bis(4-fluoropheny1)-1H-pyrazol-4-
-.-
RI = 4-fluorophenyl
33 yflmethylene)-4-oxo-2-thioxothiazolidin-3-yD Nacetic \ZN /
s ,I¨R2 R2 = (CH2)CO2H
(0
acid
R3= 4-fluorophenyl
Rl
0
S
o
R1 0
tv
cn
o.
cn
w
(Z)-2-(5-((1,3-bis(4-chloropheny1)-1H-pyrazol-4-
N 7
RI = (4-chlorophenyl) co
w
34 yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic
\ / R2= (CH2)CO2H N.,
o
acid N S cR2
R3 = (4-chlorophenyl) o
/
co
R3
'-
1')
1
R1 0
tv
(Z)-2-(541,3-bis(4-bromopheny1)-1H-pyrazol-4-
N
R1= (4-bromophenyl)
35 yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic
\7 / R2= (CH2)CO2H
acid N S C-R2
R3 = (4-bromophenyl)
/
R3 s (r)
R1 o
(Z)-2-(5-03-(4-methoxy-3-methylpheny1)-1-phenyl-
N Z
R' = (4-methoxy-3-methylphenyl)
36 1H-pyrazol-4-yl)methylene)-4-oxo-2- \N /
R2 = (CHOCO2H
thioxothiazolidin-3-yl)acetic acid s 41¨R2
R3= Ph
Rl S (I)
21
;
_
R1 0 .
(Z)-2-(5-43-(4-ethoxy-2-methylpheny1)-1-phenyl-
le = (4-ethoxy-2-methylphenyl)
37 1H-pyrazol-4-yl)methylene)-4-oxo-2- \N /
s__I¨R2
R2 = (CH2)CO2H
thioxothiazolidin-3-ypacetic acid
le = Ph
/
R3 s (I)
R1 o
(Z)-2-(54(3-(4-(4-chlorobenzyloxy)pheny1)-1-
RI = (4-chlorobenzyloxy)phenyl
38 phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-
4\1¨R2 R2 = C2H4S03H
thioxothiazolidin-3-ypethanesulfonic acid
R.3= Ph
R3/
S (I)
o
o
R1 0 "
0)
0.
R' = (2-methyl-2,3-
0,
(Z)-2-(54(3-(2-methy1-2,3-dihydrobenzofuran-5-y1)-
N , w
co
39 1-pheny1-1H-pyrazol-4-yOmethylene)-4-oxo-2- \N / N¨R2
dihydrobenzofuran-5-y1) w
1..)
R2= C
thioxothiazolidin-3-yDethanesulfonic acid
0
R3/ s
2H4S03H
R3= Ph
co
S (I)
1
'-
1')
R1 o 1
1-,
1.)
(Z)-2-(54(3-(4-methoxypheny1)-1-pheny1-1H-
N V R' = (4-methoxyphenyl)
40 pyrazol-4-yl)methylene)-4-oxo-2-thioxothiazolidin- \N /1
R2 = C2H4S03H
3-yl)ethanesulfonic acid s (--
R2 le = Ph
R3/
S (I)
R1 O.
41 4-yl)methylene)-4-oxo-2-thioxothiazolidin-3- N\ V / ''' N¨R2
/
S
R2 = C2H4S03H
yl)ethanesulfonic acid N-
R3 = Ph
R3/ s (1)
22
.>.
R1 0
` .
IV = (4-
(Z)-2-(54(3-(4-(morpholinosulfonyl)pheny1)-1-
N,
(morpholinosulfonyl)phenyl)
42 phenyl-1H-pyrazol-4-yO \
methylene)-4-oxo-2- N /
R2 = C2H4S03H
thioxothiazolidin-3-yl)ethanesulfonic acid s
N¨R2
/
R3 = Ph
R3 s
(D
R1 o
(Z)-2-(4-oxo-5-((1-pheny1-3-(4-(piperidin-1-
N V
RI = 1-(phenylsulfonyl)piperidine
43 ylsulfonyl)pheny1)-1H-pyrazol-4-yOmethylene)-2- \N /
R2 = C2H4S03H
thioxothiazolidin-3-yl)ethanesulfonic acid s 4:¨R2
R3 = Ph
Rf S
(I) 0
.
0
RI 0
t..)
0)
0.
Ri = Ph
0,
3-(1,1-Dioxo-tetrahydro-thiophen-3-y1)-541-(1,3-
w
c
R2 = -(1,1-dioxo-tetrahydro-
w
44 dipheny1-1H-pyrazol-4-y1)-meth-(Z)-ylidene]-2-
\N i thiophen-3-y1)
thioxo-thiazolidin-4-one s
..(1¨R2 R3= Ph 0
0
Rlco
S
(I) 1
'-
1')
R1 0
I
'-
1')
R' = 4-Chloro-phenyl
5-[143-(4-Chloro-pheny1)-1-pheny1-1H-pyrazol-4-
N \,,
R2 = -(1 ,1 -dioxo-tetrahydro-
45 yll-meth-(Z)-ylidene]-3-(1,1-dioxo-tetrahydro-
\")'Ç"' thiophen-3-y1)
thiophen-3-y1)-2-thioxo-thiazolidin-4-one N S
.14-----R2 R3 = Ph
Ri S
(I)
R1 0
. (Z)-2-(54(3-(4-ethoxy-3-fluoropheny1)-1-phenyl-
N ,
R' = 4-ethoxy-3-fluorophenyl
46 1H-pyrazol-4-yl)methylene)-4-oxo-2- \ /
R2 = (CH2)CO2H
. thioxothiazolidin-3-ypacetic acid N S CR2
R3 = Ph
Rl S
(I)
23
..
o
R' =H
Al
R2 = (CH2)CO2H
_
----
57 (Z)-2-(5-(benzo[b]thiophen-3-ylmethylene)-4-oxo-
R, N-R2
= Optional double bondQ,
)
--,3 present
. 2-thioxothiazolidin-3-yl)acetic acid -A2 A SA' =
CH
S
A2 = CH
(II)
A3 = S
o
R1 =Br
Al
R2 = (CH2)CO2H
(Z)-2-(54 R1
5-((5-3- ...------\s> N-
R2 ---- = Optional double bond
--
58 yOmethylene)-4-oxo-2-thioxothiazolidin-3-ypacetic s--
present
c)
acid CA2------A3
A' = CH -
s0
A2 = CH
1.)
0,
(II)
A3 = S 0.
0,
(...)
o
RI = H co
(...)
Al R2 = (CH2)CO2H
0
r
0
(Z)-2-(5((6,7-dihydrothieno[2,3-b]pyrazin-6- R1-
)N-R2
---- = Optional double bond co
1
59 yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic s
absent
11)
acid A2.'''-""-
A3 A1 = N
S
A2 = N 1.)
(II)
A3 = S
o
R1 = H
Al
R2 = (CH2)CO2H
60 (Z)-2-(5((1H-indo1-3-ypmethylene)-4-oxo-2- R1-11 '-
------'\ N R2 ---- = Optional double bond
2A(
S
present
thioxothiazolidin-3-yl)acetic acid A
A' = CH
\ s
A2 = CH
OD
A3 = NH
_
_______________________________________________________________________________
______________________________
24
..
O
Ri = Br
Al
R2= (CH2)CO2H
---C: ) N¨R 2
---- = Optional double bond
61 (Z)-2-(545-bromo-1H-indo1-3-yOmethylene) R1
-4- [I
----
oxo-2-thioxothiazolidin-3-yl)acetic acid 3 S
A2 A
present
Al = CH
S
A2= CH .
(11)
A3= NH ;
,
O
le = 2-(methyl(pyridin-2- '
yl)amino)ethoxy
Rio, R2_ H
¨
2 544-(2-(methy1(pyridin-2- I
N¨R2
---- = Optional double bond
S¨
ypamino)ethoxy)benzyp R1-= thiazolidine-2,4-dione C
A,A
absent 0
R1 Q (111) A = CH
1.)
Q = 0
0.
Oand R
w
RI
' together = 4-phenyl co
w
R2= (CH2)CO2H
49 (Z)-2-(5-(4-phenylbenzylidene)-4-oxo-2-
R1----L- ---'s N¨R2
---- = Optional double bond
0
thioxothiazolidin-3-yl)acetic acid A
S¨ Present
A = CH
0
1
1-.
1.)
RI cl (II1) Q = S i
1-,
. 1.)
o
R' and R1 together = N- ,
,
pyrrolidino
,
R2= (CH2)CO2H
,
,
50 (Z)-2-(5-(3-(pyrrolidin-1-yl)benzylidene)-4-oxo-2- I
R1-11 N¨R2 ---- = Optional double bond .
thioxothiazolidin-3-yl)acetic acid C
.A S¨
Present ,
R"A Q
A = CH ,
(III) Q=S
,
,
O RI and R' together = (1, 4-
.
diazepan-l-y1)
..,,,
R2 = (CH2)CO2H
51 (Z)-2-(5-(4-(1,4-diazepan-l-yl)benzylidene)-4-oxo-
W--I
¨ '.- - ,N
----R2
---- = Optional double bond
2- s thioxothiazolidin-3-
yoacetic acid (
Present
R1 Q om A
= CH
.
, Q=S
O RI = N-pyrazolyl
R2 = (CH2)CO2H
52 (Z)-2-(5-(3-(1H-pyrazol-1-yl)benzylidene)-4-oxo-2- Ri I N¨R2
---- = Optional double bond
thioxothiazolidin-3-yl)acetic acid Q.
/A S¨
Present 0
R1 Q
A = CH 0
010 Q=S 1..)
0,
0.
0,
O
RI and RI together = 1, 3- w
co
dioxole
w
R2 = (CH2)CO2H
1..)
0
53 (Z)-2-(5-(benzo[d][1,3]dioxo1-5-ylmethylene)-4-
R1---1=
--
N¨R2
-- = Optional double bond
0
0
oxo-2-thioxothiazolidin-3-yl)acetic acid 1L',A S
Present
A = CH
1
1-.
1..)
1
R10 Q (III) Q=S r..1
O RI and RI together = 2-
methylpyridinium
R.2 = (CH2)CO2H
54 (Z)-2-(542-methylquinolin-6-yl)methylene)-4-oxo-
R1-11I N¨R2
---- = Optional double bond
2-thioxothiazolidin-3-yl)acetic acid C A
S
Present
Ri Q inn A = CH
kw,/ Q=S
26
,
,
,
=
0
le = benzofuran
R2= (CH2)CO2H
RI = H
55 (Z)-2-(5-(dibenzofuran-6-ylmethylene)-4-oxo-2- I
Ftli N¨R2
---- = Optional double bond
thioxothiazolidin-3-yl)acetic acid LI A
A, S Present =
R1 Q A = CH
(III) Q = s
RI = Br
0
R2 = (CH2)CO2H
RI = H
56 (Z)-2-(5-((6-bromopyridin-2-yl)methylene)-4-oxo-
R1 ni N¨R2
---- = Optional double bond
2-thioxothiazolidin-3-yl)acetic acid
A S
Present 0
= A=N
R1 Q (HD Q=S 0
N.)
0,
0.
0,
0
(...)
0
1J)R1,7 x Ni.,-,N
R2 = (CH2)2S03H le = 3-nitrophenyl
1.)
9 (Z)-2-(5-05-(3-nitrophenyl)furan-2-yl)methylene)-
\ N¨R2
X = 0 o
0
0
4-oxo-2-thioxothiazolidin-3-yl)ethanesulfonic acid
Y is = CH '
Z¨Y S 1-,
Z iS =CH
1.)
1
s (W)1-,
N)
0
R' = 3-chlorophenyl
R1 x
R2 = (CH2)2S03H
= 10 (Z)-2-(545-(3-chlorophenypfuran-2-yl)methylene)- 1
N¨R2
X = 0
Y is = CH
4-oxo-2-thioxothiazolidin-3-yl)ethanesulfonic acid
Z¨Y S =
.
(1v) Z is = CH
s
> 27
...
,
p
0
111= 3,4-dichloropheriy1
R1,Xy
R2 = (CH2)2S03H
(Z)-2-(54(5-((5-2-
,.\, N¨R2 X = 0
y
11 yl)methylene)-4-oxo-2-thioxothiazolidin-3-
Y is = CH
yl)ethanesulfonic acid Z¨Y S---
Z is = CH
s (w)
0
RI = 4-chlorophenyl
R1 X
R2 = (CH2)2S03H
12 (Z)-2-(54(5-(4-chlorophenyl)furan-2-yl)methylene)- I
N¨R2 X = 0
4-oxo-2-thioxothiazolidin-3-yl)ethanesulfonic acid
Y is = CH
S (Iv)
,
0
1.)
0
R1= (2,3-dichlorophenyl) 0,
0.
x
w
(5Z)-5-11 Ftl
5-(2,3-dichloropheny1)-2- y NI,/y
N¨R2
dioxidotetrahydrothien-3-y1) c
w
14 furyll \ methylenel-3-(1,1-
dioxidotetrahydrothien-3- X = 0 "
y1)-2-thioxo-1,3-thiazolidin-4-one Z¨Y S
Y = CH 0
0
co
1
S
(Iv) Z = CH
1.)
1
o R = (2,3-dichlorophenyl)
1.)
x
(5Z)-5- {[5-(2,3-dichloropheny1)-2- Ri z
dioxidotetrahydrothien-3-y1)
15 furylimethylene}-3-(1,1-dioxidotetrahydrothien-3- \ yYN= N¨R2
X = 0
y1)-2-thioxo-1,3-thiazolidin-4-one Z¨Y S---
Y = CH
S
(IV) Z = CH
_
o
R' = 2,5-dichlorophenyl :
R2= -(tetrahydrofuran-2-
.
(Z)-54(5-(2,5-dichlorophenyl)furan-2- R1 ,..X yyN
16 yl)methylene)-3-((tetrahydrofuran-2-ypmethyl)-2- N¨R2
yl)methyl
X = 0 (Oxy)
thioxothiazolidin-4-one Z¨Y S--
Y is =CH
S
(IV) Z is =CH
28
= .
'
s
R.
I
R1 = C1
(Z)-2-(5-(1-(2-(3-chlorophenylamino)-2-oxoethyl)- (R)¨j-
62 2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-
..õ,......y.....- 0 -.
N¨R2 n 2= 1
0
R = (CH2)CO2H ,
3-yl)acetic acid s4
le = H
s
(V)
Fe
I
N
6.........'''''''''- Ir'N 4111 0 RI = CH3
2
(Z)-2-(5-(1-(2-(m-toluidino)--oxoethyl)-2- mr, 1
0
0
63 oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-
1............-
N¨R2 n 2= 1
i R = (CH2)CO2H
- i
yl)acetic acid s¨
le =H "
s
0.
0.,
w ,
(V)
co i
w
_
o 1..)
0
0
co
le Br
1
(Z)-2-(5-((7-bromo-2,3-dihydrothieno[3,4- w s -,
R2= = (CH2)CO2H
1..)
47 b][1,4]dioxin-5-yl)methylene) 4-oxo-2-
/ N-R2
R11 and R12together are 1,4-
1
1-.
thioxothiazolidin-3-ypacetic acid s¨
1..)
dioxane
R" R12 S
(VI) ,
o
,
(Z)-2-(5-05-morpholinothiophen-2-yl)methylene)- W s
R1= N-morpholino
48
,
4-oxo-2-thioxothiazolidin-3-yl)acetic acid )/ r,c7IN
N-R2
R2 = (CH2)CO2H
s
Ri2= H
R1' R12 S
(vi)
29
r
_
o
(Z)-2-(4-oxo-5-((5-(thiophen-2-yl)thiophen-2- RIS
RI = thiophene
66 ypmethylene)-2-thioxothiazolidin-3-ypacetic acid =
)' / '-- N¨R2 R2 = (CH2)CO2H
R12 = H
R11 R12 S (VI)
0
R I = (5-methylene-4-oxo-2-
{(5Z)-5-[(5-{(Z)-[4-(carboxymethyl)-3-oxo-5- RI S
thioxo-tetrahydrothiophen-3-
67 thioxodihydrothien-2(3H)-ylidene]methyl}thien-2-
)/ /
_._1¨R2 yl)acetic acid
yOmethyl ene]-4 -oxo -2-thioxo -1,3 -thiazolidin-3-
R2= (CH2)CO2H
s
yl}acetic acid
R" = H
R11 R12 S
(VI) R12 = H 0
0
R' =H
= H
0,
0.
x
18 N¨R2
R2= (CH2)CO2H
thi
0,
(Z)-2-(5-((2-(1-(tert-butoxycarbonyl)piperidin-4- V
w
co
yl)azol-4l thl 4 2
-y)meyene)--oxo-- Y
\
S----
XI: a r:b oNt exr ty-lba tuet Yipiperidine-1-
0;
co
thioxothiazolidin-3-yl)acetic acid z
RI s (VII)
Z=s 1
1-,
1.)
o 1
1-,
RI = H
1..)
x
20 (Z)-2-(5((5-(furan-2-ypisoxazol-3-yOmethylene)-4- YV
N¨R2
R2 = (CH2)CO211
oxo-2-thioxothiazolidin-3-yl)acetic acid \
s
X = N
z
Y = 0
R1 S
(VII) Z = C-Furan
. o
RI = Ph
(Z)-2-(5((2-methy1-4-phenylthiazol-5- /x =-= 14¨R2
R2= (C112)CO2H
Y
19 yOmethylene)-4-oxo-2-thioxothiazolidin-3-ypacetic \X = S
acid Z S (
Y = C-CH3
R1 µS
(VII) Z = N
o
RI = CH3
17 (Z)-2-(5-((2,5-dirriethyloxazol-4-ypmethylene)-4- ,x
N¨R2 R2 = (CH2)CO2H
oxo-2-thioxothiazolidin-3-yl)acetic acid
X = N
s
Y = C-CH3
R1 (VII)
Z
o
o
o
31
CA 02646383 2008-12-12
(
Preparation of Compounds of the Invention
The compounds of the invention can be prepared using various standard
techniques known to those skilled in the art or by the methods described in
the Examples
below. Many known compounds are commercially available from a chemical
supplier,
such as Maybridge, Chembridge and Chemnavigator (San Diego, CA).
In cases where compounds are sufficiently basic or acidic to form stable
nontoxic
acid or base salts, administration of the compounds as salts may be
appropriate.
Examples of pharmaceutically acceptable salts are organic acid addition salts
formed
with acids which form a physiological acceptable anion, for example, tosylate,
methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate,
ascorbate,
a-ketoglutarate, and a-glycerophosphate. Suitable inorganic salts may also be
formed,
including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
Pharmaceutically acceptable salts may be obtained using standard procedures
well
known in the art, for example by reacting a sufficiently basic compound such
as an amine
with a suitable acid affording a physiologically acceptable anion. Alkali
metal (for
example, sodium, potassium or lithium) or alkaline earth metal (for example
calcium)
salts of carboxylic acids can also be made.
Pharmaceutical Compositions
The compounds of formulas can be formulated as pharmaceutical
compositions and administered to a mammalian host, such as a human patient in
a variety
of forms adapted to the chosen route of administration, i.e., orally or
parenterally, by
intravenous, intramuscular, topical or subcutaneous routes.
Thus, the present compounds may be systemically administered, e.g., orally, in
combination with a pharmaceutically acceptable vehicle such as an inert
diluent or an
assimilable edible carrier. They may be enclosed in hard or soft shell gelatin
capsules,
may be compressed into tablets, or may be incorporated directly with the food
of the
patient's diet. For oral therapeutic administration, the active compound may
be combined
with one or more excipients and used in the form of ingestible tablets, buccal
tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such
compositions
and preparations should contain at least 0.1% of active compound. The
percentage of the
compositions and preparations may, of course, be varied and may conveniently
be
32
CA 02646383 2008-12-12
between about 2 to about 60% of the weight of a given unit dosage form. The
amount of
active compound in such therapeutically useful compositions is such that an
effective
dosage level will be obtained.
The tablets, troches, pills, capsules, and the like may also contain the
following:
binders such as gum tragacanth, acacia, corn starch or gelatin; excipients
such as
dicalcium phosphate; a disintegrating agent such as corn starch, potato
starch, alginic acid
and the like; a lubricant such as magnesium stearate; and a sweetening agent
such as
sucrose, fructose, lactose or aspartame or a flavoring agent such as
peppermint, oil of
wintergreen, or cherry flavoring may be added. When the unit dosage form is a
capsule,
it may contain, in addition to materials of the above type, a liquid carrier,
such as a
vegetable oil or a polyethylene glycol. Various other materials may be present
as
coatings or to otherwise modify the physical form of the solid unit dosage
form. For
instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac
or sugar and
the like. A syrup or elixir may contain the active compound, sucrose or
fructose as a
sweetening agent, methyl and propylparabens as preservatives, a dye and
flavoring such
as cherry or orange flavor. Of course, any material used in preparing any unit
dosage
form should be pharmaceutically acceptable and substantially non-toxic in the
amounts
employed. In addition, the active compound may be incorporated into sustained-
release
preparations and devices.
The active compound may also be administered intravenously or
intraperitoneally
by infusion or injection. Solutions of the active compound or its salts can be
prepared in
water, optionally mixed with a nontoxic surfactant. Dispersions can also be
prepared in
glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in
oils. Under
ordinary conditions of storage and use, these preparations contain a
preservative to
prevent the growth of microorganisms.
The pharmaceutical dosage forms suitable for injection or infusion can include
sterile aqueous solutions or dispersions or sterile powders comprising the
active
ingredient which are adapted for the extemporaneous preparation of sterile
injectable or
infusible solutions or dispersions, optionally encapsulated in liposomes. In
all cases, the
ultimate dosage form should be sterile, fluid and stable under the conditions
of
manufacture and storage. The liquid carrier or vehicle can be a solvent or
liquid
33
CA 02646383 2008-12-12
=
dispersion medium comprising, for example, water, ethanol, a polyol (for
example,
glycerol, propylene glycol, liquid polyethylene glycols, and the like),
vegetable oils,
nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity
can be
maintained, for example, by the formation of liposomes, by the maintenance of
the
required particle size in the case of dispersions or by the use of
surfactants. The
prevention of the action of microorganisms can be brought about by various
antibacterial
and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic
acid,
thimerosal, and the like. In many cases, it will be preferable to include
isotonic agents,
for example, sugars, buffers or sodium chloride. Prolonged absorption of the
injectable
compositions can be brought about by the use in the compositions of agents
delaying
absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound
in
the required amount in the appropriate solvent with various of the other
ingredients
enumerated above, as required, followed by filter sterilization. In the case
of sterile
powders for the preparation of sterile injectable solutions, the preferred
methods of
preparation are vacuum drying and the freeze drying techniques, which yield a
powder of
the active ingredient plus any additional desired ingredient present in the
previously
sterile-filtered solutions.
For topical administration, the present compounds may be applied in pure form,
i.e., when they are liquids. However, it will generally be desirable to
administer them to
the skin as compositions or formulations, in combination with a
dermatologically
acceptable carrier, which may be a solid or a liquid.
Useful solid carriers include finely divided solids such as talc, clay,
microcrystalline cellulose, silica, alumina and the like. Useful liquid
carriers include
water, alcohols or glycols or water-alcohol/glycol blends, in which the
present
compounds can be dissolved or dispersed at effective levels, optionally with
the aid of
non-toxic surfactants. Adjuvants such as fragrances and additional
antimicrobial agents
can be added to optimize the properties for a given use. The resultant liquid
compositions can be applied from absorbent pads, used to impregnate bandages
and other
dressings, or sprayed onto the affected area using pump-type or aerosol
sprayers.
34
CA 02646383 2008-12-12
Thickeners such as synthetic polymers, fatty acids, fatty acid salts and
esters, fatty
alcohols, modified celluloses or modified mineral materials can also be
employed with
liquid carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for
application directly to the skin of the user.
Examples of useful dermatological compositions which can be used to deliver
the
compounds of formula I-VII to the skin are known to the art; for example, see
Jacquet et
al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al.
(U.S. Pat.
No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
Useful dosages of the compounds of the formulas described herein can be
Generally, the concentration of the compound(s) of the formulas described
herein
in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%,
preferably from
The amount of the compound, or an active salt or derivative thereof, required
for
use in treatment will vary not only with the particular salt selected but also
with the route
of administration, the nature of the condition being treated and the age and
condition of
3 to about 50 mg per kilogram body weight of the recipient per day, preferably
in the
range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60
mg/kg/day.
25 The compound is conveniently administered in unit dosage form; for
example,
containing 5 mg to 1000 mg, conveniently 10 mg to 750 mg, most conveniently,
50 mg to
500 mg of active ingredient per unit dosage form.
Ideally, the active ingredient should be administered to achieve peak plasma
concentrations of the active compound of from about 0.5 to about 75 AM,
preferably,
30 about 1 to 50 uM, most preferably, about 2 to about 30 uM. This may
be achieved, for
example, by the intravenous injection of a 0.05 to 5% solution of the active
ingredient,
- õ-
CA 02646383 2008-12-12
optionally in saline, or orally administered as a bolus containing about 1-100
mg of the
active ingredient. Desirable blood levels may be maintained by continuous
infusion to
provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about
0.4-15
mg/kg of the active ingredient(s).
The desired dose may conveniently be presented in a single dose or as divided
doses administered at appropriate intervals, for example, as two, three, four
or more sub-
doses per day. The sub-dose itself may be further divided, e.g., into a number
of discrete
loosely spaced administrations; such as multiple inhalations from an
insufflator or by
application of a plurality of drops into the eye.
The ability of a compound of the invention to inhibit LF protease activity may
be
determined using pharmacological models which are well known to the art, or
using the
procedures described in the Examples below.
The following Examples are intended to illustrate the above invention and
should
not be construed as to narrow its scope. One skilled in the art will readily
recognize that
the Examples suggest many other ways in which the invention could be
practiced. It
should be understood that numerous variations and modifications may be made
while
remaining within the scope of the invention.
EXAMPLES
Example 1. Compounds of the Invention as Selective Protease Inhibitors
Methods And Materials
Compounds and reagents. Compounds 2-16, 29, 31, 36-46, 53, 60, 62, and 63
were purchased from Sigma Aldrich (St. Louis, MO, USA). Compound 68 was
purchased from Astatech, Inc (Bristol, PA, USA). All common chemicals,
reagents, and
buffers were purchased from Sigma-Aldrich or Acros (Geel, Belgium). Synthetic
details
of the rhodanine derivatives 17¨ 28, 30, 32-35, 47-52, 54-59, and 61 are
described in
Example 3. Compounds 1 and 64-67 were synthesized and characterized within our
laboratory as described previously (Proc. Natl. Acad. Sci. U.S.A.; 102:9499-
9504, J.
Med. Chem.; 49:27-30). Characterization of each rhodanine derivative was
obtained by
means of NMR spectroscopy, mass spectrometry, and/or elemental analysis as
reported in
Example 3. Recombinant LF, MAPKICidee, BoNT A, and SNAPtidee were purchased
36
CA 02646383 2008-12-12
=
from List Biological Laboratories (Campbell, CA, USA). The MMP-2 and MMP-9
assay
kits were purchased from Anaspec, Inc. (San Jose, CA, USA).
MAPKKide assay. The fluorescence peptide cleavage assay (100 AL) was
performed in a 96-well plate in which each reaction mixture contained MAPKKide
(4
AM) and LF (50 nM) (List Biological Laboratories) in 20 mM HEPES, pH 7.4, and
the
screening compounds. Kinetics of the peptide cleavage was examined for 30 min
by
using a fluorescence plate reader (VictorTm2 V, Perkin Elmer, Waltham,
Massachusetts,
USA) at excitation and emission wavelengths of 485 and 535 nm, respectively,
and ICso
values were obtained by dose¨response measurements. For selected compounds,
Lineweaver-Burk analysis was also carried out to verify that the compounds are
competitive against the substrate. The Km and Vmax values of the MAPKKide
cleavage
by LF were determined at 25 C by using the same experimental condition
described
above for the fluorescence screening assay but with increasing MAPKKide
concentrations (10, 8, 4, 2, and 1 AM). The Ki and Km(app) were calculated at
5 and or 10
AM inhibitor concentration.
SNAPtidee assay. The fluorescence peptide cleavage assay (50 AL) was
performed in a 96-well plates in which each reaction mixture contained
SNAPtide (30
AM) and BoNT A (20 nM) (List Biological Laboratories) in 20 mM HEPES, 0.3 mM
ZnC12, 1.25 mM DTT, 0.1% Tween-20, pH 8.0, and the screening compounds.
Kinetics
of the peptide cleavage was examined for 30 min by using a fluorescence plate
reader
(VictorTm2 V, Perkin Ehner) at excitation and emission wavelengths of 485 and
535 nm,
respectively, and ICso values were obtained by dose¨response measurements. The
Km
and Vma. values of the SNAPtide cleavage by BoTN A were determined at 25 C
by
using the same experimental condition described above for the fluorescence
screening
assay, but with increasing SNAPTide concentrations (100, 60, 30, 10, and 1
AM).
MMP-2 and MMP-9 assay. This assay was performed as outlined in the
Anaspec MMP assay kit (Cat. No. 71151/71.155). The fluorescence peptide
cleavage
assay (50 AL) was performed in a 96-well plate in which each reaction mixture
contained
5-FAM/QXLTM520 (60 AL; diluted 1:100 in assay buffer) and MMP-2 or MMP-9 (10
Ag/mL; pro-MMP-2 and pro-MMP-9 are first activated with 1 mM APMA for 20 min
or
2 h, respectively) in Enzolytemi 520 MMP-2 assay buffer, and the screening
compounds
37
CA 02646383 2008-12-12
(compound 1-6; each compound at 20 AM). Kinetics of the peptide cleavage was
examined every 5 min for 30 min by using a fluorescence plate reader
(VictorTm2 V,
Perkin Elmer) at excitation and emission wavelengths of 485 and 535 mn,
respectively,
and percent inhibition values were obtained.
ADME-TOX Studies. In vitro Evaluation of Chemical Stability of selected
LF inhibitors in PBS for 24 hours (J. Biomolecular Screening,- 8: 292-304; J.
Biomolecular Screening; 11: 40-7). Chemical stability was evaluated in PBS
(1.9 mM
NaH2PO4, 8.1 mM Na2HPO4, 150 mM NaC1, pH 7.4). An accurately weighted amount
of
analyzed compounds was dissolved in DMSO (10 mM stock), and then reconstituted
in
PBS at a 100 uM concentration following HPLC-UV analysis ("zero" point).
Samples in
PBS were incubated in closed vials at 37 C for 24 h in the dark following HPLC-
UV
analysis ("24-hours" point). Observed UV peak area ratio of each tested
compound'
versus internal standard (IS) was calculated. The percentages of the remaining
parent
compounds was defmed as the ratio of the parent compounds peak area at "zero"
point to
the peak area at "24 hours" point multiplied by 100%.
Determination of aqueous solubility by nephelometric assay (Anal. Chem.;72:
1781-7). The purpose of this study was to measure the aqueous solubility of
selected LF
inhibitors determined by using nephelometry-based method described in the
literature.
Compounds were dissolved to 10 mM in 100% DMSO. Solubility of compounds was
measured in PBS (pH 7.4), 5% DMSO at Room Temperature (around 23 C). Aqueous
solubility of acetylsalicylic acid was determined to validate the assay. It
was found to be
>100 Ag/ml at the day of experiment, which corresponds to the reported
literature value
of at least 2.17 mg/mL (The Merck index, 10th ed), 2.7 mg/mL (Sigma Aldrich
Catalog,
2004-2005).
Determination of cytotoxicity (J. Immunol. Meth.; 213: 157-67). The method is
based on the measurement of the metabolic activity of living cells using the
resazurin
system. The key component is the oxidoreduction indicator dye resazurin.
Bioreduction
of the dye by viable cells reduces the amount of its oxidized form [blue] and
concomitantly increases the amount of its fluorescent intermediate (Pharmacol.
Rep.; 58:
453-72), indicating the degree of cytotoxicity caused by the test material.
38
CA 02646383 2008-12-12
In the day of the experiment cells HepG2 (5*105 cells/nil) in medium DMEM
with 10% FBS (HyClone Standard) were seeded into all wells of 96-well plates
with the
=
exception of wells E1-H1 where is medium DMEM with 10% FBS without cells.
Controls: in wells A1-H1 ¨ 5 p.1 10 % DMSO. In all other wells except of
controls 5 1
tested compounds solutions in 10% DMSO were added following by 72 hours
incubation
at 37 C in a humidified 5% CO2 incubator. In each well 10 gl AlamarBlue were
added.
The plate were incubated 2 hours at 37 C in a humidified 5% CO2 incubator and
the
fluorescence of the AlamarBlue was measured on SAFIRE, Tecan with excitation
at 570
nm and emission 595 nm.
Determination of permeability by Parallel Artificial Membrane Permeation
Assay (J. Med. Chem.; 44: 923-30; J. Med. Chem.; 41: 1007-10). The
permeability was
determined by using PAMPA method described in the literature. The selected
inhibitors
were dissolved in DMSO to 10mM. The tested compounds and controls were diluted
with PBS to 1.67mM and mixed well by pipetting, centrifuged for 5min at 3500
rpm,
followed by the addition of 280 1 of PBS, 5% DMSO to acceptor plate. Then add
5 1 of
2% L-a-Phosphatidylcholine suspension in dodecane to the membrane of the donor
plate.
Immediately add 980 of PBS to donor plate and make the sandwich with the
acceptor
plate. Add 441 of tested compounds and controls dilutions to the acceptor
plate. Cover
the plate, place into camera and incubate for 16 hours. Make the equilibrium
plate, add
225 1 of PBS, 3.7% DMSO and 2411 of tested compounds and controls dilutions to
UV
plate. After 16 hours pull the donor plate out and transfer 250 1 from
acceptor plate to
UV plate. Scan UV plate on Safire (Tecan) plate reader from 245 to 450nM with
step
5nM. Permeabilities of 4 reference compounds were determined to validate the
assay. It
was found to be: Lucifer Yellow - Low, Furosemid - Low, Metoprolol High,
Propranolol ¨ High; at the day of experiment, which corresponds to the
reported literature
data.
Interaction studies of selected LF inhibitors with human MDR1 ABCB1/Pgp
(Pgp Calcein Transport Assay) (Blood, 91: 4480-8; Antimicrob Agents
Chemother., 42:
3157-62; FEBS Lett., 383: 99-104). The objective of this study was to evaluate
the
selected LF inhibitors to observe inhibition of P-glycoprotein mediated
transport of
39
_
CA 02646383 2008-12-12
calcein-AM out of the cell. Potency of the compounds was determined by the
dose
response experiment where the compounds were tested at 8 concentrations in
triplicate.
Cell culture: Cell line was maintained in RPMI 1640 supplemented with 10%
fetal bovine serum (HyClone, Standard), 100 units/ml of penicillin, 100 mg/ml
of
streptomycin, 2 mM glutamine, and 10 nM vincristine.
Calcein AM efflux assay: For the calcein-AM efflux assay, K562/i-S9 cells were
seeded on 96-well (Costar) tissue culture plates at cell density of 5x106
cells/well. Cells
were cultured in 100 IA of serum free RPMI 1640 without vincristine. Controls:
in wells
A1-D1: 5 121 1 mM verapamil solution; in wells E1-H1: 5 pl 20% DMSO in PBS.
Assay Protocol: 5 p.1 of tested compound solutions in 20% DMSO were added to
corresponding wells followed by 15 minutes incubation at 37 C. Then 5 pl of 2
pM
calcein-AM solution was added to each well. The plates were incubated at 37 C
for 2
hours. Then the plates were centrifuged, the supernatant was removed and the
cells were
resuspended in 100 p.1 of cold PBS (pH 7.4). The fluorescence was measured
using
Safire.(Tecan) plate reader at 490/516 nm excitation/emission. Verapamil 1050
was
determined to validate the assay. On the day of the experiment, verapamil IC50
was equal
to 4.69 M, which corresponds to the reported literature value of 2-5 M. The
rate of
calcein accumulation in the absence or presence of compounds was calculated in
Prism
software (GraphPad). Fluorescent background was subtracted from the RFU data
points.
Interaction studies of selected LF inhibitors with human CYP 2C19 (Anal.
Biochem., 248: 188-90; Biopharm. Drug Dispos., 24: 375-84; Drug Metab.
Dispos., 29:
1196-200; Drug Metab. Dispos., 28: 1440-8). The objective of this study was to
evaluate
the ability of the selected LF inhibitors to inhibit CYP 2C19 mediated
transformation of
3-cyano-7-ethoxycoumarin (CEC).
Dilute compound stock solution (10 mM) to 5 mM by 100% DMSO. Dilute this
solution by water up to 500 M just before use. Rows A-G are serial dilutions
(3-fold) of
the test compound (DMSO concentration is 10%.). Add 35 pi reaction buffer
(final
concentrations: 50 mM K.1304, pH 7.4, 1.3 mM NADP+, 3.3 mM G6P, 3.3 mM MgC12,
0.4 U/ml GPD, 0.4 mg/ml BSA, 25 ;AM CEC) to every well. Add 5 pi diluted
tested
compounds to respective wells. Add 5 .1 Tranylcypromine to the Control+
wells. Add 5
IA 10% DMSO to the Control- wells. Read fluorescence (Satire, Ex=420 nm,
Em=460
CA 02646383 2008-12-12
=
nm, bandwidth 5 nm, Gain-150, Z-position 5300 gm) (optional). Add 10 gl CYP
2C19
solution (10 pmol/ml) to every well except for blank (add 10 gl PBS into blank
wells).
Incubate for 60 minutes at 37 C. Read fluorescence at once (Safire, E=420 nm,
Eõ,=460
nm, bandwidth 5 nm, Gain-150, Z-position 5300 gm). The IC50 values were
calculated
using GraphPad Prism V. 3.03. Inhibition constant (IC50) of tranylcypromine
was
determined to validate the assay. It was found to be 0.69 gM, which
corresponds to the
reported literature data. Interaction studies of selected inhibitors with
human CYP1A2,
CYP2C9, CYP2D6, CYP3A4 were conducted similar to the procedure above.
In Vitro Metabolic Stability Assay with Rat Hepatocytes.(3, 4, 8)
1. Prepare a suspension of hepatocytes:
a) Hepatocyte preconditioning: place 10 mL of the Hepatocyte Incubation Media
to
the vial with rat hepatocytes (1.5 ml) and shake for 5 minutes in centrifuge
tube;
centrifuge for 2 minutes at 1,000 x g; remove supernatant (9 ml);
b) add 3 mL Hepatocyte Incubation Media and shake for 2-3 minutes;
c) 10 ul of cell suspension stained with methylene blue and subjected to
Goryayev
chamber to count the percentage of living cells (translucent);
d) reconstitute hepatocytes in PBS, pH 7.4 to final concentration of 1-106/mL;
2. Prepare incubation mixtures (in triplicates):
a) place the final hepatocyes mixture of 180 uL in 96-well cluster tubes;
b) add tested compound (20 uL of 500 uM stock solution) to hepatocyte mixture;
c) place in CO2-incubator at 37 C for 60 and 120 minutes;
d) at the end of each incubation period (0, 60 and 120 min at 37 C) 200 ul of
acetonitrile was added to stop reaction, the tubes were vortex-mixed for 15
min
to precipitate proteins;
3. Centrifuge the samples for 15 minutes at 6000 x g.
4. The supernatant was immediately analyzed by HPLC-UV-DAD.
In vitro Plasma Stability Assay (Eur. Pharm. Sci., 22: 25-31). Compound
stability was tested in rat plasma. The incubation mixture contained 180 uL of
plasma
and 20 uL of 500 uM drug stock solution in PBS (1.9 inM NaH2PO4, 8.1 mM
Na2HPO4,
150 inM NaCI, pH 7.4). At the end of each incubation period (0, 30 minutes at
37 .C)
200 uL of ice-cold acetonitrile 1/1 (v/v) was added, the tubes were vortex-
mixed for 20 s
41
CA 02646383 2008-12-12
to precipitate proteins, left on ice for 30 min and centrifuged for 5 min at
15,000 x g. The
supernatant was immediately analyzed by HPLC-UV-DAD. Diclofenac was used as a
positive control.
In Vitro Human/Rat Liver S9 Stability Assay (Chem. Biol. Interact., 121: 17-
35; Drug Discov. Today, 6: 357-66). Incubation solutions were prepared
containing
microsomal protein in phosphate buffer (1mg/mL) and a NADP-regenerating
system:
Reagents Per well Volume Final conc.
(A)
buffer 60
Test compound 10 50 M
protein 10 1 mg/ml
NADPH 20 2 mM
Volume of incubation 100
The reaction was initiated by addition of a test compound after preincubation
at
37 C for 2-3 minutes. At the end of each incubation period (0, 30 and 60 min
at 37 C)
100 Al of acetonitrile was added to stop reaction, the tubes were vortex-mixed
for 20 sec
to precipitate proteins, kept on ice and centrifuged for 15 min at 6,150 x g.
The
corresponding loss of parent compound was determined by HPLC-UV. Diclofenac
(50
uM) was used as a positive control.
Determination of HERG inhibition by radioligand assay (Eur. J. PharmacoL,
430: 147-8; Biophys. 1, 74: 230-41). Tested compounds were diluted by DMSO to
1.2
mM. 5 1 of 1.2 mM compounds solutions or controls in DMSO were added to the
wells
according to the assay plate layout. 950 of assay buffer was added, 50 1 of
5.8nM
Astemizole, [0-Methyl-3i] (7nCi/well) in assay buffer was added, 50 1 of
membrane
suspension in assay buffer was added. The assay plate was incubated for lh at
rt with
shaking (300rpm). The membranes were then harvested on glass fibre filter. The
filter
was dried and sealed with melt-on scintillator. The radioactivity was counted
on a
PerkinElmer Microbeta Jet.
Cell-based assay. Murine macrophage-like RAW 264.7 cells are grown to
confluence in wells of a 48-well plate in DMEM supplemented with 10% fetal
calf
serum. The cells are then replenished with fresh medium (0.1 mL per well) and
next
incubated with the increasing concentrations of inhibitors (0.1-50 p.M) for 4
h. A known
42
=
CA 02646383 2008-12-12
hydroxamate inhibitor of the LF metalloproteinase activity, GM6001 (Ki =5 AM;
46) is
included in the assay as a control. Anthrax protective antigen-83 (PA83) and
LF are then
added to the final concentration of 500 ng mL and 25 ng mL, respectively.
After
= incubation for an additional 1 h, cell viability is assessed by 3,[4,5-
dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT) staining. Cells are incubated with 0.5
mg/mL
MTT in DMEM for 45 min at 37 C; the medium is aspirated, and the blue pigment
produced by the viable cells is solubilized with 0.5% SDS/25 rnM HC1 in 90%
isopropyl
alcohol. The concentration of oxidized MTT in the samples is measured at 570
nm.
Each datum point.represents the results of at least three independent
experiments
performed in duplicate. The percentage of viable cells is calculated by using
the
following equation:
[(A570 of cells treated with LF, PA83, and inhibitor) ¨ (A570 of cells treated
with LF and PA83)] [(Am of cells treated with LF alone) ¨ (A570 of cells
treated with LF and PA83)].
= 15
Animal experiments with anthrax spores. Purification of anthrax spores and
the inhalation model of anthrax using A/J mice was described previously (47-
49). A/J
mice (eight mice per group) received B. anthraciq Sterne spores (4 H
105/animal in 20 AL
of DMSO). On the day following infection, mice received the LF inhibitor
compound 34
(25 mg/kg intraperitoneal) in DMSO and then continued to receive injections
once daily
for the remainder of the experiment. Control mice received an equal volume of
DMSO.
Mice treated with cipro received 25 mg/kg subcutaneous treatments daily
beginning on
the fourth day following infection.
Molecular modeling. Docking studies were performed with GOLD (Version 2.1,
The Cambridge Crystallographic Data Centre, Cambridge, UK) (50-52) and
analyzed
with Sybyl software (Tripos, St Louis, MO, USA). Molecular surfaces were
generated
with MOLCAD (53). The X-ray co-ordinates of BoNT A (PDB-ID 2G7N; 54) and LF
(PDB-ID 1YQY; 55) were used to dock the compounds. Molecular models were
generated with CONCORD (56) and energy minimized with Sybyl. For each
compound,
10 solutions were generated and subsequently ranked according to Chemscore
(52). Top
solutions were used to represent the docked geometry of the compounds reported
in
Figure 2.
43
CA 02646383 2008-12-12
=
Discussion
The IC50 and IC; values against LF were obtained using the same procedure as
described previously (5,6,57) and tested selected inhibitors against BoNT A
and MMP-
2/-9 (57). Several of the rhodanine compounds of the invention illustrated in
Tables 1-7
were synthesized as described in Example 3 and together with additional
commercially
available derivatives formed six subclasses. The first subclass included an
aromatic
moiety attached to a furan ring as shown in Table 1.
44
.
-
,
Table 1: Furan rhodanine derivatives and 1050 values against lethal factor
(LF), BoTN/A.
AN.,0Th___fo
- S-irikl-R
R
I-
Compd. Ar R LF BoNT/A Compd. Ar
R LF BoNT/A
1050 (pM) 1050 (pM)
1050 1050 (pM)
(PM)
02N ¨cH2c02H CI
3
.3.45 = >200 10
=lik --(cH2)2so3H 1.70 19.9
o
I CI
o
¨cH2c02H
tv
4 02N * 2.10 11.8 11
CI II
--(CH2)2S03H 1.78 11.1 cl,
o.
cl,
w
'
co
3.98 51.6 12 w
Br li ¨CFRCO2H)CH2CO2H
H2 )2S 0 >200 >2003H IV
0
0
CO
I
I-,
6 02N 41 ¨CH(CO2H)CH2CH2CO2H 4.91 148 13 i
CI lik
¨CH(Ph) CO 21-1 1.20 >200 n.)
1
tv
02N1 ci
2
7
11 ¨CH(Ph)CO2H 2.18 >200 14
* Cro >100 >200
.
,
8
. ¨CH(Ph)CO2H 4.34 127 15
02N
9*
411 --(cH2)2s03H 1.09 9.72 16
* z,00
>25 >50
a
Compounds did not inhibit MMP-2 and MMP-9 appreciably up to 50 /.1M with the
exception of compound 9 (MMP-2-1C50 = 163 AM).
CA 02646383 2008-12-12
=
These data further confirmed the importance of an acid moiety, whether it is a
carboxylic or sulfonic acid, as the absence of this group resulted in
compounds with
markedly reduced activity. The compounds of Table 1 were less effective in
targeting
BoNT/A, and also generally did not appreciably inhibit MMP-2 and MMP-9.
In further subclasses the rhodanine acetic acid and/or sulfonic acid moieties
were
fixed, but the furan ring was replaced with an oxazole/thiazole (Table 2),
isoxazole
(Table 3), or a pyrazole (Table 4).
Table 2: Oxazole/thiazole rhodanine derivatives and 1050 values against lethal
factor
(LF), BoTN/A, MMP-2, and MMP-9.
Compd. Ar LF BoNT/A MMP2 MMP9
IC50 (pM) IC50 (pM) IC50 (pM) IC, (pM)
0 cH3
17 H3c-- 100 >200 >100 >100
1820 176 >100 >100
'BocNID--4N
19 = \ 41.0 >200 >100 >100
46
=
. ,
CA 02646383 2008-12-12
,
, =
Table 3: Isoxazole rhodanine derivatives and IC50 values against lethal factor
(LF),
BoTN/A, MMP-2, and MMP-9.
Ar N1 1
1 0
S
NCO
Compd. Ar LF BoNT/A MMP2 MMP9
IC50 (pM) IC50 (pM) IC50 (pM) IC50
(pM)
20 0 17 >200 39.4 >100
21
litr 18.7 >200 >100 50.0
22 5.9 130 <10 >100
H3c *
23 H3co . 7.8 194 6.62 >100
24 . 5.2 82.7 19.2 >100
-
ci
25 F ip 12.4 >200 10.0 >100
26
Br * = 17.3 >200 >100 27.4
47
,
Table 4: Pyrazole rhodanine derivatives and 1050 values against lethal factor
(LF), BoTN/A.
Ar-ye
Sy.N-R
S
Compd. Ar . R LF BoNT/A Compd. Ar
R LF BoNT/A
IC30 (pM) IC30 (pM)
IC30 (pM) 1C30 (pM)
CH3
27 F3c lit , -NH
-CH2CO2H 24.6 >200 37 Ph.
N- \ 1,
OCH2CH3
-Cl2CO2H 4.87 130
28 a N-
\ P -cH2c0211 >100 >200
38* PhN_N --. \ it = ,M,' -c2H4s03H 2.45 >200
W a
0
H3
Ph,N,NN . Ph, _N
-CH2CO2H
.
29 \ I 3.07 160 39 N \ * 0
-C2H4S03H 2.29 >200
30
0
n.)
cn
-
.o.
0)
Ph-N4 Ph Ph, _N
W
_ -CH2co2H 6.25 111 40* N \
=
OCH3
-c21-14s0.1-1 2.04 >200 co
w
n.)
0
Ph, Ph,
0
N-Nx . 9 r- \ -CH2211 4.26 CO >200 41 14'
31 \ *
co
o
1-,
n.)
1
1-,
32* F * CH2CO2H 2.99 71.5 42
Ph.._ \ & \--\ -C2H4S03H 1.86 85.0
n.)
0
',..
PhõN 0 0
-C2H4S03H
2.07 20.1
- 0
Ph-N\11.-s, -Ph
9
34
a . VI 10 -CH2co2H 2.95 8.18 44
¨ __Cro >100 >200
Cl
Br ,N 9
35 3.35 5A5 45 N µ *
Ph, _ H3 F
-
36 lit ocH3 -CH2co3H 3.58 127
443 Ph, _ µ =
OCH2CH3
-CH2CO2H 345 >200
48
CA 02646383 2008-12-12
,r
Compounds of Tables 2-4 did not inhibit MMP-2 and MMP-9 appreciably up to 50
AM
with the exception of compounds 32 (MMP-2: ICso = 13.7 AM); 33 (MMP-2: ICso =
39.4
PM); 38 (MMP-2: ICso = 10.1 M); 40 (MMP-2: ICso = 34.5 RM); 41 (MMP-2: ICso
26.9 p.M).
As shown in Table 2, inclusion of an oxazole or a thiazole ring generally did
not
result in substantially effective compounds against any' of the proteases: On
the contrary,
the isoxazole substitution led to compounds that overall seemed to effectively
inhibit LF,
but not BoNT/A. However, compound 23 (Table 3) also inhibited MMP-2 in the low
micromolar range. With respect to the compounds of Table 3, the furan ring was
replaced with a pyrazole (Table 4) and this series was found to be most
effective against
LF. Two of these compounds also inhibited BoNT A in the low micromolar range.
Compounds 34 and 35 displayed ICso values of 8.18 AM and 5.45 AM,
respectively,
against BoNT/A. Although these two inhibitors (compounds 34 and 35) also
inhibit LF,
the most potent pyrazole derivatives against LF were 40, 42, and 43 (Table 4).
Finally, the substitutions of the furan ring were explored with various other
ring
motifs= such as indoles/thioindoles (Table 5), as well as other ring systems
shown in
Table 6.
49
CA 02646383 2008-12-12
= ,,
Table 5: Rhodanine derivatives and 1050 values against lethal factor (LF),
BoTN/A,
MMP-2, and MMP-9.
Ar e
CO ---/21-1
SYN
S
,
Compd. Ar LF BoNT/A MMP2 MMP9
IC50 (pM) IC50 (pM) 1050 (pM)
IC50 (pM)
Br
47 11.4 >200 50.6 >100
KO
L _... S
0
48 00/ S 32.5 112 47.1 64.9
"
49
GI ill 6.20 54.6 >100 >100
50 CN 4, 28.1 >200 >100 >100
51 r-s\ . >25 >50 >25 >25
H3C-NN--71
52a 27.9 >200 >100 >100
.
53 <: 0 83.3 >200 >100 >100
-54 I 401 5.26 >200 18.0 >100
H3C N
55 = 2.96 160 >100 >100
40 GI
56 Br ,,N,, 58.0 >200 >100 >100 .
I
CA 02646383 2008-12-12
Table 6: Structure-activity relationship of various ring motifs coupled with
rhodanine
acetic acid showing 1050 values against lethal factor (LF), BoTN/A, MMP-2, and
MMP-9.
11
Compd. Ar LF
BoNT/A MMP2 MMP9
IC50 (pM) IC50 (pM) IC50 (pM) IC50
(pM)
57 s
13.0 >200 >100 >100
58 S/ 10.4 74.3 46.9 64.9
Br
59 24.9 >200 >100 >100
6038.7 >200 >100 >100
N/
61
7.04 >200 >100 >100
N
Br
62 H 4.40 >200 >25 >25
Cl
N
0
0
63 H41, 5.71 >200 >100 >100
H3c
11 N
0
0
While most of the indole or thioindole derivatives showed to be effective
against
LF, this was not the case with BoNT/A. When the furan ring was substituted
with other
various ring systems (Table 6), the resulting compounds lack in activity
against both
toxins. Using a kinetic assay, Rosiglitizone (compound 2) was also tested as a
potential
protease inhibitor and found that it did not appreciably inhibit LF or BoNT A
(tested at
100 pM). Overall, compounds 9 and 13 are the two most potent inhibitors of LF
amongst
the new series with K., values of 0.1 /.LM and 1.0 M, respectively (Figure
1). Kinetic
assays in the presence of PA (one component of anthrax tripartite, which binds
LF and
51
CA 02646383 2008-12-12
allows it to enter the cytosol) were also performed to determine if the
inhibitory effect of
the compounds would change in a more biological state.
Significantly effective inhibitors of the invention include the pyrazoles
compounds 34 and 35, given that they inhibited both bacterial toxins, BoNT/A,
and LF,
but did not inhibit the human proteases MMP-2 or MMP-9.
Docking studies performed using the X-ray co-ordinates of BoNT A (PDB-ID
2G7N; 54) and LF (PDB-ID 1YQY; 55) indicate that pyrazole-rhodanine
derivatives
such as compound 34 are nicely positioned into the binding pocket of the
enzyme,
placing both the exosulfur atom of the rhodanine ring and the carboxylate
group in close
proximity to the zinc metal ion (Figure 2).
To further test the druggability of the LF inhibitors of the invention, in
vitro
ADME-Tox (absorption, distribution, metabolic, excretion, and toxicity)
studies were
performed (Table 7). From these studies, it can be concluded that compounds 1
and 65
did not show any major cytotoxicity effects, are chemically stable, did not
degrade in
plasma, and are not rapidly metabolized by hepatocytes. It was also shown that
compounds 1 and 65 had very little effect on the inhibition of the cytochrome
P450s
(CYPs) and the hERG receptor (Table 7). In contrast, compound 64 (Table 7),
was found
to inhibit CYP1A2 and showed to be metabolically unstable. In addition,
compounds 9,
34, 38, 39, and 40 were shown to protect macrophages from LF/PA-induced cell
death
when tested at 50 and 100 tiM concentration.
52
:
=
=
,
Table 7: In vitro ADME-tox experiments with selected LF compounds of the
invention. ,
_.
_______________________________________________________________________________
_________________________
StructurefiD
IC50 Chem logS Cytox. Pampa Pgp CYP CYP CYP CYP CYP
Met. Plasma Liver hERG
( M) Stab. Ug/ (c) (d) ( M) 2C19 2D6 1A2 3A4 2C9
Stab. Stab. $9 14Inhib.
(a) mL (e) IC50 IC50
IC50 IC50 IC50 tz(n'n) T=30 Stab at 30
(b)
(PM) (PM) (tirsdi) (j.M) (11M) 1714n-1os min
;2("1") liM
cc..
(f) (g) (h)
(1) (J) cells (1) 1/mIn.1 (n)
(k) 0 cells
(m)
,
Me0 100= 0.29 100% <10 Nontoxic ND A 31.3 30.5
20.9 * 28.3 tie 90% t1,2= 58
a= up to 100
37.1 . 127.7
I / \ ' i.tM
CL= CL,õ,=
21.6 5.6
ssu,.N--..µ
1 g coti
...
_ 0
a r,
MP0.26 88% <10 Nontoxic Med. A 36.9 47.6 2.79 >50 >50 Degr- 79% Degr- 67
=
up to 100 aded aded 0
ci
n.)
I / \ = PM
cn
.o.
S.seN-.1
01
64 3 C H
W
2
CF, 0.19 97% <10 Nontoxic ND >100 >100 >100 20.5 * >100 tõ,.
82% tie 45
Ills up to 100
12M
42.4
CL,,= 37.3
.
Clint=
n.)
o
O
a l i ' .
19.8 25.0 co
1
1-,
n.)
65 3 Wil
I
2.3 85% <10 Nontoxic Med. A A A A = >100 Degr- 90% Degr- 29 r. )
up to 100
aded aded
szt4-.../co2H
fl
66 s
0.32 ND <10 Nontoxic Low >100 >100
>50 >100 ' * ' >100 ND ND ND 9
up to 100
AM
67 ' .
,
=
53
CA 02646383 2008-12-12
aChemical stability; bThe apparent (kinetic) solubility; Tytotoxicity against
HepG2 cells;
dCell permeability using the PAMPA method; eInhibition of P-glycoprotein-
mediated
transport of calcein-AM out of the cell; f-'Evaluation of the ability of the
LF compounds
to inhibit human CYPs-mediated transformation of 3-cyano-7-ethoxycoutnarin;
Evaluation of the ability of LF inhibitors to inhibit CYP 3A4-mediated
transformation of
dibenzylfluorescein; kMetabolic stability with rat hepatocytes; 'Stability in
rat plasma.
'Metabolic stability in liver microsomes; nInhibition of the hERG channel was
measured
using hERG K+ radioligand; *, no dose-response curve; ND, not detected; A,
compounds
that had high absorbance at the wavelength used for the assay; LF, lethal
factor.
Compound 34 was selected for in vivo efficacy studies against anthrax (Figure
3)
given its cross-reactivity with the BoNT A toxin. When administered via
intraperitoneal
injection, compound 34 alone or in combination with cipro protected mice
against
anthrax spores in which it increased survival of infected mice to >80% (Figure
3). It is
significant that these studies were done without detailed knowledge of the
pharmacology
of the LF inhibitors and by using a single daily dose. These data support that
the
rhodanine derivatives of the invention described herein are suitable for
development into
therapeutics against bacterial toxins.
Example 2. Preparation of Aldehyde 70.
Aldehyde 70, an intermediate for the preparation of rhodanine derivative 28,
was
prepared as described by Tanaka et al.( J. Med. Chem.; 41: 2390-410).
Scheme 2.
NW" 0 HN" = H14- 0
io
N,O-Dimethylhydroxyl-
OH amine hydrochloride ,N-ocH3 LiAlHA
a
EDC/HOBt H3C THF/5 C
cl a
68 CH202/rt/48h 69 70
Acid 68 (0.898 mmol), HOBt (0.898 mmol) and N,0-dimethylhydroxylamine
hydrochloride (0.898 mmol) were dissolved in CH2C12 (5 mL) and stirred for 10
min.
EDC (0.898 mmol) in 5 mL of CH2C12 was added dropwise over 20 mins in which
the
colorless reaction mixture was stirred for 48 hrs. at room temperature under
nitrogen.
The reaction mixture was diluted with 10 mL of CH2C12, washed 2 times with
water and
= 54
- --
CA 02646383 2008-12-12
twice with brine. The organic layer was dried with Na2SO4 and concentrated to
give the
amide 69 as a light yellow solid, which was used in the next step without
further
purification.
=
To a cooled solution of LiA1H4 (0.753 mmol) in THF (5 rnL) under nitrogen,
compound 69 (0.376 mmol) was added dropwise and the reaction mixture was
allowed to
warm to room temperature over 2 hrs and was stirred overnight. The reaction
mixture
was cooled to 0 C and quenched with H20, 15% NaOH and H20. It was warmed to
room temperature in which Na2SO4 was added. After diluting with Et20, it was
filtered
and the solid was boiled in 10 mL of THF filtered and concentrated to give
aldehyde 70.
This technique can be used to prepare other desired aldehydes from known or
readily prepared acids, as would be readily understood by those of skill in
the art.
Example 3. Preparation of Compouds of the Invention
Generic synthetic schemes and methods for the synthesis of rhodanine
derivatives
of the invention 17¨ 28, 30, 32-35, 47-52, 54-59, and 61 (Tables 1-7) are
provided in
this Example. R1 represents various heteroatoms such as furan derivatives
(Tables 1 and
7), oxazole/thiazole derivatives (Table 2), isooxazole derivatives (Table 3),
pyrazole
derivatives (Table 4), indole/thioindole derivatives (Table 6) and other
groups denoted in
Table 5. Within these derivatives R2 can be a carboxylic acid derivative,
sulfonic acid
derivative, sulfolane, or a tetrahydrofuran derivative. The variables R1 and
R2 as used in
this example can be the same or difference than other similar tenns used
elsewhere in the
specification. Various aldehydes can be purchased from Sigma Aldrich, Acros or
Astatech, Inc., or prepared by standard synthetic techniques well known to
those of skill
in the art.
Scheme 3. Generic synthetic scheme for compounds listed in Tables 1-7.
0 0
co2H CO2H
F10 + N¨/ MW
_J
R1 DMF S-\cN
s s
CA 02646383 2008-12-12
=
General Procedure: To a solution of the aldehyde (0.575 nunol) in N,N-
dimethylformamide (DMF) (1 mL) was added rhodanine N-acetic acid (0.523 mmol)
and
the mixture was stirred until it became homogenous. The solution was then
placed in the
CEM microwave (Matthews, NC) for two cycles of 2 minute heating at 140 C (ramp
2
min, 300W) and 30 second cooling at 30 C (ramp 2 min, 300W). The solution was
removed from the microwave and diluted with water (20 mL) and a precipitate
was
formed. The precipitate was collected by filtration, recrystallized using
acetone/water,
and was dried to give the desired compound.
2-(545-(furan-2-Aisoxazol-3-Amethylene)-4-oxo-2-thioxothiazolidin-3-Aacetic
acid
(20) (0.170g, 82.5%). 1H NMR (300 MHz, d-DMSO) ô 8.02 (d, 1H, J=1.5), 7.82 (d,
1H,
J=1.5), 7.27 (d, 1H, J=3.6), 7.17 (d, 1H, J=1.5), 6.79 (dd, 1H, J=3.6, 1.5
Hz), 4.76 (s,
2H). MS (ESI), m/z Calcd for C13H8N205S2[M+Hr 337Ø Found: 337Ø Anal.
(C13H8N205S2) Calc.: C, 46.42; H, 2.40; N, 8.33; S, 19.07. Found: C, 46.81; H,
2.78; N,
8.44; S, 18.63.
(Z)-2-(542,5-dimethyloxazol-4-Amethylene)-4-oxo-2-thioxothiazolidin-3-
yl)acetic acid
(17) (0.188 g, 79%). 1H NMR (300 MHz, d-DMSO) 8 7.71 (s, 1H), 4.78 (s, 2H),
2.55 (s,
3H), 2.48 (s, 2H). MS (ESI), m/z Calcd for CI iHION204S2 [M+H]' 298.01 Found:
299.1.
2-(545-bromobenzo[b] thiophen-3-yOmethylene)-4-oxo-2-thioxothiazolidin-3-
yl)acetic
acid (58) (0.198g, 77%). 1H NMR (300 MHz, d-DMSO) 6 8.54 (d, 1H, J=1.2), 8.34
(s,
1H), 8.18 (s, 1H), 8.11 (d, 1H, J=8.4), 7.67 (dd, 1H, J=1.5, 8.4), 4.78 (s,
2H). MS (ESI),
m/z Calcd for C14H8BrNO3S3 [M-HI 411.9. Found: 412Ø Anal. (C14H813rNO3S3).
Calc:
C, 40.58; H, 1.95; N, 3.38; S, 23.22. Found: C, 41.12; H, 2.14; N, 3.40; S,
21.76.
2-(542-methylquinolin-6-Amethylene)-4-oxo-2-thioxothiazolidin-3-Aacetic acid
(54)
(0.139g, 69.2%, 91.0% purity by LCMS). 1H NMR (300 MHz, d-DMSO) (5 8.44 (d,
1H,
J=8.4), 8.28 (d, 1H, J=1.8), 8.06 (s, 1H), 8.05 (d, 1H, J=8.7), 7.96 (dd, 1H,
J= 1.8, 8.7
Hz), 7.53 (d, 1H, J=8.7), 4.78 (s, 2H), 2.70 (s, 3H). MS (ESI), m/z Calcd for
C16HuN203S2 [M+H]' 345Ø Found: 344.9. Anal. (C16H12N203S2) Calc: C, 55.80;
H,
3.51; N, 8.13; S, 18.62. Found: C, 44.38; H, 3.38; N, 9.54; S, 20.54.
56
CA 02646383 2008-12-12
2-(545-(4-chloropheny1)-1H-pyrazol-3-Amethylene)-4-oxo-2-thioxothiazolidin-3-
yl)acetic acid (28) (0.015g, 21%). 1H NMR (300 MHz, d-DMSO) & 7.82 (d, 2H,
J=7.8),
7.47 (d, 2H, J=7.8), 6.66 (s, 1H), 3.81 (s, 2H).
2-(542-(1-(tert-butoxycarbonyl)piperidin-4-Athiazol-4-Amethylene)-4-oxo-2-
thioxothiazolidin-3-Aacetic acid (18) (0.168g, 73%). 1H NMR (300 MHz, d-DMSO)
8.34 (s, 1H), 7.84 (s, 111), 4.73 (s, 2H), 4.03 (d, 2H, J= 12.9), 3.28 (tt,
2H, J=3.6, 10.2),
2.96 (bs, 211), 2.11 (dd, 2H, J=2.4, 10.2), 1.64 (dq, 211, J=3.6, 12.6), 1.43
(s, 9H). HRMS
(ESI), m/z Calcd for C19H23N305S3 [M+H] 470.09. Found: 470.09 Anal.
(C19H23N305S3)
Calc: C, 48.60; H, 4.94; N, 8.95; S, 20.48. Found: C, 49.52; H, 4.78; N, 8.81;
S, 19.25.
2-(4-oxo-5-(thieno[2,3-Npyrazin-6-ylmethylene)-2-thioxothiazolidin-3-yOacetic
acid
(59) (0.242g, 81%). 1H NMR (300 MHz. , d-DMSO) 5 8.89 (d, 1H, J=2.4), 8.71 (d,
1H,
J=2.4), 8.34 (s, 111), 8.39 (s, 1H), 4.78 (s, 2H). HRMS (ESI), m/z Calcd for
Ci2H7N303S3
[M+H] 336.97. Found 337.97 Anal (C12H7N303S3) Calc: C, 42.72; H, 2.09; N,
12.45; S,
28.51. Found: C, 43.04; H, 2.42; N, 12.25; S, 27.66.
2-(5-(3-(1H-pyrazol-1-yObenzylidene)-4-oxo-2-thioxothiazolidin-3-Aacetic acid
(52)
(0.136 g, 56%). 1H NMR (300 MHz, d-DMSO) (5 8.59 (d, 1H, J=2.7), 8.19 (s, 1H),
7.97
(td, 1H, J=0.9, 8.1), 7.96 (s, 111), 7.83 (d, 111, J=0.6), 7.69 (t, 1H,
J=7.8), 7.58 (d, 1H,
J=7.8), 6.61 (t, 111, J=2.4), 4.77 (s, 2H). HRMS (EST), m/z Calcd for
C15HiiN303S2
[M+H] 346.03. Found 346.03 Anal. (C15H11N303S2) Calc: C, 52.16; H, 3.21; N,
12.17;
S, 18.57. Found: C, 51.39; H, 3.33; N, 12.00; S, 17.98.
2-(5-0-(4-methoxyphenyOisoxazol-3-Amethylene)-4-oxo-2-thioxothiazolidin-3-
- yOacetic acid (23) (0.338g, 73%). 1H NMR (300 MHz, d-DMSO) 5 7.93 (d, 2H,
J=9.3),
7.76 (s, 111), 7.17 (s, 111), 7.16 (d, 2H, J=9.3), 4.93 (s, 2H), 3.93 (s, 3H)
HRMS (ESI),
m/z Calcd for CI6H12N205S2[M+H] 377.03. Found 377.03.
2-(4-oxo-2-thioxo-5-((5-p-tolylisoxazol-3-yOmethylene)thiazolidin-3-yl)acetic
acid (22)
(0.385g, 80%). 1H NMR (300 MHz, d-DMSO) 5 7.82 (d, 111, J=8.4), 7.80 (s, 1H),
7.40
(d, 1H, J=8.1), 7.34 (s, 1H), 4.76 (s, 2H), 2.39 (s, 3H) HRMS (ESI), m/z Calcd
for
57
CA 02646383 2008-12-12
C16H12N204S2[M+H] 361.0311. Found 377.0309. Anal. (C16H12N204S2) C, 53.32; H,
3.36; N, 7.77; S, 17.79 Found: C, 52.93; H, 3.77; N, 7.98; S, 16.62.
2-(545-(4-chlorophenyOisoxazol-3-yOmethylene)-4-oxo-2-thioxothiazolidin-3-
yOacetic
acid (24) (0.113g, 62%). 1H NMR (300 MHz, d-DMSO) (5 7.97 (d, 2H, J=8.4), 7.81
(s,
1H), 7.67 (d, 2H, J=8.4), 7.45 (s, 1H), 4.76 (s, 2H). HRMS (ESI), m/z Calcd
for
C15H9C1N204S2[M+H] 380.97. Found 380.98. Anal. (C15H9C1N204S2) C, 47.31; H,
2.38; N, 9.31; S, 16.84 Found: C, 46.76; H, 2.75; N, 7.42; S, 16.09.
2-(545-(4-fluorophenyOisoxazol-3-yOmethylene)-4-oxo-2-thioxothiazolidin-3-
yOacetic
acid (25) (0.170g, 89%). 1H NMR (300 MHz, d-DMSO) ô 8.00 (dd, 1H, J=8.7, 5.4),
7.81 (s, 1H), 7.44 (t, 2H, J=8.7), 7.39 (s, 1H), 4.76 (s, 2H). HRMS (ESI), m/z
Calcd for .
C15H9FN204S2[M+H] 365.00. Found 365.00. Anal. (C15H9FN204S2) C, 49.44; H,
2.49;
N, 7.69; S, 17.60 Found: C, 49.37; H, 2.73; N, 7.87; S, 17.09.
2-(541-(2,4-difluoropheny0-3-(pyridin-3-y0-1H-pyrazol-4-yOmethylene)-4-oxo-2-
thioxothiazolidin-3-yOacetic acid (32) (0.228g, 95%). NMR (300 MHz, d-DMSO)
8.89 (d, 1H, J=2.1), 8.80 (s, 1H), 8.75 (d, 1H, J=4.5), 8.13 (d, 1H, J=7.8),
8.00 (m, 1H),
7.70 (dt, 1H, J=2.7, 9.0), 7.64 (d, 1H, J=4.8), 7.62 (t, 1H, J=2.1), 7.37 (tt,
1H, J=2.7, 7.8),
4.74 (s, 2H). HRMS (ESI), m/z Calcd for C20H12F2N403S2 [M+H] 459.04. Found:
459.04. Anal. (C201112F2N403S2) C, 52.40; H, 2.64; N, 12.22; S, 13.99 Found:
C, 51.67;
H, 2.92; N, 12.19; S, 12.98.
2-(541,3-dipheny1-1H-pyrazol-4-yOmethylene)-4-oxo-2-thioxothiazolidin-3-
yOacetic
acid (30) (0.224, 88%). 1H NMR (300 MHz, d-DMSO) ô 8.89 (s, 1H), 8.11 (d, 2H,
J=8.7), 7.67 (m, 8H), 7.56 (s, 1H), 4.74 (s, 211). HRMS (EST), m/z Calcd for
C21H15N303S2[M+H] 422.06. Found: 422.06. Anal. (C211-115N303S2) C, 59.84; H,
3.59;
N, 9.97; S, 15.21. Found: C, 60.04; H, 4.06; N, 10.10; S, 14.81.
2-(541,3-bis(4-chloropheny0-1H-pyrazol-4-yOmethylene)-4-oxo-2-
thioxothiazolidin-3-
yOacetic acid (34) (0.220g, 95%). NMR (300 MHz, d-DMSO) ô 8.90 (s, 1H),
8.12 (d,
2H, J=8.4), 7.67 (m, 6H), 7.56 (s, 1H), 4.74 (s, 2H). HRMS (ESI), m/z Calcd
for
C211113C12N303S2 [M+11]+ 489.98. Found: 489.98.
58 =
CA 02646383 2008-12-12
2-(5-0-(4-bromophenyl)isoxazol-3-Amethylene)-4-oxo-2-thioxothiazolidin-3-
Aacetic
acid (26) (0.133, 79%). 1H NMR (300 MHz, d-DMSO) 5 7.90 (m, 2H, J=8.4), 7.81
(d,
2H, J=7.5), 7.78 (s, 1H), 7.47 (s, 1H), 4.77 (s, 2H). HRMS (ESI), m/z Calcd
for
C15H9BrN204S2 [M+11} 424.93. Found: 424.93. Anal. (C15H9BrN204S2) C, 42.36;
H,
2.13; N, 6.59; S, 15.08. Found: C, 42.16; H, 2.50; N, 6.61; S, 14.46.
2-(5-0,3-bis(4-bromopheny1)-1H-pyrazol-4-Amethylene)-4-oxo-2-thioxothiazolidin-
3-
Aacetic acid (35) (0.170, 80%). 1H NMR (300 MHz, d-DMSO) 5 8.91 (s, 1H),
8.06(d,
2H, J=8.7), 7.80 (d, 2H, J=6.0), 7.77 (d, 2H, J=6.6), 7.63 (d, 2H, J=8.4),
7.57 (s, 1H),
4.74 (s, 2H). HRMS (ESI), m/z Calcd for C211-113Br2N303S2 [M+H] 577.88. Found:
577.88. Anal. (C211113Br2N303S2) C, 43.54; H, 2.26; N, 7.25; S, 11.07. Found:
C, 43.65;
H, 1.77; N, 7.83; S, 10.02.
2-(541,3-bis(4-fluoropheny1)-1H-pyrazol-4-Amethylene)-4-oxo-2-
thioxothiazolidin-3-
yOacetic acid (33) (0.234g, 97%). 1H NMR (300 MHz, d-DMSO) 8.87 (s, 1H),
8.12
(dd, 2H, J=4.5, 9.0), 7.73 (d, 2H, J=5.4), 7.57 (s, 1H), 7.44 (t, 4H, J=9.0).
HRMS (ESI),
m/z Calcd for C21H13F2N303S2 [M+11]+ 458.04. Found: 458.04. Anal.
(C2iHi3F2N303S2)
C, 55.13; H, 2.86; N, 9.19; S, 14.02. Found: C, 60.87; H, 3.11; N, 10.18; S,
13.59.
2-(545-morpholinothiophen-2-Amethylene)-4-oxo-2-thioxothiazolidin-3-Aacetic
acid
(48) (0.0129g, 5%). 1H NMR (300 MHz, d-DMSO) 5 7.91 (s, 1H), 7.59 (d, 1H,
J=4.8),
6.48 (d, 2H, J=4.5), 4.87 (s, 2H), 3.87 (t, 4H, J= 4.5), 3.48 (t, 4H, J=4.8).
HRMS (ESI),
m/z Calcd for Ci4Hi4N204S3 [M+H] 371.02. Found: 371.02. Anal. (C141114N204S3)
C,
45.39; H, 3.81; N, 7.56; S, 25.97. Found: C, 45.26; H, 3.99; N, 6.92; S,
23.77.
2-(5-(4-(4-methyl-1,4-diazepan-l-Abenzylidene)-4-oxo-2-thioxothiazolidin-3-
y1)acetic
acid (51) (0.218g, 81%). 1H NMR (300 MHz, d-DMSO) 5 7.74 (s, 1H), 7.52 (d, 2H,
J=8.7), 6.93 (d, 2H, J=9.0), 4.64 (s, 211), 3.70 (m, 2H), 3.57 (t, 2H, J=6.0),
2.83 (m, 2H),
3.68 (m, 2H), 2.42 (s, 3H), 1.99 (m, 2H) HRMS (ESI), m/z Calcd for
Ci8H2IN303S2[M+H] 392.1097 Found: 392.1092.
59
CA 02646383 2008-12-12
2-(5-((2-methy1-4-phenylthiazol-5-yOmethylene)-4-oxo-2-thioxothiazolidin-3-
yOacetic
acid (19) (0.206g, 74%). '1H NMR (300 MHz, d-DMSO) (5 7.81 (s, 1H), 7.65-7.68
(m,
2H), 7.60-7.56 (m, 3H), 4.72 (s, 2H), 2.84 (s, 3H). HRMS (ESI), m/z Calcd for
C16H12N203S3 [M+Hr 377.01. Found: 377.01 Arial. (C16H12N203S3) C, =51.05; H,
3.21;
N, 7.44; S, 25.55. Found: C, 50.88; H, 3.29; N, 7.60; S, 24.99.
= 2-(54130-(4-chloropheny1)-1H-pyrazole-3-y1)methylene)-4-oxo-2-
thioxothazolidin-3-
yl)acetic acid (27) (0.198 g, 74%). 1H NMR (300 MHz, d-DMSO) (5 8.45 (br.s.,
1H),
7.94 (d, 2H, J=8.0 Hz), 7.83 (d, 211, J=8.0 Hz), 7.64 (s, 1H), 4.73 (s, 2H).
HRMS (ESI),
m/z Calcd for C16H12N203S3 [M+Hr 414.01. Found: 414.01.
2-(5((7-bromo-2,3-dihydrothieno[3,4-b][1,4] dioxin-5-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid (47) (0.208g, 82%). 1H NMR (300 MHz, d-
DMSO)
7.71 (s, 111), 4.71 (s, 2H), 4.47 (d, 2H, J=4.8), 4.42 (d, 2H, J=3.9). HRMS
(ESI), m/z
= 15 Calcd for C12H8BrNO5S3 [M+Hr 421.88. Found: 421.88.
2-(4-oxo-542-0,yrrolidin-l-Apyridin-4-yl)methylene)-2-thioxothiazolidin-3-
yl)acetic
acid (50) (0.157g, 53%). 111 NMR (300 MHz, d-C2H60) (5 8.27 (d, 1H, J=5.0),
7.73 (s,
111), 6.73 (dd, 1H, J=1.5, 5.0), 6.63 (d, 111, J=1.5), 4.92 (s, 211), 3.53 (t,
4H, J-.6), 1.32
(m, 4H). HRMS (ESI), m/z Calcd for C15H15N303S2 [M+Hr 350.06. Found: 350.06.
2-(5-(benzo[c]thiophen-1-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yOacetic
acid (57)
(0.215g, 81%) 1H NMR (300 MHz, d-DMSO) 3 8.28 (s, 1H), 8.21 (d, 1H, J=7.2),
8.14
(d, 111, J=6.6), 8.12 (s, 1H), 7.54 (m, 2), 4.78 (s, 2H). HRMS (ESI), m/z
Calcd for
C14ll9NO3S3 [M+111+ 335.98. Found: 335.98.
=
2-(5-((6-bromopyridin-2-Amethylene)-4-oxo-2-thioxothiazolidin-3-Aacetic acid
(56)
(0.252g, 87%). 1H NMR (300 MHz, d-DMSO) (5 8.02 (d, 1H, J=7.5), 7.95 (t, 1H,
J=7.8),
7.91 (s, 111), 7.5 (d, 1H, J=7.8), 4.76 (s, 2H) HRMS (ESI), m/z Calcd for
C11H7BrN203S2
[M+Hr 358.92. Found: 358.52. Anal. (C11H7BrN203S2) C, 36.78; H, 1.96; N, 7.80;
S, =
= 30 17.85 Found: C, 37.06; H, 1.92; N, 8.04; S, 17.19.
2-(5-(dibenzolb,difuran-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic
acid (55)
(0.030g, 10%). 1H NMR (300 MHz; d-DMSO) ô 8.38 (d, 1H, J=7.5), 8.25 (dist.d,
3H),
CA 02646383 2008-12-12
7.89 (d, 1H, J=8.4), 7.72 (d, 1H, J=7.8), 7.62 (m, 2H), 7.50 (t, 1H, J=7.5),
4.80 (s, 2H)
HRMS (ESI), m/z Calcd for C18H11N04S2 [M+H] 370.02. Found: 370.02. Anal.
(C18H11N04S2) C, 58.52; H, 3.00; N, 3.79; S, 17.37 Found: C, 58.79; H, 3.10;
N, 3.99; S,
16.04.
2-(545-bromoindolin-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yOacetic acid
(61)
(0.147g, 55%). 1H NMR (300 MHz, d-DMSO) 6 8.30 (s, 1H), 8.20 (s, 1H), 7.99 (d,
1H,
J=2.7), 7.49 (d, 1H, J=8.4), 7.40 (d, 1H, J=8.7), 4.74 (s, 2H). HRMS (ESI),
m/z Calcd for
C141-111BrN203S2 [M+H] 396.93. Found: 396.93. Anal. (C14ll11BrN203S2) C,
42.33; H,
2.28; N, 7.05; S, 16.14 Found: C, 40.79; H, 2.46; N, 6.97; S, 15.03.
2-(4-oxo-2-thioxo-54.5-phenylisoxazol-3-yOmethylene)thiazolidin-3-yOacetic
acid (21)
(0.136g, 75%). 1H NMR (300 MHz, d-DMSO) 5 7.94 (m, 2H), 7.81 (s, 1H), 7.60 (m,
3H), 7.41 (s, 1H), 4.76 (s, 2H). HRMS (ESI), m/z Calcd for Ci5H10N204S2 [M+H]
347.0155. Found: 347.0157. Anal. (C15H10N204S2) C, 52.01; H, 2.91; N, 8.09; S,
18.51
Found: C, 52.13; H, 3.38; N, 9.30; S, 15.93.
2-(5-biphenyl-4-ylmethylene-4-oxo-2-thioxothiazolidin-3-y1 )acetic acid (49)
(0.071g,
38.4%). 1H NMR (300 MHz, d-DMSO) (5 7.97 (s, 1H), 7.90 (d, 2H, J= 8.1 Hz),
7.80 (d,
4H, J= 8.1 Hz), 7.52 (t, 2H, J= 7.2, Hz), 7.45 (d, 1H, J= 7.2Hz) 4.76 (s, 2H).
HRMS
(ESI), m/z Calcd for C18H13NO3S2 [M+H] 356.0410. Found: 356.0409. Anal.
(C18H13NO3S2) C, 60.83; H, 3.69; N, 3.94; S, 18.04 Found: C, 61.17; H, 3.72;
N, 4.18; S,
17.22.
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The invention has been described with reference to various specific and
preferred
embodiments and techniques. However, it should be understood that many
variations and
modifications may be made while remaining within the scope of the invention.
63
