Note: Descriptions are shown in the official language in which they were submitted.
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MACROPHAGE SCAVENGER RECEPTOR AvNTAGONISTS
FIELD OF INVENTION
Cardiovascular diseases are the leading cause of death in the U.S., accounting
annually for more than one million deaths. Atherosclerosis is t:he major
contributor to
coronary heart disease and a primary cause of non-accidental death in Western
societies.
Since the prevention of atherosclerosis is an enormous unmet medical need,
considerable
effort has been made in defining the etiology and potential treatment of
atherosclerosis and
its consequences, including myocardial infarction, angina, organ failure and
stroke.
Despite this effort, there are many unanswered questions including how and
when
atherosclerotic lesions become life-threatening, the best point of
intervention, and how to
detect and monitor the progression of lesions.
There is widespread agreement that multiple risk factors contribute to
atherosclerosis including hypertension, elevated total serum cholesterol, high
levels of low
density lipoprotein ("LDL") cholesterol, low levels of high density
lipoprotein ("HDL")
cholesterol, diabetes mellitus, severe obesity, and cigarette smoking. To
date, treatment of
atherosclerosis has been narrowly focused on treating elevated <;holesterol
levels and
modifying lipids has become the major focus of treatment and research.
However, recent studies have indicated that 40% of deaths due to coronary
disease
occurred in men with total cholesterol levels of below 220 mg/dI. It is thus
obvious that too
great an emphasis is being placed on Lipid lowering. Indeed, only 30% of
patients with
atherosclerosis have elevated lipid levels, indicating that other pathogenic
factors are
involved. A logical scenario for future therapies and preventive measures
should therefore
include a multidisciplinary approach consisting of dial modification, HMG-CoA
reductase
inhibition and novel therapies aimed directly at plaque growth and stability.
The initial lesion in atherosclerosis is the fatty streak, which arises from
cholesteryl
esters maintained as lipid droplets inside macrophage-derived foam cells.
Macrophages
down-regulate their LDL receptors and instead express mRNA a.nd undergo new
protein
synthesis for a novel receptor for modified LDL. This receptor recognizes all
modified
forms of low-density lipoprotein and has come to be known as the macrophage
scavenger
receptor ("MSR"). If the macrophage is present in an environment that is
continually
generating modified LDL, it will accumulate Lipid droplets of cholosteryl
esters, continuing
until the macrophage dies from its toxic lipid burden. The released lipid then
forms the
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acellular necrotic core of the atherosclerotic lesion. Subsequent recruitment
of fibroblasts,
vascular smooth muscle cells and circulating monocytes and T-lymphocytes
complete the
inflammatory response and formation of the mature atheroscierotic plaque.
Macrophage-
derived foam cells are concentrated in the shoulders of plaque:>, where their
secreted
proteases and collagenases may contribute to plaque rupture which may lead to
a fatal
thrombotic event.
Plaque regression, a function of the dynamic balance among initiation,
progression,
stabilization and removal of plaque constituents, has been unequivocally
demonstrated in
humans as well as in numerous animal models. Multiple regression studies in
non-human
primates have shown that even relatively advanced lesions regress over time
when
atherogenic dietary stimuli are discontinued or pharmacological regimens are
initiated.
Inhibition of lipid accumulation within macrophage-de;rived foam cells by
utilizing
MSR antagonists is expected to prevent plaque initiation, retard plaque
progression, and
initiate plaque regression through the process of "reversed cholLesteroi
transport" to acceptor
HDL. Thus, MSR antagonists provide a unique approach towards the
pharmacotherapy of
cardiovascular diseases such as atherosclerosis, coronary artery disease,
renal disease,
thrombosis, transient ischemia due to clotting, stroke, myocardial infarction,
organ
transplant, organ failure, and hypercholesterolemia.
SUMMARY OF THE INVENTION
The present invention involves compounds represented) by Formula (I)
hereinbelow
and their use as macrophage scavenger receptor ("MSR") antag;onists which are
useful in
the treatment of a variety of cardiovascular diseases including but not
limited to
atherosclerosis, coronary artery disease, renal disease, thrombosis, transient
ischemia due to
clotting, stroke, myocardial infarction, organ transplant, organ failure and
hypercholesterolemia.
The present invention further provides methods for antagonizing the macrophage
scavenger receptor in animals, including humans, comprising administering to
an animal in
need of treatment an effective amount of a compound of Formula (I), indicated
hereinbelow.
The present invention further provides methods of inhibiting lipid
accumulation
within macrophage-derived foam cells.
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DETAILED DESCRIPTION OF THE INVENTION
The compounds of the present invention are selected from Formula (I)
hereinbelow:
(RZ)'" ~H (R~m
O~)" Formula (I)
wherein
R I is independently selected from the group consisting of hydrogen,
fluoroalkyl, halo,
haloaryl, aryl, and alkoxy; or R i represents a fused ring forming a
naphthalene moiety with
the six membered aryl ring it substitutes;
R2 is independently selected from the group consisting of hydrogen, R1-
benzamido, R1-
benzyi ether, R 1-benzylamino, amino, halo, hydroxy, alkoxy, a.lkyi,
fluoroaikyl, cyano,
vitro, aryioxy, nitroalkyl, aryl, and 1,2-benzo; or the R2 moiety represents a
fused ring
farming a napthalene ring with the six membered aryl ring it substitutes;
m is an integer from 1 to 4; and
n is an integer from 1 to 3.
Preferably, R 1 is selected from the group consisting of hydrogen, 5-
trifluoromethyl,
5-chloro, 5-bromo, 4-bromo, 5-bromo-4-phenyl, 5-iodo, 5-iodo-4-phenyl, 4-
phenyl, 5-
phenyl and 5-methoxy. More preferably, R1 is hydrogen, 5-trifluoromethyl or 5-
bromo.
Preferably, any R2 aryl substituents are selected from t;he group consisting
of
hydroxy, halo, aryl, aIkyi, cyano, vitro, R 1- benzamidyl, alkoxy and aryloxy.
More
preferably, R2 is selected from the group consisting of 2-chloro~, 3,4-
dichloro, 2,3-dichloro,
3-methoxy, 2-isopropyl, 3-cyano, 4-butyl, 2-vitro, 2-phenoxy, ~;-vitro-4-
methyl, 2-phenyl,
4-phenyl, 2-benzamidyl, 1,2-benzo. Most preferably, R2 is 3,4.-dichloro, 4-
bromo, 4-
phenyl or 4-butyi.
As used herein, "alkyl" refers to an optionally substitutE;d hydrocarbon group
joined
together by single carbon-carbon bonds. Preferred alkyl substituents are as
indicated
throughout. The alkyl hydrocarbon group may be linear, brandied or cyclic,
saturated or
unsaturated.
As used herein, "aryl" refers to an optionally substituted aromatic group with
at
least one ring having a conjugated pi-electron system, containing up to two
conjugated or
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fused ring systems. "Aryl" includes carbocyclic aryl, heteroc:yclic aryl and
biaryl groups,
ail of which may be optionally substituted. Preferred aryl substituents are as
indicated
throughout.
The compounds of the present invention may contain one or more
asymmetric carbon atoms and may exist in racemic and optically active forms.
All
of these compounds and diastereomers are contemplated to be. within the scope
of
the present invention.
Particularly preferred compounds useful in the present invention include:
bis-N-(4-biphenyl}-5-bromo-2-hydroxyisophthalic dicarboxannide,
bis-N-(4-biphenyl)-S-trifluorornethyl-2-hydroxyisophthalic dicarboxamide, and
bis-N-(4-
biphenyl)- 2-hydroxyisophthalic dicarboxamide,
bis-N-(3,4-dichlorophenyl)-2-hydroxyisophthalic dicarboxamiide,
bis-N-(3;4-dichlorophenyl}-5-bromo-2-hydroxyisophthalic dic:arboxamide,
bis-N-(3,4-dichlorophenyl-2-hydroxy-S-trifluoromethyIisaphtlhalic
dicarboxamide,
bis-N-(4-bromophenyl)-S-bromo-2-hydroxyisophthalic dicarboxamide,
bis-N-(4-bromophenyl)-2-hydroxyisophthalic dicarboxamide, and
bis-N-(4-bromophenyl-2-hydroxy-S-trifluoromethylisophthaiic dicarboxamide.
The present compounds can also be formulated as pharmaceutically acceptable
salts and complexes thereof. Pharmaceutically acceptable salts are non-toxic
salts in the
amounts and concentrations at which they are administered.
Pharmaceutically acceptable salts for use when basic groups are present
include
acid addition salts such as those containing sulfate, hydrochloride, fumarate,
maleate,
phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate,
ethanesulfonate,
benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate anti quinate.
Pharmaceutically
acceptable salts can be obtained from acids such as hydrochloric acid, malefic
acid, sulfuric
acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid,
tartaric acid,
malonic acid, methanesuifonic acid, ethanesulfonic acid, benze;nesulfonic
acid, p-
toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid.
Pharmaceutically acceptable salts also include basic adldition salts such as
those
containing benzathine, chloroprocaine, choiine, diethanolaminc:,
ethylenediamine,
meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium,
ammonium, alkylamine, and zinc, when acidic functional groulps, such as
carboxylic acid or
phenol are present.
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The present invention provides compounds of Formula (I) above which can be
prepared using standard techniques. An overall strategy for preparing
preferred compounds
described herein can be carried out as described in this sectiom. The example
which follows
illustrates the synthesis of specific compounds. Using the protocols described
herein as a
model, one of ordinary skill in the art can readily produce other compounds of
the present
invention.
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Scheme I
-- _
OH O O ; CI O'~ O
(COCI)2
I \ OH ~.. ~ O~ ~ CI
/I
L
2
CI ~ NH2
CI /
3
O O ~ O t:l
CI / \ H ~ H ~ ~ CI
4
BBr3
r
H CI
CI O O~ O
CI / \ H ~ H ~ ~ ~CI
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Compound 1 is commercially available (AIdrich). The carboxylic acids can be
activated
towards condensation with amines by a variety of means. One: of the most
convenient is
conversion to the bis-acid chlorides such as 2 by oxalyl chloride. Many other
methods of
making acid halides are well known as are procedures for preparing esters and
especially
activated esters. Many procedures are well known for the cleavage of aromatic
methyl
ethers including hydrohalic acids and nucleophiles such as sulivr and selenium
compounds.
An especially effective reagent is boron tribromide or an equivalent such as
boron
trichloride-sodium iodide.
Examyle 1
Bis-N,N'-(3,4-Dichlorophenyi)-2-hydroxyisophthalic dicarbox;amide (5)
A suspension of 0.89 g (4.61 mmol) of 2-methoxyisoplhthalic acid in 25 ml of
CH2C12 was treated with 4.68 g (36.8 mmol) of oxalyl chloride and a few drops
of DMF.
After 2.5 hr the reaction mixture was filtered, concentrated under vacuum,
toluene added,
and again concentrated under vacuum to a small volume. The :residue was
stirred with 150
ml of toluene and treated with 50 ml of a toluene solution containing 2.27 g {
14 mmol) of
3,4-dichloroaniline and 3.2 ml {23.05 rnmol) of triethylamine. After 18 hr.
the reaction
mixture was filtered and the collected solids washed with fresh toluene. The
solid was
dissolved in EtOAc, the organic solution washed with water and then brine, and
dried over
MgS04. Concentration under vacuum gave bis-N,N=(3,4-dichlorophenyl)-2-
methoxyisophthalic dicarboxarnide (4} as a creamy white powder, mp 205-206C.
A suspension of 4 {250 mg, 0.52 rnmol) in 25 ml of CI f2C12 under argon was
treated with 1.3 ml of a 1 M solution of boron tribrornide ( i .3 rr~mol).
After 4 hr 15 mi of
MeOH was added and after 20 min concentrated under vacuum to give a solid
which was
recrystaIlized from toluene. This gave bis-N,N =(3,4-dichlorophenyl)-2-
hydroxyisophthalic
dicarboxamide as straw yellow crystals, mp 232-235C.
By a similar procedure using 4-aminobiphenyl in place of 4, bis-N,N =(4-
biphenyl)-
2-hydroxyisophthalic dicarboxamide, mp 200-2020 C is obtained. Similarly using
4-
bromoaniline in place of 4 gives bis-N,N=(4 bromophenyl)-2-hydroxyisophthalic
dicarboxamide mp 189-191°. Using 4-bromo-2-methoxy-isophthalic acid in
place of 1
gives 4-bromo-bis-N,N'-(3,4-dichlorophenyl)-2-hydroxyisophtliaiic
dicarboxamide, mp
292-295o C, 4-bromo-bis-N,N =(4-bromophenyl)-2-hydroxyisophthalic
dicarboxamide
soften 169-1700 C, mp 230-240o and 4-bromo-bis-N,N'-(4-biphenyl)-2-
hydroxyisophthalic
dicarboxamide mp 216-219o C.. Using 4-trifluoromethyl-2-mf;thoxy-isophthaIic
acid in
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place of 1 gives 4-trifluoromethyl-bis-N;N'-(3,4-dichlorophenyl)-2-
hydroxyisophthalic
dicarboxamide, 4-trifluoromethyl-bis-N,N'-(4-bromophenyl)-2-hydroxyisophthalic
dicarboxamide, and 4-trifluoromethyl-bis-N,N'-(4-biphenyl)-2-
hydroxyisophtkialic
dicarboxamide. Other 2-methoxyisophthalic acids and anilines may also be used
to give
the corresponding anticipated products. Refluxing a mixture of 2,4-
dihydroxyisophthalic
acid, PC13, and 4-chloroaniline in chlorobenzene gives N,N'-bi.s-(4-
chlorophenyl)-2,4-
dihydroxyisophthalamide.
Unsymmetrical 2-hydroxyisophthalanilides may be made by stepwise reaction of 2-
methoxyisophthalic diacid chloride to give a monoanilide, hydrolyzing the
unreacted acid
chloride, and then reacting the resulting acid with an aniline using standard
amide forming
reagents. Finally the methoxy group is cleaved to give the required 2-hydroxy
derivative,
Sd~eme 2
CI O ~ O (~ ~ C! O ~ O
f HzN~ ~ O .~N~
o~~ a
ESN I ,J H i.
2 6
/ I
\ ~/ ~i O O~ ~ ~8r ~ pl"E O~ O I~&'
H ~~ ~~ ~~~ ~~ H
7
~3
\ / I O O.HO / ~ Br
~N~~~N \
i
H
9
_g_
SUBSTITUTE SHEET (RULE 21~)
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Example 2
1-[N-(4-Bromophenyl)]-2-hydroxy-3-[N=(4-biphenyl)]isophthalic dicarboxamide
(9)
A solution of 0.44 g (2.6 mmol) of 4-bromoaniline and 0.36 ml (2.5 mmol) of
triethylamine in
25 mI of dry toluene was added dropwise to a solution of 2-me;thoxyisophthalic
diacid chloride
0.59 g (2.55 mmol) in 40 ml of toluene. After stirring for 18 hr at 25°
C the volatiles were
removed under vacuum and the residue dissolved in 30 ml of EtOAc. The solution
was washed
in turn with water, dil HCI, water, and 10% acqueous NaOH. .Addition of HCl to
bring the pH
of the alkaline extract to I gave the monoanilide 7, mp 193 - 196° C
which had the anticipated
MS.
I O A mixture of 0.35 g ( 1 mmol) of 9, 0.148 g ( 1.1 mmol) of H013T, 0.211 g
( 1.1 mmol) of
EDC.HCI, and 0.38 ml (2.2 mrnol) of DIEA in 1-methyl-2-pyrrolidinone was
stirred at 25° for
0.5 h and then 0.22g (2.2 mrnol) of 4-aminobiphenyl and 0.018 g (0.15 mmol) of
DMAP added.
After stirring at ambient for 18 hr the mixture was diluted with water,
extracted with EtOAc
and the extract washed with water, 10 % aqueous HCl, water, :i% Na2C03, water,
and brine. It
I 5 was dried over MgS04~ concentrated under vacuum, and the reaidue
recrystallized from
methanol fo give 8, mp 205-208°C.
A solution of 0.31 g (0.61 mmol) of 8 in 30 ml of CH2Cl2 was. treated with a
1.OM solution of
BBr3 in CH2Cl2 (1.52 ml, 1.52 mmol) at 25°C for 18 hr. Met)H was added
cautiously to
20 decompose excess reagent, and then the solution was concentrated under
vacuum to give a
yellow residue which was dissolved in MeOH, fltered, concentrated under
vacuum, and
triturated with hexane to give a yellow solid. This was dissolved in CH2C12,
filtered, and
diluted with hexane to give a solid which was dissolved in CHCl3 and filtered
through a short
silica column. Concentration gave 9, mp 214-2I8°C which gave
satisfactory elemental
25 analyses for C, H, N, and Br.
A similar procedure gave 3-{N-(4-bromophenyl)]-1-[N-(3,4-dichlorophenyl)]-2-
hydroxyisophthalic dicarboxamide, mp 218-220°C.
30 Example 3
N,N'-Bis-(4-chlorophenyl)-2>4-dihydroxyisophthalarnide
A mixture of 0.4529 g (2.4 mmol) of 2,4-dihydroxyisoophthalic acid, 0.62 g
(4.84
mmoI) of 4-chloroaniline, and 0.22 rni (2.2 mmol) of PCl3 in 10 ml of
chlorobenzene was
refluxed for 3 hr. The hot reaction mixture was filtered and on cooling
filtration gave the
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desired product which on purification by preparative HPLC (C:18, 20 - 95%
acetonitrile - 0.1 %
aqueous TFA) gave saatisfactory elemental analyses for C,H, ;end N.
With appropriate manipulation and protection of any chemical functionality,
synthesis of the remaining compounds of Formula (I) is accomplished by methods
analogous to those above and to those described in the Experimental section.
In order to use a compound of Formula (I) or a pharmaceutically acceptable
salt
thereof for the treatment of humans and other mammals, it is normally
formulated in
accordance with standard pharmaceutical practice as a pharmaceutical
composition.
The present compounds can be administered by different routes including
intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical
(transdermal), or
transmucosal administration. For systemic administration, oral administration
is preferred.
For oral administration, for example, the compounds can be formulated into
conventional
oral dosage forms such as capsules, tablets, and liquid preparations such as
syrups, elixirs,
and concentrated drops.
Alternatively, injection (parenteral administration) may be used, e.g.,
intrarnuscular, intravenous, intraperitoneal, and subcutaneous. For injection,
the
compounds of the invention are formulated in liquid solutions, preferably, in
physiologically compatible buffers or solutions, such as saline solution,
Hank's solution, or
Ringer's solution. In addition, the compounds may be formulated in solid form
and re-
dissolved or suspended immediately prior to use. Lyophilized forms can also be
produced.
Systemic administration can also be by transmucosal o~r transdermal means. For
transmucosal or transdermal administratian, penetrants appropriate to the
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art, and
include, for example, for transmucosal administration, bile salts and fitsidic
acid
derivatives. In addition, detergents may be used to facilitate permeation.
Transmucosal
administration, for example, may be through nasal sprays, rect;~l
suppositories, or vaginal
suppositories.
For topical administration, the compounds of the invention can be formulated
into
ointments, salves, gels, or creams, as is generally known in the art.
The amounts of various compounds to be administered can be determined by
standard procedures taking into account factors such as the compound ICSg,
EC50, the
biological half life of the compound, the age, size and weight o~f the
patient, and the disease
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or disorder associated with the patient. The importance of these and other
factors to be
considered are known to those of ordinary skill in the art.
Amounts administered also depend on the routes of administration and the
degree
of oral bioavailability. For example, for compounds with low oral
bioavailability,
relatively higher doses will have to be administered.
Preferably the composition is in unit dosage form. For oral application, for
example, a tablet, or capsule may be administered, for nasal application, a
metered aerosol
dose may be administered, for transdermal application, a topical formulation
or patch may
be administered and for transmucosal delivery, a buccal patch may be
administered. In
each case, dosing is such that the patient may administer a single dose.
Each dosage unit for oral administration contains suiW bly from 0.01 to 500
mglKg,
and preferably from O.I to 50 mg/Kg, of a compound of Formula {I) or a
pharmaceutically
acceptable salt thereof, calculated as the free base. The daily dosage for
parenteral, nasal,
oral inhalation, transmucosai or transdermai rautes contains suitably from
0.01 mg to 100
mglKg, of a compound of Formula (I). A topical formulation contains suitably
0.01 to
5.0% of a compound of Formula (I). The active ingredient ma.y be administered
from 1 to
6 times per day, preferably once, sufficient to exhibit the desired activity,
as is readily
apparent to one skilled in the art.
As used herein, "treatment" of a disease includes, but its not limited to
prevention,
retardation and prophylaxis of the disease.
The MSR receptors described in the present application belong to a recently
classified group designated the SR-A group and exist in two forms, type A-I
and type A-II,
which arise through differential exon splicing of a single gene. The terms
"MSR" and "SR-
A" are used interchangeably in the present application.
Diseases and disorders which might be treated or prevented, based upon the
affected cells, include atherosclerosis, coronary artery disease, renal
disease, thrombosis,
transient ischemia during clotting, stroke, organ transplant, organ failure,
myocardial
infarction and hyperchoiesterolemia.
Composition of Formula (I) and their pharmaceuticall~~ acceptable salts which
axe
active when given orally can be formulated as syrups, tablets, capsules and
lozenges. A
syrup formulation will generally consist of a suspension or solution of the
compound or salt
in a liquid earner far example, ethanol, peanut oil. olive oil, glycerine or
water with a
flavoring or coloring agent. Where the composition is in the form of a tablet,
any
pharmaceutical carrier routinely used for preparing solid formulations may be
used.
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Examples of such carriers include magnesium stearate, terra alba, talc,
gelatin, acacia,
stearic acid, starch, lactose and sucrose. Where the composition is in the
form of a capsule,
any routine encapsulation is suitable, for example using the aforementioned
Garners in a
hard gelatin capsule shell. Where the composition is in the form of a soft
gelatin shell
capsule any pharmaceutical carrier routinely used for preparin;; dispersions
or suspensions
may be considered, for example aqueous gums, celluloses, silicates or oils,
and are
incorporated in a soft gelatin capsule shell.
Typical parenteral compositions consist of a solution or suspension of a
compound
or salt in a sterile aqueous or non-aqueous carrier optionally containing a
parenterally
acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone,
lecithin, arachis oil
or sesame oil.
Typical compositions for inhalation are in the form of a solution, suspension
or
emulsion that may be administered as a dry powder or in the form of an aerosol
using a
conventional propellant such as dichlorodifluoromethane or
trichlorofluoromethane.
A typical suppository formulation comprises a compound of Formula (I) or a
pharmaceutically acceptable salt thereof which is active when administered in
this way,
with a binding andlor lubricating agent, for example polymeric, glycols,
gelatins, cocoa-
butter or other low melting vegetable waxes or fats or their synthetic
analogs.
Typical dennal and transdenmal formulations comprise a conventional aqueous or
non-aqueous vehicle, for example a cream, ointment, lotion or paste or are in
the form of a
medicated plaster, patch or membrane.
Preferably the composition is in unit dosage form, for example a tablet,
capsule or
metered aerosol dose, so that the patient may administer a single dose.
No unacceptable toxological effects are expected when compounds of the present
invention are administered in accordance with the present invention.
The biological activity of the compounds of Formula (I) are demonstrated by
the
following tests.
Assays of MSR activity, both degradation and binding~~internalization, were
adapted from Goldstein et al., "Receptor-mediated Endocytosis of Low-density
Lipoprotein
in cultured Cells," Methods Enzymol., 98:241-260 ( 1983); incorporated herein
in its entirety
by reference. Briefly, 293 cells transfected with MSRI or II are seeded at 105
celis/ml/well
in a 24-well dish in Eagle's Minimal Essential Medium with 2 mM glutamine, 10%
FCS
and 0.4 mg/ml geneticin. After 2 days, the medium is replacedl with 5001.11
fresh serum-free
medium containing 2 mg/ml BSA and 125[I]-AcLDL (iodinate;d acetylated low
density
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lipoprotein) at Suglml, and cells are incubated at 37C for 5 hours. After this
suitable period
for ligand degradation, cells are removed to a 4C cold room. ;supernatant is
removed into
trichloroacetic acid, and the mixture is centrifuged. The supernatant is
chloroform-
extracted in order to isolate 125[I]-monoiodotyrosine, the degradation product
of 125[I]-
AcLDL, and portions are counted to determine degradative activity. To
determine cell-
associated ligand, cell monolayers are washed and incubated at 4C with ice-
cold buffer "A"
containing 150 mM NaCI, 50 mM Tris-HCI, and 2 mglml BSA, pH 7.4, to eliminate
nonspecifically bound counts. Cells are washed three times rapidly with 1 ml,
incubated
twice for 10 min each on a rotary shaker in 1 mI buffer A, then washed twice
rapidly in 1
ml buffer A without BSA. After aspiration of all wash buffer, cells are lysed
in O.1N NaUH
and removed to counting vials for determination of binding/uptake and
subsequent protein
determination (Pierce BCA protein assay). The present active; yield IC50
values of <50
um in degradation assays and <100um in binding/uptake assays.
The fluorescent compound DiI-AcLDL { 1,1'-dioctadec;yl-3,3,3',3'-
tetramethylindocarbocyanine perchlorate-labeled LDL) has al~~o been shown to
be a useful
tool in assessing activity of the macrophage scavenger receptor (Freeman et
al., Proc. Nail.
Acad. Sci., USA, 88:4931-4935 ( 1991 ); Penman et aL, J. Biol. ~Chem.,
266:23985-23993
(1991)). We also utilized an assay for MSR antagonists based on the uptake of
DiI-AcLDL
by the transfected HEK 293 cells. For most assays, HEK 293 cells transfected
with SR-AI
were used, although both SR-AI and SR-All appeared to have equivalent activity
in all
studies performed. Briefly, HEK 293 cells were seeded at 2 x 104 cellsl well
in a 96-well
plate in EMEM with 2mM glutamine, 10%FBS and 0.4mg/ml geneticin. The assay was
standardized and optimized, and testing was performed in serum-free EMEM
containing
2mg/ml bovine serum albumin. Confluent cells were incubated with DiI-AcLDL
(final
concentration 2uglml) In the absence and presence of inhibitors (quadruplicate
wells) for 4
hours at 37C. Following aspiration of solution and a Locke's buffer wash,
results were
quantified with a fluorescence plate reader at 530nm exc/590mm em.
All publications, including but not limited to patents and patent applications
cited
in this specification are herein incorporated by reference as if each
individual publication
were specifically and individually indicated to be incorporated by reference
as though fully
set forth.
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