Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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IL-8 RECEPTOR ANTAGONISTS
FIELD OF THE INVENTION
This invention relates to novel sulfonamide substituted Biphenyl urea
compounds, pharmaceutical compositions, processes for their preparation, and
use
thereof in treating IL-8, GROG, GROG, GROy, NAP-2, and ENA-78 mediated
diseases.
BACKGROUND OF THE INVENTION
Many different names have been applied to Interleukin-8 (IL-8), such as .
neutrophil attractant/activation protein-1 (NAP-1), monocyte derived
neutrophil
to chemotactic factor (MDNCF), neutrophil activating factor (N.AF), and T~eell
lymphocyte chemotactic factor. Interleukin-8 is a chemoattractant for
neutrophils,
basophils, and a subset of T-cells. It.is produced by a majority of nucleated
cells
including macrophages, fibroblasts, endothelial and epithelial cells exposed
to
TNF, TL-loc, IL-1(3 or LPS, and by neutrophils themselves when exposed to LPS
or
15 chemotactic factors such as FMLP. M. Baggiolini et al., J. Clin. Tnvest.
h4, 1045
(1989); J. Schroder et al, J. Tmmunol.139, 3474 (1987) and J. Tmmunol.144,
2223 (1990) ; Strieter, et al., Science 243, 1467 (1989) and J. Biol. Chem.
264,
10621 (1989); Cassatella et al., J. Immunol. 14~, 3216 (1992).
GROoc, GROG, GROy and NAP-2 also belong to the chemokine family.
20 Like IL-8 these chemokines have also been referred to by different names.
For
instance GROG, (3, y have been referred to as MGSAa, (3 and y respectively
(Melanoma Growth Stimulating Activity), see Richmond et al., J. Cell Ph siy
olo~y_
129, 375 (1986) and Chang et al., J. Immunol 148, 451 (1992). All of the
chemokines of the oc-family which possess the ELR motif directly preceding the
25 CXC motif bind to the IL-8 B receptor (CXCR2).
TL-8, GROoc, GROG, GROy, NAP-2, and ENA-78 stimulate a number of
functions in vitro. They have all been shown to have chemoattractant
properties
for neutrophils, while IL-8 and GROa have demonstrated T-lymphocytes, and
basophilic chemotactic activity. In addition IL-8 can induce histamine release
3o from basophils from both normal and atopic individuals. GRO-a and IL-8 can
in
addition, induce lysozomal enzyme release and respiratory burst from
neutrophils.
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IL-8 has also been shown to increase the surface expression of Mac-1
(CDllb/CD18) on neutrophils without de novo protein synthesis. This may
contribute to increased adhesion of the neutrophils to vascular endothelial
cells.
Many known diseases are characterized by massive neutrophil infiltration. As
IL-
8, GROoc, GR0~3, GROy and NAP-2 promote the accumulation and activation of
neutrophils, these chemokines have been implicated in a wide range of aeute
and
chronic inflammatory disorders including psoriasis and rheumatoid arthritis,
Baggiolini et al., FEBS Lett. 307, 97 (I992); Miller et al., Crit. Rev.
Immunol. l2,
17 (1992); Oppenheim et al., Annu. Rev. Immunol. 9, 617 (1991); Seitz et al.,
J.
l0 Clin. Invest. 87, 463 (1991); Miller et al., Am. Rev. Respir. Dis.146, 427
(1992);
Donnely et al., Lancet 341, 643 (1993). In addition the ELR chemokines (those
containing the amino acids ELR motif just prior to the CXC motif) have also
been
implicated in angiostasis, Strieter et al., Science 258, 1798 (1992).
In vitro, IL-8, GROoc, GROG, GROy and NAP-2 induce neufrophil shape
change, chemotaxis, granule release, and respiratory burst, by binding to and
activating receptors of the seven-transmembrane, G-protein-linked family, in
particular by binding to IL-8 receptors, most notably the IL-8(3 receptor
(CXCR2).
Thomas et al., J. Biol. Chem. 266, 14839 (1991); and Holmes et al., Science
253,
1278 (1991). The development of non-peptide small molecule antagonists for
2o members of this receptor family has precedent. For a review see R.
Freidinger in
Progress in Dn ~ Research, Vol. 40, pp. 33-98, Birkhauser Verlag, Basel 1993.
Hence, the IL-8 receptor represents a promising target for the development of
novel anti-inflammatory agents.
Two high affinity human IL-8 receptors (77% homology) have been
characterized: IL-BRa, which binds only IL-8 with high affinity, and IL-BR~i,
which has high affinity for IL-8 as well as for GROa, GROG, GRO~y and NAP-2.
See Holmes et aL, supra; Murphy et al., Science 253, 1280 (1991); Lee et al.,
J.
Biol. Chem. 267, 16283 (1992); LaRosa et al., J. Biol. Chem. 267, 25402
(1992);
and Gayle et al., J. Biol. Chem. 268, 7283 (1993).
3o There remains a need for treatment, in this field, for compounds, which are
capable of binding to the IL-8 a or (3 receptor. Therefore, conditions
associated
with an increase in IL-8 production (which is responsible for chemotaxis of
-2-
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neutrophil and T-cells subsets into the inflammatory site) would benefit by
compounds, which are inhibitors of IL-8 receptor binding.
SUMMARY OF THE INVENTION
This invention provides for a method of treating a chemokine mediated
disease, wherein the chemokine is one which binds to an IL-8 a or b receptor
and
which method comprises administering an effective amount of a compound of
Formula (I) or a pharmaceutically acceptable salt thereof. In particular the
chemokine is IL-8.
This invention also relates to a method of inhibiting the binding of IL-8 to
1o its receptors in a mammal in need thereof which comprises administering to
said
mammal an effective amount of a compound of Formula (I).
The present invention also provides for the novel compounds of Formula
(I), and pharmaceutical compositions comprising a compound of Formulae (I),
and a
pharmaceutical carrier or diluent.
Compounds of Formula (I) useful in the present invention are represented
by the structure:
R14 R15
R14
OH
H Y
(Rb)2NS(O)2 ~ N II H
R14 R15
(R1)m
(I)
2o wherein
Rb is independently selected rom the group consisting of hydrogen, NR6R~, OH,
ORa,
C 1 _5 alkyl, aryl, arylC 1 _q.alkyl, aryl C2_q.alkenyl; cycloalkyl,
cycloalkyl C 1 _5
alkyl, heteroaryl, heteroarylCl_q.alkyl, heteroarylC2_q. alkenyl,
heterocyclic,
heterocyclic C1_q.alkyl, and a heterocyclic C~_q.alkenyl, all of which
moieties may
be optionally substituted one to three times independently by a substituent
selected
from the group consisting of halogen, nitro, halosubstituted Cl_q. alkyl,
C~_q. alkyl,
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amino, mono or di-C1_q. alkyl substituted amine, ORa, C(O)Ra, NRaC(O)ORa,
OC(O)NRgR~, hydroxy, NR9C(O)Ra, S(O)m~Ra, C(O)NR6R~, C(O)OH,
C(O)ORa, S(O)2NR6R~, NHS(O)2Ra, or, the two Rb substituents join to form a 3-
membered ring, optionally substituted and containing, in addition to carbon,
5 independently, 1 to 3 moieties selected from the group consisting of NRa, O,
S,
SO, or SO2; and wherein the substituent can be optionally unsaturated;
Ra is selected from the group consisting of alkyl, aryl, arylCl_q.alkyl,
heteroaryl,
heteroaryl C1_q.alkyl, heterocyclic, COOR13, and a heterocyclic C1_q.alkyl
moiety,
all of which moieties may be optionally substituted;
l0 m is an integer having a value of 1 to 3;
m' is 0, or an integer having a value of 1 or 2;
n is an integer having a value of 1 to 3;
q is 0, or an integer having a value of 1 to 10;
t is 0, or an integer having a value of 1 or 2;
is s is an integer having a value of 1 to 3;
R1 is independently selected from the froup consisting of hydrogen, halogen,
vitro,
cY~o= C 1-10 ~kYh halosubstituted C 1 _ 10 alkyl, C~_ 1 p alkenyl, C 1 _ l p
alkoxy,
halosubstituted C1_lpalkoxy, azide, S(O)tRq., (CRgRg)q S(O)tRq., hydroxy,
hydroxy
substituted C 1 _q.alkyl, aryl, aryl C 1 _q. alkyl, aryl C2_ 10 alkenyl,
aryloxy, aryl C 1 _q.
alkyloxy, heteroaryl, heteroarylalkyl, heteroaryl C2_10 alkenyl, heteroaryl
C1_4
alkyloxy, heterocyclic, heterocyclic C1_q.alkyl, heterocyclicCl_q.alkyloxy,
heterocyclicC2_lp alkenyl, (CRgRg)q NRq.RS, (CRgRg)qC(O)NRq.RS, C2_10
alkenyl C(O)NRq.RS, (CRgRg)q C(O)NRq.RlO, S(O)3Rg, (CRgRg)q C(O)R11,
C2-10 ~kenyl C(O)R11, C~_10 alkenyl C(O)OR11, (CRgRg)q C(O)OR11,
(CRgRg)q OC(O)R11~ (CRgRg)qNRq.C(O)Rll, (CRgRg)q C(NRq.)NR4R5,
(CRgRg)q NRq.C(NR5)R11, (CRgRg)q NHS(O)ZR13, and (CRgRg)q S(O)2NRq.RS;
or two R1 moieties together may form O-(CH~)s0 or a 5 to 6 membered saturated
or
unsaturated ring, and wherein the alkyl, aryl, arylalkyl, heteroaryl,
heterocyclic
moieties may be optionally substituted;
_q._
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R4 and RS are independently selected from the group consisting of hydrogen,
optionally substituted C1_4 alkyl, optionally substituted aryl, optionally
substituted
aryl C1_q.alkyl, optionally substituted heteroaryl, optionally substituted
heteroaryl
C 1 _q.alkyl, heterocyclic, and heterocyclicC 1 _q. alkyl; or R4 and R5
together with
the nitrogen to which they are attached form a 5 to 7 member ring which may
optionally comprise an additional heteroatom selected from O, N and S;
R( and R~ are independently selected from the group consisting of hydrogen, C
1-4
alkyl, heteroaryl, aryl, aklyl aryl, and alkyl C1_4 heteroalkyl; or R( and R~
together with the nitrogen to which they are attached form a 5 to 7 member
ring
l0 which ring may optionally contain an additional heteroatom is selected from
oxygen, nitrogen and sulfur; wherein the ring may be optionally substituted;
Y is selected from the group conisting of CR14C15, NR14~ O~ CO, and S(O)t
Rg is hydrogen or C1_4 alkyl;
R9 is C 1 _q. alkyl;
Rlp is C1_10 alkyl C(O)2Rg;
R11 is selected from the group consisting of hydrogen, optionally substituted
Cl_4
alkyl, optionally substituted aryl, optionally substituted aryl Cl_4alkyl,
optionally
substituted heteroaryl, optionally substituted heteroarylCl_4alkyl, optionally
substituted heterocyclic, and optionally substituted heterocyclicCl_4alkyl;
and
R1~, is selected from the group consisting of hydrogen C1_4 alkyl, aryl, aryl
C1_4alkyl,
heteroaryl, heteroarylCl_4alkyl, heterocyclic, and heterocyclicCl_4alkyl;
R13 is selected from the group consisting of C1_4 alkyl, aryl, aryl
C1_q.alkyl,
heteroaryl, heteroarylCl_4alkyl, heterocyclic, and heterocyclicCl_4alkyl;
R14 and R15 are, independently, selected from the group consisting of
hydrogen,
optionally substituted C1_4 alkyl group, ORa, and NR4R5; or R14 and R1~
together with the atom (s) to which they are attached may form a 4 to 7 member
ring which may optionally contain an additional heteroatorn which heteroatom
is
selected from the group consisting of oxygen, nitrogen and sulfur; wherein the
ring
maybe optionally substituted;
or a pharmaceutically acceptable salt thereof.
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DETAILED DESCRIPTION OF THE INVENTION
The compounds of Formula (I), may also be used in association with the
veterinary treatment of mammals, other than humans, in need of inhibition of
IL-8
or other chemokines which bind to the IL-8 oc and (3 receptors. Chemokine
mediated diseases for treatment, therapeutically or prophylactically, in
animals
include disease states such as those noted herein in the Methods of Treatment
section.
Suitably, Rb is independently hydrogen, NR(R~, OH, ORa, C1_q.alkyl, aryl,
arylCl_q.alkyl, aryl C2_q.alkenyl, heteroaryl, heteroarylCl_q.alkyl,
heteroarylC~_q.
alkenyl, heterocyclic, heterocyclic C 1-q.alkyl, or a heterocyclic
C2_q.alkenyl moiety, all
of which moieties may be optionally substituted one to three times
independently by
halogen, nitro, halosubstituted C 1 _q. alkyl, C 1 _q. alkyl, amino, mono or
di-C 1 _q. alkyl
substituted amine, cycloalkyl, cycloalkyl C1_5 alkyl, ORa, C(O)Ra, NRaC(O)ORa,
OC(O)NRgR~, aryloxy, aryl Cl_q. oxy, hydroxy, C1_q. alkoxy, NR9C(O)Ra,
S(O)m~Ra, C(O)NR~R~, C(O)OH, C(O)ORa, S(O)~NRgR~, NHS(O)~Ra.
Alternatively, the two Rb substituents can join to form a 3-10 membered ring,
optionally substituted and containing, in addition to carbon, independently, 1
to 3
NR9, O, S, SO, or SO~, moities which can be optionally substituted.
Suitably, Ra is an alkyl, aryl, arylCl_q.alkyl, heteroaryl, heteroaryl
C1_q.alkyl,
heterocyclic, or a heterocyclic Cl_q.alkyl moiety, all of which moieties may
be
optionally substituted.
Suitably, R1 is independently selected from hydrogen; halogen; nitro; cyano;
halosubstituted C1-10 alkyl, such as CF3, C1_10 alkyl, such as methyl, ethyl,
isopropyl, or n-propyl, C2_ l p alkenyl, C 1 _ 10 alkoxy, such as methoxy, or
ethoxy;
halosubstituted C1-10 alkoxy, such as trifluoromethoxy, azide, (CRBRg)q
S(O)tRq.,
wherein t is 0, 1 or 2, hydroxy, hydroxy Cl_q.alkyl, such as methanol or
ethanol,
aryl, such as phenyl or naphthyl, aryl C 1 _q. alkyl, such as benzyl, aryloxy,
such as
phenoxy, aryl C1_q. alkyloxy, such as benzyloxy; heteroaryl, heteroarylalkyl,
heteroaryl C1_q. alkyloxy; aryl C~_10 alkenyl, heteroaryl C~_10 alkenyl,
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heterocyclic C2_10 alkenyl, (CRgRg)qNRq.RS, C~,_10 alkenyl C(O)NRq.RS,
(CRgRg)qC(O)NRq.RS, (CRgRg)qC(O)NR4Rlp, S(O)3H, S(O)3Rg,
(CRgRg)qC(O)R11, C2-10 ~kenyl C(O)R11, C2-10 ~kenyl C(O)OR11, (CRgRg)q
C(O)R11, (CRgRg)qC(O)ORll, (CRgRg)q OC(O)R11, (CRgRg)qNRq.C(O)R11,
(CRgRg)qC(NRq.)NRq.R.S, (CRgRg)q NRq.C(NR5)R11, (CRgRg)qNHS(O)2R13,
(CRgRg)qS(O)ZNR4R5. All of the aryl, heteroaryl, and heterocyclic-containing
moieties may be optionally substituted as defined herein below.
For use herein the term "the aryl, heteroaryl, and heterocyclic containing
moieties" refers to both the ring. and the alkyl, or if included, the alkenyl
rings,
1o such as aryl, arylalkyl, and aryl alkenyl rings. The term "moieties" and
"rings"
may be interchangeably used throughout.
Suitably, Rq. and R5 are independently hydrogen, optionally substituted C1_
4 alkyl, optionally substituted aryl, optionally substituted aryl C1_q.alkyl,
optionally substituted heteroaryl, optionally substituted heteroaryl
C1_q.alkyl,
heterocyclic, heterocyclicCl_q. alkyl, or Rq. and R5 together with the
nitrogen to
which they are attached form a 5 to 7 member ring which may optionally
comprise
an additional heteroatom selected from O, N and S.
Suitably R( and R~ are hydrogen, or a C1_q. alkyl, heteroaryl, alkyl C1_4
heteroalkyl or Rg and R~ together with the nitrogen to which they are attached
2o form a 5 to 7-member ring which ring may optionally contain an additional
heteroatom that is selected from oxygen, nitrogen or sulfur, and which ring
may be
optionally substituted;
Suitably, Rg is independently hydrogen or Cl_q. alkyl.
Suitably, R9 is hydrogen or a C 1 _q. alkyl;
Suitably, q is 0 or an integer having a value of 1 to 10.
Suitably, R10 is C1-10 alkyl C(O)~Rg, such as CHZC(O)~H or
CH~C(O)2CH3.
Suitably, R11 is hydrogen, C1_4 alkyl, aryl, aryl C1_q. alkyl, heteroaryl,
heteroaryl C1_q.a.lkyl, heterocyclic, or heterocyclic C1_q.alkyl.
Suitably, R1~, is hydrogen, C1_10 alkyl, aryl or arylalkyl.
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Suitably, R13 is C1_4alkyl, aryl, arylalkyl, heteroaryl, heteroarylCl_q.alkyl,
heterocyclic, or heterocyclicCl_q.alkyl, wherein all of the aryl, heteroaryl
and
heterocyclic containing moieties may all be optionally substituted.
R14 and R15 are suitably hydrogen, optionally substituted C1_q. alkyl group,
ORa, NRq.RS or Rlq. and R15 together with the atom (s) to which they are
attached
may form a 4 to 7 member ring which may optionally contain an additional
heteroatom
which is selected from oxygen, nitrogen or sulfur; this ring maybe optionally
substituted.
Suitably, Y is CRlq.RlS, NRlq., O
1o Suitably, Ra is an alkyl, aryl C1_q. alkyl, heteroaryl, heteroaryl-
Cl_q.alkyl,
heterocyclic, or a heterocyclicCl_q. alkyl, wherein alI of these moieties may
all be
optionally substituted.re-
As used herein, °'optionally substituted" unless specifically
defined shall
mean such groups as halogen, such as fluorine, chlorine, bromine or iodine,
hydroxy; hydroxy substituted Cl-lO~kYl, Cl-10 ~koxy, such as methoxy or
ethoxy, S(O)m° Cl-10 alkyl, wherein m' is 0, 1 or 2, such as methyl
thio, methyl
sulfinyl or methyl sulfonyl; amino, mono & di-substituted amino, such as in
the
NRgRl~ group, NHC(O)R13, C(O)NRgRl2, C(O)OH, S(O)~NRgRl2,
NHS(O)2R13, C1-10 alkyl, such as methyl, ethyl, propyl, isopropyl, or t-butyl,
halosubstituted Cl-10 alkyl, such CF3, an optionally substituted aryl, such as
phenyl, or an optionally substituted arylalkyl, such as benzyl or phenethyl,
optionally substituted heterocylic, optionally substituted heterocyclicalkyl,
optionally substituted heteroaryl, optionally substituted heteroaryl alkyl,
wherein
these aryl, heteroaryl, or heterocyclic moieties may be substituted one to two
times
by halogen; hydroxy; hydroxy substituted alkyl, C 1 _ 10 alkoxy; S (O)m~C 1 _
l 0 alkyl;
amino, mono & di-substituted alkyl amino, such as in the NR(R~ group; C 1 _ l
0
alkyl, or halosubstituted C1_1p alkyl, such as CF3.
Suitable pharmaceutically acceptable salts are well known to those skilled
in the art and include basic salts of inorganic and organic acids, such as
hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methane
sulphonic acid, ethane sulphonic acid, acetic acid, malic acid, tartaric acid,
citric
_g_
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acid, lactic acid, oxalic acid, succinic acid, fumaric acid, malefic acid,
benzoic acid,
salicylic acid, phenylacetic acid and mandelic acid. In addition,
pharmaceutically
acceptable salts of compounds of Formula (I) may also be formed with a
pharmaceutically acceptable cation. Suitable pharmaceutically acceptable
cations
are well known to those skilled in the art and include alkaline, alkaline
earth,
ammonium and quaternary ammonium canons.
The following terms, as used herein, refer to:
"halo" - all halogens, that is chloro, fluoro, bromo and iodo.
~ "Cl-l0~ky1" or "alkyl" - both straight and branched chain moieties of 1
to IO carbon atoms, unless the chain length is otherwise limited, including,
but not
limited to, methyl, ethyl, h-propyl, iso-propyl, n-butyl, sec-butyl, iso-
butyl, tert-
butyl, n-pentyl and the like.
~ "cycloalkyl" is used herein to mean cyclic moiety, preferably of 3 to 8
carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl,
and the
like.
~ "alkenyl" is used'herein at all occurrences to mean straight or branched
chain moiety of 2-10 carbon atoms, unless the chain length is limited thereto,
including, but not limited to ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-
propenyi,
1-buteriyl, 2-butenyl and the like.
~ "aryl" - phenyl and naphthyl;
~ "heteroaryl" (on its own or in any combination, such as "heteroaryloxy",
or "heteroaryl alkyl") - a 5-10 membered aromatic ring system in which one or
more rings contain one or more heteroatoms selected from the group consisting
of
N, O or S, such as, but not limited, to~pyrrole, pyrazole, furan, thiophene,
quinoline, isoquinoline, quinazolinyl, pyridine, pyrimidine, oxazole,
tetrazole,
thiazole, thiadiazole, triazole, imidazole, or benzimidazole.
~ "heterocyclic" (on its own or in any combination, such as
"heterocyclicalkyl") - a saturated or partially unsaturated 4-10 membered ring
system in which one or more rings contain one or more heteroatorns selected
from
the group consisting of N, O, or S; such as, but not limited to, pyrrolidine,
piperidine, piperazine, morpholine, tetrahydropyran, thiomorpholine, or
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imidazolidine. Furthermore, sulfur may be optionally oxidized to the sulfone
or
the sulfoxide.
~ "arylalkyl" or "heteroarylalkyl" or "heterocyclicalkyl" is used herein to
mean C1-10 alkyl, as defined above, attached to an aryl, heteroaryl or
heteroeyclic
moiety, as also defined herein, unless otherwise indicated.
~ "sulfinyl" - the oxide S (O) of the corresponding sulfide, the term "thio"
refers to the sulfide, and the term "sulfonyl" refers to the fully oxidized
S(O)2
moiety.
~ "wherein two R1 moieties may together form a 5 or 6 membered
1o saturated or unsaturated ring" is used herein to mean the formation of an
aromatic
ring system, such as naphthalene, or is a phenyl moiety having attached a 6
membered partially saturated or unsaturated ring such as a C( eyeloalkenyl,
i.e.
hexene, or a C5 cycloalkenyl moiety, such as cyclopentene.
Illustrative compounds of Formula (I) include:
N-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N'-cyelohexylurea;
N-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N'-( 1-adamantyl)urea;
N-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N'-(tetrahydro-2-pyranyl)ur ea;
N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N'-(3-tetrahydrofuryl)urea;
6-Chloro-2-hydroxy-3-[3-(2-methyl-cyclopropyl)-ureido]-benzenesulfonamide;
2o N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N'-cyclohexylurea;
6-Chloro-2-hydroxy-3-[3-(2,2,3,3-tetrmethyl-cyclopropyl)-ureido]-
benzenesulfonamide;
6-Chloro-2-hydroxy-3-(3-piperidin-4-yl-ureido)-benzenesulfonamide;
N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N'-(4-methyl-cyclohexyl) urea;
6-Chlora-2-hydroxy-3-[3-(3-methoxy-eyclohexyl)-ureido]-benzenesulfonamide;
N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N'-cyclopentylurea;
N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N'-cyclobutylurea;
N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N'-cyclopropylurea;
4.-[6-Chloro-3-(3-cyclopentyl-ureido)-2-hydroxy-benzenesulfonyl]-piperazine-1-
3o carboxylic acid tert butyl ester;
1-[4-Chloro-2-hydroxy-3-(piperazine-1-sulfonyl)-phenyl]-3-cyclopentyl-urea;
10-
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4-[6-Chloro-3-(3-cyclobutyl-ureido)-2-hydroxy-benzenesulfonyl]-piperazine-1-
carboxylic acid tert-butyl ester;
3-[3-(( 1 S,2S)-2-Benzyloxy-cyclohexyl)-ureido]-6-chloro-2-hydroxy-
benzenesulfonamide; and
6-Chloro-3-(3-cyclobutyl-ureido)-2-hydroxy-N,N'-dimethyl-benzenesulfonamide.
METHODS OF PREPARATION
The compounds of Formulas (I) may be obtained by applying synthetic
procedures, some of which are illustrated in the Schemes below. The synthesis
provided for in these Schemes is applicable for the producing compounds of
Formulas (I), having a variety of different R, Rb, and Y groups which are
reacted,
employing optional substituents which are suitably protected, to achieve
compatibility with the reactions outlined herein. Subsequent deprotection, in
those
cases, then affords compounds of the nature generally disclosed. Once the urea
nucleus has been established, further compounds of these formulas may be
prepared by applying standard techniques for functional group interconversion,
well known in the art.
Scheme 1
SH S02NR'R" SOZNR'R"
CI / CI a CI / CI b CI / CI
--~. -
N+.. O
1 2 3 I
O
S02NR'R" S02NR'R" S02NR'R"
CI / CI a CI ~ OAc d CI / ON
\ N+-. O \ ~ +.. O ~ \ +._ O
I N N
3 O_ 4 I 5 I_
O- O
S02NR'R" SOZNR'R" S02NR'R"
CI / OH a CI / OH f CI / OH O
\ ~ +..O
\ ~ \ ~ ~ ,.R
N \NH2 \H H
5 O_ 6
7
-II-
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a)i)NCS, AcOH, H20, ii NR~2"H, pyr b)H2SOq., HN03 c)NaOAc, 18-crown-6
d)H2SOq., MeOH e) Pd/C, H~ fjRCNO, DMF
The desired 4-chloro N-(3-sulfonamido-2-hydroxy phenyl)-N"-cycloalkyl
urea can synthesized from the commercially available 2, 6-dichloro thiophenol
using the procedure elaborated above in Scheme 1. The thiol can be oxidized to
the corresponding sulfonyl halide using a halogenating agent, such as NCS,
NBS,
Cl2 or Br2, in the presence of a protic solvent, such as water, acetic acid,
or an
alcohol or combination. The yield may be increased if a buffering agent, such
as
l0 sodium or potassium acetate is included in the reaction mixture, and the
reaction is
conducted at or below room temperature. The coiTesponding sulfonyl halide can
then be condensed with an amine in presence of a base such as pyridine,
triethyl
amine, potassium carbonate or sodium hydride to form the analogous sulfonamide
2-scheme 1. The dichlorosulfonamide 2.-scheme 1 can be nitrated using strong
nitrating conditions such as nitric acid in sulfuric acid to form the aromatic
vitro
compound 3-scheme 1. The chlorine ortho to the vitro group can be selectively
hydrolyzed using acetate salt such as sodium acetate in the presence of a
crown
ether, such as 18-crown-6, to form the acetate 4-scheme 1. The acetate group
can
be hydrolyzed under acidic conditions in an alcohol solvent such as methanol
or
ethanol with a catalytic amount of acid to form the phenol 5-scheme 1. The
vitro
can be reduced by conditions well known in the art such as hydrogen and
palladium on carbon, tin chloride in methanol, zinc in acetic acid or thiol to
form
the corresponding aniline 5-scheme 1. The aniline can then be coupled with a
commercially available isocyanate or thioisocyanate to form the desired urea
or
thio urea. Alternatively the desired isocyanates can be made by condensing the
amine with triphosgene in the presence of base (such as potassium carbonate)
or by
reacting the carboxylic acid with diphenyl phosphoryl azide in the presence of
a
base (such as triethyl amine).
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Scheme 2
S02NH~ S02NR'H S02NR'R"
CI / OAc a Cl / OAc b CI / OAc
\ N+..O _ \ ~ +..O \ ~ +..O
I N N
O- 2 O_ g O_
a)NaH, R~ b)NaH R"X
If the sulfonamide 1-scheme 2 (3-scheme 1) is unfunctionalized R'--R"=H
then it can be functionalized as required herein, by alkylation. The
sulfonamide is
deprotonated using a base such as sodium hydride and then alkylated using an
alkyl
halide such as benzyl bromide or methyl iodide form 2-scheme 2. The
sulfonamide
can then be alkylated a second time using sodium hydride and another alkyl
halide
to form 3-scheme 2. This compound can then be converted to the desired urea
to using the process elaborated in scheme 1.
Scheme 3
SH SOzNa SOzH
CI / CI a CI / CI b CI / CI
\ l \ \ l N+..
1 2
O
SOzH S02Na S02NR'R"
CI / CI c CI / CI d a CI / CI
N+'- O \ ~ +._ O \ ~ +.- O
I N N
3 O_ 4 10_ 5 ~_
15 a)i)NCS, AcOH, HBO ii)NaOH MeOH b)H2S04, HN03 c)NaOH MeOH d) PC15,
POCl3 e)NHR~2", Et3N
An alternative route to 5-scheme 3 (3-scheme 1) is outlined above, in
scheme 3 wherein the commercially available 2.,6-dichloro thiol can be
oxidized to
the sulfonyl halide using a halogenating agent such as NCS, NBS, chlorine or
2o bromine in the presence of a erotic solvent such as alcohol, acetic acid or
water.
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The sulfonyl halide can be hydrolyzed by using a metal hydroxide such as
sodium
or potassium hydroxide to form the corresponding sulfonic acid salt. The
sulfonic
acid salt can then be nitrated under nitration conditions such as nitric acid
in a
solvent of strong acid such as sulfuric acid to form the nitro phenyl sulfonic
acid 3-
scheme 3. The sulfonic acid 3-scheme 3 can be converted to the sulfonamide
5-scheme 3 using a three step procedure involving the formation of the metal
salt
using a base such as sodium hydroxide, sodium hydride or sodium carbonate to
form 4-scheme 3. The sulfonic acid salt is then converted to the sulfonyl
chloride
using PC15 with POCl3 as a solvent. The sulfonyl chloride can then be
converted
1o to the corresponding sulfonamide using the desired amine HNR~" in triethyl
amine at temperatures ranging from -78 oC to 60 oC to form the corresponding
sulfonamide 5-scheme 3 ( 3-scheme 1). The sulfonamide 5-scheme 3 can be
further elaborated by the methods contained in scheme 1. This method is not
limited to the 2,6-dichloro thiol it can also be applied to the 2,6-diflouro
thiol, 2,6-
15 dibromo thiol and the 2,6-diiodo thiol. The halogens in these compounds can
be
converted to the corresponding cyano, amino, thiol, or alkoxy compounds by
nucleophilic displacement reactions using nucleophiles such as alkyl
thiolates,
alkoxides, amine and cyanides. The halogens can also be further functionalized
by
palladium coupling and carbonylation reactions, well known in the art, to form
the
20 corresponding amido, carbonyl, alkenyl, alkyl, phenyl and heterocyclic
substituted
products as required by Formula (~.
SYNTHETIC EXAMPLES
The invention will now be described by reference to the following examples
which are merely illustrative and axe not to be construed as a limitation of
the scope of
25 the present invention. All temperatures are given in degrees centigrade,
all solvents
are highest available purity and all reactions run under anhydrous conditions
in an
argon atmosphere unless otherwise indicated.
In the Examples, all temperatures are in degrees Centigrade (°C). Mass
spectra
Were performed upon a VG Zab mass spectrometer using fast atom bombardment,
unless
30 otherwise indicated. 1H-NMR (hereinafter "NMR") spectra were recorded at
250 MHz
using a Bruker AM 250 or Am 400 spectrometer. Multiplicities indicated are:
s=singlet,
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d=doublet, t=triplet, q=quartet, m=multiplet and br indicates a broad signal.
Sat.
indicates a saturated solution, eq indicates the proportion of a molar
equivalent of
reagent relative to the principal reactant.
General synthesis of 2-hydroxy-3-amino-6-chlorobenzenesulfonamide
a) 2,6-dichlorobenzenesulfonyl chloride
Into a mixture of 200 milliliters (hereinafter "mL") of acetic acid, water and
dichloromethane (3/1/4, v/v/v), 2,6 dichlorobenzenethiol (10.0 grams
(hereinafter
to "g"), 55.8 millimoles (hereinafter "mmol"), N-chlorosuccinimide (37.28 g,
279
mmol) and potassium acetate (2.29 g, 27.9 mmol) were added. The resulting
mixture was stirred at 0°C, then warmed to room temperature overnight.
The
mixture was then diluted with 200 mL of dichloromethane, and washed with water
(100 mL x 3). The organic layer was dried (NaZSO~) and concentrated to give
the
desired product (11 g, 80%). 'H NMR (CDC13): ~ 7.57 (d, 2H), 7.47 (t, 1H).
b) 2,6-dichlorobenzenesulfonamide
A solution of 2,6-dichlorobenzenesulfonyl chloride (10.50 g, 42.77 mmol) in
100
mL of pyridine was added into 100 mL of pyridine dropwise while anhydrous
ammonia gas was passing through the solution simultaneously for 4 hours at
0°C.
The mixture was acidified to pH >1 with 6N aq. HCl, then extracted with ethyl
acetate. The combined organic layer was then dried (Na~S04) and concentrated
to
give the desired product (8.69 g, 90%). EI-MS (m/z) 225.0, 227.1 (M-).
c) 2,6-dichloro-3-nitrobenzenesulfonamide
Into a solution of 2,6-dichlorobenzenesulfonamide (7.8 g, 34.5 mmol) in 30 mL
of
concentrated sulfuric acid at 0°, nitric acid (1.74 mL, 41.4 mmol) was
added
dropwise. The mixture was stirred at 0°C for 2 hours, then 200 mL of
water was
added to produce a precipitate. The resulting mixture was filtered. The white
solid
was collected, washed with water and dried iia vacuo to give the desired
product
(7.17 g, 76%). 'H NMR (DMSO-db): S 8.25 (s, 2H), 8.20 (d, 1H), 7.92 (d, 1H).
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d) 2-acetoxy-3-nitro-6-chlorobenzesulfonamide
A solution of 2,6-dichloro-3-nitrobenzenesulfonamide (2.04 g, 7.5 mmol),
potassium acetate (2.21 g, 22.5 mmol) and 18-crown-6 (5.95 g, 22.5 mmol) in SO
mL of dimethyl sulfoxide was heated to 45°C for 7 days. The mixture was
acidified with 1N aq. HCI, and extracted with ethyl acetate. The organic layer
was
concentrated to give the crude. Column chromatography on silica gel, eluting
with
ethyl acetate/hexane/acetic acid (50/49/1, v/v/v), gave the desired product
(1.67 g,
76%). EI-MS (m/z) 293.1, 295.1 (M-).
to e) 2-hydroxy-3-nitro-6-chlorobenzesulfonamide
A solution of 2-acetoxy-3-nitro-6-chlorobenzesulfonamide ( I .72 g, 5.83
rnmol),
chlorotrimethylsilane (2 mL) and fuming sulfuric acid (0.5 mL) in methanol was
heated to reflux for 20 hours. The solvent was evaporated. The residue was
diluted with ethyl acetate and washed with water. The organic layer was then
dried
(Na~S04) and concentrated to give the desired product (1.0 g, 68%). EI-MS
(m/z)
251.1, 253.2 (M-).
f) 2-hydroxy-3-amino-6-chlorobenzenesulfonamide
To a solution of 2-hydroxy-3-nitro-6-chlorobenzenesulfonamide (1.I g, 4.36
mmol) in ethyl acetate, was added 10 % PdIC (500 mg). The mixture was flushed
with hydrogen, and then stirred under a hydrogen atmosphere at balloon
pressure
for 4 hours at room temperature. The mixture was filtered through celite and
the
celite was washed with methanol. The solvent was evaporated to give the
desired
product (91 %). EI-MS (mlz) 221.1, 223.1 (M-).
Example 1
Standard yrocedure for the condensation of an aniline with an isocyante.
Preparation of N-(3-aminosulfonyl-4-chloro-2-hydroxyuhenyl)-N'-cyclohexyl
urea
3o A solution of 3-amino-6-chloro-2-hydroxybenzenesulfonamide (150 mg,
0.67 mmol) and cyclohexyl isocyanate (95 ~.L., 0.74 mmol) in 1 mL of N,N-
dimethyl-formamide was stirred at room temperature for 20 hours. The mixture
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was diluted with ethyl acetate and washed with water to give the crude.
Purification upon column chromatograph on silica gel, eluting with ethyl
acetate/hexane (50/50, v/v), followed by recrystallization from acetone and
hexane,
gave the desired product (121 mg, 52%). LC-MS (m/z) 348.0 (M+).
Example 2
Preparation of N-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N'-(1-
adamantyl) urea
A solution of 3-amino-6-chloro-2-hydroxybenzenesulfonarnide (120 mg,
0.54 mmol) and 1-adamantyl isocyanate (115 mg, 0.64 mmol) in 1 mL of N,N-
dimethyl-formamide was stirred at room temperature for 20 hours. The mixture
was diluted with ethyl acetate and washed with water to give the crude.
Purification upon column chromatograph on silica gel, eluting with ethyl
acetate/hexane (50/50, v/v), gave the desired product (122 mg, 56%). LC-MS
(m/z)
400.0 (M+).
Examule 3
Preparation of N-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N'-
(tetrahydro-2-pyranyl) urea
2o A solution of 3-amino-6-chloro-2-hydroxybenzenesulfonamide (170 mg,
0.76 mrnol) and tetrahydro-2-pyranyl isocyanate (117 mg, 0.92 mmol) in 1.5 mL
of
N,N-dimethyl-formamide was stirred at room temperature for 20 hours. The
mixture was diluted with ethyl acetate and washed with water to give the
crude.
Purification upon column chromatograph on silica gel, eluting with ethyl
acetate/hexane (60/40, v/v), followed by separation with Gilson HPLC, gave the
desired product (28 mg, 10%). LC-MS (m/z) 350.2 (M+).
Example 4
Standard procedure for the synthesis of ureas by coupling carboxylic acids
3o with an aniline. Synthesis of N-f4-chloro-2-hydroxy-3-sulfamylnhen~Il-N'-(3-
tetrahydrofuryl) urea
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To a solution of Tetrahydro-furan-3-carboxylic acid (0.093 mL, 1.0 mmol)
in DMF (0.5 mL) was added DPPA (0.25 mL, 1.2 mmol) and TEA (0.25 mL, 1.8
mmol) and the reaction heated at 70°C. After l8hrs, 3-Amino-6-chloro-2-
hydroxy-
benzenesulfonamide (1.0 mmol) was added and the reaction heated at
70°C. After
l8hrs, the reaction mixture was quenched with water and extracted with ethyl
acetate. The organic layers were dried over anhydrous magnesium sulfate, and
concentraed under reduced pressure. The crude residue was purified via HPLC to
give 18 mg (5%) of N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N'-(3-
tetrahydrofuryl)urea. LC-MS (m/z) 336 (M+).
to
Example 5
Synthesis of 6-Chloro-2-hydroxy-3-f3-(2-methyl-cyclonrouyl)-ureidol-
benzenesulfonamide
To a solution of 2-Methyl-cyclopropanecarboxylic acid (0.097 mL, 1.0
mmol) in DMF (0.5 mL) was added DPPA (0.25 mL, 1.2 mmol) and TEA (0.25
mL, 1.8 mmol) and the reaction heated at 90°C. After l8hrs, 3-Amino-6-
chloro-2-
hydroxy-benzenesulfonamide ( 1.0 mmol) was added and the reaction heated at
90°C. After l8hrs, the reaction mixture was quenched with water and
extracted
with ethyl acetate. The organic layers were dried over anhydrous magnesium
sulfate, and concentraed under reduced pressure. The crude residue was
purified
via HPLC to give 29 mg ( 11 %) of 6-Chloro-2-hydroxy-3-[3-(2-methyl-
cyclopropyl)-ureido]-benzenesulfonamide. LC-MS (m/z) 320 (M+).
Examule 6
Synthesis of 6-Chloro-2-hvdroxv-3-f3-(2,2,3,3-tetrmethvl-cvclonronvl)-
ureidol-benzenesulfonamide
To a solution of 2,2,3,3-tetramethyl-cyclopropanecarboxylic acid (0.142
mL, 1.0 mmol) in DMF (0.5 mL) was added DPPA (0.25 mL, 1.2 mmol) and TEA
(0.25 mL, 1.8 mmol) and the reaction heated at 80°C. After l8hrs, 3-
Amino-6-
3o chloro-2-hydroxy-benzenesulfonamide (1.0 rnmol) was added and the reaction
heated at 80°C. After l8hrs, the reaction mixture was quenched with
water and
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extracted with ethyl acetate. The organic layers were dried over anhydrous
magnesium sulfate, and concentraed under reduced pressure. The crude residue
was purified via HPLC to give 96 mg of 6-Chloro-2-hydroxy-3-[3-(2,2,3,3-
tetrmethyl-cyclopropyl)-ureido)-benzenesulfonamide. LC-MS (m/z) 347 (M+).
Example 7
Synthesis of 6-Chloro-2-hydroxy-3-(3-piperidin-4-yl-ureido)-
benzenesulfonamide
To a solution of piperidine-1,4-dicarboxylic acid mono-tert-butyl ester
(0.23g, 1.o mmol) in DMF (0.5 mL) was added DPPA (0.215 mL, 1.0 mmol) and
TEA (0.139 mL, 1.0 mmol) and the reaction heated at 80°C. After
l8hrs, 3-
Amino-6-chloro-2-hydroxy-benzenesulfonamide (1.0 mmol) was added and the
reaction heated at 80°C. After l8hrs, the reaction mixture was quenched
with
water and extracted with ethyl acetate. The organic layers were dried over
anhydrous magnesium sulfate, and concentraed under reduced pressure. The crude
residue was purified via HPLC to give 12 mg of 6-Chloro-2-hydroxy-3-(3-
piperidin-4-yl-ureido)-benzenesulfonamide. LC-MS (mlz) 349 (M+).
Example 8
2o Synthesis of N-f 4-chloro-2-hydroxy-3-sulfamylphenyll-N'-(4-methyl-
cyclohexyl) urea
To a solution of 4-Methyl-cyclohexanecarboxylic acid (0.141 mL) in DMF
(0.5 mL) was added DPPA (0.215 mL) and TEA (0.139 mL) and the reaction
heated at 80°C. After l8hrs, 3-Amino-6-chloro-2-hydroxy-
benzenesulfonamide
(0.150 g) was added and the reaction heated at 80°C. After l8hrs, the
reaction
mixture was quenched with water and extracted with ethyl acetate. The organic
layers were dried over anhydrous magnesium sulfate, and concentraed under
reduced pressure. The crude residue was purified via HPLC to give 15 mg of N-
[4-
chloro-2-hydroxy-3-sulfamylphenyl)-N'-(4-methyl-cyclohexyl) urea. LC-MS (m/z)
347 (M+).
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Example 9
Snthesis of 6-Chloro-2-hydroxy-3-f3-(3-methoxy-cyclohexyl)-ureidol-
benzenesulfonamide
To a solution of 3-Methoxy-cyclohexanecarboxylic acid (0.143 mL) in
DMF (0.5 mL) was added DPPA (0.25 mL) and TEA (0.25 mL) and the reaction
heated at 70°C. After l8hrs, 3-Amino-6-chloro-2-hydroxy-
benzenesulfonamide
(0.222 g) was added and the reaction heated at 70°C. After l8hrs, the
reaction
mixture was quenched with water and extracted with ethyl acetate. The organic
to layers were dried over anhydrous magnesium sulfate, and concentraed under
reduced pressure. The crude residue was purified via HPLC to give 3 mg of 6-
Chloro-2-hydroxy-3-[3-(3-methoxy-cyclohexyl)-ureido]-benzenesulfonamide. LC-
MS (m/z) 378 (M+)
Example 10
N-(3-arninosulfonyl-4-chloro-2-hydroxyphenyl)-N'-cyclonentyl urea
A solution of 3-amino-6-chloro-2-hydroxybenzenesulfonamide (200 mg,
0.90 mmol) and cyclopentyl isocyanate (100 mg, 0.90 mmol) in 1.5 mL of N,N-
dimethyl-formamide was stirred at room temperature for 20 hours. Purification
2o upon column chromatograph on silica gel, eluting with ethyl acetate/hexane
(20/80,
v/v), followed by recrystallization from diethyl ether and hexane, gave the
desired
product ( 130 mg, 43 %). LC-MS (m/z) 334.2 (M+)
Example 11
N-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N'-cycIobutyl urea
Under Ar, the mixture of cyclobutanecarboxylic acid (250 mg, 2.50 mmol),
diphenylphosphoryl azide (756 mg, 2.74mmo1), and triethyl amine (0.38mL,
2.74mmol) in 3mL of N,N-dimethyl-formamide was heated to 80°C for 2
hours.
The mixture was cooled to room temperature, 3-amino-6-chloro-2-
hydroxybenzenesulfonamide (556mg, 2.50mmo1) was added. The resulting
mixture was stirred at room temperature for 20 hours. Purification upon Gilson
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HPLC, eluting with acetonitrile/water (10/90, v/v to 90/10, v/v, over lOmin),
gave
the desired product (137mg, 17°70). LC-MS (m/z) 320.0 (M+)
Example 12
N-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N'-cyclonropanyl urea
Under Ar, the mixture of cyclopropanecarboxylic acid (500 mg, 5.81
mmol), diphenylphosphoryl azide (1.58g, 5.81mmol), and triethyl amine (0.81mL,
5.81mmol) in 3mL of N,N-dimethyl-formamide was heated to 80°C for 3
hours.
The mixture was cooled to room temperature, 3-amino-6-chloro-2-
hydroxybenzenesulfonamide (200mg, 0.89mmol) was added. The resulting
mixture was stirred at.room temperature for 20 hours. Purification upon Gilson
HPLC, eluting with acetonitrile/water (10/90, v/v to 90/10, v/v, over lOmin),
gave
the desired product (74mg, 27%). LC-MS (m/z) 306.2 (M+)
Example 13
N-(3-(N'-Boc-piperazine)-aminosulfonyl-4-chloro-2-hydroxynhenyl)-N"-
cyclouentyl urea
A solution of 3-N-(Boc-piperazine)amino-6-chloro-2-hydroxy
benzenesulfonamide (200 mg, 0.51 mmol) and cyclopentyl isocyanate (57 ~,L,
0:51
2o mmol) in 2 mL of N,N-dimethyl-formamide was stirred at room temperature for
20
hours. Purification upon column chromatograph on silica gel, eluting with
ethyl
acetate/hexane (30/70, v/v), followed by recrystallization from
dichloromethane
and hexane, gave the desired product (180 mg, 70%). LC-MS (rn/z) 503.2 (M+)
Example 14
N-(3-(N'-niperazine)-aminosulfonyl-4-chloro-2-hydroxynhenyl)-N"-
cyclouentyl urea hydrochloride
The solution of N-(3-(N'-Boc-piperazine)-aminosulfonyl-4-chloro-2-
hydroxyphenyl)-N"-cyclopentylurea (140mg, 0.27mmo1) in 2mL of 4N HCl in 1,4-
3o dioxane was stirred at room temperature for 1 hour. The mixture was
concentrated.
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Recrystallization from acetonitrile gave the desired product (I05mg, 88%). LC-
MS
(m/z) 403.2 (M+).
Examule 15
N-(3-(N'-Boc-niperazine)-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N"-
cyclobutyl urea
Under Ar, the mixture of cyclobutanecarboxylic acid (500mg, 4.99mmo1),
diphenylphosphoryl azide (1.18mL, 5.49mmol), and triethyl amine (0.76mL,
5.49mmol) in 3mL of N,N-dimethyl-formamide was heated to 80°C for 2
hours.
l0 The mixture was cooled to room temperature, 3-N-(Boc-piperazine)amino-6-
chloro-2-hydroxybenzenesulfonamide (1.95g, 4.97mmo1) was added. The
resulting mixture was stirred at room temperature for 20 hours. Purification
upon
column chromatograph on silica gel, eluting with ethyl acetate/hexane (30/70-
50150, vlv), gave the desired product (500 mg, 21 %). LC-MS (m/z) 489.2 (M+).
Example 16
Synthesis of N-(3-aminosulfonyl-4-chloro-2-hydroxyuhenyl)-N'-f (1S,2S)-(-)-2-,
benzyloxycyclohexyll urea
(1S,2S)-(-)-2-benzyloxycyclohexyl isocynate
2o Under Ar, the solution of (1S,2S)-(-)-2-benzyloxycyclohexylamine (100
mg, 0.49mmo1) in 5 mL of dichloromethane was added to a solution of di-tert-
butyl dicarbonate (148mg, 0.68mmo1) and 4-dimethylaminopyridine (87mg,
0.49mmol) in 5 mL of dichloromethane. The mixture was stirred fox 15 min at
room temperature, then concentrated to give the crude material. FT-IR: 2237.92
cm-1.
N-(3-aminosulfonyl-4-chloro-2-hydroxynhenyl)-N'-f (1S,2S)-(-)-2-
benzyloxycyclohexyll urea
A solution of 3-amino-6-chloro-2-hydroxybenzenesulfonamide (109 mg,
0.49mmo1) and the crude (1S,2S)-(-)-2-benzyloxycyclohexyl isocynate in 2 mL of
'
3o N,N-dimethyl-formamide was stirred at room temperature for 20 hours.
Purification upon Gilson HPLC, eluting with acetonitrile/ water (10/90, v/v to
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90/10, v/v, over lOmin), gave the desired product (60mg, 27% two steps). LC-MS
(m/z) 454.0 (M+).
Example 17
Synthesis of N-(3~N'-dimethyl)-aminosulfonyl-4-chloro-2-hydroxyphenyl)-
N"-cyclobutyl urea hydrochloride
N-(3-(N'-dimethyl)-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N-Boc-N"-
cyclobutyl urea
Under Ar, the mixture of cyclobutylamine (14~,L, 0.16mmo1), trimethyl
aluminum (80~,L, 0.16mmo1) in 3mL of dichloromethane was stirred at room
temperature for 10 min. The solution of 7-(N-dirnethyl)aminosulfonyl-3-Boc-5-
.
chloro-2-benzoxazolinone (60mg, 0.16mmo1) in 3 mL of dichloromethane was
added. The resulting mixture was stirred at room temperature for 20 hours.
Purification upon column chromatograph on silica gel, eluting with ethyl
acetate/hexane (20/80, v/v), gave the desired product (36 mg, 51 %). LC-MS
(m/z)
448.2 (M+).
N-(3-(N'-dimethyl)-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N"-cyclobutyl
urea hydrochloride
2o The solution of N-(3-(N'-dimethyl)-aminosulfonyl-4-chloro-2-
hydroxyphenyl)-N-Boc-N"-cyclobutylurea (36mg, 0.08mmo1) in 2mL of 4N HCl
in 1,4-dioxane was stirred at room temperature for 1 hour. The mixture was
concentrated. Recrystallization from acetone and hexane gave the desired
product
(20mg, 65%). Elemental analysis (%) theory: C 44.89, H 5.22, N 12.08,
experiment C44.85, H 5.00, N11.91.
METHOD OF TREATMENT
The compounds of Formula (I), or a pharmaceutically acceptable salt
thereof can be used in the manufacture of a medicine for the prophylactic or
therapeutic treatment of any disease state in a human, or other mammal, which
is
exacerbated or caused by excessive or unregulated IL,-8 cytokine production by
such mammal's cell, such as but not limited to monocytes and/or macrophages,
or
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other chemokines which bind to the IL-8 oc or (3 receptor, also referred to as
the
type I or type II receptor.
Accordingly, the present invention provides a method of treating a
chemokine mediated disease, wherein the chemokine is one which binds to an IL-
8
a or (3 receptor and which method comprises administering an effective amount
of
a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In
particular, the chemokines are IL-8, GROa,, GROG, GRO~y, NAP-2 or ENA-78.
The compounds of Formula (I) are administered in an amount sufficient to
inhibit cytokine function, in particular IL-8, GROa, GRO[3, GRO~y, NAP-2 or
ENA-78, such that they are biologically regulated down to normal levels of
physiological function, or in some case to subnormal levels, so as to
ameliorate the
disease state. Abnormal levels of IL-8, GROa, GROG, GROy, NAP-2 or ENA-78
for instance in the context of the present invention, constitute: (i) levels
of free IL-8
greater than or equal to 1 picogram per mL; (ii) any cell associated IIJ-8,
GROoc,
GROG, GROy, NAP-2 or ENA-78 above normal physiological levels; or (iii) the
presence of IL-8, GROG, GR0~3, GRO~y, NAP-2 or ENA-78 above basal levels in
cells or tissues in which IL-8, GROoc, GRO~i, GROy, NAP-2 or ENA-78
respectively, is produced.
The compounds of Formula (I), in generally have been shown to have a
' longer t1/2 and improved oral. bioavailabilty over the compounds disclosed
in WO
96/25157 and WO 97/29743 whose disclosures are incorporated herein by
reference.
There are many disease states in which excessive or unregulated IL-8
production is implicated in exacerbating and/or causing the disease. Chemokine
mediated diseases include psoriasis, atopic dermatitis, osteo arthritis,
rheumatoid
arthritis, asthma, chronic obstructive pulmonary disease, adult respiratory
distress
syndrome, inflammatory bowel disease, Crohn's disease, ulcerative colitis,
stroke,
septic shock, multiple sclerosis, endotoxic shock, gram negative sepsis, toxic
shock
syndrome, cardiac and renal reperfusion injury, glomerulonephritis,
thrombosis,
graft vs. host reaction, Alzheimer's disease, allograft rejections, malaria,
restenosis,
angiogenesis, atherosclerosis, osteoporosis, gingivitis and undesired
hematopoietic
stem cells release and diseases caused by respiratory viruses, herpes viruses,
and
hepatitis viruses, meningitis, cystic fibrosis, pre-term labor, cough,
pruritus, multi-
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organ dysfunction, trauma, strains, sprains, contusions, psoriatic arthritis,
herpes,
encephalitis, CNS vasculitis, traumatic brain injury, CNS tumors, subarachnoid
hemorrhage, post surgical trauma, interstitial pneumonitis, hypersensitivity,
crystal
induced arthritis, acute and chronic pancreatitis, acute alcoholic hepatitis,
necrotizing enterocolitis, chronic sinusitis, uveitis, polymyositis,
vasculitis, acne,
gastric and duodenal ulcers, celiac disease, esophagitis, glossitis, airflow
obstruction, airway hyperresponsiveness, bronchiolitis obliterans organizing
pneumonia, bronchiectasis, bronchiolitis, bronchiolitis obliterans, chronic
bronchitis, cor pulmonae, dyspnea, emphysema, hypercapnea, hyperinflation,
, hypoxemia, hyperoxia-induced inflammations, hypoxia, surgical lung volume
reduction, pulmonary fibrosis, pulmonary hypertension, right ventricular
hypertropy, sarcoidosis, small airway disease, ventilation-perfusion
mismatching,
wheeze, colds and lupus.
These diseases are primarily characterized by massive neutrophil
infiltration, T-cell infiltration, or neovascular growth, and are associated
with
increased IL-8, GROoc, GROG, GRO~y, NAP-2 or ENA-78 production which is
responsible for the chemotaxis of neutrophils into the inflammatory site or
the
directional growth of endothelial cells. In contrast to other inflammatory
cytokines
(IL-1, TNF, and IL-6), IL-8, GROoc, GR0J3, GRO~y, NAP-2 or ENA-78 have the
unique property of promoting neutrophil chemotaxis, enzyme release including
but
not limited to elastase release as well as superoxide production and
activation. The
a-chemokines but particularly, GROG, GROG, GRO~y, NAP-2 or ENA-78, working
through the IL-8 type I or II receptor can promote the neovascularization of
tumors
by promoting the directional growth of endothelial cells. Therefore, the
inhibition
of IL-8 induced chemotaxis or activation would lead to a direct reduction in
the
neutrophil infiltration.
Recent evidence also implicates the role of chemokines in the treatment of
HIV infections, Littleman et al., Nature 381, pp. 661 (1996) and Koup et al.,
Nature 381, pp. 667 (1996).
3o Present evidence also indicates the use of IL-8 inhibitors in the treatment
of
atherosclerosis. The first reference, Boisvert et al., J. Clin. Invest, 1998,
101:353-
363 shows, through bone marrow transplantation, that the absence of IL-8
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receptors on stem cells (and, therefore, on monocytes/macrophages) leads to a
reduction in the development of atherosclerotic plaques in LDL receptor
deficient
mice. Additional supporting references are: Apostolopoulos, et al.,
Arterioscler.
Thromb. Vasc. Biol. 1996, 16:1007-1012; Liu, et al., Arterioscler. Thromb.
Vasc.
Biol, 1997, 17:317-323; Rus, et al., Atherosclerosis. 1996, 127:263-271.; Wang
et
al., J. Biol. Chem. 1996, 271:8837-8842; Yue, et al., Eur. J. Pharmacol. 1993,
240:81-84; Koch, et al., Am. J. Pathol., 1993, 142:1423-1431.; Lee, et al.,
Immunol. Lett., 1996, 53, 109-113.; and Terkeltaub et al., Arterioscler.
Thromb.,
1994, 14:47-53.
to The present invention also provides for a means of treating, in an acute
setting, as
well as preventing, in those individuals deemed susceptible to, CNS injuries
by the
chemokine receptor antagonist compounds of Formula (I).
CNS injuries as defined, herein include both open or penetrating head
trauma, such as by surgery, or a closed head trauma injury, such as by an
injury to
the head region. Also included within this definition is ischemic stroke,
particularly to the brain area.
Ischemic stroke may be defined as a focal neurologic disorder that results
from insufficient blood supply to a particular brain area, usually as a
consequence
of an embolus, thrombi, or local atheromatous closure of the blood vessel. The
2o role of inflammatory cytokines in this area has been emerging and the
present
invention provides a mean for the potential treatment of these injuries.
Relatively
little treatment, for an acute injury such as these has been available.
TNF-oc is a cytokine with proinflammatory actions, including endothelial
leukocyte adhesion molecule expression. Leukocytes infiltrate into ischemic
brain
lesions and hence compounds which inhibit or decrease levels of TNF would be
useful for treatment of ischemic brain injury. See Liu et al., Stroke, Vol.
25., No.
7, pp. 1481-88 (1994) whose disclosure is incorporated herein by reference.
Models of closed head injuries and treatment with mixed 5-LOlCO agents
is discussed in Shohami et al., J. of Vaisc & Clinical Physi_ 'olog.
3o Pharmacolo~y, Vol. 3, No. 2, pp. 99-107 (1992) whose disclosure is
incorporated
herein by reference. Treatment, which reduced edema formation, was found to
improve functional outcome in those animals treated.
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The compounds of Formula (I) are administered in an amount sufficient to
inhibit IL-8, binding to the IL-8 alpha or beta receptors, from binding to
these
receptors, such as evidenced by a reduction in neutrophil chemotaxis and
activation. The discovery that the compounds of Formula (I) are inhibitors of
IL-8
binding is based upon the effects of the compounds of Formulas (I) in the i~c
vitro
receptor binding assays which are described herein. The compounds of Formula
(I) have been shown to be inhibitors of type IT II,-8 receptors.
As used herein, the term "IL-8 mediated disease or disease state" refers to
any and all disease states in which IL-8, GROoc, GROG, GRO~y, NAP-2 or ENA-78
l0 plays a role, either by production of TL-8, GROoc, GROG, GROy, NAP-2 or ENA-
78 themselves, or by IL-8, GROG, GROG, GRO~y, NAP-2 or ENA-78 causing
another monokine to be released, such as but not limited to IL,-1; IL-6 or
TNF. A
disease state in which, for instance, IL-1 is a major component, and whose
production or action, is exacerbated or secreted in response to IL-8, would
therefore be considered a disease state mediated by IL-8.
As used herein, the term "chemokine mediated disease or disease state"
refers to any and all disease states in which a chemokine which binds to an IL-
8 a,
or (3 receptor plays a role, such as but not limited to IL-8, GRO-oc, GRO-(3,
GRO~,
NAP-2 or ENA-78. This would include a disease state in which, IL-8 plays a
role,
either by production of IL-8 itself, or by IL-8 causing another monokine to be
released, such as but not limited to IL-1, IL-6 or TNF. A disease state in
which, for
instance, IL-1 is a major component, and whose production or action, is
exacerbated or secreted in response to IL-8, would therefore be considered a
disease stated mediated by IL,-8.
As used herein, the term "cytokine" refers to any secreted polypeptide that
affects the functions of cells and is a molecule, which modulates interactions
between cells in the immune, inflammatory or hematopoietic response. A
cytokine
includes, but is not limited to, monokines and lymphokines, regardless of
which
cells produce them. For instance, a monokine is generally referred to as being
produced and secreted by a mononuclear cell, such as a macrophage and/or
monocyte. Many other cells however also produce monokines, such as natural
killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain
astrocytes,
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bone marrow stromal cells, epideral keratinocytes and B-lymphocytes.
Lymphokines are generally referred to as being produced by lymphocyte cells.
Examples of cytokines include, but are not limited to, Interleukin-1 (IL-1),
Interleukin-6 (IL-6), Interleukin-8 (IL,-8), Tumor Necrosis Factor-alpha (TNF-
a)
and Tumor Necrosis Factor beta (TNF-13).
As used herein, the term "chemokine" refers to any secreted polypeptide
that affects the functions of cells and is a molecule which modulates
interactions
between cells in the immune, inflammatory or hernatopoietic response, similar
to
the term "cytokine" above. A chemokine is primarily secreted through cell
transmembranes and causes chemotaxis and activation of specific white blood
cells
and leukocytes, neutrophils, monocytes, macrophages, T-cells, B-cells,
endothelial
cells and smooth muscle cells. Examples of chemokines include, but are not
limited to IL-8, GRO-a, GRO-(3, GRO-y, NAP-2, ENA-78, IP-10, MIP-la, MIP-
(3, PF4, and MCP 1, 2, and 3.
In order to use a compound of Formula (I) or a pharmaceutically acceptable
salt thereof in therapy, it will normally be formulated into a pharmaceutical
composition in accordance with standard pharmaceutical practice. This
invention,
therefore, also relates to a pharmaceutical composition comprising an
effective,
non-toxic amount of a compound of Formula (I) and a pharmaceutically
acceptable
carrier or diluent.
Compounds of Formula (I), pharmaceutically acceptable salts thereof and
pharmaceutical compositions incorporating such may conveniently be
administered
by any of the routes conventionally used for drug administration, for
instance,
orally, topically, parenterally or by inhalation. The compounds of Formula (I)
may
be administered in conventional dosage forms prepared by combining a compound
of Formula (I) with standard pharmaceutical carriers according to conventional
procedures. The compounds of Formula (I) may also be administered in
conventional dosages in combination with a known, second therapeutically
active
compound. These procedures may involve mixing, granulating and compressing or
dissolving the ingredients as appropriate to the desired preparation. It will
be
appreciated that the form and character of the pharmaceutically acceptable
character or diluent is dictated by the amount of active ingredient with which
it is
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to be combined, the route of administration and other well-known variables.
The
carriers) must be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the recipient thereof.
The pharmaceutical carrier employed may be, for example, either a solid or
liquid. Exemplary of solid carriers are lactose, terra albs, sucrose, talc,
gelatin,
agar, pectin, acacia, magnesium stearate, stearic acid and the Iike. Exemplary
of
liquid carriers are syrup, peanut oil, olive oil, water and the Iike.
Similarly, the
carrier or diluent may include time delay material well known to the art, such
as
glyceryl mono-stearate or glyceryl distearate alone or With a wax.
A wide variety of pharmaceutical forms .can be employed. Thus, if a solid
carrier is used, the preparation can be tableted, placed in a hard gelatin
capsule in
powder or pellet form or in the form of a troche or lozenge. The amount of
solid
carrier will vary widely but preferably will be from about 25mg to about 1 g.
When
a liquid carrier is used, the preparation will be in the form of a syrup,
emulsion,
soft gelatin capsule, sterile injectable liquid such as an ampule or
nonaqueous
liquid suspension.
Compounds of Formula (I) may be administered topically, that is by non-
systemic administration. This includes the application of a compound of
Formula,
(I) externally to the epidermis or the buccal cavity and the instillation of
such a
2o compound into the ear, eye and nose, such that the compound does not
significantly enter the blood stream. In contrast, systemic administration
refers to
oral, intravenous, intraperitoneal and intramuscular administration.
Formulations suitable for topical administration include liquid or semi-
liquid preparations suitable for penetration through the skin to the site of
inflammation such as liniments, lotions, creams, ointments or pastes, and
drops
suitable for administration to the eye, ear or nose. The active ingredient may
comprise, for topical administration, from 0.001 % to IO% w/w, for instance
from
1 % to 2% by weight of the Formulation. It may however comprise as much as
10% w/w but preferably will comprise less than 5% w/w, more preferably from
0.1 % to 1 % w/w of the Formulation.
Lotions according to the present invention include those suitable for
application to the skin or eye. An eye lotion may comprise a sterile aqueous
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solution optionally containing a bactericide and may be prepared by methods
similar to those for the preparation of drops. Lotions or liniments for
application to
the skin may also include an agent to hasten drying and to cool the skin, such
as an
alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as
castor oiI
or arachis oil.
Creams, ointments or pastes according to the present invention are semi-
solid formulations of the active ingredient for external application. They may
be
made by mixing the active ingredient in finely-divided or powdered form, alone
or
in solution or suspension in an aqueous or non-aqueous fluid, with the aid of
suitable machinery, with a greasy or non-greasy base. The base may comprise
hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a
metallic
soap; a mucilage; an oil of natural origin such as almond, corn, arachis,
castor or
olive oil; wool fat or its derivatives or a fatty acid such as steric or oleic
acid
together with an alcohol such as propylene glycol or a macrogel. The
formulation
may incorporate any suitable surface active agent such as an anionic, cationic
or
non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative
thereof. Suspending agents such as natural gums, cellulose derivatives or
inorganic
materials such as silicaceous silicas, and other ingredients such as lanolin,
may also
be included.
Drops according to~the present invention may comprise sterile aqueous or
oily solutions or suspensions and may be prepared by dissolving the active
ingredient iri a suitable aqueous solution of a bactericidal and/or fungicidal
agent
and/or any other suitable preservative, and preferably including a surface
active
agent. The resulting solution may then be clarified by filtration, transferred
to a
suitable container which is then sealed and sterilized by autoclaving or
maintaining
at 98-100°C for half an hour. Alternatively, the solution may be
sterilized by
filtration and transferred to the container by an aseptic technique. Examples
of
bactericidal and fungicidal agents suitable for inclusion in the drops are
phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and
chlorhexidine acetate (0.01 %). Suitable solvents for the preparation of an
oily
solution include glycerol, diluted alcohol and propylene glycol.
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Compounds of formula (I) may be administered parenterally, that is by
intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal
or
intraperitoneal administration. The subcutaneous and intramuscular forms of
parenteral administration are generally preferred. Appropriate dosage forms
for
such administration may be prepared by conventional techniques. Compounds of
Formula (I) may also be administered by inhalation that is by intranasal and
oral
inhalation administration. Appropriate dosage forms for such administration,
such
as an aerosol formulation or a metexed dose inhaler, may be prepared by
conventional techniques.
1o For all methods of use~disclosed herein for the compounds of Formula (I)
the daily oral dosage regimen will preferably be from about 0.01 to about 80
mg/kg
of total body weight. The daily parenteral dosage regimen about 0.001 to about
80
mg/kg of total body weight. The daily topical dosage regimen will preferably
be
from 0.1 mg to 150 mg, administered one to four, preferably two or three times
daily. The daily inhalation dosage regimen will preferably be from about 0.01
mg/kg to about 1 mg/kg per day. It will also be recognized by one of skill in
the
art that the optimal quantity and spacing of individual dosages of a compound
of
Formula (I) or a pharmaceutically acceptable salt thereof will be determined
by the
nature and extent of the condition being treated, the form, route and site of
administration, and the particular patient being treated, and that such
optimums can
be determined by conventional techniques. Tt will also be appreciated by one
of
skill in the art that the optimal course of treatment, i.e., the number of
doses of a
compound of Formula (I) or a pharmaceutically acceptable salt thereof given
per
day for a defined number of days, can be ascertained by those skilled in the
art
using conventional course of treatment determination tests.
The invention will now be described by reference to the following
biological examples which are merely illustrative and are not to be construed
as a
limitation of the scope of the present invention.
BIOLOGICAL EXAMPLES
3o The IL-8, and GRO-oc chemokine inhibitory effects of compounds of the
present invention are determined by the following iya vitro assay:
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Receptor Binding Assays:
[125I] IL-8 (human recombinant) is obtained from Amersham Corp.,
Arlington Heights, IL, with specific activity 2000 Ci/mmol. GRO-oc is obtained
from NEN- New England Nuclear. All other chemicals are of analytical grade.
High levels of recombinant human IL-8 type a and (3 receptors were
individually
expressed in Chinese hamster ovary cells as described previously (Holmes, et
al.,
Science, 1991, 253, 1278). The Chinese hamster ovary membranes were
homogenized according to a previously described protocol (Haour, et al., J.
Biol.
Chem., 249 pp 2195-2205 (1974)). Except that the homogenization buffer is
changed to lOmM Tris-HCL,'1mM MgS04, 0.5xnM EDTA (ethylene-diaminetetra-
acetic acid), lxnM PMSF (oc-toluenesulphonyl fluoride), 0.5 mg/L Leupeptin, pH
7.5. Membrane protein concentration is determined using Pierce Co. micro-assay
kit using bovine serum albumin as a standard. All assays are performed in a 96-
well micro plate format. Each reaction mixture contains 1251 ~,_g (0.25 nM) or
1251 GRO-oc and 0.5 ~glmL of IL-8Roc or 1.0 ~g/mL of IL-8R(3 membranes in 20
mM Bis-Trispropane and 0.4 mM Tris HCl buffers, pH 8.0, containing 1.2 mM
MgS04, 0.1 mM EDTA, 25 mM Na and 0.03% CHAPS. In addition, drug or
compound of interest is added which has been pre-dissolved in DMSO so as to
reach a final concentration of between O.OlnM and I00 uM. The assay is
initiated
by addition of 125I_IL-8. After 1 hour at room temperature the plate is
harvested
using a Tomtec 96-well harvester onto a glass fiber filtermat blocked with 1 %
polyethyleniniine/ 0.5% BSA and washed 3 times with 25 mM NaCI, 10 mM
TrisHCl, 1 mM MgS04, 0.5 mM EDTA, 0.03 % CHAPS, pH 7.4. The filter is
then dried and counted on the Betaplate liquid scintillation counter. The
recombinant IL-8 Roc, or Type I, receptor is also referred to herein as the
non-
permissive receptor and the recombinant IL-8 R(3, or Type II, receptor is
referred to
as the permissive receptor.
Representative compounds of Formula (I), Examples 1 to 106 have
exhibited positive inhibitory activity in this assay at IC50 levels < 30 uM.
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Chemotaxis Assay:
The in vitro inhibitory properties of these compounds are determined in the
neutrophil chemotaxis assay as described in Current Protocols in Immunology,
vol.
I, Suppl 1, Unit 6.12.3., whose disclosure is incorporated herein by reference
in its
entirety. Neutrophils where isolated from human blood as described in Current
Protocols in Immunology Vol. I, Suppl 1 Unit 7.23.1, whose disclosure is
incorporated herein by reference in its entirety. The chemoattractants IL-8,
GRO-
oc, GRO-(3, GRO-y and NAP-2 are placed in the bottom chamber of a 48 multiwell
chamber (Neuro Probe, Cabin John, MD) at a concentration between 0.1 and 100
nM. The two chambers :are separated by a 5 uM polycarbonate filter. When
compounds of this invention are tested, they are mixed with the cells (0.001 -
1000
nM) just prior to the addition of the cells to the upper chamber. Incubation
is
allowed to proceed for between about 45 and 90 min at about 37°C in a
humidified
incubator with 5% CO2. At the end of the incubation period, the polycarbonate
membrane is removed and the top side washed, the membrane then stained using
the Diff Quick staining protocol (Baxter Products, McGaw Park, IL, USA). Cells
which have chemotaxed to the chemokine are visually counted using a
microscope.
Generally, four fields are counted for each sample, these numbers are averaged
to
give the average number of cells which had migrated. Each sample is tested in
triplicate and each compound repeated at least four times. To certain cells
(positive control cells) no compound is added, these cells represent the
maximum
chemotactic response of the cells. In the case where a negative control
(unstimulated) is desired, no chemokine is added to the bottom chamber. The
difference between the positive control and the negative control represents
the
chemotactic activity of the cells.
Elastase Release Assay:
The compounds of this invention are tested for their ability to prevent
Elastase release from human neutrophils. Neutrophils are isolated from human
blood as described in Current Protocols in Immunology Vol. I, Suppl 1 Unit
7.23.1.
PMNs 0.88 x 106 cells suspended in Ringer's Solution (NaCl 118, KCl 4.56,
NaHC03 25, KH2P04 1.03, Glucose 11.1, HEPES 5 mM, pH 7.4) are placed in
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each well of a 96 well plate in a volume of 50 uI. To this plate is added the
test
compound (0.001 - 1000 nM) in a volume of 50 u1, Cytochalasin B in a volume of
50 u1 (20ug/ml) and Ringers buffer in a volume of 50 u1. These cells are
allowed to
warm (37 oC, 5% C02, 95% RH) for 5 min before IL-8, GROG, GROG, GROyor
NAP-2 at a final concentration of 0.01 - 1000 nM was added. The reaction is
allowed to proceed for 45 min before the 96 well plate is centrifuged (800 xg
5
min.) and 100 u1 of the supernatant removed. This supernatant is added to a
second
96 well plate followed by an artificial elastase substrate (MeOSuc-Ala-Ala-Pro-
Val-
AMC, Nova Biochem, La Jolla, CA) to a final concentration of 6 ug/mI dissolved
in
to phosphate buffered saline. Immediately, the plate is placed in a
fluorescent 96 well
plate reader (Cytofluor 2350; Millipore, Bedford, MA) and data collected at 3
min
intervals according to the method of Nakajima et al J. Biol. Chem. 254 4027
(1979).
The amount of Elastase released from the PMNs is calculated by measuring the
rate
of MeOSuc-Ala-Ala-Pro-Val-AMC degradation.
TNF-oc in Traumatic Brain Injury Assay
The present assay provides for examination of the expression of tumor
necrosis factor mRNA in specific brain regions, which follow experimentally,
induced lateral fluid-percussion traumatic brain injury (TBI) in rats. Adult
Sprague-Dawley rats (n=42) were anesthetized with sodium pentobarbital (60
rng/kg, i.p.) and subjected to lateral fluid-percussion brain injury of
moderate
severity (2.4 atm.) centered over the left ternporaparietal cortex (n=18), or
"sham"
treatment (anesthesia and surgery without injury, n=18). Animals are
sacrificed by
decapitation at 1, 6 and 24 hr. post injury, brains removed, and tissue
samples of
left (injured) parietal cortex (LC), corresponding area in the contralateral
right
cortex (RC), cortex adjacent to injured parietal cortex (LA), corresponding
adjacent
area in the right cortex (RA), Ieft hippocampus (LH) and right hippocampus
(RH)
are prepared. Total RNA are isolated and Northern blot hybridization is
performed
and quantitated relative to an TNF-oc positive control RNA (macrophage =
100%).
A marked increase of TNF- oc mRNA expression is observed in LH (104~17% of
3o positive control, p < 0.05 compared with sham), LC (105~21%, p< 0.05) and
LA
(69~8%, p < 0.01) in the traumatized hemisphere 1 hr. following injury. An
increased TNF- oc mRNA expression is also observed in LH (46~8%, p < 0.05), LC
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(30~3%, p < 0.01) and LA (32~3%, p < 0.01) at 6 hr which resolves by 24 hr
following injury. In the contralateral hemisphere, expression of TNF- oc mRNA
is
increased in RH (46~2%, p < 0.01), RC (4~3%) and RA (22~8%) at 1 hr and in RH
(28~11%), RC (7~5%) and RA (26~6%, p < 0.05) at 6 hr but not at 24 hr
following
injury. In sham (surgery without injury) or naive animals, no consistent
changes in
expression of TNF- cc mRNA are observed in any of the 6 brain areas in either
hemisphere at any times. These results indicate that following parasagittal
fluid-
percussion brain injury, the temporal expression of TNF-oc mRNA is altered in
specific brain regions, including those of the non-traumatized hemisphere.
Since
TNF-oc is able to induce nerve growth factor (NGF) and stimulate the release
of
other cytokines from activated astrocytes, this post-traumatic alteration in
gene
°expression of TNF-a plays an important role in both the acute and
regenerative
. response to CNS trauma.
CNS Injury model for IL-1(3 mRNA
This assay characterizes the regional expression of interleukin-lf3 (IL-lf3)
mRNA in specific brain regions following experimental lateral fluid-percussion
traumatic brain injury (TBT) in rats. Adult Sprague-Dawley rats (n=42) are
anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and subjected to
lateral
fluid-percussion brain injury of moderate severity (2.4 atm.) centered over
the left
temporaparietal cortex (n=18), or "sham" treatment (anesthesia and surgery
without
injury). Animals are sacrificed at 1, 6 and 24 hr. post injury, brains
removed, and
tissue samples of left (injured) parietal cortex (LC), corresponding area in
the
contralateral right cortex (RC), cortex adjacent to injured parietal cortex
(LA),
corresponding adjacent area in the right cortex (RA), left hippocampus (LH)
and
right hippocampus (RH) are prepared. Total RNA is isolated and Northern blot
hybridization was performed and the quantity of brain tissue IL-113 mRNA is
presented as percent relative radioactivity of IL-1!3 positive macrophage RNA
which was loaded on the same gel. At 1 hr following brain injury, a marked and
significant increase in expression of IL-113 mRNA is observed in LC (20.0~0.7%
of
3o positive control, n=6, p < 0.05 compared with sham animal), LH (24.5~0.9%,
p < 0.05) and LA (21.5~3.1 %, p < 0.05) in the injured hemisphere, which
remained
elevated up to 6 hr. post injury in the LC (4.0~0.4%, n=6, p < 0.05) and LH
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(5.0~1.3%, p < 0.05). In sham or naive animals, no expression of IL-113 mRNA
is
observed in any of the respective brain areas. These results indicate that
following
TBI, the temporal expression of IL-113 mRNA is regionally stimulated in
specific
brain regions. These regional changes in cytokines, such as IL-lf3 play a role
in the
post-traumatic.
All publications, including but not limited to patents and patent
applications, cited in this specification are herein incorporated by reference
as if
each individual publication were specifically and individually indicated to be
incorporated by reference herein as though fully set forth.
The above description fully discloses the invention including preferred .
embodiments thereof. Modifications and improvements of the embodiments
specifically disclosed herein are within the scope of the following claims.
Without
further elaboration, it is believed that one skilled in the art can, using the
preceding
description, utilize the present invention to its fullest extent. Therefore
the
Examples herein are to be construed as merely illustrative and not a
limitation of
the scope of the present invention in any way. The embodiments of the
invention
in which an exclusive property or privilege is claimed are defined as follows.
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