Note: Descriptions are shown in the official language in which they were submitted.
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NAPHTHYL GLYOXAMIDES AS sPLA2 INHIBITORS
BACKGROUND OF THE INVENTION
This invention relates to novel naphthyl
glyoxamides useful for inhibiting sPLA2 mediated release of
fatty acids for conditions such as septic shock. These
compounds are also known as naphthyl oxyalamides or
naphthyl oxamides.
The structure and physical properties of human
non-pancreatic secretory phospholipase A2 (hereinafter
called, UsP~A2'') has been thoroughly described in two
articles, namely, ~Cloning and Recombinant Expression of
Phospholipase A2 Present in Rheumatoid Arthritic Synovial
Fluid~ by Seilhamer, Jeffrey J.; Pruzanski, Waldemar; Vadas
Peter; Plant, Shelley; Miller, Judy A.; Kloss, Jean; and
Johnson, Lorin K.; The Journal of Bioloaical ChemistrY,
Vol. 264, No. 10, Issue of April 5, pp. 5335-5338, 1989;
and "Structure and Properties of a Human Non-pancreatic
Phospholipase A2" by Kramer, Ruth M.; Hession, Catherine;
Johansen, Berit; Hayes, Gretchen; McGray, Paula; Chow, E.
Pingchang; Tizard, Richard; and Pepinsky, R. Blake; The
Journal of Bioloaical Chemistr~, Vol. 264, No. 10, Issue of
April 5, pp. 5768-5775, lg89; the disclosures of which are
incorporated herein by reference.
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It is believed that sPLA2 is a rate limiting
enzyme in the arachidonic acid cascade which hydrolyzes
membrane phospholipids. Thus, it is important to develop
compounds which inhibit sPLA2 mediated release of fatty
acids (e.g., arachidonic acid). Such compounds would be of
value in general treatment of conditions induced and/or
maintained by overproduction of sPLA2; such as septic
shoc~, adult respiratory distress syndrome, pancreatitis,
trauma, bronchial asthma, allergic rhinitis, rheumatoid
arthritis, and etc.
The Communication to the Editor by Hayden G. Beaton
et al., Journal of Medicinal Chemistry, 1994, Vol. 37, No. 5,
describes various novel ~naphthylthio)methyl analogs of non-
phospholipid sPLA2 inhibitors.
It is desirable to develop new compounds and
treatments for sPLA2 induced diseases.
This invention is a novel use of compounds known
as naphthyl glyoxamide compounds as depicted in the general
formula I:
Y~ ~~
Rl
X ~_ R2
where Rl, R2, X and Y are as defined below.
These naphthyl glyoxamide compounds are effective
in inhibiting human sPLA2 mediated release of fatty acids.
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-3-
This invention is also a novel class of naphthyl
glyoxamides having potent and selective effectiveness as
inhibitors of human sPLA2.
This invention is also a process for preparing
the novel class of naphthyl glyoxamide compounds.
This invention is also a pharmaceutical
composition containing a naphthyl glyoxamide compound.
This invention is also a method of preventing and
treating septic shock, adult respiratory distress syndrome,
la pancreatitis, trauma, bronchial asthma, allergic rhinitis,
rheumatoid arthritis, and related diseases by contact with
a therapeutically effective amount of the naphthyl
glyoxamide.
Defi n itions:
The napthyl glyoxamides of the invention employ
certain defining terms as follows:
The term, ~alkyl~' by itself or as part of another
substituent means, unless otherwise defined, a straight or
branched chain monovalent hydrocarbon radical such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, tertiary
butyl, isobutyl, sec-butyl, n-pentyl, and n-hexyl.
The term, ~alkenyl n employed alone or in
combination with other terms means a straight chain or
2~ branched monovalent hydrocarbon group having the stated
number range of carbon atoms, and typified by groups such
as vinyl, propenyl, crotonyl, isopentenyl, and various
butenyl isomers.
The term ~C2-C6 alkynyl" refers to straight and
branched chains of 2 to 6 carbon atoms, both inclusive,
having a triple bond. As such, the term includes acetylene,
propyne, 1-butyne, 2-butyne, 1-pentyne, 2-pentyne, 3-methyl-
1-butyne, 1-hexyne, 2-hexyne, 3-hexyne and the like.
The term, Uhalo'' means fluoro, chloro, bromo, or
iodo.
The term, ~non-interfering substituent~, refers to
radicals suitable for substitution on the phenyl ring
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attached to the naphthalene ring. Illustrative non-
interfering radicals are C1-C6 alkyl, C2-C6 alkenyl, C2-C6
alkynyl, C7-C~2 aralkyl, C7-C12 alkaryl, C3-Cg cycloalkyl,
C3-Cg cycloalkenyl, phenyl, toluyl, xylenyl, biphenyl, C1-C6
alkoxy, Cl-C6 alkenyloxy, Cl-c6 alkynyloxy, C2-C12
alkoxyalkyl, C2-C12 alkoxyalkyloxy, C2-C12 alkylcarbonyl, C2-
C12 alkylcarbonylamino, C2-cl2 alkoxyamino, C2-C12
alkoxyaminocarbonyl, C1-C12 alkylamino, C1-C6 alkylthio, C2-
C12 alkylthiocarbonyl, C1-C6 alkylsulfinyl, C1-C6
alkylsulfonyl, Cl-C6 haloalkoxy, C1-C6 haloalkylsulfonyl, C1-
C6 haloalkyl, Cl-C6 hydroxyalkyl, -C(O)O~Cl-C6 alkyl),
-(CH2)n-0-(Cl-C6 alkyl), benzyloxy, phenoxy, phenylthio,
-(CONHS02R), -CHO, amino, amidino, bromo, carbamyl, carboxyl,
ethoxycarbonyl, -(CH2)n-CO2H, chloro, cyano, cyanoguanidinyl,
fluoro, guanidino, hydrazide, hydrazino, hydrazido, hydroxy,
hydroxyamino, iodo, nitro, phosphono, -SO3H, thioacetal,
thiocarbonyl, and C1-C6 carbonyl; where n is from 1 to 8.
ComDoun~ of the Invention
Compounds of the invention are represented by
formula I
Y' O ~ NH2
~ Rl
X~ R2
wherein:
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R1 and R2 are each independently hydrogen or a non-
interfering substituent with the proviso that at least one
of R1 or R2 must be hydrogen;
X is -CH2- or -0-; and
Y is (CH2)nZ where n is a number from 1-3 and Z is an
acid group selected from the group consisting of -CO2H, -SO3H
or -PO~OH)2-
Preferred Co~ounds
A preferred subclass of compounds of-formula I are
those wherein R1 and R2 are hydrogen or phenyl.
Another preferred subclass of compounds of formula
I are those wherein Y is (CH2jnZ where n is 1.
More partlcularly preferred are compounds wherein
R1 and R2 are hydrogen or phenyl, X is oxygen or -CH2- and Y
is -CH2C02H .
Specific preferred compounds and all
pharmaceutically acceptable salts, solvates and prodrug
derivatives thereof which are illustrative of the compounds
of the invention include the following:
~q~O O~NH'
HO
~'13
The salts of the above naphthyl glyoxamide
compounds are an additional aspect of the invention. In
those instances where the compounds of the invention possess
acidic or basic functional groups various salts may be formed
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which are more water soluble and physiologically suitable
than the parent compound. Representative pharmaceutically
acceptable salts, include but are not limited to, the alkali
and alkaline earth salts such as lithium, sodium, potassium,
calcium, magnesium, alllmi nt~m and the like. Salts are
conveniently prepared from the free acid by treating the acid
in solution with a base or by exposing the acid to an ion
exchange resin.
Included within the definition of pharmaceutically
acceptable salts are the relatively non-toxic, inorganic and
organic base addition salts of compounds of the present
invention, for example, ammonium, quaternary ammonium, and
amine cations, derived from nitrogenous bases of sufficient
basicity to form salts with the compounds of this invention
(see, for example, S. M. Berge, et al., ~'Pharmaceutical
Salts,~ J. Ph~r. Sci., 66: 1-19 (1977)). Moreover, the basic
group(s) of the compound of the invention may be reacted with
suitable organic or inorganic acids to form salts such as
acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate,
bitartrate, borate, bromide, camsylate, carbonate, chloride,
clavulanate, citrate, chloride, edetate, edisylate, estolate,
esylate, fluoride, fumarate, gluceptate, gluconate,
glutamate, glycolylarsanilate, hexylresorcinate, bromide,
chloride, hydroxynaphthoate, iodide, isothionate, lactate,
lactobionate, laurate, malate, malseate, mandelate, mesylate,
methylbromide, methylnitrate, methylsulfate, mucate,
napsylate, nitrate, oleate, oxalate, palmitate, pantothenate,
phosphate, polygalacturonate, salicylate, stearate,
subacetate, succinate, tannate, tartrate, tosylate,
trifluoroacetate, trifluoromethane sulfonate, and valerate.
Prodrugs are derivatives of the compounds of the
invention which have chemically or metabolically cleavable
groups and become by solvolysis or under physiological
conditions the compounds of the invention which are
pharmaceutically active in vivo. The prodrug derivative form
often offers advantages of solubility, tissue compatibility,
or delayed release in a mammalian organism (see, sundgard,
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H., Desian of Pro~ru~s, pp. 7-9, 21-24, Elsevier, Amsterdam
1985). Prodrugs include acid derivatives well known to
practitioners of the art, such as, for example, esters
prepared by reaction of the parent acidic compound with a
suitable alcohol, or amides prepared by reaction of the
parent acid compound with a suitable amine. Simple aliphatic
or aromatic esters derived from acidic groups pendent on the
compounds of this invention are preferred prodrugs. In some
cases it is desirable to prepare double ester type prodrugs
such as (acyloxy) alkyl esters or ((alkoxycarbonyl)oxy)alkyl
esters.
Synthesls Methods
Compounds of formula I where X is oxygen can be
prepared by the following reaction Scheme I.
Scheme I
HO OK+ CH
2 equ valen~ dlmethvl-
2) HCl
1 equivalent
OH o~ OH
(1) (2) (3)
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OH OCH3
appropriately
substituted
HOAC ~ phenol
~ K2CO3/CuO
(5) ~ (4) ~ R2
oxayl
chloride
~ o O AlCl~
4-dimethylamine ~ // 1) NaNH~ o
pyridene 1~ ~ 2~ BrCH2CO?CH
~o~ Rz (~/ )\~/
Step A
~ ~ O ~ CONH2 0 ~ ~ CONH2
l 1) NaOH
HO ~ ~ 2) HCl ~ ~ ~
~ R2 Step B (8) ~ / R2
In the above depicted reaction scheme, the 1, 5-
dihydroxy napthalene starting material (1) ls dispersed in
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water and then treated with 2 equivalents of potassium
hydroxide. The resultant solution is chilled in an ice bath
and one equivalent of a strong mineral acid, such as
hydrochloric acid, is added to produce the potassium salt
(2).
Alkylation of the radical (2) can then be
accomplished by treatment with a methylating agent such as
dimethyl sulfate to prepare the ether (3).
Preparation of (4) is achieved by reacting the
ether (3) with an appropriately substituted phenol in an
Ullman-type reaction using potassium carbonate and cupric
oxide.
De-methylatlon of ~4) can be accomplished by
treating (4) with a 40% HBr/HOAC solution at reflux in a
protic polar solvent such as acetic acid, to prepare (5).
Reflux of compound ~5) with oxalyl chloride and 4-
demethylamino pyridine, in an alkylhalide solvent such as
methylene chloride, prepares the oxalyl chloride (6).
Internal cycli~ation of (6) can be achieved under
Friedel-Crafts condition using aluminum chloride or other
similar metal halide as the catalyst. The reaction can be
conveniently conducted in an alkyl halide solvent, such as 1,
2-dichloro ethane.
Alkylation and hydrolysis of the cyclized
compound (7) can be achieved by reacting (7) with an
alkaliamide base, such as sodium amide, followed by treatment
with an alkylating agent, such as methyl bromoacetate, using
potassium iodide as a catalyst.
Finally, the acid (9) is achieved by treating the
ester (8) with an alkali base, such as aqueous sodium
hydroxide, followed by treatment with a dilute aqueous
mineral acid such as hydrochloric acid The acid compound (9)
is then extracted with an organic solvent such as ethyl
acetate.
The final product (9) can be purified using
standard recrystallization procedures in a suitable organic
solvent such as methylene ch~oride~hexane.
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X-9512 (EPO~ -10-
Compounds of formula I where X is methylene can be
prepared as shown in the following Scheme II
s
Scheme II
Br OCH3
l1) Mg, ET2O~
R2 j~ ~ ~,1 R2
CH30
3) aqueous acid
(la)
NaBH4
CF3C02H
H3CO
R2
-
(2a)
Using an appropriately substituted phenyl bromide,
a Grignard reagent is prepared. The phenyl Grignard is then
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reacted with 4-methoxy naphthylnitrile and the resultant
compound is hydrolyzed with a dilute acid such as
hydrochloric acid to form the benzoyl naphthylene compound
(la).
Reduction of (la) to form compound (2a) is
accomplished by treatment with a reducing agent such as
sodium borohydride. The reaction is conducted in a solvent-
catalyst such as trifluoroacetic acid and inltiated in an ice
bath which is allowed to warm to room temperature as the
reaction proceeds.
The deslred naphthyl glyoxamide may then be
prepared from (2a) according to the procedure in Scheme I
starting with the chloromethylation step.
It will be readily appreciated by a person skilled
in the art that the substituted benzyl bromide, substituted
phenol and substituted naphthylnitrile compounds of Schemes I
and II are either commercially available or can be readily
prepared by known techniques from commercially available
starting materials.
All other reactants used to prepare the compounds
in the instant invention are commercially available.
Process of the Invention
The process of the present invention provides a
method for synthesizing novel compounds of formula I
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~ o ~~ NH
R 1
X~, R2
wherein:
X is -O- or -CH2- which comprises reacting a compound of
the formula III
~ (III)
IRl
X~13 R2
wherein X, R1 and R2 are as defined above, with an alkaiamide
base;
alkylating with an alkylating agent to form a compound of the
formula II
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q~O ~
I
~ ~ ~ (II)
X~, R2
where Rl, R2 and X are as defined above and R3 is C~-C4 alkyl
and;
hydrolyzing the compound of formula II.
The process of the invention is illustrated in Scheme I,
steps A and B.
According-to the process of the present invention,
the a-ketolactone starting material ~compound III) is
dissolved in an aprotic polar solvent, preferably
dimethylformamide (DMF). Other suitable aproctic solvents
include tetrahydrofuran (THF), dimethyl sulfoxide ~DMSO) and
the like. The amount of solvent used should be sufficient to
ensure that all compounds stay in solution until the desired
reaction is complete. The solution is chilled in an ice bath
to from about -10~C to about -30~C, preferably -20~C.
After the starting material has been dissolved, an
aklaliamide base, such as sodium amide, is added to the
reaction mixture and the reaction is allowed to proceed, with
stirring for about 30 minutes to an hour, preferably an hour.
Preferably one mo~e of amide per mole of starting material
III is employed.
A mole of an alkylating agent, such as
methylbromoacetate, per mole of starting material is added to
the reaction mixture, along with a small amount of a
catalyst, preferably potassium iodide to prepare the ester
II. The reaction is allowed to proceed another 30 minutes to
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an hour, preferably for an additional hour, while maintaining
the tempera~ure below -10~C in an ice bath.
Preparation of the acid I is accomplished by
treating the ester III with an alkali base, such as sodium
hydroxide, follower by treatment with a strong inorganic acid
such as hydrochloric acid. The reaction is preferably
conducted in a low molecular weight alcohol such as methanol
at temperatures of from about 0~C to about 40~C, preferably
25~C.
The following examples further illustrate the
preparation of the compounds of this invention as well as the
compounds used in the method of this invention. The examples
are illustrative only and are not intended to limit the scope
of the invention in any way.
EX~PT~F. 1
Preparation of 8-carboxymethoxy-4-phenoxynaphth-l-yl
glyoxamide
A Preparation of the mono-potassium salt of the
dihydroxynaphthalene
Into a 3-neck flask fitted with a mechanical
stirrer and thermometer, 150 mL water, followed by 140g
potassium hydroxide was added. To the flask was added 128g
1,5-dihydroxynapthalene under an argone atmosphere, and the
mixture was stirred for 5 minutes. The mixture was cooled in
an ice bath, and 105 mL of concentrated (37%) hydrochloric
acid was added over 15 minutes, producing the mono-potassium
salt of the dihydroxynaphthalene starting material.
B. Preparation of l-hydroxy-5-methoxynaphthalene
The solution of the potassium salt, prepared as
above, was treated with 126g dimethyl sulfate, added dropwise
so as not to exceed 30~C. The reaction was stirred for an
additional 4 hours at room temperature, and maintained at
70~C for 20 min. After cooling, the reaction mixture was
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filtered, and the precipitate was washed with aqueous
potassium hydroxide, combining the washings with the
filtrate. The combined filtrate was acidified with
concentrated hydrochloric acid, and the precipitated 1-
hydroxy-5-methoxynaphthalene was f ltered, washed with water,
and dried under vacuum at 50~C, yielding 82.2g (59%).
M.P. : 127-8~C
Elemental Analysis for CllH1002:
Calculated: C, 75.84i H, 5.79; O, 18.37;
Found: C, 75.58; H, 5.79; O, 18.30.
C. Preparation of 1-methoxy-5-phenoxynaphthalene
In a 3-neck flask fitted with a mechanlcal stirrer,
26.lg of the 1-hydroxy-5-methoxynaphthalene prepared as above
was treated with 16 ml bromobenzene, 41.4g potassium
carbonate, and 24.g of copper(I)oxide in 300 ml pyridine at
reflux under argone. After 16 hours, an additional 3.2 mL of
bromobenzene and 4.8g of copper oxide were added and the
reaction was continued for 4 hours. The reaction was cooled
and filtered over supercel with an ethyl acetate washing. The
combined filtrate was concentrated under vacuum, diluted with
ethyl acetate, and shaken twice with cold dilute hydrochloric
acid. The organic layer was dried over magnesium sulfate and
evaporated under vacuum to an oil. The crude product was
dissolved in 30 ml methylene chloride, and the resulting
solution filtered and diluted with hexane to cloudiness. On
cooling in the freezer, 3.98g (11%) of crystalline 1-methoxy-
5-phenoxynaphthalene was obtained.
M.P. : 65-66~C
Elemental AnalysiS for C17H1402:
Calculated: C, 81.58; H, 5.64; O, 12.78;
Found: C, 80.01; H, 5.66; O, 13.47.
D. Preparation of l-hydroxy-5-phenoxynaphthalene
Into 50 mL of glacial acetic acid, 3.7g of 1-
methoxy-5-phenoxynaphthalene prepared as above, followed by
20 mL of 40% aqueous hydrogen bromide was s~irred. The
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mixture was heated at 95-100~C for 16 hours. The solvent was
removed under vacuum, and the residue was redissolved in
ethyl acetate and washed three times with brine. The organic
layer was dried over magnesium sulfate and filtered. After
5 removing solvent, the residue was chromatographed over silica
gel with a gradient elution of 0 to 30% ethyl acetate in
hexane, providing 2.10g (59%) of 1-hydroxy-5-
phenoxynaphthalene.
M.P.: 82-83~C
Elemental Analysis for C16 H12 02:
Calculated: C, 81.34i H, 5.12; û, 13.54;
Found: C, 81.04; H, 5.23; 0, 13.69.
E. Preparation of 5-phenoxy-1-naphthyl oxalylchloride
Into 100 mL of chloroform, 2.0 g of the 1-hydroxy-
5-phenoxynaphthalene prepared as above, followed by 30 mg 4-
dimethylaminopyridine ~DAP) and 1.78 ml of oxalyl chloride,
was dissolved. The mixture was refluxed for 16 hours, and the
solvent was removed under vacuum, providing the desired
oxalyl chloride intermediate, which was characterized by NMR.
F. Preparation of 5-phenoxy-8-hydroxy-naphthyl glyoxylic acid
lactone
All of the oxalyl chloride as prepared above was
redissolved in 75 mL methylene chloride and added dropwise
over 15 minutes to 3.38g of aluminum chloride in 75 mL
methylene chloride cooled by an ice bath. The reaction
mixture was stirred in ice bath for 1 hour and then allowed
to warm to room temperature for 30 minutes. The reaction
mixture was poured into 300 mL of 2:1 mixture of ice and
concentrated hydrochloric acid with stirring. The organic
layer was separated and washed with brine, dried over
magnesium sulfate, and filtered. After removing solvent under
vacuum, the residue was chromatographed over silica gel with
a gradient of 0 to 100% ethyl acetate in hexane followed by 0
to 20g~ methanol in ethyl acetate, providing 1.4g (54%) of the
1-oxalic acid intermediate as an oil. All of this
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intermediate was dissolved in 50 mL methylene chloride, and
treated with excess oxalyl chloride and a catalytic amount of
dimethylformamide (DMF) while the solution was cooled by an
ice bath. After 30 minutes, the reaction was allowed to warm
to room temperature for 1 hour. The solvent was removed under
vacuum, providing the lactone as a solid, which gave 0.883g
(67~ of crystals from a concentrated methylene chloride
solution.
M.P. : 195-6~C
Elemental Analysis for C18 H10 04 :
Calculated: C, 74.48; H, 3.47; O, 22.05;
Found: C, 74.49; H, 3.56; O, 21.76.
G. Preparation of 8-carbomethoxy-4-phenoxy-naphth-yl-
glyoxamide
Into 20 m~ of dimethylformamide G.290g of the
lactone prepared as above was dissolved. After providing for
argone atmosphere and cooling the solution to -20~C,
0.049g sodium amide was added in one portion, and stirring
was continued for 15 minutes. To the reaction mixture, 0.118
mL methyl bromoacetate was added, and the temperature was
maintained at -10~C for 1 hour and then at 0~C for 1 hour.
The reaction mixture was poured into dilute, cold brine
containing some hydrochloric acid, and resulting mixture was
extracted with ethyl acetate. The organic layer was washed
two times with cold brine containing hydrochloric acid, dried
over magnesium sulfate, and filtered. After removing solvent
under vacuum, the residue was chromatographed over silica gel
with a gradient elution of 0 to ~30% ethyl acetate in hexane,
giving 186mg ~49~) of sub-titLed compound as a crystalline
solid. M.P. : 103-5~C
Elemental Analysis for C21 H17 NO6 :
Calculated: C22.49; H, 4.52i N, 3.69;
Found: C, 66.30; H, 4.45; N, 3.60.
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H. Preparation of 8-carboxymethoxy-4-phenoxynaphth-1-yl
glyoxamide
In 10 mL methanol, 180 mg of the methyl ester amide
above was treated with 0.96 mL of 0.5N sodium hydroxide for
16 hours at room temperature. Most of the methanol was
removed under vacuum, and the reaction mixture was diluted
with 50 ml cold water. The mixture was extracted with ethyl
acetate, and the resulting aqueous layer was acidified with
dilute hydrochloric acid, producing 120 mg ~69%), after
drying, of title compound
M.P. : 204-6~C
Elemental Analysis for C20 H15 NO6 :
Calculated: C, 65.75; H, 4.14; N, 3.83;
Found: C, 66.01; H, 4.03; N, 3.76.
Thera~eutic Use of NA~hthyl GlyoxAmide
Naphthyl glyoxamide compounds described herein
are believed to achieve their beneficial therapeutic action
principally by direct inhibition of human sPLA2, and not by
acting as antagonists for arachidonic acid, nor other
active agents below arachidonic acid in the arachidonic
acid cascade, such as 5-lipoxygenases, cyclooxygenases, and
etc.
The method of the invention for inhibiting sPLA2
mediated release of fatty acids comprises contacting sPLA2
with an therapeutically effective amount of naphthyl
glyoxamide its salt or a prodrug derivative thereof.
The compounds of the invention may be used in a
method of treating a m~mmAl ~e.g., a human) to alleviate
the pathological effects of septic shock, adult respiratory
distress syndrome, pancreatitus, trauma, bronchial asthma,
allergic rhinitis, and rheumatoid arthritis; wherein the
method comprises administrating to the mammal a naphthyl
glyoxamide compound of formula I in a therapeutically
effective amount. A therapeutically effective amount is an
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-19 -
amount sufficient to inhibit sPLA2 mediated release of
fatty acid and to thereby inhibit or prevent the
arachidonic acid cascade and lts deleterious products. The
therapeutic amount of compound of the invention needed to
inhibit sPLA2 may be readily determined by taking a sample
of body fluid and assaying it for sPLA2 content by
conventional methods.
The specific dose of a compound administered
according to this invention to obtain therapeutic or
prophylactic effects will, of course, be determined by the
particular circumstances surrounding the case, including, for
example, the compound administered, the route of
administration and the condition being treated. Typical
daily doses will contain a non-toxic dosage level of from
about O.Ol mg/kg to about 50 mgfkg of body weight of an
active compound of this invention.
Ph~r~ceutical Forml~lations of the Invention
As previously noted the compounds of this
invention are useful for inhibiting sPLA2 mediated release
of fatty acids such as arachidonic acid. By the term,
"inhibiting~ is meant the prevention or therapeutically
significant reduction in release of sPLA2 initiated fatty
acids by the compounds of the lnvention. By
"pharmaceutically acceptable~' it is meant the carrier,
diluent or excipient must be compatible with the other
ingredients of the formulation and not deleterious to the
recipient thereof.
Preferably the pharmaceutical formulation is in
unit dosage form. The unit dosage form can be a capsule or
tablet itself, or the appropriate number of any of these. The
quantity of active ingredient in a unit dose of composition
may be varied or adjusted from about O.l to about lO00
milligrams or more according to the particular treatment
involved. It may be appreciated that it may be necessary to
make routine varia~ions to the dosage depending on the age
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and condition of the patient. The dosage will also depend on
the route of administration.
The compound can be administered by a variety of
routes including oral, aerosol, rectal, transdermal,
subcutaneous, intravenous, intramuscular, and intranasal.
Pharmaceutical formulations of the invention are
prepared by combining ~e.g., mixing) a therapeutically
effective amount of a naphthyl glyoxamide compound of
formula I together with a pharmaceutically acceptable
carrier or diluent therefor. The present pharmaceutical
formulations are prepared by known procedures using well
known and readily available ingredients.
In making the compositions of the present
invention, the active ingredient will usually be admixed
with a carrier, or diluted by a carrier, or enclosed within
a carrier which may be in the form of a capsule, sachet,
paper or other container. When the carrier serves as a
diluent, it may be a solid, semi-solid or liquid material
which acts as a vehicle, or can be in the form of tablets,
pills, powders, lozenges, elixirs, suspensions, emulsions,
solutions, syrups, aerosols (as a solid or in a llquid
medium), or ointment, containing, for example, up to 10% by
weight of the active compound. The compounds of the
present invention are preferably formulated prior to
administration.
For the pharmaceutical formulations any suitable
carrier known in the art can be used. In such a formulation,
the carrier may be a solid, liquid, or mixture of a solid and
a liquid. Solid form formulations include powders, tablets
and capsules. A solid carrier can be one or more substances
which may also act as flavoring agents, lubricants,
solubilisers, suspending agents, binders, tablet
disintegrating agents and encapsulating material.
Tablets for oral administration may contain
suitable excipients such as calcium carbonate, sodium
carbonate, lactose, calcium phosphate, together with
disintegrating agents, such as maize, starch, or alginic
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acid, and/or binding agents, for example, gelatin or
acacia, and lubricating agents such as magnesium stearate,
stearic acid, or ~alc.
In powders the carrier is a finely divided solid
which is in admix~ure with the finely divided active
ingredient. In tablets the accive ingredient is mixed with a
carrier having the necessary binding properties in suitable
proportions and compacted in the shape and size desired. The
powders and tablets preferably contain from about 1 to about
99 weight percent of the active ingredient which is the novel
compound of this invention. suitable solid carriers are
magnesium carbonate, magnesium stearate, talc, sugar lactose,
pectin, dextrin, starch, gelatin, tragacanth, methyl
cellulose, sodium carboxymethyl cellulose, low melting waxes,
and cocoa butter.
Sterile liquid form formulations include
suspensions, emulsions, syrups and elixirs.
The active ingredient can be dissolved or suspended
in a pharmaceutically acceptable carrier, such as sterile
water, sterile organic solvent or a mixture of both. The
active ingredient can often be dissolved in a suitable
organic solvent, for instance aqueous propylene glycol.
Other compositions can be made by dispersing the finely
divided active ingredient in aqueous starch or sodium
carboxymethyl cellulose solution or in a suitable oil.
The following pharmaceutical formulations 1 through
8 are illustrative only and are not intended to limit the
scope of the invention in any way. 'lActive ingredient~,
refers to a compound according to Formula I or a
pharmaceutically acceptable salt, solvate, or prodrug
thereof.
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Formulation 1
Hard gelatin capsules are prepared using the
following ingredients:
Quantity
(m~capsule)
Active ingredient 250
Starch, dried 200
Magnesium stearate 10
Total 460 mg
Formul~tion 2
A tablet is prepared using the ingredients below:
Quantity
(ma/t~hlet)
Active ingredient 250
Cellulose, microcrystalline 400
Silicon dioxide, fumed 10
Stearic acid 5
Total 66 5 mg
The components are blended and compressed to form tablets
each weighing 6 65 mg
Form~ tion 3
An aerosol solution is prepared containing the following
15 components:
Weiaht
Active ingredient 0.25
Ethanol 25.75
Propellant 22 (Chlorodifluoromethane) 74.00
Total 100.00
The active compound is mixed with ethanol and the
mixture added to a portion of the propellant 22, cooled to
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~3_
-30~C and transferred to a filling device. The required
amount is then fed to a stainless steel container and diluted
with the remainder of the propellant. The valve units are
then fitted to the container.
Formulation 4
Tablets, each containing 60 mg of active
ingredient, are made as follows:
Active ingredient 60 mg
Starch 45 mg
Microcrystalline cellulose 35 mg
Polyvinylpyrrolidone (as 10% solution in water) 4 mg
Sodium carboxymethyl starch 4.5 mg
Magnesium stearate 0.5 mg
Talc 1 m~
Total 150 mg
The active ingredient, starch and cellulose are
passed through a No. 45 mesh IT.S. sieve and mixed thoroughly.
The aqueous solution containing polyvinylpyrrolidone is mixed
with the resultant powder, and the mixture then is passed
through a No. 14 mesh U.5. sieve. The granules so produced
are dried at 50~C and passed through a No. 1~ mesh U.S.
sieve. The sodium carboxymethyl starch, magnesium stearate
and talc, previously passed through a No. 60 mesh U.S. sieve,
are then added to the granules which, a~ter mixing, are
compressed on a tablet machine to yield tablets each weighing
150 mg.
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Formulation 5
Capsules, each containing 80 mg of active
ingredient, are made as follows:
Active ingredient 80 mg
Starch 59 mg
Microcrystalline cellulose 59 mg
Magnesium stearate 2 ma
Total 200 mg
The active ingredient, cellulose, starch, and
magnesium stearate are blended, passed through a No. 45 mesh
U.S. sieve, and filled into hard gelatin capsules in 200 mg
quantities.
Formulation 6
Suppositories, each containing 225 mg of active
ingredient, are made as follows:
Active ingredient 225 mg
Saturated fatty acid glycerides2,000 ma
Total 2, 225 mg
The active ingredient is passed through a No. 60
mesh U.S. sieve and suspended in the saturated fatty acid
glycerides previously melted using the minimum heat
necessary. The mixture is then poured into a suppository mold
of nominal 2 g capacity and allowed to cool.
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Formulation 7
Suspensions, each containing 50 mg of active
ingredient per 5 ml dose, are made as follows:
Active ingredient 50 mg
Sodium carboxymethyl cellulose 50 mg
Syrup l.25 ml
senzoic acid solution O.lO ml
Flavor q.v.
Color q.v.
Purified water to total 5 ml
The actlve ingredient lS passed through a No. 45
mesh U.S. sieve and mixed with the sodium carboxymethyl
cellulose and syrup to form a smooth paste. The benzoic acid
solution, flavor and color are diluted with a portion of the
water and added, with stirring. Sufficient water is then
added to produce the required volume.
Formulation 8
An intravenous formulation may be prepared as
follows:
Active ingredient lO0 mg
Isotonic saline l,000 ml
The solution of the above ingredients generally is
administered intravenously to a subject at a rate of l ml per
20 minute.
Assav Procedures
C~romo~enic Assav
25The following chromogenic assay procedure was
used to identify and evaluate inhibitors of recombinant
human secreted phospholipase A2. The assay described
herein has been adapted for high volume screening using 96
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well microtiter plates. A general description of this
assay method is found in the article, ~Analysis of Human
Synovial Fluid Phospholipase A2 on Short Chain
Phosphatidylcholine-Mixed Micelles: Development of a
Spectrophotometric Assay Suitable for a Microtiterplate
Reader", by Laure J. Reynolds, Lori L. Hughes, and Edward A
Dennis, Analvtical Biochemistrv, 204, pp. 190-137, 1992
(the disclosure of which is incorporated herein by
reference):
10 Reagents:
REACTION BUFFER
CaCl2.2H20 (1.47 g/L)
KCl (7.455 g/L)
Bovine Serum Albumin (fatty acid free~ (1 g/L)
(Sigma A-7030, product of Sigma Chemical Co.
St. Louis MO, USA)
TRIS HCl ~3.94 g/L)
pH 7.5 (adjust with NaOH)
ENZYME BUFFER -
0.05 NaOAc.3H20, pH 4.5
0.2 NaCl
Adjust pH to 4.5 with acetic acid
DTNB - 5,5'-dithiobis-2-nitrobenzoic acid
RACEMIC DIHEPTANOYL THIO - PC
racemic 1,2-bis(heptanoylthio)-1,2-dideoxy-sn-
glycero-3-phosphorylcholine
TRITON X-100TM prepare at 6.24g mgiml in
reaction buffer to equal 10uM.
REACTION MIXTURE
A measured volume of racemic dipheptanoyl thio PC
supplied in chloroform at a concentration of 100 mg/ml is
taken to dryness and redissolved in 10 millimolar TRITON X-
l00TM nonionic detergent aqueous solution. Reaction Buffer
is added to the solution, then DTNB to give the Reaction
Mixture.
The reaction mixture thus obtained contains lmM
diheptanoly thio-PC substrate, 0.29 mm Triton X-100TM
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detergent, and 0.12 mm DTMB in a buffered aqueous solution
at pH 7.5.
AssaY Procedure:
1. Add 0. 2 ml reaction mixture to all wells;
2. Add 10 ul test compound (or solvent blank~ to
appropriate wells, mix 20 seconds;
3. Add 50 nanograms of sPLA2 (10 microliters) to
appropriate wells;
4. Incubate plate at 40~C for 30 minutes;
5. Read absorbance of wells at 405 nanometers with an
automatic plate reader.
All compounds were tested in triplicate.
Typically, compounds were tested at a final concentration
of 5 ug/ml. Compounds were considered active when they
exhibited 40% inhibition or greater compared to uninhihited
control reactions when measured at 405 nanometers. Lack of
color development at 405 nanometers evidenced inhibition.
Compounds initially found to be active were reassayed to
confirm their activity and, if sufficiently active, ICso
values were determined. Typically, the ICso values (see,
Table I, below) were determined by diluting test compound
serially two-fold such that the final concentration in the
reaction ranged from 45 ug/mL to 0.35 ug/ml. More potent
inhibitors required significantly greater dilution. In all
cases, % inhibition measured at 405 nanometers generated by
enzyme reactions containing inhibitors relative to the
llninhihited control reactions was determined. Each sample
was titrated in triplicate and result values were averaged
for plotting and calculation of ICso values. ICso were
determined by plot~ing log concentration versus inhibition
values in the range from 10-90% inhibition.
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Results of Human Secreted Phospholipase A2 Inhibition Tests
Inhibition of human
secreted PLA2
Compound of mM IC50 + mean
Exam~le number devi~tion(3-4 tests~
1 0.69 + 0.051
While the present invention has been illustrated
above by certain specific embodiments, it is not intended
that these specific examples should limit the scope of the
invention as described in the appended claims.
