Note: Descriptions are shown in the official language in which they were submitted.
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G-PROTEIN COUPLED HEPTAHELICAL RECEPTOR BINDING
COMPOUNDS AND METHODS OF USE THEREOF
Background of the Invention
The chemokine family of peptides is defined on the basis of sequence homology
and on the presence of variations on a conserved cysteine motif (Schall (1996)
Cytokine
3:165-183; and Oppenheim et al. ( 1991 ) Arrnu. Rev. Immunol. 9:617-648). The
family
can be subdivided on the basis of this motif into two major subfamilies, in
which
members of each contain four characteristic cysteine residues. This
subdivision
therefore defines the CC or ~3-chemokine family in which the first two
cysteines are
juxtaposed, and the CXC or a-chemokine family in which there is an intervening
amino
acid between the first two cysteines. Two other subfamilies have subsequently
been
described which have variations in the number of amino acids between the first
two
cysteine residues (Kelner et al. (1994) Science 266:1395-1399; Dorner et al.
(1997) J.
Biol. Chem. 272:8817-8823; and Bazan et al. (1996) Nature 385:640-644.)
Chemokines display a range of in vitro and in vivo functions ranging from
proinflammatory activities on a range of cell types to proliferative
regulatory activities.
All of the functions of the chemokine family are believed to be signaled into
a
responsive cell using members of the G protein-coupled heptahelical receptor
family.
To date several a-chemokine and (3-chemokine receptors have been described.
(e.g.,
Neote et al. (1993) Cell 72:415-425; Ponath et al. (1996) J. Exp. Med.
183:2437-2448;
and Power et al. (1995) J. Biol. Chem. 270:19495-19500.)
Summary of the Invention
The present invention is based, at least in part, on the discovery of
compounds
which interact with G-protein coupled heptahelical receptors. The compounds of
the
invention can be used to treat chemokine mediated disorders, e.g.
neurological,
immunological, inflammatory and cancer related disorders.
The present invention provides a G-protein coupled heptahelical receptor
(GPCR) binding compound of the formula:
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J-M (I)
where J is an aromatic moiety and M is a G-protein coupled heptahelical
receptor pocket
interacting moiety. Preferably, the compound interacts with a (3-chemokine
receptor,
e.g. CCR2, CCR3, CCR4, CCRS, CCR6, CCR7, CCRB, or CCRIO. In a preferred
aspect, the compound modulates recruitment of at least one cell type, e.g.
leukocytes,
e.g. macrophages or eosinophils, associated with inflammation in a subject.
In another aspect, the invention pertains to a method for treating a chemokine
mediated disorder in a subject. The method involves administering an effective
amount
of a G-protein coupled heptahelical receptor binding compound such that the
disorder is
treated, e.g. at least one symptom of the disorder is diminished or
alleviated. The
chemokine mediated disorder may be a neurological disorder (e.g. multiple
sclerosis,
Alzheimer's disease, or Parkinson's disease), an immunological disorder (e.g.
AIDS,
arthritis, or lupus), cancer, or an inflammatory disorder (e.g. asthma).
In another aspect, the invention pertains to a compound represented by the
formula:
A-L I -B-L2-E (II)
wherein
A is selected from the group consisting of branched and straight chain alkyl,
aryl,
alkenyl, alkynyl, and heteroaryl moieties optionally substituted by NR'R", CN,
N02, F,
Cl, Br, I, CF3, CC13, CHF2. CHC12, CONR'R", S(O)NR'R", CHO, OCF3, OCC13,
SCF3, SCC13, COR', C02R', and OR' and wherein R' and R" are each independently
hydrogen, C I -C6 alkyl, C2-C6 alkenyl, or optionally substituted aryl;
L 1 is a linker moiety selected from the group consisting of a bond, O, S,
CHOH,
CHSH, CHNH2, CHNHR, CHNRR', NH, NR, (CH2)n, O(CH2)n, and (CH2)n0(CH2)n,
an optionally substituted ring moiety of 4 to 7 atoms containing up to three
heteroatoms,
a chain of 1 to 5 atoms optionally substituted by C 1-C6 alkyl, halogens,
wherein n is
either 0, I , 2, or 3, and R and R' are each independently substituted or
unsubstituted C 1-
C6 branched or straight chain alkyl, C1-C6 branched or straight chain alkenyl,
aryl, C4-
C~ ring, optionally substituted with up to three heteroatoms;
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B is an aromatic moiety containing from 0 to 3 heteroatoms and containing ~ to
7
members optionally substituted by NR'R", cyano, nitro, halogen. CFA, CHF2,
CONR'R",
S(O)NR'R", CHO, OCF3, SCF~, COR', C02R', OR' where R' and R" are each
independently hydrogen, halogen, C1-C6 alkyl, optionally substituted aryl or
optionally
substituted aryl;
L2 is a second linking moiety selected from the group consisting of a
bond, CH2C=O, NHC=O, OC=O, C=O, CH2NHC=O, CHOH, (CH2)n, O, NH,
O(CH2)n, NH(CH2)n, CH2CHOH and NRC=O; and
E is a G-protein coupled heptahelical receptor pocket interacting moiety.
In another aspect, the invention also relates to a compound represented by the
formula:
R~
R Z' Z2~Li ~Z3 Z4~L,~Rs
~~N / _N
Rz R3 Ra Rs R6
(III)
wherein
Z1, Z2, Z3, and Z4 are each independently N or C;
R1, R2, R3, R4, R5, R6, R~, and Rg are each independently hydrogen,
C1-C6 branched or straight chain alkyl, alkoxy, thioalkyl, hydroxyalkyl, halo,
haloalkyl,
amino, alkylamino, or carboxyl;
L1 is O, S, NH, NR~, (CH2)n, CO, CHOH, O(CH2)n, and
(CH2)n0(CH2)n wherein n is either 1,2, or 3 and R~ is C1-C6 branched or
straight
chain alkyl, alkoxy, thioalkyl, hydroxyalkyl, halo, haloalkyl, amino,
alkylamino, or
carboxyl; and
L2 is a second linking moiety selected from the group consisting of a
bond, CH2C=O, NHC=O, OC=O, C=O, CH2NHC=O, CHOH, (CH2)n, O, NH,
O(CH2)n, NH(CH2)n, CH2CHOH and NRC=O.
In yet another aspect, the invention relates to a pharmaceutical preparation
comprised of the G-protein coupled heptahelical receptor binding compound and
a
pharmaceutically acceptable carrier. The invention also pertains to a packaged
G-
protein coupled heptahelical receptor binding compound containing instructions
for
using the compound for treating a chemokine mediated disorder.
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Brief Description of the Drawings
Figure 1 depicts a binding curve of Compound A in a DB Assay (see Example 3
below).
Figure 2 depicts a binding curve of Compound CU in a DB Assay (see Example
3 below).
Figure 3 depicts a binding curve of Compound CV in a DB Assay (see Example
3 below).
Figure 4 is a graph depicting the blockage of THP.1 cell migration by compound
B in a CBIR Assay (see Example 4 below).
Figure 5 is a graph depicting the blockage of THP.l cell migration by compound
C in a CBIR Assay (see Example 4 below).
Figure 6 shows MCP-5 induced peritoneal eosinophil recruitment in mice after
administering compounds B and C in a MIR Assay (see Example 5 below).
Detailed Description of the Invention
The present invention pertains to a G-protein coupled heptahelical receptor
binding compound of the formula:
J-M (I)
wherein J is an aromatic moiety and M is a G-protein coupled heptahelical
receptor pocket interacting moiety.
The language "G protein coupled heptahelical receptor" includes receptors for
which belong to the G-protein coupled receptor (GPCR) superfamily of seven-
transmembrane domain heptahelical receptors. G-protein coupled receptors
(GPCRs),
along with G-proteins and effectors (intracellular enzymes and channels which
are
modulated by G-proteins), are the components of a modular signaling system
that
connects the state of intracellular second messengers to extracellular inputs.
These
genes and gene-products are potential causative agents of disease (Spiegel et
al. ( 1993)
J. Clin. Invest. 92:1119-1125; McKusick and Amberger (1993) J. Med. Genet.
30:1-26).
Specific defects in the rhodopsin gene and the V2 vasopressin receptor gene
have been
shown to cause various forms of autosomal dominant and autosomal recessive
retinitis
pigmentosa (see Nathans et al. (1992) Annu. Rev. Genet. 26:403-424),
nephroaenic
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diabetes insipidus (Holtzman et al. (1993) Hum. Mol. Genet. 2:1201-1204 and
references
therein). These receptors are of critical importance to both the central
nervous system
and peripheral physiological processes. Evolutionary analyses suggest that the
ancestor
of these proteins originally developed in concert with complex body plans and
nervous
systems.
The GPCR protein superfamily now contains over 250 types of paralogues,
receptors that represent variants generated by gene duplications (or other
processes), as
opposed to orthologues, the same receptor from different species. The
superfamily can
be broken down into five families: Family I, receptors typified by rhodopsin
and the
beta2-adrenergic receptor and currently represented by over 200 unique members
(reviewed by Dohlman et al. (1991) Annu. Rev. Biochem. 60:653-688 and
references
therein); Family II, the recently characterized parathyroid
hormone/calcitonin/secretin
receptor family (Juppner et al. ( 1991 ) Science 254:1024-1026; Lin et al. (
1991 ) Science
254:1022-1024); Family III, the metabotropic glutamate receptor family in
mammals
(Nakanishi (1992) .Science 258:597-603); Family IV, the cAMP receptor family,
important in the chemotaxis and development of D. discoideum (Klein et al.
(1988)
Science 241:1467-1472); and Family V, the fungal mating pheromone receptors
such as
STE2 (reviewed by Kurjan (1992) Annu. Rev. Biochem. 61:1097-1129).
Examples of GPCRs include chemokine receptors which are expressed in
specific tissues and leukocyte subtypes. Many chemokine receptors can bind to
and be
activated by more than one chemokine, and many chemokines can bind and
activate
more than one receptor in the nanomolar or subnanomolar range (MacKay ( 1996)
J.
Exp. Med. 184:522-549; Wells et al. ( 1996) Chem. Biol. 3:603-609). Chemokine
receptors can be classified generally into three groups: a-chemokine
receptors, (3-
chemokine receptors and a-(3 chemokine receptors. Preferably, the GPCR binding
compounds of the present invention interact with receptors of the (3-chemokine
receptor
family (Bonini et al. DNA Cell Biol. (1997) 16(10):1249-1256). Examples of ~3-
chemokine receptors include CCR2. CCR3, CCR4, CCRS, CCR6, CCR7, CCRB, and
CCR10.
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The (3-chemokine receptors (CCRs) are characterized by their ability to bind
to
CC chemokines (also referred to as (3-chemokines). The CC chemokines are
characterized by a conserved cysteine motif, in which the first t«-o cysteines
are
juxtaposed. The CCR family includes both specific and non-specific receptors.
For
example, CCR1 is known to bind macrophage inflammation protein-la (MIP-la),
RANTES (regulation on activation normal T cell expressed and secreted), and
monocyte
chemoattractant protein-3 (MCP-3) (Neote et al., (1993) Cell 72:415-425). CCR2
binds
MCP-1, MCP-3, and MCP-4 (Myers et al. (1995) J. Biol. Chem. 270:5786-5792;
Garcia-
Zepeda et al. (1996) J. Immunol. 157:5613), whereas CCR3 recognizes eotaxin
and
MCP-4 (Kitaura et al. (1996) J. Biol. Chem. 271:7725; Garcia-Zepada et al.
(1996) J.
Immunol. 157:5613). CCR4 is activated by macrophage inflammatory protein-la
(MIP-
la), RANTES, and MCP-1 (Power et al. (1995) J. Biol. Chem. 270:19495). CCRS
was
found to bind to and be activated by RANTES , MIP-la, and MIP-lb (Raport et
al.
(1996) J. Biol. Chem. 271: 17161). CCR6 and CCR7 have recently been discovered
and
specifically bind to liver and activation regulated chemokine (LARC) and EBII-
ligand
chemokine (ELC) respectively (Baba et al, 1997; Yoshida et al. 1997). It is
known that
CCR10 binds MCP-1 and MCP-3 with high affinity.
The language "aromatic moiety" includes groups with aromaticity, e.g. moieties
that have at least one aromatic ring. For example, 5- and 6-membered single-
ring
aromatic groups that may include from zero to four heteroatoms, for example,
benzene,
pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole,
tetrazole,
pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl
groups also
include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl,
and the
like. Those aryl groups having heteroatoms in the ring structure may also be
referred to
as "aryl heterocycles", "heteroaryls" or "heteroaromatics". The aromatic ring
can be
substituted at one or more ring positions with such substituents as described
above, as
for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano,
amino
(including alkyl amino, dialkylamino, arylamino, diarylamino, and
alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and
ureido),
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amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkylaryl, or
an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged
with
alicyclic or heterocyclic rings which are not aromatic so as to form a
polycycle (e.g.,
tetralin). In a preferred embodiment, the aromatic moiety of the present
invention
comprises two aromatic rings, e.g. pyridyl or pyrimidyl rings, e.g. two
pyrimidyl rings
connected with an ether linkage.
The language "G-protein coupled heptahelical receptor pocket" refers to a
region
of the GPCR which is capable of interacting with, e.g. binding to. the GPCR
pocket
binding moiety. Not wishing to be bound by theory, it is believed that the
GPCR pocket
may be a cavity in the GPCR lined with hydrophobic amino acid residues.
The language "G-protein coupled heptahelical receptor pocket interacting
moiety" refers to a group which interacts with a GPCR pocket. The group may be
substituted or unsubstituted aromatic, alkyl, alkenyl, cycloalkyl, etc. The
interaction
between the GPCR pocket interacting moiety and the pocket includes any
interaction
which allows the compound to perform its intended function, e.g., the
interaction is
hydrophobic, ionic, covalent, or combinations thereof.
In one aspect of the invention, the GPCR binding compound modulates the
recruitment of at least one inflammatory cell type in a subject. The ability
of a GPCR
binding compound to modulate the recruitment of at least one inflammatory cell
type
can be measured or observed using art-recognized techniques or assays.
Examples of
such assays are the Murine Inflammatory Cell Recruitment Assay (referred to
herein as
the MICR Assay) and the Cell Based Inflammatory Recruitment Assay (referred to
herein as the CIBR Assay) as described in Examples 4 and 5, respectively.
The term "subject" includes any animal which expresses GPCRs, for example,
mammals e.g., mice, rats, cows, sheep, pigs, horses, monkeys, dogs. cats and,
preferably,
humans.
The language "inflammatory cell type" includes cell types associated with a
chemokine mediated disorder characterized by inflammation. Examples of these
cell
types include, but are not limited to, leukocytes, e.g., eosinophils,
neutrophils, basophils,
fibroblasts, monocytes, T lymphocytes, and macrophages. Leukocytes are white
blood
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cells which are involved in nonspecific resistance against pathogenic
microorganisms
and inflammatory response. Monocytes are particularly important in the
nonspecific
immune response, while lymphocytes are especially important in the specific
immune
response. Neutrophils are the most abundant phagocytic cells in blood and are
continuously produced in circulating blood, affording protection against the
entry of
foreign materials. These leukocytes exhibit chemotaxis and are attracted to
foreign
substances, including invading microorganisms, which they engulf and digest
along with
particulate matter. Eosinophils are leukocytes which react with the acidic dye
eosin.
Basophils are leukocytes which stain with basic dyes. Macrophages are large
ameboid
mononuclear phagocytic cells.
The present invention also pertains to a GPCR binding compound which is an
antagonist of a G-protein coupled heptahelical receptor, e.g., a (3-chemokine
receptor. In
one embodiment, the IC;o of the GPCR binding compound to the GPCR is about 10
~M
or less, e.g., about 5 ~M or less, e.g., about 1 ~M or less, e.g., about 50 nM
or less.
The term "antagonist" includes compounds which bind to the GPCR such that
the binding of a second compound to the GPCR is modulated.
The ability of a compound to bind to a GPCR can be determined through using
art-recognized techniques and assays. Examples of such assays include the Time
Resolved Fluorescence assay (herein referred to as the TRF assay) and the
Direct
Binding Assay (herein referred to as the DB Assay), described in Examples 2
and 3,
respectively. The TRF assay determines the binding affinity of a compound to a
receptor by over expressing the receptor in a culture of cells. The TRF assay
determines
the binding affinity of a compound to a receptor using a cell line engineered
to
overexpress a GPCR, e.g. CCR10. The cells are exposed simultaneously to the
test
compound and a fluorescently labeled ligand specific for the receptor. After a
predetermined amount of time, excess ligand and test compound is removed. The
amount of fluorescence is measured and the percent inhibition of binding of
the ligand is
calculated. By repeating this experiment at multiple concentrations of test
compound, it
is possible to generate a dose-response curve from which an IC50 can be
determined.
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In another aspect, the invention pertains to a method for treating a chemokine
mediated disorder, (e.g., a neurological disorder, an immunological disorder.
a disorder
characterized by inflammation, or a disorder characterized by unwanted
cellular
proliferation) in a subject. The method includes administering an effective
amount of a
G-protein coupled heptahelical receptor binding compound to a subject. For
example,
the disorder may be treated through modulation of a (3-chemokine receptor,
e.g., CCR2,
CCR3, CCR4, CCRS, CCR6, CCR7, CCRB, or CCR10. Examples of preferred
disorders include AIDS, multiple sclerosis, asthma. cancer, and lupus. The
disorder may
also be characterized by abnormal cellular signal transduction, or amounts of
chemokine
stimulated chemotaxis.
The term "administering" includes routes of administration which allow the
GPCR binding compound to perform its intended function, e.g. interacting with
GPCRs
and/or treating a chemokine mediated disorder. Examples of routes of
administration
which can be used include parental injection (e.g., subcutaneous, intravenous,
and
intramuscular), intraperitoneal injection, oral, inhalation, and transdermal.
The injection
can be bolus injections or can be continuous infusion. Depending on the route
of
administration, the GPCR binding compound can be coated with or disposed in a
selected material to protect it from natural conditions which may
detrimentally effect its
ability to perform its intended function. The GPCR binding compound can be
administered alone or with a pharmaceutically acceptable carrier. Further, the
GPCR
binding compound can be administered as a mixture of GPCR binding compounds,
which also can be coadministered with a pharmaceutically acceptable carrier.
The
GPCR binding compound can be administered prior to the onset of a chemokine
mediated disorder, or after the onset of a chemokine mediated disorder. The
GPCR
binding compound also can be administered as a prodrug which is converted to
another
form in vivo.
The term "treatment" includes the diminishment or alleviation of at least one
symptom associated or caused by the disorder being treated. For example,
treatment can
be diminishment of several symptoms of a disorder or complete eradication of a
disorder.
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The language "chemokine mediated disorder" includes a disorder characterized
by the participation of chemokines or association with chemokines. The
language also
includes disorders characterized by aberrant chemokine expression. Chemokines
have a
wide variety of functions. They are able to elicit chemotactic migration of
distinct cell
types, such as monocytes, neutrophils, T lymphocytes, basophils and
fibroblasts. Many
chemokines have proinflammatory activity and are involved in multiple steps
during an
inflammatory reaction. These activities include stimulation of histamine
release,
lysosomal enzyme and leukotriene release, increased adherence of target immune
cells
to endolethial cells, enhanced binding of complement proteins, induced
expression of
granulocyte adhesion molecules and complement receptors, and respiratory
burst. In
addition to their involvement in inflammation, certain chemokines have been
shown to
exhibit other activities. For example, macrophage inflammatory protein -1 (MIP-
1) is
able to suppress hematopoietic stem cell proliferation, platelet factor-4 is a
potent
inhibitor of endolethial cell growth, interleukin-8 (IL-8) promotes
proliferation of
keratinocytes, and GRO is an autocrine growth factor for myeloma cells. .
Chemokines
have been proposed to participate in a number of physiological and disease
conditions,
including, for example, lymphocyte trafficking, wound healing, hemapoietic
regulation
and immunological disorders such as asthma and arthritis.
The language "chemokine mediated disorder characterized by inflammation"
includes a disorder having inflammation as at least one of its symptoms.
Examples of
such disorders include anaphylaxis, systemic necrotizing vasculitis, systemic
lupus
erthyematosus, serum sickness syndromes, psoriasis, rheumatoid arthritis,
adult
respiratory distress syndrome CARDS), allergic rhinitis, atopic dermatitis,
asthma and
other allergic responses, and reperfusion injury occurring after periods of
ischemia such
as in myocardial infarction or shock. Preferably, the disorder is asthma.
Other groups of possible chemokine mediated disorders include neurological
related disorders, immunological related disorders and disorders characterized
by
unwanted cellular proliferation, e.g. cancer.
The language "neurological related disorders" includes disorders of the
nervous
system, including, but not limited to those involving the brain, the central
and peripheral
nervous system, and the interfaces between muscles and the nerves. Some
examples of
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neurological related disorders include Alzheimer's disease, demential related
to
Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy
diffuse body
diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive
supranuclear
palsy, epilepsy, and Jakob-Creutzfieldt disease. "Neurological related
disorders" also
includes neurological disorders associated with inflammation, e.g. stroke,
traumatic
injury to the brain, traumatic injury to the spinal cord, spinal crush, and
central and
peripheral nervous system trauma.
The language "immunological related disorder" includes both organ-specific and
systemic immunological disorders. Some examples of immunological disorders
include
immune thyroiditis, hyperthyroidism, type I diabetes mellitus, insulin related
diabetes,
Addison's disease, autoimmune oophoritis, autoimmune orchids, AIDS, autoimmune
hemolytic anemia, paroxysmal cold hemoglobinuria, autoimmune thrombocytopenia,
autoimmune neutropenia, pernicious anemia, autoimmune coagulopathies,
myasthenia
gravis, multiple sclerosis, experimental allergic encephalomyelitis, pemphigus
and other
bullous diseases, rheumatic carditis, Goodpasture's syndrome, postcardiotomy
syndrome, systemic lupus erythematosus, rheumatoid arthritis, keratitis,
parotids,
polymositis, dermatomyositis, and scleroderma. Preferably, the immunological
disorder
is AIDS, multiple sclerosis, rheumatoid arthritis, or lupus.
In another embodiment, the invention pertains to a pharmaceutical preparation
comprised of an effective amount of a G-protein coupled heptahelical receptor
binding
compound and a pharmaceutically acceptable carrier. In a preferred aspect, the
effective
amount is an effective amount to treat a (3-chemokine mediated disorder, e.g.,
asthma.
The language "pharmaceutically acceptable carrier" includes substances capable
of being coadministered with the GPCR binding compound(s), and which allows
both to
perform their intended function, e.g., treating a chemokine mediated disorder
or
preventing a chemokine mediated disorder. Examples of such carriers include
solutions,
solvents, dispersion media, delay agents, emulsions and the like. The use of
such media
for pharmaceutically active substances are well known in the art. Any other
conventional carrier suitable for use with the GPCR binding compound also fall
within
the scope of the present invention.
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Furthermore, the language "pharmaceutically acceptable carrier" is art
recognized and includes a pharmaceutically acceptable material. composition or
vehicle,
suitable for administering compounds of the present invention to mammals. The
carriers
include liquid or solid filler, diluent, excipient, solvent or encapsulating
material,
involved in carrying or transporting the subject agent from one organ, or
portion of the
body, to another organ, or portion of the body. Each carrier must be
"acceptable" in the
sense of being compatible with the other ingredients of the formulation and
not injurious
to the patient. Some examples of materials which can serve as pharmaceutically
acceptable carriers include: sugars, such as lactose, glucose and sucrose;
starches. such
as corn starch and potato starch; cellulose, and its derivatives, such as
sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered
tragacanth:
malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes;
oils, such as
peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and
soybean oil;
glycols, such as propylene glycol; polyols, such as glycerin, sorbitol,
mannitol and
polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar;
buffering agents,
such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free
water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and
other non-toxic compatible substances employed in pharmaceutical formulations.
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and
magnesium stearate, as well as coloring agents, release agents, coating
agents,
sweetening, flavoring and perfuming agents, preservatives and antioxidants can
also be
present in the compositions.
Examples of pharmaceutically acceptable antioxidants include: water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate,
sodium
metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as
ascorbyl
palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
lecithin,
propyl gallate, a-tocopherol, and the like; and metal chelating agents, such
as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric
acid, and the
like.
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Formulations of the present invention include those suitable for oral, nasal,
topical, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral
administration.
The formulations may conveniently be presented in unit dosage form and may be
prepared by any methods well known in the art of pharmacy. The amount of
active
ingredient which can be combined with a carrier material to produce a single
dosage
form will generally be that amount of the compound which produces a
therapeutic
effect. Generally, out of one hundred per cent, this amount will range from
about 1 per
cent to about ninety-nine percent of active ingredient, preferably from about
5 per cent
to about 70 per cent, most preferably from about 10 per cent to about 30 per
cent.
Methods of preparing these formulations or compositions include the step of
bringing into association a compound of the present invention with the carrier
and,
optionally, one or more accessory ingredients. In general, the formulations
are prepared
by uniformly and intimately bringing into association a compound of the
present
invention with liquid carriers, or finely divided solid carriers, or both, and
then, if
necessary, shaping the product.
Formulations of the invention suitable for oral administration may be in the
form
of capsules, cachets, pills, tablets, lozenges (using a flavored basis,
usually sucrose and
acacia or tragacanth), powders, granules. or as a solution or a suspension in
an aqueous
or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion,
or as an
elixir or syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or
sucrose and acacia) and/or as mouth washes and the like, each containing a
predetermined amount of a compound of the present invention as an active
ingredient. A
compound of the present invention may also be administered as a bolus,
electuary or
paste.
In solid dosage forms of the invention for oral administration (capsules,
tablets,
pills, dragees, powders, granules and the like), the active ingredient is
mixed with one or
more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium
phosphate, and/or any of the following: fillers or extenders, such as
starches, lactose,
sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for
example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose
and/or acacia;
humectants, such as glycerol; disintegrating agents. such as agar-agar,
calcium
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carbonate, potato or tapioca starch, alginic acid, certain silicates, and
sodium carbonate;
solution retarding agents, such as paraffin; absorption accelerators. such as
quaternary
ammonium compounds; wetting agents, such as, for example, cetyl alcohol and
glycerol
monostearate; absorbents, such as kaolin and bentonite clay; lubricants, such
a talc,
calcium stearate, magnesium stearate, solid polyethylene glycols, sodium
lauryl sulfate,
and mixtures thereof; and coloring agents. In the case of capsules. tablets
and pills, the
pharmaceutical compositions may also comprise buffering agents. Solid
compositions of
a similar type may also be employed as fillers in soft and hard-filled gelatin
capsules
using such excipients as lactose or milk sugars, as well as high molecular
weight
polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more
accessory ingredients. Compressed tablets may be prepared using binder (for
example,
gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent,
preservative,
disintegrant (for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets
may be
made by molding in a suitable machine a mixture of the powdered compound
moistened
with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions
of
the present invention, such as dragees, capsules, pills and granules. may
optionally be
scored or prepared with coatings and shells, such as enteric coatings and
other coatings
well known in the pharmaceutical-formulating art. They may also be formulated
so as to
provide slow or controlled release of the active ingredient therein using. for
example,
hydroxypropylmethyl cellulose in varying proportions to provide the desired
release
profile, other polymer matrices, liposomes and/or microspheres. They may be
sterilized
by, for example, filtration through a bacteria-retaining filter, or by
incorporating
sterilizing agents in the form of sterile solid compositions which can be
dissolved in
sterile water, or some other sterile injectable medium immediately before use.
These
compositions may also optionally contain opacifying agents and may be of a
composition that they release the active ingredients) only, or preferentially,
in a certain
portion of the gastrointestinal tract, optionally, in a delayed manner.
Examples of
embedding compositions which can be used include polymeric substances and
waxes.
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The active ingredient can also be in micro-encapsulated form, if appropriate,
with one or
more of the above-described excipients.
Liquid dosage forms for oral administration of the compounds of the invention
include pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions,
syrups and elixirs. In addition to the active ingredient, the liquid dosage
forms may
contain inert diluent commonly used in the art, such as, for example, water or
other
solvents, solubilizing agents and emulsifiers, such as ethyl alcohol.
isopropyl alcohol,
ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene
glycol, 1,3-
butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ,
olive, castor and
sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and
fatty acid esters
of sorbitan, and mixtures thereof.
Besides inert dilutents, the oral compositions can also include adjuvants such
as
wetting agents, emulsifying and suspending agents, sweetening, flavoring,
coloring,
perfuming and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending
agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene
sorbitol and
sorbitan esters, microcrystalline cellulose, aluminum metahydroxide,
bentonite, agar-
agar and tragacanth, and mixtures thereof.
Formulations of the pharmaceutical compositions of the invention for rectal or
vaginal administration may be presented as a suppository, which may be
prepared by
mixing one or more compounds of the invention with one or more suitable
nonirritating
excipients or carriers comprising, for example, cocoa butter, polyethylene
glycol, a
suppository wax or a salicylate, and which is solid at room temperature, but
liquid at
body temperature and, therefore, will melt in the rectum or vaginal cavity and
release the
active compound.
Formulations of the present invention which are suitable for vaginal
administration also include pessaries, tampons, creams, gels, pastes, foams or
spray
formulations containing such carriers as are known in the art to be
appropriate.
Dosage forms for the topical or transdermal administration of a compound of
this
invention include powders, sprays, ointments, pastes, creams, lotions, gels,
solutions,
patches and inhalants. The active compound may be mixed under sterile
conditions with
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a pharmaceutically acceptable carrier. and with any preservatives, buffers, or
propellants
which may be required.
The ointments, pastes, creams and gels may contain, in addition to an active
compound of this invention, excipients, such as animal and vegetable fats.
oils. waxes,
paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols,
silicones.
bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to a compound of this invention,
excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium
silicates and
polyamide powder, or mixtures of these substances. Sprays can additionally
contain
customary propellants, such as chlorofluorohydrocarbons and volatile
unsubstituted
hydrocarbons, such as butane and propane.
Transdermal patches have the added advantage of providing controlled delivery
of a compound of the present invention to the body. Such dosage forms can be
made by
dissolving or dispersing the compound in the proper medium. Absorption
enhancers can
I 5 also be used to increase the flux of the compound across the skin. The
rate of such flux
can be controlled by either providing a rate controlling membrane or
dispersing the
active compound in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the Like, are
also contemplated as being within the scope of this invention.
Pharmaceutical compositions of this invention suitable for parenteral
administration comprise one or more compounds of the invention in combination
with
one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile powders which may
be
reconstituted into sterile injectable solutions or dispersions just prior to
use, which may
contain antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic
with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers which may be employed
in the pharmaceutical compositions of the invention include water, ethanol.
polyols
(such as glycerol, propylene glycol, polyethylene glycol, and the like), and
suitable
mixtures thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as
ethyl oleate. Proper fluidity can be maintained, for example, by the use of
coating
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materials, such as lecithin. by the maintenance of the required particle size
in the case of
dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting
agents, emulsifying agents and dispersing agents. Prevention of the action of
microorganisms may be ensured by the inclusion of various antibacterial and
antifungal
agents, for example, paraben, chlorobutanol, phenol sorbic acid. and the like.
It may also
be desirable to include isotonic agents, such as sugars, sodium chloride, and
the like into
the compositions. In addition, prolonged absorption of the injectable
pharmaceutical
form may be brought about by the inclusion of agents which delay absorption
such as
aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to
slow the
absorption of the drug from subcutaneous or intramuscular injection. This may
be
accomplished by the use of a liquid suspension of crystalline or amorphous
material
having poor water solubility. The rate of absorption of the drug then depends
upon its
I S rate of dissolution which, in turn, may depend upon crystal size and
crystalline form.
Alternatively, delayed absorption of a parenterally-administered drug form is
accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of the
subject compounds in biodegradable polymers such as polylactide-polyglycolide.
Depending on the ratio of drug to polymer, and the nature of the particular
polymer
employed, the rate of drug release can be controlled. Examples of other
biodegradable
polymers include poly(orthoesters) and poly(anhydrides). Depot injectable
formulations
are also prepared by entrapping the drug in liposomes or microemulsions which
are
compatible with body tissue.
The preparations of the present invention may be given orally, parenterally,
topically. or rectally. They are of course given by forms suitable for each
administration
route. For example, they are administered in tablets or capsule form, by
injection,
inhalation, eye lotion, ointment, suppository, etc. administration by
injection, infusion or
inhalation; topical by lotion or ointment; and rectal by suppositories. Oral
administration
is preferred.
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The phrases "parenteral administration" and "administered parenterally" as
used
herein means modes of administration other than enteral and topical
administration,
usually by injection, and includes, without limitation, intravenous,
intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,
intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular,
subcapsular,
subarachnoid, intraspinal and intrasternal injection and infusion.
The phrases "systemic administration," "administered systemically,"
"peripheral
administration" and "administered peripherally" as used herein mean the
administration
of a compound, drug or other material other than directly into the central
nervous
system, such that it enters the patient's system and, thus, is subject to
metabolism and
other like processes. for example, subcutaneous administration.
These compounds may be administered to humans and other animals for therapy
by any suitable route of administration, including orally, nasally, as by, for
example, a
spray, rectally, intravaginally, parenterally, intracisternally and topically,
as by powders,
1 ; ointments or drops. including buccally and sublingually.
Regardless of the route of administration selected, the compounds of the
present
invention, which may be used in a suitable hydrated form, and/or the
pharmaceutical
compositions of the present invention, are formulated into pharmaceutically
acceptable
dosage forms by conventional methods known to those of skill in the art.
Actual dosage levels of the active ingredients in the pharmaceutical
compositions
of this invention may be varied so as to obtain an amount of the active
ingredient which
is effective to achieve the desired therapeutic response for a particular
patient,
composition, and mode of administration, without being toxic to the patient.
The selected dosage level will depend upon a variety of factors including the
activity of the particular compound of the present invention employed, or the
ester, salt
or amide thereof, the route of administration, the time of administration, the
rate of
excretion of the particular compound being employed, the duration of the
treatment,
other drugs, compounds and/or materials used in combination with the
particular
compound employed, the age, sex, weight, condition, general health and prior
medical
history of the patient being treated, and like factors well known in the
medical arts.
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A physician or veterinarian having ordinary skill in the art can readily
determine
and prescribe the effective amount of the pharmaceutical composition required.
For
example, the physician or veterinarian could start doses of the compounds of
the
invention employed in the pharmaceutical composition at levels lower than that
required
in order to achieve the desired therapeutic effect and gradually increase the
dosage until
the desired effect is achieved.
If desired, the effective daily dose of the active compound may be
administered
as two, three, four, five, six or more sub-doses administered separately at
appropriate
intervals throughout the day, optionally, in unit dosage forms.
While it is possible for a compound of the present invention to be
administered
alone, it is preferable to administer the compound as a pharmaceutical
composition.
As set out above, certain embodiments of the present compounds can contain a
basic functional group, such as amino or alkylamino, and are, thus, capable of
forming
pharmaceutically acceptable salts with pharmaceutically acceptable acids. The
term
"pharmaceutically acceptable salts" is art recognized and includes relatively
non-toxic,
inorganic and organic acid addition salts of compounds of the present
invention. These
salts can be prepared in situ during the final isolation and purification of
the compounds
of the invention, or by separately reacting a purified compound of the
invention in its
free base form with a suitable organic or inorganic acid, and isolating the
salt thus
formed. Representative salts include the hydrobromide, hydrochloride, sulfate,
bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate,
laurate,
benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,
tartrate,
napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts
and the
like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci.
66:1-19).
In other cases, the compounds of the present invention may contain one or more
acidic functional groups and, thus, are capable of forming pharmaceutically
acceptable
salts with pharmaceutically acceptable bases. The term "pharmaceutically
acceptable
salts" in these instances includes relatively non-toxic, inorganic and organic
base
addition salts of compounds of the present invention. These salts can likewise
be
prepared in situ during the final isolation and purification of the compounds,
or by
separately reacting the purified compound in its free acid form with a
suitable base, such
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as the hydroxide. carbonate or bicarbonate of a pharmaceutically acceptable
metal
cation, with ammonia, or with a pharmaceutically acceptable organic primary.
secondary
or tertiary amine. Representative alkali or alkaline earth salts include the
lithium,
sodium, potassium, calcium, magnesium, and aluminum salts and the like.
Representative organic amines useful for the formation of base addition salts
include
ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine,
piperazine
and the like.
The term "pharmaceutically acceptable esters" refers to the relatively non-
toxic,
esterified products of the compounds of the present invention. These esters
can be
prepared in situ during the final isolation and purification of the compounds,
or by
separately reacting the purified compound in its free acid form or hydroxyl
with a
suitable esterifying agent. Carboxylic acids can be converted into esters via
treatment
with an alcohol in the presence of a catalyst. Hydroxyls can be converted into
esters via
treatment with an esterifying agent such as alkanoyl halides. The term also
includes
lower hydrocarbon groups capable of being solvated under physiological
conditions,
e.g., alkyl esters, methyl, ethyl and propyl esters. (See, for example, Berge
et al., supra.)
A preferred ester group is an acetomethoxy ester group.
The language "effective amount" of the compound is that amount necessary or
sufficient to treat or prevent a chemokine mediated disorder, e.g. prevent the
various
morphological and somatic symptoms of a chemokine mediated disorder. The
effective
amount can vary depending on such factors as the size and weight of the
subject, the
type of illness, or the particular GPCR binding compound. For example. the
choice of
the GPCR binding compound can affect what constitutes an "effective amount".
One of
ordinary skill in the art would be able to study the aforementioned factors
and make the
determination regarding the effective amount of the GPCR binding compound
without
undue experimentation. An in vivo assay as described in Example 5 below or an
assay
similar thereto (e.g., differing in choice of cell line or type of illness)
also can be used to
determine an "effective amount" of a GPCR binding compound. The ordinarily
skilled
artisan would select an appropriate amount of a GPCR binding compound for use
in the
aforementioned in vivo assay.
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The regimen of administration can affect what constitutes an effective amount.
The GPCR binding compound can be administered to the subject either prior to
or after
the onset of a chemokine mediated disorder. Further, several divided dosages,
as well as
staggered dosages, can be administered daily or sequentially, or the dose can
be
continuously infused, or can be a bolus injection. Further, the dosages of the
GPCR
binding compounds) can be proportionally increased or decreased as indicated
by the
exigencies of the therapeutic or prophylactic situation.
In yet another aspect, the invention features a protein coupled heptahelical
receptor binding compound comprising a G-protein coupled heptahelical receptor
binding compound packaged with instructions for using said compound for
treating a (3-
chemokine mediated disorder.
The invention also features a method of using a G-protein coupled heptahelical
receptor binding compound to modulate the binding of a second compound to a G-
protein coupled heptahelical receptor.
In another aspect, the invention pertains to a compound represented by the
formula:
A-L 1-B-L2-E (II)
where A is selected from the group consisting of branched and straight chain
alkyl, aryl, alkenyl, alkynyl, and heteroaryl moieties optionally substituted
by NR'R",
CN, N02, F, Cl, Br, I, CF3, CCl3, CHF2, CHC12, CONR'R", S(O)NR'R", CHO, OCF3,
OCC13, SCF3, SCCI;, COR', C02R', and OR' and wherein R' and R" are each
independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, or optionally substituted
aryl;
L 1 is a linker moiety selected from the group consisting of a bond, O, S,
CHOH,
CHSH, CHNH2, CHNHR, CHNRR', NH, NR, (CH2)n, O(CH2)n, and (CH2)n0(CH2)n,
an optionally substituted ring moiety of 4 to 7 atoms containing up to three
heteroatoms,
a chain of 1 to ~ atoms optionally substituted by C 1-C6 alkyl. halogens,
wherein n is
either 0, 1, 2, or 3, and R and R' are each independently substituted or
unsubstituted C1-
C6 branched or straight chain alkyl, C1-C6 branched or straight chain alkenyl,
aryl, C4-
C~ ring, optionally substituted with up to three heteroatoms;
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B is an aromatic moiety containing from 0 to 3 heteroatoms and containing 5 to
7
members optionally substituted by NR'R", cyano. nitro. halogen. CFA, CHF~.
CONR'R",
S(O)NR'R". CHO, OCF~, SCF~, COR', C02R', OR' where R' and R" are each
independently hydrogen, halogen, C1-C6 alkyl, optionally substituted aryl or
optionally
substituted aryl;
L2 is a second linking moiety selected from the group consisting of a bond.
CH2C=O, NHC=O, OC=O, C=O, CH2NHC=O, CHOH, (CH2)n, O. NH, O(CH2)n,
NH(CH2)n, CH2CHOH and NRC=O; and
E is a G-protein coupled heptahelical receptor pocket interacting moiety.
In one aspect, L, is S, NH, CH,, or O. In another aspect. L, is NHC=O.
In one embodiment, A is represented by the following formula:
Z~z~
R,'~
R / N Rs
wherein
Z, and Z, each independently represent N or C;
R,, R2, and R3 are independently selected from the group
comprised of hydrogen, C,-C,~ branched or straight chain alkyl. alkoxy,
thioalkyl,
hydroxyalkyl, halo, haloalkyl, amino, alkylamino, or carboxyl.
In one embodiment, both Z, and Z, are carbon, R, is alkyl (e.g.. methyl).
halogen
(e.g., bromine, chlorine or fluorine), or alkoxy and the L, linker is located
in the meta
position. For example, A may be represented by the following formula:
R, R,
w ~ w
N / N /
or
In another embodiment, R, is carbonyl (e.g., a ketone, an aldehyde. an ester,
or
an amide.). Furthermore, R, may be substituted with a cyclic moiety such as
piperazine,
furan, or phenyl.
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In yet another embodiment, A is substituted or unsubstituted phenyl. Examples
of substituents includes substituted or unsubstituted alkyl (e.g., methyl),
alkenyl, aryl
and heteroaryl moieties. Furthermore. A may be substituted with halogens
(e.g.,
chlorine).
In one embodiment, L, is O.
In another aspect, the invention features a compound wherein B is represented
by
the following formula:
N
Rs
wherein
Z~ and Za each independently represent N or C;
R4 and R; are independently selected from the group comprised of
hydrogen, C,-C~ branched or straight chain alkyl, alkenyl, alkynyl, alkoxy,
thioalkyl,
hydroxyalkyl, halo, haloalkyl, amino, alkylamino, or carboxyl. In one
embodiment, R~
is alkyl and R; is hydrogen.
In one embodiment, B is a substituted or unsubstituted pyridyl or pyrimidyl
moiety, wherein B may be represented by the following formula:
~ ~ ,~'I
~N
or
In an aspect, the invention features a compound wherein E is represented by
the
formula below:
wherein R6 is an electron withdrawing moiety and the aryl ring is additionally
optionally substituted with zero to four halogen atoms. Preferably, E is
substituted with
at least one fluorine atom, e.g. two or more fluorine atoms. For example, R6
may be
alkyl, alkoxy, haloalkyl, nitro, halo. alkylamino, hydroxyalkyl, or carboxyl.
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In an embodiment. E is a para substituted aryl moiety represented by the
formula
below:
w
i
In a further embodiment, RG is a halogenated alkyl moiety, e.g. a fluorinated
alkyl moiety, e.g. a trifluoromethyl or pentafluoroethyl. Other examples of
R~, include
substituted or unsubstituted alkoxy moieties (e.g., methoxy, trifluoromethoxy)
or
thioether moieties. R6 can be alkenyl or alkynyl (e.g., ethenyl).
Furthermore, in another embodiment E is heterocyclic, e.g., substituted or
unsubstituted furanyl, imidazolyl, benzothiophenyl, benzylfuranyl, quinolinyl,
isoquinolinyl, benzodioxazolyl, benzoxazolyl, benzothiazolyl,
benzylimidazolyl,
thiazolyl, isothiaozolyl, oxazolyl, benzylthiazolyl, isooxazolyl,
methylenedioxyphenyl,
indolyl, thienyl, pyrimidyl, pyrazinyl, purinyl, or deazapurinyl.
In yet another embodiment, E is branched or straight chain alkenyl or alkynyl.
Examples include ethynyl trimethyl silane and alkenes (e.g., dimes, trienes).
In another aspect, the invention features a compound represented by the
formula
below:
R~
R~ Zi Z2~L.i Z3 Z4~L=~Rs
R~N IZ, R~N RS R~
- (III)
wherein
Z,, Z,, Z3, and Z~ are each independently N or C;
R,, R,, R;, R4, R5, R6, R,, and Rx are each independently hydrogen, C,-C~,
branched or straight chain alkyl, alkenyl, alkynyl, alkoxy, thioalkyl,
hydroxyalkyl, halo,
haloalkyl, amino, alkylamino, or carboxyl;
L, is O, S, NH, NR,, (CHR,)n, CO, CR,OH, O(CHR,)n, and
(CHR,)n0(CHR,)n wherein n is either 1,2, or 3;
L, is a second linking moiety selected from the group consisting of a
bond, CH2C=O, NHC=O, OC=O, C=O, CH2NHC=O, NHC=OCH,. CHOH, (CH2)n, O,
NH, O(CH2)",. NH(CH2)~,, CH2CHOH and NRC=O. wherein m is 0, 1, 2, or 3
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Furthermore, the compound may be represented by the formula below:
R~
4 ~, 7~
R Z ~ Z'~L, Z3 Z ~Ls~R$
R; N R; R~~' R; R~
- (IV)
wherein
Z1, Z2. Z3, and Z4 are each independently N or C;
R1, R2, R;, R4, R5, R6, R~, and Rg are each independently hydrogen.
C1-C6 branched or straight chain alkyl, alkoxy, thioalkyl, hydroxyalkyl, halo.
haloalkyl,
amino, alkylamino, or carboxyl;
L 1 is O, S, NH, NR~, (CH2)n, CO, CHOH, O(CH2)n, and
(CH2)n0(CH2)n wherein n is either 1,2, or 3 and R~ is C1-Cg branched or
straight
chain alkyl, alkoxy, thioalkyl, hydroxyalkyl, halo, haloalkyl, amino,
alkylamino, or
carboxyl;
L2 is a second linking moiety selected from the group consisting of a
bond, CH2C=O, NHC=O, OC=O, C=O, CH2NHC=O, CHOH, (CH2)n, O, NH,
O(CH2)n, NH(CH2)n, CH2CHOH and NRC=O.
In one embodiment, Z, and ZZ are both carbon. In another, R, is methyl, and RZ
and R3 are hydrogen. In another aspect, L, is O. In an yet another embodiment,
R~ and
R; are both hydrogen. In yet another embodiment, L, is NHC=O. In a preferred
aspect,
R6 is a halogenated alkyl moiety, e.g. a fluorinated alkyl moiety, e.g. a
trifluoromethyl or
pentafluoroethyl moiety. In another aspect, R, and Rs are each independently
fluorine or
hydrogen.
In yet another aspect, the invention can be represented by the structure
below:
R~
R L,-~~ L, ~ Rs
i ~
R / _N Rs R.~ N Rs Rb
The invention also features compounds of the formula:
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Rt
N ~ Lt N
i ~ I L
R6
In one embodiment, L, is O and L, is NHCO. In a further embodiment. L 1 is O,
L~ is NHC=O, and R6 is a halogen, a halogenated alkyl group, e.g.
trifluoromethyl or
pentafluoroethyl), or an alkoxy group, e.g. a halogenated alkoxy group, e.g. a
trifluoromethoxy group. Furthermore, R~ also can be ethenyl or a thioether
moiety (e.g.
-S-CF3).
The invention also features binding compounds represented by the following
structures:
O
N ~ O N I O I ~ N I O
N ~ N N /
H
H w CFA CF;
Cl ~ O
N . I .H
( N CH3 N
N. I O ~ F
N N O
C F;
O ~ ~ O
C~ N . I .H ~ N . I .H
N CH N N N
3
O, w I O, w I
OCF3 OCF;
O ~ ~ O
N . I . H ~~~ N . I . H
N N H;C N N
O, w I O~ w
C F; C F;
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~O
N , ~ .H C~ N , ~ .H
H;C N N N CH N
OCF3 tBu
/O , ~ O
N. ~ .H C~ N. I .H
N N N CH3 N
tBu CF(CF3)2
O ~ ~ O
N . I .H ~C -~ N . I .H
N N N CH3 N
O, w I O~ w
COH(CF3)2 COH(CF3)z
w O ~ C~O i
H C I~ N . I N.H N / N ~ I .H
3
N
O
O
COH(CF3)2
CF3
C~O ~ i O i
N N . I .H ~ I N . I .H
N
N
O i ~ O, i
'CI
OCF;
O O
N I .H / I N I .H
N CH3 N N N
O, \ I O
N02 CF~CF,CF3
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O O
N ~ .H / I N ~ .H
N N N N
O / CI O i
CI ~ N02
O O
N ~ .H / I N ~ .H
N CH3 N N CH3 N
O, w I O, w
I Br
O o
/ O ~ i O
i N~ I _ I i N~
N Cl ~' NH \ I N OMe ~ NII
CF3 CF3
o
O / O I ~ i
N' ' N ~ I NH / N Me NH
Me
fl
0 0
I o
N Me N ~ NH ~ I N Me N \ NIA
S
I
CF;
O O
O I / N I O
N Me N NH ~ I N Me \ Nf-I
CF,_
I
CF3
The term "alkyl" includes saturated aliphatic groups, including straight-chain
alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups,
alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The
term alkyl
further includes alkyl groups, which can further include oxygen, nitrogen,
sulfur or
phosphorous atoms replacing one or more carbons of the hydrocarbon backbone,
e.g.,
oxygen, nitrogen, sulfur or phosphorous atoms. In preferred embodiments, a
straight
chain or branched chain alkyl has 10 or fewer carbon atoms in its backbone
(e.g., C 1-
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C I O for straight chain, C3-C 1 p for branched chain), and more preferably 6
or fewer.
Likewise, preferred cycloalkyls have from 4-7 carbon atoms in their ring
structure, and
more preferably have 5 or 6 carbons in the ring structure.
Moreover, the term alkyl includes both "unsubstituted alkyls" and "substituted
alkyls", the latter of which refers to alkyl moieties having substituents
replacing a
hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents
can
include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,
phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and
alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,
thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,
azido, silyl,
trialkylsilyl, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety. It will be
understood by those skilled in the art that the moieties substituted on the
hydrocarbon
chain can themselves be substituted, if appropriate. Cycloalkyls can be
further
substituted, e.g., with the substituents described above. An "alkylaryl"
moiety is an
alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
The term "aryl" includes aryl groups, including 5- and 6-membered single-ring
aromatic groups that may include from zero to four heteroatoms, for example,
benzene,
pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole,
tetrazole,
pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl
groups also
include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl,
and the
like. Those aryl groups having heteroatoms in the ring structure may also be
referred to
as "aryl heterocycles", "heteroaryls" or "heteroaromatics". The aromatic ring
can be
substituted at one or more ring positions with such substituents as described
above, as
for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano,
amino
(including alkyl amino, dialkylamino, arylamino, diarylamino, and
alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and
ureido),
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amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates.
sulfonato,
sulfamoyl, sulfonamido, vitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkvlaryl, or
an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged
with
alicyclic or heterocyclic rings which are not aromatic so as to form a
polycycle (e.g..
tetralin).
The terms "alkenyl" and "alkynyl" include unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls described above.
but that
contain at least one double or triple bond, respectively.
Unless the number of carbons is otherwise specified, "lower alkyl" as used
herein
means an alkyl group, as defined above, but having from one to three carbon
atoms in its
backbone structure. Likewise, "lower alkenyl" and "lower alkynvl" have similar
chain
lengths.
The terms "alkoxyalkyl", "polyaminoalkyl" and "thioalkoxvalkyl" include alkyl
groups, as described above, which further include oxygen, nitrogen or sulfur
atoms
replacing one or more carbons of the hydrocarbon backbone, e.g.. oxygen.
nitrogen or
sulfur atoms.
The terms "polycyclyl" or "polycyclic radical" refer to two or more cyclic
rings
(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls)
in which two
or more carbons are common to two adjoining rings, e.g., the rings are "fused
rings".
Rings that are joined through non-adjacent atoms are termed "bridged" rings.
Each of
the rings of the polycycle can be substituted with such substituents as
described above.
as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,
phosphinato.
cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and
alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,
thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, vitro, trifluoromethyl. cyano,
azido,
heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.
The term "heteroatom" as used herein means an atom of anv element other than
carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen. sulfur and
phosphorus.
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It will be noted that the structure of some of the compounds of this invention
includes asymmetric carbon atoms. It is to be understood accordingly that the
isomers
arising from such asymmetry (e.g., all enantiomers and diastereomers) are
included
within the scope of this invention, unless indicated otherwise. Such isomers
can be
obtained in substantially pure form by classical separation techniques and by
stereochemically controlled synthesis.
In one embodiment. the present invention includes GPCR binding compounds
and/or methods of using the same which are encompassed by the formulae set
forth
herein and which are not described in Brombridge, et al., J. Med Chenz (1997)
40:3494,
U.S. 3,499,898, EP358 118, EP 344 634, and/or WO 96/23783. The contents of
each of
which are expressly incorporated herein by reference.
In another embodiment, E or the G-protein coupled heptahelical pocket
interacting moiety is not:
i
N p
H ,
and/or unsubstituted phenyl.
In another embodiment, L, is O, L, is NHCO, and E or the G-protein coupled
heptahelical pocket interacting moiety is not:
i
N p
H ' , and/or unsubstituted phenyl.
In another embodiment, E or the G-protein coupled heptahelical pocket
interacting moiety is a phenyl group having at least one substituent. In yet
another
embodiment, E is a phenyl group having at least one substituent in the para
position. In
a further embodiment, E is a phenyl group having at least two substituents.
The compounds of the present invention can be synthesized using standard
methods of chemical synthesis and/or can be synthesized using schemes
described
herein. Synthesis of specific compounds is discussed in detail in the Example
sections
below. An example of a general synthesis is outlined in the scheme below:
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Scheme 1: General Synthesis of Test Compounds
,Y OH ,Y O ~Y.X R
X,Y/ NOz R -X~' ~ NaH, dioxane R3~ ~ ~~~ ~,
R1~ ~Rz + ' ~N ~ N ~ N "I NOz
C 1 N ~t R~
MeOH, H20
Fe, AcOH
O
R7 w C1
O Y. '
R3~Y~~ N X O ~ i Rg ,Y O Y.X
N ~N ~ CHZCIz R;~ , ~ Rz
Rz ~~ N ~ N H
R~ H R7 N ~ z
R~
Rs
Hydroxypyridine (or other hydroxyaryl precursor) is dissolved in dioxane and
1.5 equivalents of 95% sodium hydride is added. The mixture is stirred at room
temperature for 20 minutes and 1 equivalent of 2-chloro-5-nitropyridine (or
other a-
chloro-heterocycle) is then added. The mixture is subsequently brought to
reflux for 3
hours and cooled. The reaction mixture is then quenched by addition of
saturated
ammonium chloride solution. Silica gel is added to the solution and the
mixture is
rotovapped to dryness. The product is eluted from the silica gel and flash
chromatographed using a mixture of ethyl acetate/hexane.
The nitro group is reduced by dissolving the nitropyridine in 1:1
methanol:water.
Acetic acid and iron powder is then added and the mixture is brought to reflux
for 3
hours. After cooling, the iron is precipitated by addition of 20% NaOH and
subsequently
filtered through Celite. The methanol is removed by rotary evaporation and the
remaining aqueous mixture is extracted with methylene chloride. The organic
layer is
dried and the solvent removed by rotary evaporation to give product.
The dipyridyl ether is then dissolved in methylene chloride followed the
addition
of polymer bound morpholine (Booth, et al., J. Am. Chem. Soc., 119, 1997, 4882-
4886).
1.3 equivalents of acid chloride is added and the mixture shaken overnight.
The excess
acid chloride is scavenged using polymer bound tris-2-aminoethylamine.
Filtration
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followed by flash chromatography of the filtrate using ethyl acetate:hexane
gives the
product.
Further examples of syntheses of compounds of the invention are included in
the
Example section.
The invention is further illustrated by the following examples which in no way
should be construed as being further limiting. The contents of all references,
pending
patent applications and published patent applications, cited throughout this
application
are hereby incorporated by reference. It should be understood that the animal
models
used throughout the examples are accepted animal models and that the
demonstration of
efficacy in these animal models is predictive of efficacy in humans.
EXAMPLE 1: Synthesis of GPCR Binding Compounds
A. General Procedures
Many GPCR binding compounds of the invention were made by the general
reaction scheme, outlined below:
.Y OH ,y O ~Y.
R1 X'Y R 02 R3,~ ~ NaH,~ R3~ ~~ N X R,
C~N~ 2 + N N' ~N02
R2
MeOH, H20
Fe, AcOH
O
R~ w CI
.Y O ~Y~X R6
R ~ ' ~ i O i Rg O Y.
~Ra N i X -~' ~ ~ X R2
N ~'I~N C~ R3 , R4 N .
Ri
R1 H R N ~NHZ
vR 7 R~
s
I: Nucleophilic displacement of 2-chloroheterocycles
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Method A:
50 mmol of hydroxypyridine (or other hydroxyaryl precursor) is dissol~~ed in
500
mL of dioxane. 1.5 equivalents of 95% sodium hydride is added and the mixture
stirred
at room temperature for 20 minutes. 1 equivalent of 2-chloro-~-nitropyridine
(or other a-
chloro-heterocycle) is added and the mixture is brought to reflux for 3 hours.
After
cooling, the reaction mixture is quenched by addition of 2 mL of saturated
ammonium
chloride solution. 10 g of silica gel is added to the solution and the mixture
is
rotovapped to dryness. The product is eluted from the silica gel and flash
chromatographed using a mixture of ethyl acetate/hexane.
Method B:
50 mmol of hydroxypyridine is dissolved in 150 mL of dry DMF. The mixture is
cooled to 0 C and 1.5 equivalents of sodium hydride added. The mixture is
allowed to
warm to room temperature over 20 minutes, followed by addition of the a-chloro
heterocycle. The reaction mixture is stirred at room temperature for 16 hours,
and then
partitioned between ethyl acetate/water in a separatory funnel. The organic
layer is dried
and the solvent removed to give product which is typically > 90% pure.
II: Reduction of Nitro group
Method A:
40 mmol of nitropyridine is dissolved in 200 mL of methanol followed by the
addition of 200 mL of water. Acetic acid (8.3 mL) and iron powder (17 g) is
added and
the mixture is brought to reflux for 3 hours. After cooling, the iron is
precipitated by
addition of 25 mL of 20% NaOH and the mixture filtered through Celite. Removal
of the
methanol by rotary evaporation is followed by extraction of the remaining
aqueous
mixture with methylene chloride. The organic layer is dried and the solvent
removed by
rotary evaporation to give product.
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Method B:
40 mmol of nitropyridine is dissolved in 2S0 mL of ethanol and treated with a
mixture of tin(II) chloride (30 g) in conc. HC1 (6S mL). The mixture is heated
to SO C
for 1 hour. After cooling, the mixture is basified with 40% NaOH and the
product
S extracted into ethyl acetate. The solution is dried over sodium sulfate and
the solvent
removed to give product.
III: Acylation of the amino-heterocycle
1 mmol of the dipyridyl ether is dissolved in 12 mL of methylene chloride
followed by
the addition of 0.4 g of polymer bound morpholine (loading 4 mmol/g) (Booth,
et al. J.
Am. Chem. Soc., 119, 1997, 4882-4886). 1.3 equivalents of acid chloride is
added and
the mixture shaken overnight. The excess acid chloride is scavenged using
polymer
bound tris-2-aminoethylamine. Filtration followed by flash chromatography of
the
1 S filtrate using ethyl acetate:hexane gives the product.
B. Synthesis of the GPCR Binding Compounds
NOZ OH O
I . + I ~ N~ I ~ N
C1 N
N Dioxane N N02
1
Fe/AcOH
3a R=CF3 MeOH/water
3b R=OCF3
3c R= Br
I ~ O ~ I O 3d R=tBu
NJ~ N~N , 3e=CF(CF3)2 ~ O
I
H \ R ~ N ~ N NH2
2
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(1) 2-methyl-3-hydroxy pyridine (5 g, 46 mmol) and 2-chloro-5-nitro pyridine
were
suspended in 500 mL of dioxane. Sodium hydride (1.7 g of 95%, 1.~ eq) was
added and
the mixture refluxed under argon for 3 hours. After cooling, the excess sodium
hydride
was quenched by addition of 2 mL of saturated ammonium chloride solution and
10 g
of silica gel added to the solution. The solvents were removed by rotary
evaporation
leaving the product adsorbed on silica. Purification was achieved by flash
chromatography using 1:1 ethyl acetate/hexanes as eluent. Yield: 9.3 g, 87%.
LC-MS
(diode array detection at 210-300 nM) showed that the product was > 95% pure
and had
the expected M.W. of 232 (M+H+). 1H NMR (CDC13, shifts relative to the solvent
peak
at 7.24 ppm): 8 8.98 (d, 1 H) 8 8.52 (dd, 1 H) 8 8.42 (dd, 1 H) 8 7.30 (dd, 1
H) 8 7.2 (m,
1H) b 7.08 (d, 1H) 8 2.38 (s, 3H).
(2) Compound (1) (9.3 g) was suspended in 400 mL of 1/1 methanol/water. Acetic
acid
(8.3 mL) and powdered iron ( 17 g) were added and the mixture brought to
reflux for 3
hours. After cooling, the iron was precipitated by addition of 25 mL of 20%
NaOH. The
mixture was filtered through Celite and the filter cake rinsed with methanol.
The
methanol was removed by rotary evaporation and the product partitioned into
methylene
chloride in a separatory funnel. Removal of the solvent by rotary evaporation
gave 4.1 g
(51 %)of product. LC-MS showed the product to be > 95% pure and to have the
expected M. W. of 202 (M+H+). 1 H NMR (CDC13, shifts relative to the solvent
peak at
7.24 ppm): 8 8.24 (d, 1 H) 8 7.60 (d, 1 H) 8 7.24 (dd, 1 H) 8 7.08 (m, 2H) 8
6.88 (d, 1 H) 8
3.52 (br s, 2H) 8 2.38 (s, 3H).
(3a)Compound (2) (202 mg, 1 mmol) was dissolved in 10 mL of methylene chloride
in a
20 mL scintillation vial. Polystyrene bound morpholine (0.4 g, loading 4
mmol/g) was
added followed by 1.3 mmol of 4-trifluoromethylbenzoyl chloride. The mixture
was
shaken at room temperature for 12 hours followed by the addition if 0.5 g of
polystyrene
supported tris-2-aminoethylamine (loading 4 mmol/g). The mixture was shaken
for an
additional 12 hours and filtered through a polystyrene frit. The reaction
mixture was
applied to a 90 g cartridge of silica gel and the product eluted with ethyl
acetate.
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Removal of the solvent gave 248 mg of product. LC-MS indicated a purity >
97%and
the expected M. W. of 374 (M+H+). 1 H NMR (CDC13, shifts relative to the
solvent peak
at 7.24 ppm): 8 8.79 (s, 1 H) b 8.19 (m, 2H) b 7.99 (d, 1 H) 8 7.84 (d, 2H) 8
7.53 (d, 2H) 8
7.21 (d, 1 H) b 7.06 (m, 1 H) 8 6.81 (d, 1 H) 8 2.20 (s, 3H). Anal. Calcd for
C 19H 14F3N302 : C, 61.13 H, 3.78 N, 11.26 Found: C, 61.27 H, 3.76 N, 11.17.
A number of similar analogs were made from intermediate (2) by acylation with
other
substituted benzoyl chlorides:
(3b) LC-MS showed the product to be > 95% pure and to have the expected M.W.
of
390 (M+H+). 1H NMR (CDC13, shifts relative to the solvent peak at 7.24 ppm) 8
9.02
(s, 1H)88.18(d,H)88.06(d, 1H)87.97(d, 1H)87.8(m,2H)87.21 (d, 1H)87.05
(m, 3H) 8 6.8 (d, 1H) b 2.20 (s, 3H).
(3c) LC-MS showed the product to be > 95% pure and to have the expected M.W.
of
385 (M+H+). 1H NMR (CDC13, shifts relative to the solvent peak at 7.24 ppm): 8
9.18
(s, 1 H) 8 8.24 (d, 1 H) 8 8.18 (d, 1 H) 8 8.04 (s, 1 H) 8 7.66 (d, 2H) 8 7.45
(d, 2H) 8 7.27
(d, 1 H) 8 7.08 (m, 1 H) ~ 6.80 (d, 1 H) 8 2.21 (s, 3H).
(3d) LC-MS showed the product to be > 95% pure and to have the expected M.W.
of
362 (M+H+). 1H NMR (CDC13, shifts relative to the solvent peak at 7.24 ppm): 8
9.18
(d, 1H)88.22(d, 1H)88.19(d, 1H)88.17(d, 1H)87.78(d,2H)87.36(d,2H)87.26
(d, 1 H) 8 8 7.05 M, 1 H) 8 6.8 (d, 1 H) b 2.22 (s, 3H) 8 1.20 (s, 9H).
(3e) 4-(perfluoroisopropyl)benzoyl chloride was made as follows: 4-iodo
benzoic acid (5
g, 20.2 mmol) was dissolved in DMF along with perfluoroisopropyl iodide (6 g,
20.3
mmol). Copper powder (6.35 g) and DMF (25 mL) were added and the mixture was
heated at 140 C in a sealed tube for 8 hours. After cooling, the reaction
mixture was
partitioned between diethyl ether and 1 N HCI. The organic layer was separated
and
dried and the resulting solid chromatographed on silica (ethyl acetate as
eluent) to give a
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mixture of compounds containing the desired product (by LC-MS.) The crude
mixture
was treated with oxalyl chloride (3 mL) in 25 mL of methylene chloride. After
3 hours
of stirring, the solvents were removed by rotary evaporation and residual
oxalyl chloride
removed by azeotroping with toluene. The crude acid chloride was used to
acylate
compound (2) according to the general procedure given above. The product was
purified
by preparative TLC (5:4:1 methylene chloride:ethyl acetate:methanol). LC-MS
showed
the product to be > 95% pure and to have the expected M.W. of 474 (M+H+) 1H
NMR
(CDCl3, shifts relative to the solvent peak at 7.24 ppm): 8 8.25 (m, 3H) 8
8.19 (s, 1H) 8
8.05 (d, 2H) ~ 7.79 (d, 2H) 8 7.4 (d, 1 H) 8 7.2 (m, 2H) 8 6.99 (d. 1 H) 8 2.2
(s, 3H).
C. Synthesis of "Extended Bridge" GPCR Binding Compounds
I ~ OH
N ~ NaH, Dioxane
~N02
Fe
I ~ N02 ( w O N ~ HOAc
Cl N N
4
H ~ CF3 \ NHZ
w N I i
O N
O N O E I ~ s
I , N
N
6
(4) 3-pyridinol (Sg, 45.8 mmol) was dissolved in 500 mI. of dioxane. Sodium
hydride
(1.7 g, 1.5 equivalents) was added and the mixture stirred for 10 minutes. 2-
chloro-5-
nitro pyridine (7.27 g, 45.8 mmol) was added and the mixture was brought to
reflux for
3 hours. After cooling, the excess sodium hydride was quenched with 2 mL of
saturated
aqueous ammonium chloride and 10 g of silica added to the mixture. Removal of
the
solvent left the product adsorbed on silica gel. Elution with ethyl acetate:
hexane ( 1:1 )
combined with flash chromatography gave 5.4 g of product. LC-MS indicated a
purity
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of > 95%. 1 H NMR (CDC13, shifts relative to the solvent peak at 7.24 ppm): 8
9.08 (d,
1 H) 8 8.65-8.8 (br d, 2H) 8 8.32 (dd, 1 H) 8 7.79 (d, 1 H) b 7.27 (m, 1 H) 8
6.81 (d, 1 H) 8
5.44 (s, 2H). MW=231. M+H+=232.
(5) The product from above (4) was dissolved in 45 mL of methanol and 45 mL of
water
added. Iron powder (3.6 g) and acetic acid (1.72 mL) were added and the
mixture
brought to reflux for 3 hours. After cooling, the iron was precipitated by
addition of 20%
NaOH and the mixture filtered through Celite. Removal of the methanol by
rotary
evaporation was followed by extraction of the product into methylene chloride.
Removal
of the solvent gave 1.08 g of product (5). LC-MS indicated a purity of >95%. 1
H NMR
(CDC13, shifts relative to the solvent peak at 7.24 ppm): 8 8.43 (d, 1 H) 8
8.25 (d, 1 H) 8
7.5 8 (d, 1 H) 8 7.42 (d, 1 H) 8 7.02 (dd, 1 H) 8 6.8 (dd, 1 H) ~ 6.4 (d, 1 H)
8 5.1 (s, 2H) 8
3.2 (br s, 2H). MW=201. M+H+=202.
(6) Compound (5) from above (1 mmol, 202 mg) was dissolved in 12 mL of
methylene
chloride. 0.4 g of polymer bound morpholine was added, followed by 1.3 mmol of
4-
trifluoromethylbenzoyl chloride. The mixture was shaken at room temperature
for 12
hours, at which time 0.5 g of polymer bound tris-2-aminoethylamine was added
to
scavenge excess acid chloride. After an additional 12 hours of shaking, the
reaction
mixture was filtered through a polystyrene frit and chromatographed on silica
using
ethyl acetate as eluent. Yield: 340 mg. LC-MS indicated the product was > 95%
pure.
1H NMR (CDC13, shifts relative to the solvent peak at 7.24 ppm): 8 8.86 (s,
1H) 8 8.55
(d, 1 H) 8 8.25, (s, 1 H) 8 8.07 (s, 1 H) b 7.99 (m, 3H) 8 7.81 (d, 1 H) b
7.71 ( d, 2H) 8 7.27
(m, 1H) 8 6.80 (d, 1H) 8 5.39 (s, 2H). MW=373
D. Synthesis of A-ring Pyrimidyl GPCR Binding Compounds
The general procedure is analogous to those described above, the only
difference being
that the starting material was 5-hydroxypyrimidine rather than 3-
hydroxypyridine.
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~~Br Nao~ N ~ OMe K~ ~~OH C
N i
N N N NOz
6 7
Na
O
N NOZ
s
O
N \ ~ O Fe/AcOH
N N ~ I ~~O i
H ~ CF
3 N N H2
5
(6) 5-bromo pyrimidine (8 g, 50.6 mmol) was dissolved in 100 mL of methanol
containing 3.14 g of sodium methoxide. The mixture was sealed in a glass
pressure
vessel and heated to 130 C for 3 hours. After cooling, 10 g of silica was
added to the
mixture and the solvent removed by rotary evaporation. The product was eluted
from the
10 silica and flash chromatographed using 1:1 hexanes/ethyl acetate. Yield:
2.5 g
LC-MS showed the product was > 95% pure and had the correct M.W. (M+H+= 111.0)
1H NMR (CDC13, shifts relative to the solvent peak at 7.24 ppm): 8 8.82 (s,
1H) 8 8.40
(s, 2H) 8 3.85 (s, 3H).
(7) 5-methoxy pyrimidine (4.0 g, 36.4 mmol) was dissolved in 30 mL of dry
ethylene
glycol. Powdered KOH (10 g) was added and the mixture refluxed under argon for
3
hours. Excess ethylene glycol was removed by rotary evaporation at 0.5 mm
Hg/130 C.
The product was extracted from the residue using several portions of boiling
dioxane.
Removal of the dioxane gave a viscous solution which, upon cooling, deposited
white
needles of 5-hydroxypyrimidine. LC-MS showed the product to be > 95% pure and
of
the expected M.W. (M+H+=97). 1H NMR (CDCl3, shifts relative to the solvent
peak at
7.24 ppm): 8 8.51 (s, 1 H) 8 8.20 (s, 2H).
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(8) 5-hydroxy pyrimidine (0.35 g, 3.64 mmol) was dissolved in I S mL of dry
DMF.
Sodium hydride (0.14 g, 1.6 eq) was added and the mixture stirred for '/2 hour
at 0 C. 2-
chloro-5-nitro pyridine ((0.867 g, l.~ eq) was added and the mixture stirred
at room
temperature for 12 hours. The reaction mixture was partitioned between ethyl
acetate
and sodium bicarbonate solution and the organic layer separated and mixed with
10 g of
silica gel. Removal of the solvent left the product adsorbed on silica. The
product was
eluted onto a flash column with ethyl acetate and fractions containing product
combined
and rotovapped to dryness. Yield: 0.7 g. LC-MS showed the product to be > 95%
pure
but the expected parent ion was not observed. NMR was consistent with the
expected
product: ~ 9. I 9 (s, I H) 8 9.02 (s, I H) d 8.89 (br s, 2H) 8 8.~8 (dd, 1 H)
8 7.25 (d, 1 H).
(9) Nitropyrimidyl-pyridine (8) was dissolved in 40 mL of 50% water/methanol
containing 0.77 g of acetic acid. 1.54 g of powdered iron was added and the
mixture
brought to reflux for 3 hours. The iron oxides were precipitated by addition
of 2 mL of
20% NaOH . Filtration of the reaction mixture through Celite was followed by
removal
of the methanol by rotary evaporation. The product was extracted into
methylene
chloride and the solvent removed by rotary evaporation. Yield: 0.37 g. LC-MS
showed
the product to be > 90% pure and of the expected M.W. (M+H+=189). IH NMR
(CDC13, shifts relative to the solvent peak at 7.24 ppm) 8 8.98 (s, 1H) 8 8.60
(s, 2H) 8
7.60 (d, 1 H) 8 7.18 (dd, 1 H) 8 6.80 (dd, 1 H) 8 3.60 (br s, 2H).
(10) pyrimidyl-pyridine (9) (189 mg, 1 mmol) was dissolved in 12 mL of
methylene
chloride along with 0.4 g of polymer bound morpholine. 4-
trifluoromethylbenzoyl
chloride (270 mg, 1.3 mmol) was added and the mixture shaken at room
temperature for
12 hours. Polymer bound tris-2-aminoethylamine (0.~ g) was added and the
mixture
shaken for an additional 12 hours. Filtration of the reaction mixture followed
by flash
chromatography using ethyl acetate as eluent gave 230 mg of the expected
product. LC-
MS showed a purity of > 95% and the expected M. W. (M+H+=361 ). 1 H NMR
(CDC13,
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shifts relative to the solvent peak at 7.24 ppm): 8 9.01 (s, 1H) 8 8.68 (m,
3H) 8 8.35 (dd,
1 H) 8 8.22 (d, 1 H) 8 8.01 (d, 2H) 8 7.75 (d, 1 H) 8 7.20 (d, 1 H).
Example 2: Identification of GPCR Binding Compounds which Interact with a
GPCR Using a Time Resolved Fluorescence (TRF) Assay
High-Throughput Receptor Binding Screening for Antagonists of CCR10
A. Tissue Culture and Production of CCR10 Cells
CCR10 cells are stable recombinant K293 cells overexpressing the CCR10
receptor. The cells are routinely cultured and passaged in a growth medium
composed
of DMEM base medium: 10% fetal bovine serum (FBS), 1X Glutamine, and 0.4 mg/ml
6418. 1 % Pen/Strep is also included in the media when the cells are seeded
into the
plates on Day 1.
B. Labeling of rhMCP-1 with a Europium Chelate
rhMCP-1 (carrier free) was supplied as a frozen stock solution in PBS at a
concentration of 0.65 mg/ml. 250 ~g (385 ~1) was buffer exchanged on a 2.8 ml
column
of Sephardex G-25 (fine), equilibrated with I OmM Borax, pH 9. Fractions (250
ql) were
collected and 2 ~l aliquots were analyzed using a Bradford protein assay. Four
fractions
eluting immediately after the void volume contained the bulk of the protein
and were
pooled (approx. 1 ml). 1 mg of Eu-labeling reagent (1.5 q,mol) was dissolved
in 200 pl
of water and 100 ul of this solution was added to the protein pool. The final
relative
concentration ratios of Eu:MCP-1 was about 26:1. The ratio of Eu:NH2 was about
3:1.
The mixture was incubated for 20 hours at 4°C and then desalted on
a 15 mL
column of Sephardex G-25 (fine) equilibrated with 20 mM HEPES (hemi-sodium
salt),
pH 7.5, and 0.9% NaCI. 0.~ mL fractions were collected and 1 ~l aliquots were
added to
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100 ~l of enhancement solution and counted on the VICTOR. The five fractions
that
were eluted immediately after the void volume showed a peak of Eu containing
material
and were pooled. 60 ~l of the pool was set aside for protein assay. 36 ~l of
0.1 % BSA
(heavy metal free; Wallac) was added as a stabilizer to the remainder. The
material was
then stored at 4°C. The total volume was about 2.5 ml.
The MCP-1 concentration was determined by a BCA protein assay with BSA as
a standard to be 8.5 ~M (74 ~g/ml).
The Eu concentration of the material was measured on the VICTOR with a
standard curve prepared from the Eu standard solution provided from the
vendor,
Wallac. The Eu concentration was 12 ~M.
The calculated stoichiometry was 1.4 Eu chelate per MCP-1 molecule.
C. Preparation of Compounds for Dispensation onto Cells
The compounds were provided as a dried film on a polystyrene "Master Plate"
containing 1 ~g of compound per well.
The compounds were dispensed into cell plates by preparing a 3X stock solution
in binding buffer from the dried films. The cell plates were prepared
according to the
following method.
Prior to the day of dispensation of compounds onto cells, the "Master Plates"
were allowed to reach room temperature. 50 ~l each of 1X Binding Buffer
(0.125%
BSA in deionized water) and MCP-1 stock solution (40nM and 600 nM in 1X
Binding
Buffer) was added to each well. The plates were subsequently stored at
4° C overnight.
D. Time-resolved fluorescence (TRF) assay
Day 1
Each 96-well tissue culture plate (poly-D-lysine coated) was seeded with
40,000
cells in complete media containing 0.4 mg/ml 6418 (200 ~l per well). The
plates were
incubated at 37° C and 6% C02 and left overnight.
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Day 2
The cells were approximately 90% confluent in the wells by Day 2. The cells
were washed twice with Binding Buffer (no BSA) using 200 ~l per wash. 40 ~l of
Binding Buffer was left behind following the second wash. To this final 40 ~l.
100 ~l of
Binding Buffer was then added. Excess Binding Buffer from each well was
removed
leaving about 15 ~1 in each well. 15 ~l of compound solution was dispensed
into each
well followed by 15 ~.1 of 30 nM Eu-MCPI working solution. The plates were
then
incubated at room temperature for 3 hours.
The plates were then washed twice with 150 ~l of Wash Buffer (250 mM Hepes,
pH 7.4; 1mM CaCl2; 1.15 M NaCI in deionized water). 15 yl remained in each
well
following the washes. Then, the plates were washed ten times with Wash Buffer
using
200 ~1 per wash. After the final wash, about 40 ~l of Wash Buffer was left
behind. To
this residual Wash Buffer, 100 ~1 of Wash Buffer was added. The excess Wash
Buffer
was then removed, leaving about 15 ~l behind. 100 ~1 of Enhancement Solution
was
added to each well. Time-resolved fluorescence was read by the Wallac/Victor
Plate
Reader 60 minutes later. Time-resolved fluoremetry measurements were taken
from 50
sec to 400 sec.
E. Method of Identification of GPCR Interacting Compounds
Controls were included on each 96-well assay plate. The controls were ( l )
wells
with no compounds and no unlabeled MCP1, (2) wells with no compounds and
unlabeled MCP1 (13.3 nM) corresponding to the ICSO, and (3) wells with no
compounds
and unlabeled MCP1 (200 nM) corresponding to complete inhibition. Each control
was
set up in duplicates.
Hits were defined by compounds that reduce binding of Eu-MCPl below the
level of the 50% inhibition control containing unlabeled MCP1 at the IC~o
concentration.
Percent inhibition was expressed by the following formula:
inhibition = [ 1 - (test well cps - background cps) = (no inhibition control
cps -
background cps)] x 100%;
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where background is defined as cps from control set #3 (wells with complete
inhibition) and the no inhibition control is defined as cps from control set
#1 (wells with
no compounds or unlabeled MCP 1 ).
Hits showing greater than 50% inhibition were retested using other assay
methods.
The results of this assay are summarized in the Tables below.
In Table I, *** represents IC;°'s less than 5 ~M, ** represents
IC;°'s of between
5 and 1 ~ pM, * represents IC;°'s greater than 15 ~M.
In Table 2, * * * represents very high binding affinity. * * represents high
binding
affinity, and * represents some binding affinity for the CCR10 receptor.
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Table 1: Time-resolved fluorescence (TRF) assay Results for Vew High Binding
Affinity Compounds
Compound ICSp Rating
- A ***
E ***
F ***
CU ***
CW ***
CV ***
DG ***
DO ***
EO ***
EQ ***
B **
C **
G **
T **
U **
**
DR **
DS **
DP
H
I
Y
AB
DH
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Table 2: Time Resolved Fluorescence (TRF) Assay Results
O
I N
N R~ N
I R
6
Compound # R, R6 Affinity
A Me CF3 ***
B H CF; ***
C H Cl ***
E Me OCF3
F H OCF3 ***
G Me t-Bu
H H t-Bu ***
I Me CF(CF3)z
J H COH(CF3)z
K Me COH(CF3)z
L Me NOz
M H NOz
N H (CFz)zCF3
O H Me
P H N(Me)3C1
Q Me N(Me)3C1
R H CN
CV Me I ***
CW Me Br ***
CY CONHz CF3
DG CI CF, ***
DH OMe CF; ***
DO Me Cl ***
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DP Me CHCH
DQ Me H **
DR Me CH,CH3
DS Me CH(CH3),. ***
DT Me CCI3 **
DX Me Me
EA Br CFA **
EO Me S-CF3
EQ Me CF,CF3 ***
L,
O
RZ N R~ N
R
6
Comp.# Z1 R1 R2 L1 R6 Affinity
S N H H O CFA **
T CH H Me O CFA ***
U CH H Me O OCF~ * * *
V CH H Me O CoH(CF3),
W CH H H CH20 CF3 ***
X CH H H CH20 OCF3
CX N H H O OCF3
CZ N H H CHZN(CO CF3
(P-CF3_
C6tI4))
DA N H H CH,NH CF;
EB CH I Me O CF3
ED CH H H NH CF; **
EJ CH H CH,-Ph O CF;
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R3 w ~ i ~ R6
IZ~ RZ3. I L I ~ R
> > z
R8
Comp. Z1 Rl R3 Z3 L2 R6 R'7 R8 Affinity
Y CH H H N N~tCO Cl H H ***
Z N H H N NHCO C1 H Cl
CU N Me H N NHCO CF; F H
AA N Me H N NHCO Cl H Cl
AB N H H N NHCO Cl Cl H ***
AC N H H N NHCH, CF; H H *
AD N H H N NHC''Z Cl H H
AE CH H H N NHCO CF; H H **
AF CH H H CH NHCO Cl H H
AG CH H H CH NHCO CF3 H H
AH N H H N CONH CF3 H H
AI N H H N NHSOZ Me H H
AJ N H Cl N NHCH H H H
(COOMe)
NHCO
AK N H H N NricoNH H H H
AL N H H N NHCONH CI H H
AM N H H N NHSOZ F Cl H
AN N H Cl N NHSOZ H C1 H
AO N H H N cNHCO), H H H
AP N H H N NHSO2 C1 H H
AQ N H H N NHS°~ Cl Cl H
AR N H H N NHCO o~cHZ); H H
Me
AS N H H N NHCO H H H
cH,o
IAT IN IH IH IN INHCOIH ICN IH-
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AU CH H H CH NHCO CF; H H
AV CH H H CH NHCO ph H H
AW CH H H CH NHCO occH,);H H
Me
AX CH H H CH NHCO H H H
CH~O
AY CH H H CH NHCO H CN H
DC CH H H CH NHCO t-Bu H H
DD CH H H CH N1-1COCN H H
DE N H H N NcMe)coCFA H H
DI N I-I cooMe N NHCO CF3 H H
DJ N H cooF~ N NHCO CF3 H H
DV N Me H N rrHCO H H H
CHI
DY N H c-cN- N NHCO CF3 H H
piprazinyl)
EF N H H N cH'-N"CF3 H H
co
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R3 ~ O ~ R4
N
N R~ Lz-E
comp RI R3 R4 12 E Affinity
BA H Cl H NHCOMe -
BB H H Me NHCO p-CFA-C6H4 **
BC H H Me NHCO p-OCF~-C6H4 **
BD H H H NHSO, 4-CF3-
cyclohex[ 1 ]enyl
BE H Cl H NHCO(CH,),COOH -
BF H H H NO, -
BG H H H NHCHzCH(CN)COOEt -
BH H Cl H NCHN(Me)2 -
BI H C1 H NHCH2CH(CN)COOEt -
BJ H Cl H NHS02Me -
BK H Cl H NHCHNOH -
BL H Cl H 1-pyrolyl -
BM H Cl H NHCOEt -
BN H CI H NHCO- _
transCH=CHCOOH
BO H Cl H NHCONHCO m-Cl-C6H4
BP H Cl H NHCONHCO 2,6
dimethoxyphenyl
BQ H H H NHCO 3-pyridyl
BR H H H NHCO 3-phenyl phenyl
BS H H H NHCO 3-napthyl
BT H H H NHCO 3,5-(CF;)~-C6H3
BU H H H NHCO cyclohexyl
BV H C1 H NHCO m-C1-C6H4
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DN H H H NHCO
~t-Bu
DU Me H H NHCOCH, - ~S
DW Me H H NHCO - ~
DZ H H H NHCO
- \'CF3
EL Me H H NHCO
EM Me H H NHCO ~s'~
EN Me H H NHCO ~ ~ ~
ER Me O H NHCO /
s\
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I
L
-E
3
2
comp R3 L2 E Affinity
B H NHCO 2-pyridyl
W
BX H NHCO 3-napthyl
BY H NHCO 3,5-(CF;)2-C6H3
BZ H NHCO cyclohexyl
CA CF3 OCO p-tBu-C6Hq
CB CF3 OCO 3-pyridyl
CC CF3 OCO (p_C6H5)-C6H4
CD CFA OCO 3-napthyl
CE CF3 OCO p-(O(CH2)SCH3)-C6H4
CF CF; oCOCH~o phenyl
CG CF3 OCO m-cyanophenyl
CH CF3 OCO p-cyanophenyl
CI CF3 OCO 3,5-(CF;)2-C6H3
CJ CF3 OCO cyclohexyl
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0
A/ ~ ~ O
N ~ / 'E
~N
H
Compound A E Affinity
CK Me p-tBu-phenyl
CL Me 3-pyridyl
CM Me (p-C6H5)-C6H4
CN Me 3-napthyl
CO Me p-(O(CH2)5
CH3)-C6H4
CP Me -CH2-O-C6H5
CQ Me m-CN-C6H4
CR Me p-CN-C6H4
CS Me 3,5-(CF3)2-C6H3
CT Me cyclohexyl
DF ~ I ~ p-CF3-phenyl
\ N
DM \ °~~,f'~ p-CFA-phenyl
EG p-CF3-phenyl
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N\ O ~ O
N R~ N
H
R6
Compound R, R6 Affinity
DB H CF3
DL Me O-CF3
0
Z~ R~ N
H
CF3
Compound Z, ZZ R, L, Z3 Z4 Affinity
DK CH N H S N CH
EC CH CC1 H CH~ N N
EH N CH Me O N CMe
EI N CH H O N CMe
EK CH Cowte H / I ", N N
N"u
EP CH coM~ H ~J'NH N N
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Example 3: Direct Binding Assay (DB Assay) for Identification of GPCR Binding
Compounds
CCRIO Manual Binding Assay Method
CCR10 receptors were expressed in stably transfected K293 cells, and the cells
were maintained in DMEM base medium, 10% FBS, 1X glutamine, and 0.4 mg/ml
6418, and in the incubator set at 37 oC, 6.0 % C02 and 90% relative humidity.
The day
before the experiment, the cells were trypsinized and 200 pl of the cell
suspension
(150,000 cells/ml) was deposited into 96-well Biocoat plates (poly-D-lysine-
coated).
The binding assay was performed 24 hours later.
8-point dose response curves were generated as follows: compounds to be tested
were dissolved in DMSO at 10 mg/ml concentration and diluted to 100 pg/ml into
n-
butanol. 0.75, 1.5, 3, 6, 12, 18, 24, and 30 pl of the compounds were
dispensed into 8
wells of a 96-well Costar plate. In the case of more potent compounds, further
dilution
was applied to generate 10 pg/ml compound solutions in n-butanol, and the
solutions
were used to make the compound plates by dispensing 1, 2, 4, 8, 10, 20 pl of
the diluted
solutions, and 4 and 8 pl of the 100 pg/ml solutions, into 8 wells of the
plate. The
compound plates were placed in the hood overnight to evaporate the butanol,
leaving
dried films of the compounds. On the day of the binding assay, 50 ~l of
filtered binding
buffer (25 mM HEPES, pH 7.4, 75 pM EDTA, I 1.5 mM KCI, 115 mM NaCI, 6 mM
MgS04, and 1.8 mM CaCl2) was added into each well, and the plates were stored
at 4
oC for about two hours. Before performing the binding assay, the compounds
were
thoroughly resuspended in the binding buffer.
The cells were washed three times with the binding buffer by adding and then
decanting the medium or binding buffer and drying the plates on paper towels.
Then the
buffer was added slowly to the side wall of the wells to avoid disturbing the
cells. After
the third wash, the plates were dried over paper towels to leave about 5 ~l of
the binding
buffer in each well. An additional 10 ~l of binding buffer was added to each
well. 15 ~l
of the compound solutions, and then I 5 pl of the Eu3+-labeled MCP-1 ligand
solution
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( 15 nM) in binding buffer with 0.1 % BSA was added. The binding reaction
mixtures
were maintained at RT for three hours.
After three-hour of incubation, 150 ~l of the wash buffer (2~ mM HEPES, pH
7.4, 0.1 mM CaCl2 and 115 mM NaCI) was added into each well and the solution
was
decanted and the plates were dried over paper towels. Subsequently, three sets
of three
washes were performed. Fresh wash buffer was used at each step. and pipette
tips were
also changed to avoid cross-contamination of the Eu3+-labeled ligand. The
buffer
solution of the final wash was decanted and the plate thoroughly dried on a
paper towel.
For each set, 100 ~1 of the enhancement buffer (Wallac) was added, and
incubated with
the cells at RT for one hour. Time-resolved fluorescence (excitation
wavelength 320
nM, emission 615 nM) was measured on the Wallac Victor fluorescence reader
Inhibition of MCP-1 binding was determined according to the following formula:
inhibition = [1 - (test well cps - background cps) ( (no inhibition control
cps -
background cps)] (100%)
Figures 1, 2, and 3 depict the binding curves for Compounds A, CU and CV,
respectively.
Example 4: Cell Based Inflammatory Recruitment Assay (CBIR Assay)
THP-1 Cell Motility Assays
A THP-1 cell line was used that expresses both CCR-10 and CCR-2. The cells
were
placed in the top half of a chamber (Transwell plate, 24-well, 5 ~M pore size,
purchased
from Costar) separated in the middle by a membrane. A gradient of chemokine
(MCP-1
or MCP-3, purchased from R&D) was established which, in the absence of
inhibitors,
leads to migration of cells across the membrane. Quantitation of cell
migration was done
using a FACScan machine. The % migration was calculated as the number of
migrated
cells divided by the number of input cells. As shown in Figures 4 and 5,
compounds B
and C blocked chemokine induced migration of cells.
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Example 5: Modulation of Recruitment of Inflammatory Cell Types using GPCR
Binding Compounds B and C in a Murine Inflammatory
Recruitment Assay (MIR Assay)
Mouse Peritoneal Infiltration Studies
CCR-10 and its ligands MCP-1, MCP-3 (MCP-5 in mice) have been demonstrated to
mediate the recruitment of eosinophils and a variety of other leukocytes to
tissue where
it is expressed. Antibodies against CCR-10 have been shown to block the
effects of
these ligands. This example demonstrates that compounds B and C are capable of
blocking MCP-5 induced peritoneal eosinophil recruitment.
Mice and in vivo procedures:
8-10-wk-old C57BL/6J mice were purchased from the Jackson laboratory (Bar
Harbor, ME) and kept in Millennium Pharmaceuticals Inc. Specific Pathogen Free
mouse facility.
Peritoneal recruitment assays in vivo with MCP-5 or mMCP-1 (mJE) protein
were performed after injection of lmg/mouse i.p. of either MCP-5 or mMCP-1.
Two
hours after chemokine injection, peritoneal lavage was performed and
leukocytes from
this organ were collected and enumerated. In one series of blocking
experiments, mice
were injected i.v. either with 50 nmol/Kg (7 mM/mouse) or 100 nmol/Kg (15
mM/mouse) of compound B or C 30 min before MCP-5 or mMCP-1 administration.
Immunohistochemical phenotyping and quantitation of leukocytes.
Total peritoneal cell counts were performed and aliquots (5x105 cells/slide)
were
pelleted onto glass slides by cytocentrifugation. To determine the number of
eosinophils
and mononuclear cells, slides were stained with Wright-Giemsa (Fisher
Diagnostics,
Pittsburgh, PA). T-lymphocytes, B-lymphocytes and mononuclear phagocytes were
identified by Thy 1.2 (53-2.1 ) (PharMingen, San Diego, CA), IgM (II/41 )
(PharMingen,
San Diego, CA) and Moma-2 (Biosource Int. Camarillo, CA) staining. Percentage
of
eosinophils, lymphocytes, neutrophils and macrophages was determined by
counting
their number in eight high power fields (40x magnification; total area 0.5
mm2) per area
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randomly selected and dividing this number by the total number of cells per
high power
field. To obtain the absolute number of each leukocyte subtype in the lavage,
these
percentages were multiplied by the total number of cells recovered from the
peritoneal
fluid.
As shown in Figure 6, mice pretreated with compounds B and C prior to MCP-5
challenge showed significantly reduced levels of eosinophil recruitment than
untreated
mice. Control experiments with eotaxin demonstrate that the compounds are
acting
through CCR-10 rather than by inhibition of cytoskeletal function.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments and
methods
described herein. Such equivalents are intended to be encompassed by the scope
of the
following claims.
