PLA.sub.2 inhibitors selected from the group consisting of ##STR1## wherein A is selected from the group consisting of ##STR2## and Z, W, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, X, X' and Y are as defined herein.

BACKGROUND OF THE INVENTION 
This invention relates to PLA.sub.2 inhibitors. In one aspect, this 
invention relates to a method of inhibiting PLA.sub.2 using the PLA.sub.2 
inhibitors of the invention. In another aspect, this invention relates to 
a method of inhibiting calcium-independent PLA.sub.2 using the PLA.sub.2 
inhibitors of the invention. 
Phospholipases A.sub.2 are a diverse group of esterases which specifically 
hydrolyse the sn-2 ester of membrane and other phospholipids. Both calcium 
dependent and independent examples are known. These enzymes are 
principally responsible for the release of arachidonic acid during signal 
transduction in mammalian cells. As such their modulation and inhibition 
are of potential therapeutic value in treating many diseases and 
conditions where arachidonic acid, lysophospholipids, and their 
metabolites (prostaglandins, leukotrienes, thromboxanes, platelet 
activating factor, etc.,) are responsible for the deleterious effects of 
these conditions and diseases. 
In some circumstances it may be beneficial to inhibit more than one 
phospholipase A.sub.2, particularly in cases where inhibition of the 
production of early as well as late mediators can be important in 
controlling pathology, or where inhibition of signalling events can be 
combined with inhibition of release of arachidonic acid stores. 
##STR3## 
Evidence has accumulated for a potential role of PLA.sub.2 in myocardial 
injury to the ischemic heart. 
Lysophospholipids have also been implicated as potential mediators of 
sudden cardiac death, Corr et al, "Lethal Arrhythmias Resulting from 
Myocardial Ischemia and Infarction", Rosen & Patti, eds , Kluwer Academic 
Publishers, Boston, 91-014 (1989). The addition of lysophospholipids to 
normoxic myocardial tissue in vitro induces electrophysiological 
alterations that are similar to those observed in the ischemic heart in 
vivo Corr et al, Circ. Res, 55, 135-54 (1984). 
Most importantly, lysophospholipid accumulation in the ischemic dog heart 
in vivo has been correlated with the frequency of cardiac arrhythmias, 
Kinnaird et al, Lipids, 23, 32-35 (1988). Furthermore, it is known that 
the carnitine acyltransferase 1 inhibitor, 
2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (POCA), prevents the 
onset of ventricular fibrillation and ventricular tachycardia and inhibits 
the accumulation of lysophospholipids (and long-chain acylcarnitines) in 
the ischemic cat heart in vivo, Corr et al, J. Clin. Invest., 83, 927-36 
(1989). 
Accelerated phospholipid catabolism by PLA.sub.2 has also been implicated 
as a cause of infarct damage in the ischemic heart. In the ischemic heart, 
ATP levels decrease. Treatment of rat neonatal myocytes with the 
glycolytic inhibitor iodoacetate lowers the levels of ATP which results in 
the release of arachidonic acid and morphological alterations of the 
myocytes, Chien et al, J. Clin. Invest., 75, 1770-80 (1985). One PLA.sub.2 
inhibitor (U26,384) prevented the release of arachidonic acid, 
phospholipid degradation, sarcolemmal membrane defects and the release of 
creatine kinase that was induced by the treatment of rat neonatal myocytes 
with iodoacetate, Sen et al, J. Clin. Invest., 82, 1333-38 (1988). 
LTB.sub.4 is an arachidonic acid metabolite which is produced by the 
5-lipoxygenase pathway. Pharmacologically, LTB.sub.4 is an important 
mediator of inflammation. LTB.sub.4 is known to induce chemotaxis, 
chemokinesis, aggregation, and degranulation of leukocytes in vitro, and 
to induce accumulation of polymorphonuclear leukocytes, and increase 
vascular permeability and edema formation in vivo. Particularly high 
levels of LTB.sub.4 are detected in lesions in inflammatory diseases such 
as rheumatoid or spondylarthritis, gout, psoriasis, ulcerative colitis, 
Crohn's disease, multiple sclerosis and some respiratory diseases. Since 
the compounds herein inhibit PLA.sub.2 and thereby LTB.sub.4 synthesis, 
the compounds of the present invention are useful in treating inflammatory 
conditions in mammals such as rheumatoid arthritis, inflammatory bowel 
disease, psoriasis and the like. 
Therefore, compounds which inhibit PLA.sub.2 provide potential therapeutic 
approaches to the prevention of arrhythmia, infarct damage, sudden death, 
and inflammatory conditions, i.e., conditions mediated by inflammatory 
mediators such as prostaglandins and leukotrienes. 
SUMMARY OF THE INVENTION 
It is an object of the invention to provide novel PLA.sub.2 inhibitors. It 
is a further object of the invention to provide a method for inhibiting 
PLA.sub.2 using the PLA.sub.2 inhibitors of the invention. It is a still 
further object of the invention to provide a method for inhibiting 
calcium-independent PLA.sub.2 using the PLA.sub.2 inhibitors of the 
invention. It is yet a further object of the invention to provide novel 
intermediates useful in the preparation of the PLA.sub.2 inhibitors of the 
invention. 
According to the invention, PLA.sub.2 inhibitors are provided comprising a 
compound selected from the group consisting of 
##STR4## 
wherein A is selected from the group consisting of 
##STR5## 
Z is selected from the group consisting of a direct bond and substituted 
or unsubstituted alkynyl, alkenyl, alkyl and dienyl groups wherein the 
substituent is selected from the group consisting of --COR, hydroxy alkyl, 
--SO.sub.2 R and --PO(OR)(OR') groups; 
W is selected from the group consisting of hydrogen and substituted or 
unsubstituted alkyl, alkenyl, alkynyl, aryl, alkaryl and heteroaryl groups 
wherein the substituent is selected from the group consisting of --COR, 
hydroxy, halogen, trifluoromethyl, --NHCOR, --NR'COR, amino, --NR'SO.sub.2 
R and --NHSO.sub.2 R groups; 
wherein the sum of the number of carbon atoms in Z and W is at least 3, 
preferably about 8 to about 20; R.sub.1, R.sub.2, R.sub.3, R.sub.4 and 
R.sub.5 are independently selected from the group consisting of hydrogen, 
halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkaryl, alkoxy, 
thioalkyl, --CHO, --COR, --COOH, --NH.sub.2, --NHR, --NRR', --SH, --OH, 
--COOR, --SO.sub.2 R, --SOR, --SO.sub.2 OR, --P(O)(OR)(OR') and 
--OP(O)(OR)(OR'); 
X and X' are independently selected from the group consisting of oxygen and 
sulfur atoms; 
Y is selected from the group consisting of hydrogen, --CHO, --COOH, --COOR, 
--CONH.sub.n R.sub.2-n, --CONHOH, --CN, --COSH, --COSR, --CSOH and --CSOR 
wherein R and R' are independently selected from the group consisting of 
alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, cycloalkyl, cycloalkenyl 
and cycloalkynyl groups and n is an integer from 0 to 2; 
R.sub.6 and R.sub.7 are independently selected from the group consisting of 
alkyl, alkenyl, alkynyl, alkoxy, aryloxy, hydroxy, amino, alkylamino, 
--SH, thioalkyl, thioaryl, halogen and --OM wherein M is a 
pharmaceutically acceptable cation or R.sub.6 and R.sub.7 can form a 
cyclic or bicyclic structure; 
R.sub.8 and R.sub.9 are independently selected from the group consisting of 
alkyl, alkenyl, alkynyl, alkoxy, aryloxy, hydroxy, amino, alkylamino, 
--SH, thioalkyl, thioaryl, halogen and --OM or R.sub.8 and R.sub.9 can 
form a cyclic or bicyclic structure; and pharmaceutically acceptable salts 
thereof. 
The number of carbon atoms in R6, R.sub.7, R.sub.8 or R.sub.9 is 0 to about 
8, preferably 0 to 2, and most preferably 1 to 2. 
Further according to the invention, a method of inhibiting PLA.sub.2 is 
provided which comprises utilizing an effective inhibitory amount of a 
PLA.sub.2 as defined herein. 
Still further according to the invention, intermediates for use in the 
preparation of PLA.sub.2 are provided comprising a compound selected from 
the group consisting of 
##STR6## 
wherein A, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are as defined herein and 
X" is a halogen. 
DETAILED DESCRIPTION OF THE INVENTION 
A first embodiment of the invention relates to compounds selected from the 
group consisting of 
##STR7## 
wherein A, Z, W, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are as defined 
above. 
As utilized herein, the term "alkyl" means a linear or branched alkyl 
radical having 1 to about 20 carbon atoms, preferably about 2 to about 15 
carbon atoms. The term "alkenyl" means a linear or branched alkyl radical 
having 2 to about 20 carbon atoms having ethylenic unsaturation. The term 
"alkynyl" means a linear or branched alkyl radical having 2 to about 20 
carbon atoms having ethynyl unsaturation. The term "dienyl" means a linear 
or branched diene having cumulated or noncumulated double bonds. Examples 
of such alkyl, alkenyl, alkynyl and allenyl radicals include ethyl, 
n-propyl, isobutyl, t-butyl, sec-butyl, n-butyl, pentyl, isoamyl, hexyl, 
octyl, methyl, 1-propenyl, 2-propenyl, 2-isobutenyl, 1-pentenyl, 
1-hexenyl, 1-octenyl, 1-tridecenyl, ethynyl, 1-butynyl, 1-hexynyl, 
1-octynyl, 1-tridecynyl, 1,3-decadienyl, 1,3-butadienyl, 1,4-pentadienyl, 
2-methyl-1,3-butadienyl, 1,2-propadienyl and 2,3-pentadienyl The term 
"aryl", alone or in combination, means a phenyl or naphthyl radical. The 
term "alkaryl" means an aryl radical as defined above which is substituted 
by an alkyl radical as defined above. The term "heteroaryl" is an aromatic 
monocyclic, bicyclic or tricyclic heterocycle which contains one or more 
heteroatoms. Examples of such heteroaryl radicals include pyridyl, 
quinolyl, furyl, thienyl and oxazolyl. The terms "alkoxy" and "aryloxy" 
means an alkyl or aryl ether radical wherein the terms alkyl and aryl have 
the meanings given above. Examples of alkoxy and aryloxy radicals include 
methoxy, ethoxy, phenoxy and 2-naphthyloxy The term "cycloalkyl" alone or 
in combination, means a cycloalkyl radical containing from 3 to about 10 
carbon atoms. The term "cycloalkenyl", alone or in combination, means a 
cycloalkyl radical having 1 or more double bonds The term "cycloalkynyl", 
alone or in combination, means a cycloalkyl radical having one or more 
triple bonds. Examples of cycloalkyl, cycloalkenyl and cycloalkynyl 
radicals include cyclopentyl, cyclohexyl, cyclooctyl, cyclopentenyl, 
cyclohexenyl, cyclooctenyl, cyclopentadienyl, cyclohexadienyl and 
cyclooctynyl. The term "halogen" means chlorine, bromine, fluorine and 
iodine, preferably bromine and iodine. 
The term "pharmaceutically acceptable cation" as used herein refers to 
cations such as ammonium, sodium, potassium, lithium, calcium, magnesium, 
ferrous, zinc, copper, manganous, aluminum, ferric, manganic, 
pentaalkyl-ammonium, and the like. The currently preferred 
pharmaceutically acceptable cation is sodium. 
The subject compounds useful as PLA.sub.2 inhibitors can be prepared 
utilizing the methods set forth below in Examples 1-3, 5, 6, 8-15, 17 and 
18. 
A second embodiment of the invention relates to a method of inhibiting 
PLA.sub.2 comprising contacting the PLA.sub.2 with an effective inhibitory 
amount of a compound selected from the group consisting of 
##STR8## 
wherein A, Z, W, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are as defined 
above. 
A preferred class of compounds for use in the inhibition of PLA.sub.2 is 
one wherein at least one of Z and W contains at least one carbon-carbon 
double bond or carbon-carbon triple bond, such as wherein Z is alkenyl or 
alkynyl and W is hydrogen. 
A more preferred class of compounds for use in the method of the present 
invention is the class wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are 
hydrogen. 
A most preferred class of compounds for use in the present invention is the 
class wherein Y is selected from the group consisting of hydrogen, --COOR 
and --COOH, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are independently 
selected from the group consisting of hydroxy, alkoxy and --ONa. 
The compounds of the present invention for inhibiting PLA.sub.2 can be 
administered in such oral dosage forms as tablets, capsules, soft gels, 
pills, powders, granules, elixirs or syrups. The compounds can also be 
administered intravascularly, intraperitoneally, subcutaneously, 
intramuscularly, or topically using forms known to the pharmaceutical art. 
Moreover, they can be administered rectally or vaginally, in such forms as 
suppositories or bougies. In general, the preferred form of administration 
is oral. 
For the orally administered pharmaceutical compositions and methods of the 
present invention, the foregoing active ingredients will typically be 
administered in admixture with suitable pharmaceutical diluents, 
excipients or carriers (collectively referred to herein as "carrier" 
materials) suitably selected with respect to the intended form of 
administration and consistent with conventional pharmaceutical practices. 
For example, for oral administration in the form of tablets or capsules, a 
therapeutically or prophylactically effective amount of one or more 
compounds of the present invention can be combined with any oral non-toxic 
pharmaceutically acceptable inert carrier such as lactose, starch, 
sucrose, cellulose, magnesium stearate, dicalcium phosphate, calcium 
sulfate, mannitol, and the like, or various combinations thereof. For oral 
administration in liquid forms, such as in soft gels, elixirs, syrups, 
drops and the like, a therapeutically or prophylactically effective amount 
of the active drug components can be combined with any oral nontoxic 
pharmaceutically acceptable inert carrier such as water, saline, ethanol, 
polyethylene glycol, propylene glycol, corn oil, cotton seed oil, peanut 
oil, sesame oil, benzyl alcohol, various buffers, and the like, or various 
combinations thereof. Moreover, when desired or necessary, suitable 
binders, lubricants, disintegrating agents, and coloring agents can also 
be incorporated in the mixture. Suitable binders include starch, gelatin, 
natural sugars, corn sweeteners, natural and synthetic gums such as 
acacia, sodium alginate, carboxymethyl cellulose, polyethylene glycol, and 
waxes, or combinations thereof. Lubricants for use in these dosage forms 
include boric acid, sodium benzoate, sodium acetate, sodium chloride, and 
the like, or combinations thereof. Disintegrators include, without 
limitation, starch, methyl cellulose, agar, bentonite, guar gum, and the 
like, or combination thereof. Sweetening and flavoring agents and 
preservatives can also be included where appropriate. 
For intravascular, intraperitoneal, subcutaneous or intramuscular 
administration, one or more compounds of the present invention can be 
combined with a suitable carrier such as water, saline, aqueous dextrose, 
and the like. For topical administration, therapeutically effective 
amounts of one or more compounds of the present invention can be combined 
with pharmaceutically acceptable creams, oils, waxes, gels, and the like. 
Regardless of the route of administration selected, the compounds of the 
present invention are formulated into pharmaceutically acceptable dosage 
forms by conventional methods known to those skilled in the art. The 
compounds can also be formulated using pharmacalogically acceptable base 
addition salts. Moreover, the compounds or their salts may be used in a 
suitable hydrated form. 
Regardless of the route of administration selected, a nontoxic but 
therapeutically or prophalactically effective quantity of one or more 
compounds of this invention is employed in any treatment associated with 
the inhibition of PLA.sub.2. 
As used herein, the term "pharmaceutically acceptable salts" refers to 
pharmacologically acceptable base addition salts derived from 
pharmaceutically acceptable nontoxic inorganic or organic bases. Among the 
inorganic bases employed to produce pharmaceutically acceptable salts are 
the hydroxide bases of the "pharmaceutically acceptable cations" disclosed 
above. Among the organic bases employed to produce pharmaceutically 
acceptable salts are the pharmaceutically acceptable nontoxic bases of 
primary, secondary and tertiary amines. Especially preferred nontoxic 
bases are isopropyl amine, diethyl amine, ethanol amine, dicyclohexyl 
amine, choline and caffeine. 
All of the pharmaceutically acceptable nontoxic addition salts are prepared 
by conventional processes which are well known to those of ordinary skill 
in the art. 
A third embodiment of the invention relates to intermediates for use in the 
preparation of the PLA.sub.2 inhibitors of the invention. The 
intermediates are compounds selected from the group consisting of 
##STR9## 
wherein A, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are as defined above and 
X" is a halogen, preferably bromine or iodine. 
A preferred class of intermediates for use in the present invention is one 
wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are hydrogen. 
A most preferred class of intermediates is the class wherein Y is selected 
from the group consisting of hydrogen, --COOR and --COOH, and R.sub.6, 
R.sub.7, R.sub.8 and R.sub.9 are independently selected from the group 
consisting of hydroxy, alkoxy and --ONa. 
The subject intermediates can be prepared utilizing the methods set forth 
below in Examples 4, 7 and 16.

In the following examples, all reagents were used as received without 
purification. All proton and carbon NMR spectra were obtained on either a 
Varian VXR-300 or VXR-400 nuclear magnetic resonance spectrometer. 
EXAMPLES 
Example 1 
Tetraethyl[2-[3-(1-tridecynyl)phenyl]ethenylidene]bisphosphonate 
In a three neck round bottom flask fitted with an argon inlet, dropping 
funnel, magnetic stir bar, and septum, tetrahydrofuran (anhydrous, 50 ml) 
was introduced and cooled to 0.degree. C. Titanium tetrachloride (2.73 ml, 
24.9 mmol) dissolved in dry carbon tetrachloride (6.8 ml) was added 
dropwise over a 20 minute period, producing a copious yellow precipitate. 
3-(1-Tridecynyl)benzaldehyde prepared according to the method described in 
EP 195 097 A1, which is incorporated by reference herein, (3.52 g, 12.37 
mmol) was then added via cannula over a ten minute period. A few 
milliliters of tetrahydrofuran was used to rinse the aldehyde containing 
flask into the reaction flask. Tetraethylmethylenediphosphonate (3.55 g, 
12.37 mmol) was added via cannula over a two to three minute period. 
N-Methyl morpholine (anhydrous, 5.46 ml, 49.7 mmol) in dry tetrahydrofuran 
(8.4 ml) was added dropwise over a forty five minute period. The reaction 
was stirred for 3.5 hours at 0.degree. C. At the end of this time, water 
(14 ml) was added dropwise over a few minutes to the reaction mixture at 
0.degree. C. Ether (25 ml) was added to the mixture which was shaken, and 
the layers were separated. The aqueous layer was extracted with two more 
portions of ether (25 ml). The combined organic layers were washed with 
saturated aqueous sodium chloride solution (25 ml), saturated aqueous 
sodium bicarbonate solution (25 ml), and with saturated sodium chloride 
solution (25 ml). After drying (MgSO.sub.4), the organic phase was 
filtered and stripped down to a brown-orange liquid. This liquid was 
chromatographed on silica (0.4 kg) eluting with 80% hexane, 15% ethyl 
acetate, and 5% ethanol to give the product as a pale yellow liquid, 4.65 
g, 8.38 mmol, 67.8% yield; .sup.1 H NMR (CDCl.sub.3) .delta.0.88 (m, 3H), 
1.12 to 1.67 (several m, 30H), 2.37 (t, J=7.1 Hz, 2H), 4.06 (m, 4H), 4.21 
(m, 4H), 7.31 (m, 1H), 7.41 (d, J=7.7 Hz, 1H), 7.68 (m, 2H), 8.25 (dd, 
J=47.5, 28.9 Hz, 1H); FAB mass spectrum m/z 561 (M+Li).sup.+. 
Example 2 
[2-[3-(1-Tridecynyl)phenyl]ethenylidene]bisphosphonic acid 
Tetraethyl[2-[3-(1-tridecynyl)phenyl]ethenylidene]bisphosphonate (3.247 g, 
5.85 mmol) prepared according to Example 1 was dissolved in a mixture of 
CH.sub.2 Cl.sub.2 -CD.sub.2 Cl.sub.2 (25 ml, 4:1), and stirred under argon 
while trimethylsilyl bromide (3.5 ml, 26.5 mmol) was added. After the 
reaction was stirred for 6.5 hours, the solvent was removed under reduced 
pressure and the residue was briefly placed on the vacuum line. Hydrolysis 
was effected in tetrahydrofuran-water (9:1, 10 ml), stirring for 39 hours 
at room temperature. After stripping off the solvent, the residue was 
recrystallized from heptane-ether to give the product as a white solid, 
0.416 g, 0.94 mmol, 16% yield; .sup.1 H NMR (CD.sub.3 OD) .differential. 
0.89 (t, J=6.9 Hz, 3H), 1.21 to 1.65 (several m, 18H), 2.40 (t, J=7.0 Hz, 
2H), 7.55 (m, 2H), 7.71 (m, 2H), 8.03 (dd, J=46.1, 29.2 Hz, 1H); HRMS 
calcd for (M+H).sup.+ C.sub.21 H.sub.33 O.sub.6 P.sub.2 m/z 443.1752, 
found 443.1700. 
Example 3 
Disodium[2-[3-(1tridecynyl)phenyl]ethenylidene]bisphosphonate 
[2-[3-(1Tridecynyl)phenyl]ethenylidene]bisphosphonic acid (0.103 g, 0.223 
mmol) prepared according to Example 2 was dissolved in dry tetrahydrofuran 
(10 ml) and stirred while sodium ethoxide in ethanol (0.177M, 2.58 ml, 
0.457 mmol) was added. After one hour, the solvent was removed under 
reduced pressure leaving a brownish yellow solid. This solid was powered 
and washed successively with anhydrous ether, tetrahydrofuran, 
acetonitrile, and ethanol leaving a white solid, 43.3 mg, 0.089 mmol, 38% 
yield; .sup.1 H NMR (D.sub.2 O, relative to HOD peak at 4.64) 
.differential. 0.76 (t, J=6.9 Hz, 3H), 1.05 to 1.58 (several m, 18H), 2.35 
(t, J=7.0 Hz, 2H), 7.30 (m, 2H), 7.55 (dd, J=4.38, 27.0 Hz, 1H), 7.69 (m, 
2H); FAB mass spectrum m/z 443 (M-2Na+3H).sup.+, 465 (M-Na+2H).sup.+, 487 
(M+H).sup.+. 
Example 4 
Ethyl 3-(2-bromophenyl)-2-E-(diethoxyphosphinyl)propenoate 
2-Bromobenzaldehyde (18.5 g, 100.0 mmol) was added to a 500 ml round bottom 
flask along with triethyl phosphonoacetate (21 ml, 105.9 mmol). Dry 
benzene (200 ml) was added, and benzoic acid (0.60 g, 4.9 mmol) and 
piperidine (0.60 ml, 6.07 mmol). The flask was equipped with a magnetic 
stir bar, Dean-Stark trap, and reflux condenser. The apparatus was inerted 
by several cycles of vacuum followed by argon, then connected to a 
bubbler, and reflux started. Collection of water in the Dean-Stark trap 
was very slow and gradual. Additional quantities of piperidine (0.2 ml, 2 
mmol) were added on days 2, 5, 10, 12, 19, 21, and 25 (0.1 ml, 1 mmol); 
Additional quantities of benzoic acid (0.20 g, 1.64 mmol) were added on 
days 10, 12, 21 and 25 (0.10 g, 0.82 mmol). Reflux was stopped after 26.7 
days. After cooling, the reaction mixture was poured into water. The 
organic phase was washed successively with 0.1N HCl (2.times.150 ml), 
water (150 ml), saturated aqueous sodium bicarbonate (2.times.150 ml), and 
water (2.times.150 ml). After stripping down to an orange oil, the residue 
was chromatographed on silica eluting with 1:1 ethyl acetate-hexanes. Tlc 
on silica developed in the same solvent system gave an R.sub.f of 0.22. 
The product was obtained as a liquid, 7.84 g, 20.0 mmol, 20% yield. .sup.1 
H NMR (C.sub.6 D.sub.6) .differential. 0.73 (t, J=7 Hz, 3H), 1.12 (m, 6H), 
3.89 (q, J=7.1 Hz, 4H), 6.59 (t, J=7.7 Hz, 1H), 6.76 (t, J=7.6 Hz, 1H), 
7.21 (d, J=8.1 Hz, 1H), 7.32 (d, J=7.7 Hz, 1H), 8.16 (d, J=22.7 Hz, 1H). 
Example 5 
Ethyl 2-(diethoxyphosphinyl)-3-[2-(1-tridecynyl)phenyl]-2-E-propenoate 
Ethyl 3-(2-bromophenyl)-2-E-(diethoxyphosphinyl)propenoate (7.84 g, 20.0 
mmol) prepared according to Example 4 was placed in a three neck round 
bottom flask equipped with a condenser, stir bar, internal thermometer, 
and argon inlet. 1-Tridecyne (6.30 g, 34.9 mmol) and triphenylphosphine 
(0.161 g, 0.614 mmol) were added along with triethylamine (110 ml) and the 
system was degassed by repeated vacuum-argon cycles. Under an argon 
atmosphere, palladium acetate (0.048, 0.214 mmol) was added, and heating 
in an oil bath (100.degree.-105.degree. C.) was started. The internal 
temperature stayed in the range of 89.degree.-92.degree. C. during the 
reaction. Within a few hours a light precipitate started forming. After 
about 42 hours, additional tridecyne (1.0 g, 5.5 mmol) was added. The 
reaction was stopped after 53 hours. After cooling the reaction mixture 
was filtered and the precipitate was washed with triethylamine. The 
filtrate was stripped down to a reddish oil. Multiple chromatographies on 
silica eluting with ethyl acetate in hexane (32% ethyl acetate was best) 
yielded a yellow liquid, 2.43 g, 5.08 mmol, 25.3% yield; .sup.1 H NMR 
(CDCl.sub.3) .differential. 0.88 (m, 3H), 1.17 (t, J=7.2 Hz, 3H), 1.21 to 
1.48 (several m, 22H), 1.64 (quintet, J=7.5 Hz, 2H), 2.43 (t, J=7.2 Hz, 
2H), 4.22 (m, 4H), 7.19 to 7.48 (several m, 4H), 8.06 (d, J=23.7 Hz, 1H); 
CI mass spectrum m/z 491 (M+H).sup.+. 
Example 6 
Ethyl E-2-phosphono-3-[2-(1-tridecynyl)phenyl]propenoate 
Ethyl 2-(diethoxyphosphinyl)-3-[2-(1-tridecynyl)phenyl]-2-E-propenoate 
(0.50 g, 1.02 mmol) prepared according to Example 5 was dissolved in dry 
CH.sub.2 Cl.sub.2 (3 ml) and CD.sub.2 Cl.sub.2 (2 ml, dried over activated 
neutral alumina) and cooled to 0.degree. C. Bromotrimethylsilane (0.31 ml, 
2.35 mmol) was added over a two to three minute period. The progress of 
the reaction was monitored by .sup.31 P NMR, to follow disappearance of 
the starting material. After about 5 hours, the solvent was removed under 
reduced pressure. Acetone-heptane (5.5 ml, 10:1) containing 0.045 ml (2.5 
mmol) water was added and the reaction was stirred for 1.5 hours. At the 
end of this time the solvent was stripped. Since NMR indicated incomplete 
reaction, acetone (5 ml) containing water (0.05 ml, 2.8 mmol) was added 
and stirring was continued for two hours. After stripping the solvent, the 
reaction was still incomplete. It required 2.5 more hours under these 
conditions to complete the reaction. The solvent was stripped off and the 
residue was chromatographed on silica eluting with 10% chloroform and 20% 
methanol in heptane. Fractions containing product were recrystallized from 
heptane yielding a white solid, 0.211 g, 0.486 mmol, 47.6% yield; mp 
68.degree.-69.degree. C.; .sup.1 H NMR (CDCl.sub.3) .differential. 0.86 
(m, 3H), 1.09 to 1.63 (several m, 21H), 2.40 (t, J=7.1 Hz, 2H), 4.19 (q, 
J=7.1 Hz, 2H), 7.17 to 7.44 (several m, 4H), 8.15 (d, J=24.7 Hz, 1H), 8.81 
(br s, 2.7H); FAB mass spectrum m/z 434 (M+H).sup.+. 
Example 7 
Diethyl-3-iodophenylphosphonate 
m-Diiodobenzene (15.15 g, 45.92 mmol) was placed in a 3 neck round bottom 
flask with a stirring bar. Tetrakis(triethyl phosphite)nickel (14.2 mg, 
0.0196 mmol) was added to the flask in the dry box and adapters ending in 
a receiving flask were added, so as to make a small air cooled 
distillation assembly. Under argon, the reaction flask was placed in an 
oil bath at 160.degree. C. The m-diiodobenzene rapidly melted. The 
receiving flask was placed in a dry ice bath. Triethyl phosphite (8.6 ml, 
50.15 mmol) was placed in a syringe for addition in portions through a 
septum on the reaction flask. A few drops of triethyl phosphite were 
added, immediately turning the reaction mixture dark. A pale yellow color 
rapidly returned, at which point more triethyl phosphite was added. This 
process was repeated until the color change was no longer observed. At 
this point another charge of catalyst was added, then more triethyl 
phosphite. Several additional quantities of catalyst were used. When all 
the phosphite had been added, the reaction mixture was stirred for a few 
minutes longer, then cooled to room temperature. Purification was effected 
by chromatography on silica, eluting with hexanes, followed by 20% ethyl 
acetate in hexanes, then 30% ethyl acetate in hexanes. The product was 
obtained as a colorless liquid, 4.84 g, 14.23 mmol, 31% yield. .sup.1 H 
NMR (CDCl.sub.3) .differential. 1.33 (m, 6H), 4.15 (m, 4H), 7.22 (m, 1H), 
7.77 (m, 1H), 7.87 (m, 1H), 8.15 (dm, J=13.2 Hz, 1H); FAB mass spectrum 
m/z 341 (M+H).sup.+. 
Example 8 
Diethyl[3-(1-tridecynyl)phenyl]phosphonate 
Diethyl-3-iodophenylphosphonate prepared according to Example 7 (2.0 g, 
5.88 mmol) and 1-tridecyne (1.79 g, 9.97 mmol) were dissolved in triethyl 
amine (40 ml) in a two neck round bottom flask equipped with a condenser 
and a nitrogen dispersing tube. The flask was inerted. Triphenylphosphine 
(0.047 g, 0.179 mmol) and palladium acetate (0.015 g, 0.0668 mmol) were 
then added and the flask was heated in an oil bath to 100.degree. to 
105.degree. C. for 40 hours. After cooling to room temperature and 
filtering, the triethylamine was removed on the rotary evaporator. The 
residue was extracted several times with acetonitrile, and the combined 
extracts were stripped down to a reddish brown liquid. Chromatography on 
silica eluting with 20% hexane in ethyl acetate gave the product as a pale 
yellow oil, 0.410 g, 1.045 mmol, 17.7% yield; .sup.1 H NMR (CDCl.sub.3) 
.differential. 0.88 (t, J=6.8 Hz, 3H), 1.18 to 1.50 (several m, 22H), 1.60 
(quint, J=7.6 Hz, 2H), 2.40 (t, J=7.1 Hz, 2H), 4.11 (m, 4H), 7.38 (m, 1H), 
7.55 (br d, J=7.8 Hz, 1H), 7.71 (dd, J=13.2, 7.6 Hz, 1H), 7.83 (d, J=14.1 
Hz, 1H); FAB mass spectrum m/z 399 (M+Li).sup.+. 
Example 9 
Tetraethyl[2-[2-(1-tridecynyl)phenyl]ethenylidene]bisphosphonate 
In a three neck round bottom flask fitted with an argon inlet, dropping 
funnel, magnetic stir bar, and septum, tetrahydrofuran (anhydrous, 70 ml) 
was introduced and cooled to 0.degree. C. Titanium tetrachloride (3.9 ml, 
35.6 mmol) dissolved in dry carbon tetrachloride (9.5 ml) was added 
dropwise over a 30 minute period, producing a copious yellow precipitate. 
2-(1-Tridecynyl)benzaldehyde (5.00 g, 17.58 mmol) prepared according to 
the method described in EP 195 097 A1, which is incorporated by reference 
herein, was then added via cannula over a ten minute period. A few 
milliliters of tetrahydrofuran was used to rinse the aldehyde containing 
flask into the reaction flask. Tetraethylmethylenediphosphonate (5.08 g, 
17.62 mmol) was added via cannula over a two to three minute period. 
N-Methyl morpholine (anhydrous, 7.8 ml, 70.9 mmol) with dry 
tetrahydrofuran (12 ml) was added dropwise over a forty minute period. The 
reaction was stirred for four hours and twenty minutes at 0.degree. C. At 
the end of this time, water (20 ml) was added dropwise over a few minutes 
to the reaction mixture at 0.degree. C. Ether (25 ml) was added to the 
mixture which was shaken, and the layers were separated. The aqueous layer 
was extracted with two more portions of ether (25 ml). The combined 
organic layers were washed with saturated aqueous sodium chloride solution 
(25 ml), saturated aqueous sodium bicarbonate solution (25 ml), and with 
saturated sodium chloride solution (25 ml). After drying (MgSO.sub.4), the 
organic phase was filtered and stripped down to a brown-orange liquid. 
This liquid was chromatographed on silica (0.57 kg) eluting first with 82% 
hexane, 13% ethyl acetate, and 5% ethanol, then with 80% hexane, 15% ethyl 
acetate, and 5% ethanol to give the product as a pale yellow oil, 8.51 g, 
15.34 mmol, 87% yield; .sup.1 H NMR (CDCl.sub.3) 0.88 (t, 6.7 Hz, 3H), 
1.12 (t, J=7.1 Hz, 6H), 1.20 to 1.48 (m+t, J=7.1 Hz, 22H), 1.61 (m, 2H), 
2.41 (t, J=7.2 Hz, 2H), 3.99 (m, 4H), 4.22 (m, 4H), 7.30 (m, 2H), 7.40 
(m, 1H), 7.85 (m, 1H), 8.54 (dd, J=47.7 Hz, 28.5 Hz, 1H); HRMS calcd for 
(M+H).sup.+ C.sub.29 H.sub.49 O.sub.6 P.sub.2 m/z 555.3004, found 
555.3002. 
Example 10 
[2-[2-(1-Tridecynyl)phenyl]ethenylidene]bisphosphonic acid 
Tetraethyl[2-[2-(1-tridecynyl)phenyl]ethenylidene]bisphosphonate (1.26 g, 
2.28 mmol) prepared according to Example 9 was dissolved in a mixture of 
CH.sub.2 Cl.sub.2 -CD.sub.2 Cl.sub.2 (12 ml, 1.73:1). Trimethylsilyl 
bromide (1.3 ml, 9.85 mmol) was added, and the reaction was stirred for 5 
hours. At this time additional trimethylsilyl bromide (0.2 ml, 1.5 mmol) 
was added. The progress of the reaction was monitored by .sup.1 H and 
.sup.31 P NMR, until no additional change occurred. .sup.1 H NMR was more 
useful in this regard, particularly the low field doublet of doublets. At 
the end of 8 hours, the solvent was removed under reduced pressure and the 
resulting oil was placed on the vacuum line. Tetrahydrofuran (10 ml) and 
water (0.25 ml, 13.9 mmol) were added and stirred for 20 hours, then 
removed under reduced pressure. NMR showed at least two species to be 
present, so the tetrahydrofuran-water treatment was repeated. After 
stripping, the residue was recrystallized from heptane as a white solid, 
0.793 g, 1.79 mmol, 78.5% yield; mp 141.degree.-142.degree. C.; .sup.1 H 
NMR (CD.sub.3 OD) .differential. 0.89 (m, 3H), 1.20 to 1.55 (m, 16H), 1.63 
(quintet, J=7.4 Hz, 2H), 2.46 (t, J=7.1 Hz, 2H), 7.24 to 7.42 (m, 3H), 
7.98 (m, 1H), 8.42 (dd, J=46.4, 29.0 Hz, 1H); FAB mass spectrum m/z 443 
(M+H).sup.+. 
Example 11 
Disodium[2-[2-(1-tridecynyl)phenyl]ethenylidene]bisphosphonate 
[2-[2-(1-Tridecynyl)phenyl]ethenylidene]bisphosphonic acid (0.101 g, 0.228 
mmol) prepared according to Example 10 was dissolved in dry 
tetrahydrofuran (10 ml) and stirred while sodium ethoxide in ethanol 
(0.177M, 2.58 ml, 0.457 mmol) was added. After 20 minutes, the solvent was 
removed under reduced pressure to give a yellowish solid. This solid was 
pulverized and washed successively with dry ether, tetrahydrofuran, 
acetone, acetonitrile, and ethanol, then dried on the vacuum line, 0.104 
g, 0.214 mmol, 94% yield. .sup.1 H NMR (D.sub.2 O) .differential. 0.74 (m, 
3H), 1.17 (m, 14H), 1.35 (m, 2H), 1.52 (m, 2H), 2.39 (t, J=7.1H, 2H), 7.24 
(m, 2H), 7.37 (m, 1H), 7.92 (dd+m, J=43.8, 27.0 Hz, 2H); FAB mass spectrum 
m/z 443 (M-2Na+3H).sup.+, 465 (M-Na+2H).sup.+, 487 (M+H).sup.+. 
Example 12 
Tetrasodium[2-[2-(1-tridecynyl)phenyl]ethenylidene]bisphosphonate 
[2-[2-(1-Tridecynyl)phenyl]ethenylidene]bisphosphonic acid (0.102 g, 0.23 
mmol) prepared according to Example 10 was dissolved in dry 
tetrahydrofuran (10 ml) and stirred while sodium ethoxide in ethanol 
(0,177M, 5.19 ml, 0.92 mmol) was added. After 20 minutes, the solvent was 
removed under reduced pressure to give a light solid. The solid was 
powdered and washed with dry ethanol, then dried under vacuum, 40.8 mg, 
0.084 mmol, 36.5% yield; mp darkens above 233.degree. C. under N.sub.2 ; 
.sup.1 H NMR (D.sub.2 O) .differential. 0.89 (m, 3H), 1.21 to 1.57 (m, 
16H), 1.67 (m, 2H), 2.55 (t, J=7.1 Hz, 2H), 7.35 (m, 2H), 7.51 (m, 1H), 
7.90 (dd, J=42.4, 26.3 Hz, 1H), 8.19 (d, J=7.7 Hz, 1H). 
Example 13 
Diethyl E-2-[2-(1-tridecynyl)phenyl]ethenylphosphonate 
Tetraethyl[2-[2-(1-tridecynyl)phenyl]ethenylidene]bisphosphonate (2.0 g, 
3.61 mmol) prepared according to Example 9 was dissolved in 
tetrahydrofuran (26 ml). Lithium hydroxide (0.152 g, 3.62 mmol) was 
dissolved in water (10 ml) and added to the tetrahydrofuran solution. 
After stirring the reaction overnight, ether was added, and the aqueous 
phase was extracted with ether. The combined ether layers were dried 
(MgSO.sub.4) and stripped down. The residue was chromatographed on silica 
eluting with 3% ethanol in hexane. Tlc on silica, developed in 5% ethanol 
in hexane, was used to identify product, R.sub.f =0.04. Impure fractions 
were combined and rechromatographed as necessary to give 1.21 g, 2.89 
mmol, 80% yield of the product as a pale yellow liquid; .sup.1 H NMR 
(CDCl.sub.3) .differential. 0.88 (m, 3H), 1.15 to 1.81 (several m, 24H), 
2.46 (t, J=7.1 Hz), 4.15 (m, 4H), 6.37 (t, J=18 Hz, 1H), 7.26 (m, 2H), 
7.42 (m, 1H), 7.54 (m, 1H), 7.95 (dd, J=23.0, 17.7 Hz, 1H); EI mass 
spectrum m/z 419 (M+H).sup.+. 
Example 14 
2-[2-(1-Tridecynyl)phenyl]ethenylphosphonic acid 
Diethyl E-2-[2-(1-tridecynyl)phenyl]ethenylphosphonate (0.521 g, 1.25 mmol) 
prepared according to Example 13 was dissolved in CH.sub.2 Cl.sub.2 : 
CD.sub.2 Cl.sub.2 (1:1, 9 ml) and stirred while bromotrimethylsilane (0.38 
g, 2.48 mmol) was added. The reaction was monitored by .sup.1 H and 
.sup.31 P NMR. Additional bromotrimethylsilane (0.1 ml, 0.76 mmol) was 
added after 6.5 hours. After stirring for 30 hours, the solvent was 
removed under reduced pressure. Acetone (5 ml) was added, along with water 
(0.15 ml, 8.3 mmol). The reaction mixture was stirred for about 42 hours, 
then stripped. NMR indicated that the reaction was incomplete. Acetone (3 
ml) and water (0.2 ml, 11.1 mmol) were added back and the reaction was 
stirred overnight. The solvent was then removed, and the residue was 
recrystallized from heptane, as a white crystalline solid, 0.348 g, 0.96 
mmol, 76.8% yield; mp 82.degree.-83.degree. C.; .sup.1 H NMR (CDCl.sub.3) 
.differential. 0.85 (t, J=6.7 Hz, 3H), 1.10 to 1.45 (m, 16H), 1.59 (m, 
2H), 2.43 (t, J=7.1 Hz, 2H), 6.44 (t, J=18.5 Hz, 1H), 7.26 (m, 2H), 7.41 
(m, 1H), 7.54 (m, 1H), 8.04 (dd, J=24.2, 17.6 Hz, 1H), 10.46 (br s, 2H); 
FAB mass spectrum m/z 363 (M+H).sup.+. 
Example 15 
Tetraethyl[2-[2-(1-Z-tridecenyl)phenyl]ethenylidene]bisphosphonate 
Tetraethyl[2-[2-(1-tridecynyl)phenyl]ethenylidene]bisphosphonate (1.5 g, 
2.7 mmol) prepared according to Example 9 was dissolved in ethanol (17 
ml), and quinoline (0.043 ml) and 5% palladium on calcium carbonate 
poisoned with lead (13 mg) was added. A balloon filled with hydrogen was 
attached to the flask after purging with hydrogen. After stirring for 3 
hours the hydrogen pressure was released, and the reaction mixture was 
filtered through Celite. The filtrate was stripped. .sup.1 H NMR showed 
that only small amount of starting material had been converted to product. 
The hydrogenation was repeated using twice the amount of catalyst and 
quinoline for 4.5 hours, then workup as before. .sup.1 H NMR showed that a 
considerable amount of starting material remained. Three additional 
hydrogenations totaling 47 hours were done before the conversion was 
greater than 90%. Chromatography on silica eluting with 5% ethanol in 
petroleum ether (90.degree.-110.degree. C. boiling range) gave the 
product, 0.459 g, 0.825 mmol, 30.5 % yield; .sup.1 H NMR (CDCl.sub.3) 
.differential. 0.88 (t, J=6.7 Hz, 3H), 1.03 to 1.45 (several m, 30H), 2.09 
(m, 2H), 3.96 (m, 4H), 4.20 (m, 4H), 5.79 (dt, J=11.5, 7.4 Hz, 1H), 6.37 
(d, J =11.4 Hz, 1H), 7.28 (m, 3H), 7.78 (d, J=7.3 Hz, 1H), 8.31 (dd, 
J=47.8, 28.4 Hz, 1H); CI mass spectrum m/z 557 (M+H).sup.+. 
Example 16 
Methyl 3-(2-bromophenyl)-2-E-(diethoxyphosphinyl)-2-propenoate 
2-Bromobenzaldehyde (25.89 g, 139.9 mmol) and methyl diethyl 
phosphonoacetate (32.55 g, 148.7 mmol) were combined with piperidine (0.83 
ml, 8.4 mmol) and benzoic acid (0.856 g, 7.01 mmol) in dry benzene (225 
ml). Under argon, this mixture was refluxed for 10.5 days using a 
Dean-Stark trap to collect the water liberated. At the end of this time, 
the reaction mixture was cooled to ambient temperature and poured into 
water (200 ml). The organic layer was washed with water (200 ml), 0.1N HCl 
(2.times.150 ml), water (150 ml), saturated NaHCO.sub.3 (2.times.150 ml), 
and water (150 ml). After drying (MgSO4), the organic layer was stripped 
down to an orange-red oil, 41.47 g. This was chromatographed on silica, 
eluting first with 60:40 petroleum ether (boiling range 
37.degree.-52.degree. C.): CH.sub.2 Cl.sub.2, then with CH.sub.2 Cl.sub.2, 
then with hexane-ethyl acetate mixtures to pure ethyl acetate. Fractions 
containing a spot at R.sub.f =0.06 (silica tlc developed with 68% hexane, 
32% ethyl acetate) were combined and rechromatographed on silica eluting 
with 68% hexane, 32% ethyl acetate. Fractions containing the desired spot 
were combined and stripped down to a viscous liquid, 25.5 g, 64.85 mmol, 
46.4% yield. .sup.1 H NMR (CDCl.sub.3) .differential. 1.39 (t, J=7.1 Hz, 
6H), 3.68 (s, 3H), 4.24 (quint, J=7.3 Hz, 4H), 7.19 to 7.36 (several m, 
3H), 7.61 (dm, J=7.5 Hz, 1H), 7.85 (d, J =23.2 Hz, 1H); EI mass spectrum 
m/z 377, 379 (M+H).sup.+. 
Example 17 
Methyl 3-[2-(1-tridecynyl)phenyl]-2-E-(diethoxyphosphinyl)-2-propenoate 
Methyl 3-(2-bromophenyl)-2-E-(diethoxyphosphinyl)-2-propenoate (11.31 g, 
29.99 mmol) prepared according to Example 16 was mixed with 1-tridecyne 
(9.50 g, 52.68 mmol) and triphenylphosphine (0.214 g, 0.82 mmol) in a 500 
ml four-neck round bottom flask equipped with a magnetic stir bar, 
condenser topped with an argon inlet, and an internal thermometer. Dry 
triethylamine (156 ml) was added, and the flask was closed. The system was 
inerted and a bubbler was connected to the argon line. Palladium acetate 
(0.0733 g, 0.33 mmol) was added, and the reaction mixture was heated in an 
oil bath (T=100.degree. to 105.degree. C.). The reaction was refluxed for 
50 hours. At the end of this time, the reaction mixture was cooled to room 
temperature and was filtered. The white precipitate was washed with 
triethyl amine (100 ml). The filtrates were stripped down to a dark oil. 
This oil was extracted three times with acetonitrile (50 ml). The combined 
acetonitrile extracts were stripped down to an orange oil which was 
chromatographed on silica eluting with petroleum ether-dichloromethane 
(68:32) increasing to pure dichloromethane, then with hexanesethyl acetate 
(1:1) increasing to pure ethyl acetate. Fractions containing a spot with 
R.sub.f =0.20 on silica developed with hexane-dichloromethane (68:32) were 
combined and stripped. Reverse phase chromatography on C.sub.18 silica, 
eluting with 10% water in methanol, was done. Fractions were combined 
which showed the above R.sub.f. These fractions were rechromatographed on 
silica eluting with hexanes-ethyl acetate (66:34) and progressing to 
hexanes-ethyl acetate (60:40). Additional rechromatography of impure 
fractions gave 4.18 g (8.77 mmol, 29% yield) of pure product as a pale 
yellow oil. .sup.1 H NMR (CDCl.sub.3) .differential. 0.88 (t, J=6.7 Hz, 
3H), 1.17 to 1.50 (several m, 22H), 1.63 quintet, J=7.6 Hz, 2H), 2.43 (t, 
J=7.2 Hz, 2H), 3.72 (s, 3H), 4.21 (m, 4H), 7.19 to 7.36 (several m, 3H), 
7.44 (dm, J=7.6 Hz, 1H), 8.05 (d, J=23.8 Hz); EI mass spectrum m/z 476 
(M.sup.+). 
Example 18 
Tetraethyl[2-[2-[3-[(1-oxononyl)amino]-1-propynyl]phenyl]ethenylidene]bisph 
osphonate 
A. Preparation of N-Propynylnonanamide Propynylamine (10.4 ml, 151.2 mmol) 
was mixed with 150 ml of triethylamine in a round bottom flask immersed in 
a cold water bath, and vigorously stirred while nonanoyl chloride (27.0 
ml, 149.75 mmol) was added in portions from a dropping funnel over a 30 
minute period. Additional triethylamine (100 ml) was added to facilitate 
stirring. At the end of the addition, the water bath was removed and the 
reaction was stirred for 30 minutes, then filtered. The filtrate was 
stripped down to a yellow solid. The solid was partitioned between ether 
and aqueous NaOH, requiring a considerable quantity of ether to dissolve. 
The combined ether extracts were stripped and recrystallized from heptane 
to give product as a white crystalline solid, 18.12 g, 92.8 mmol, 62% 
yield; mp. 78.degree.-79.5.degree. C.; .sup.1 H NMR (CDCl.sub.3) 
.differential. 0.88 (t, J=6.0 Hz, 3H), 1.28 (m, 10H), 1.63 (m, 2H), 2.19 
(t, J=7.6 Hz, 2H), 2.22 (t, J=2.7 Hz, 1H), 4.06 (dd, J=5.2, 2.6 Hz), 5.62 
(br s, 1H); EI mass spectrum m/z 196 (M+H).sup.+. 
B. N-[3-(2-formylphenyl)-2-propynyl]nonanamide 2-Iodobenzaldehyde (4.06 g, 
17.50 mmol) and N-propynylnonanamide (10.12 g, 51.82 mmol) were added to a 
three neck round bottom flask equipped with an argon inlet and magnetic 
stir bar, along with cuprous iodide (0.622 g, 3.27 mmol) and 
tetrakis(triphenylphosphine)palladium (2.0 g, 1.7 mmol). Triethylamine 
(4.9 ml, 35.6 mmol) was added, then anhydrous N,N-dimethylformamide (90 
ml). The reaction was vigorously stirred for 2 hours and 50 minutes. At 
this time 1 ml of 1:1 CH.sub.3 OH: CH.sub.2 Cl.sub.2 was added and the 
solvent was removed under reduced pressure with slight heating 
(.ltoreq.40.degree. C.). The dark liquid was partitioned between CH.sub.2 
Cl.sub.2 and water. The organic layer was washed several times with water, 
then dried over K.sub.2 CO.sub.3 and stripped down to an orange-red solid. 
Chromatography on silica eluting with hexane-ethyl acetate mixtures gave 
product which was recrystallized from ether-heptane to yield product, 
1.21 g, 4.05 mmol 23% yield; mp 65.5.degree.-66.5.degree. C.; .sup.1 H NMR 
(CDCl.sub.3) .differential. 0.87 (m, 3H), 1.29 (m, 10H), 1.67 (m, 2H), 
2.24 (t, J=7.6 Hz, 2H), 4.35 (d, J=5.3 Hz, 2H), 5.78 (br s, 1H), 7.41 to 
7.56 (2 m, 3H), 7.90 (d, J=7.7 Hz), 10.47 (s, 1H); CI mass spectrum m/z 
300 (M+H).sup.+. 
C. 
Tetraethyl[2-[2-[3-[(1-oxononyl)amino]-1-propynyl]phenyl]ethenylidene]bisp 
hosphonate In a three neck round bottom flask equipped with a magnetic stir 
bar, argon inlet, and septum, dry tetrahydrofuran (13.5 ml) was added 
under argon. The flask was placed in an ice bath. Titanium tetrachloride 
(0.74 ml, 6.75 mmol) was dissolved in dry carbon tetrachloride (1.85 ml), 
and added to the stirred tetrahydrofuran over a fifteen minute period, 
producing a bright yellow precipitate. To the stirred mixture, 
N-[3-(2-formylphenyl)-2-propynyl]nonanamide (1.017 g, 3.40 mmol) was 
added via cannula over a ten minute period, and the flask which contained 
it was washed with tetrahydrofuran which was added to the reaction. 
Tetraethylmethylenediphosphonate (0.98 g, 3.4 mmol) was then added over a 
three minute period. A solution of N-methylmorpholine (1.50 ml, 13.64 
mmol) in 2.26 ml of tetrahydrofuran was added over a 45 minute period. 
After stirring for 3.75 hours at 0.degree. C., water (3.8 ml) was added. 
The flask was removed from the ice bath and the mixture was extracted with 
ether (3.times.12 ml). The ether layers were combined and washed 
successively with saturated aqueous sodium chloride solution (12 ml), 
saturated aqueous sodium bicarbonate solution (12 ml), and again with 
saturated aqueous sodium chloride solution (12 ml). After drying 
(MgSO.sub.4) and filtration, the solvent was stripped off. The residue was 
chromatographed on silica eluting with 20% isopropanol in ethyl acetate, 
giving 1.08 g, 1.90 mmol, 55.9% yield of a yellow liquid; .sup.1 H NMR 
(CDCl.sub. 3) .differential. 0.87 (t, J=6.7 Hz, 3H), 1.11 to 1.44 (several 
m, 22H), 1.66 (m, 2H), 2.28 (t, J=7.6 Hz, 2H), 4.03 (m, 4H), 4.12 to 4.31 
(2 m, 6H), 7.19 (br s, 1H), 7.38 (several m, 3H), 7.99 (m, 1H), 8.71 (dd, 
J=48.1, 30.1 Hz, 1H); CI mass spectrum m/z 570 (M+H).sup.+. 
The chemical reactions described above are generally disclosed in terms of 
their broadest application to the preparation of the compounds utilized to 
practice the method of this invention. Occasionally, the reactions may not 
be applicable as described to each compound included within the disclosed 
scope. The compounds for which this occurs will be readily recognized by 
those skilled in the art. In all such cases, either the reactions can be 
successfully performed by conventional modifications known to those 
skilled in the art, e.g., by appropriate protection of interfering groups, 
by changing to alternative conventional reagents, by routine modification 
of reaction conditions, and the like, or other reactions disclosed herein 
or otherwise conventional, will be applicable to the preparation of the 
corresponding compounds of this invention. In all preparative methods, all 
starting materials are known or readily preparable from known starting 
materials. 
The following example demonstrates the effectiveness of the PLA.sub.2 
inhibitors of the invention utilizing the PLA.sub.2 inhibition assays 
described below. 
Example 19 
The PLA.sub.2 inhibitors of Examples 1-3, 5, 6, 8-15, 17 and 18 were 
utilized in the following PLA.sub.2 inhibition assays. 
The primary assay shows inhibition of purified calcium-independent canine 
myocardial PLA.sub.2. This assay consists of determining the amount of 
radiolabeled fatty acid which is liberated from the radiolabeled 
phospholipid substrate by purified calcium-independent canine myocardial 
PLA.sub.2 in the presence of varying concentrations of the inhibitor. The 
concentration of inhibitor which decreases the activity of the enzyme to 
50% of the activity observed in the absence of that inhibitor is 
determined. This concentration is defined as the IC.sub.50. 
Several other PLA.sub.2 assays were also utilized to demonstrate the 
effectiveness of the PLA.sub.2 inhibitors of the invention. Specifically, 
one micromolar calcium-dependent mammalian PLA.sub.2 assay was used, i.e., 
sheep platelet PLA2, and two millimolar calcium-dependent mammalian 
PLA.sub.2 assays were used, i.e., human synovial fluid PLA.sub.2 and 
porcine pancreatic PLA.sub.2. The PLA.sub.2 enzymes represent different 
groups of PLA.sub.2 enzymes. Myocardial PLA.sub.2, platelet PLA.sub.2 and 
secreted PLA.sub.2 enzymes (human synovial fluid PLA.sub.2 and porcine 
pancreatic PLA.sub.2) have very different calcium requirements, which may 
be typical of their respective classes. 
An additional whole cell assay was also performed. In this assay, HL-60 
cells are induced with the calcium ionophore A23187 in the presence of 
varying amounts of inhibitor, and the amounts of the prostaglandin E.sub.2 
(PGE.sub.2) and leukotriene B.sub.4 (LTB.sub.4) which form are measured. 
Calcium ionophore A23187 activates a calcium-dependent PLA.sub.2 which 
leads to the enhanced production of the arachidonic acid metabolites 
PGE.sub.2 and LTB.sub.4. The concentrations of inhibitor which decrease 
the production of PGE.sub.2 and LTB.sub.4 by 50% relative to controls are 
expressed as IC.sub.50 values. This assay, when inhibition is observed, 
suggests that the inhibitor enters the cell and inhibits PLA.sub.2 or 
other enzyme in the arachidonate metabolic pathway which produces 
PGE.sub.2 and LTB.sub.4. 
Calcium-Independent PLA.sub.2 Assay 
Purified canine myocardial cytosolic PLA.sub.2 was isolated from an 
ATP-agarose eluate as described by Wolf, R. A., and Gross, R. W., J. Biol. 
Chem., 260, 7295-7303 (1985). The calcium-independent myocardial PLA.sub.2 
was incubated with selected concentrations (10.sup.-10 to 10.sup.-4 M) of 
test compound in 168 mM Tris-Cl, 6.4 mM EGTA (a calcium chelator) (pH 7.0) 
for 5 min. at 25.degree. C. Appropriate controls were performed in the 
absence of test compound. Catalysis was initiated by injection of 1 .mu.M 
radiolabeled substrate (1-O-(Z)-(1'-hexadecenyl)-2-[9,10.sup.-3 
H]-oleoyl-3-phosphorylcholine). After a 5 min. incubation at 37.degree. 
C., reaction products were extracted with n-butanol, separated by thin 
layer chromatography, and quantified by scintillation spectrometry. 
PLA.sub.2 activity was compared in the presence and absence of test 
compound and the IC.sub.50 was determined. 
Micromolar Calcium-Dependent PLA.sub.2 Assay 
Homogeneous sheep platelet cytosolic PLA.sub.2 was prepared as described by 
Loeb, L. A., and Gross, R. W., J. Biol. Chem., 261, 10467-10470 (1986). 
The purified calcium-dependent platelet PLA.sub.2 was preincubated in 70 
mM Tris-Cl (pH 7.2) containing 1 .mu.M CaCl.sub.2 and test compound for 5 
min. at 25.degree. C. Catalysis was initiated by injection of 1 .mu.M 
radiolabeled substrate (1-O-(Z)-(1'-hexadecenyl)-2-[9,10.sup.3 
H]-oleoyl-3-phosphorylcholine). After a 10 min. incubation period at 
37.degree. C., reaction products were extracted with n-butanol, separated 
by thin layer chromatography, and quantified by scintillation 
spectrometry. PLA.sub.2 activity was compared in the presence and absence 
of test compound and the IC.sub.50 was determined 
While calcium is known to be sufficient for activation of sheep platelet 
PLA.sub.2, it has been found that calcium is not necessary for activation. 
See Zupan, L. A., et al, "Calcium is sufficient but not necessary for 
activation of sheep platelet cytosolic phospholipase A.sub.2 ", FEBS 
Letters, Vol 284, No 1, 27-30 (1991). 
Millimolar Calcium-Dependent PLA.sub.2 Assays 
Purified human synovial fluid PLA.sub.2 (purified by the procedure of 
Fawzy, A. A. and Franson, R. C. Biophys. J. 49, 533a [1986]) was obtained 
from R. C. Franson (Medical College of Virginia, Richmond, Va.). PLA.sub.2 
activity was measured by using [.sup.14 C]-oleate labeled autoclaved E. 
coli as substrate as described by Franson, R. C., Patriarca, P., and 
Elsbach, P. J., Lipid Res. 15, 380-388 (1974). The assay was performed at 
37.degree. C. for 30 min. in a final volume of 100 .mu.L 50 mM Hepes 
buffer (pH 7.0) containing 150 mM NaCl, 5 mM CaCl.sub.2, and E. coli cells 
(corresponding to 10 nmol phospholipid). The test compound or control 
vehicle was preincubated with PLA.sub.2 for 5 min., followed by adding E. 
coli to initiate the reaction. The reaction was terminated by adding 2 mL 
tetrahydrofuran. The reaction product, [.sup.14 C]-oleic acid, was 
isolated using a 1 mL Bond Elute-NH.sub.2 column and counted by liquid 
scintillation spectrometry. 
Purified porcine pancreatic PLA.sub.2 was obtained from Sigma (St. Louis, 
Mo.). PLA.sub.2 activity was measured by using [.sup.14 C]-oleate labeled 
autoclaved E. coli as described by Franson, Patriarca and Elsbach 
(referenced above). The assay was performed at 37.degree. C. for 5 min. in 
a final volume of 100 .mu.L Tris-HCl buffer (100 mM, pH 8.0) containing 1 
mM EDTA, 10 mM CaCl.sub.2, and E. coli (containing 10 nmol phospholipid). 
The test compound or control vehicle was preincubated with PLA.sub.2 for 5 
min. followed by adding E. coli to initiate the reaction. The reaction was 
terminated by adding 2 mL tetrahydrofuran. The reaction product [.sup.14 
C]-oleic acid, was isolated using a 1 mL Bond Elute-NH.sub.2 column and 
counted by liquid scintillation spectrometry. 
HL-60 Cell Assay for LTB.sub.4 and PGE.sub.2 Production 
HL-60 cells grown exponentially in culture were induced to differentiate 
into granulocytes by a four day incubation with 0.8% (v/v) 
N,N-dimethylformamide. Prior to assay, differentiated HL-60 cells were 
washed once with Hanks' balanced salt solution containing 0.35 mg/mL 
sodium bicarbonate and 10 mM Hepes, pH 7.3 (HBSS) and resuspended in HBSS 
at a 3.times.10.sup.6 cells/mL concentration. DMSO or test compounds 
solubilized in DMSO were added at 1:100 dilution to 1.0 mL HL-60 cell 
suspensions (3.times.10.sup.6 cells) and preincubated at 37.degree. C. for 
10 min. in a shaking water bath. After an additional 5 min. incubation 
with 5.times.10.sup.-6 M calcium ionophore, A23187 (Calbiochem, LaJolla, 
Calif.), the cells were centrifuged at 12,800.times.6 for 15 seconds and 
the supernatant (0.8 mL) removed and stored at -20.degree. C. for 
LTB.sub.4 and PGE.sub.2 quantitation by radioimmunoassay (kits obtained 
from Amersham, United Kingdom and NEN Research Products, N. Billerica, 
Mass.). 
Two conventions are used in Table 1 to indicate an approximate degree of 
inhibition when an IC.sub.50 was not reached at the highest concentration 
of inhibitor: One greater than (&gt;) symbol before the number indicates that 
inhibition was observed, but not quite enough for an IC.sub.50. Two 
greater than symbols (&gt;&gt;) indicate little or no inhibition at the highest 
concentration used. Activation of an enzyme is indicated by a +X % which 
gave that activation of X %. 
The results in Table 1 illustrate the effectiveness of the compounds of the 
invention in inhibiting PLA.sub.2, specifically calcium-independent 
PLA.sub.2 and micromolar calcium-dependent PLA.sub.2 as evidenced by the 
results of the compounds in the canine heart PLA.sub.2 and sheep platelet 
PLA.sub.2 assays. 
TABLE 1 
__________________________________________________________________________ 
Canine 
Sheep Porcine 
Example 
Heart Platelet 
HSF Pancreatic 
HL-60 HL-60 
No. PLA.sub.2 
PLA.sub.2 
PLA.sub.2 
PLA.sub.2 
PGE.sub.2 
LTB.sub.4 
__________________________________________________________________________ 
1 0.56 0.18 &gt;&gt;100 
100.sup.a 
3.0 1.2 
2 0.45 11 31 
&gt;&gt;100 &gt;&gt;10 &gt;&gt;10 
3 0.36 3.2 38 
&gt;&gt;100 &gt;&gt;10 &gt;&gt;10 
5 0.34 2.4 &gt;100 
&gt;&gt;100 2.3 1.3 
6 5.6 1.4 16 
2.5 3.5 4.4 
8 0.36 0.22 -- -- -- -- 
9 1.5 0.16 &gt;&gt;100 
-- 4.4 2.6 
10 0.71 50 50 
&gt;&gt;100 &gt;10 10.sup.b 
11 1.9 2.8 25 
&gt;&gt;100 &gt;&gt;10 &gt;&gt;10 
12 2.8 7.1 -- -- -- -- 
13 3.0 1.1 -- -- -- -- 
14 4.5 2.9 -- -- -- -- 
15 1.33 0.251 -- -- -- -- 
17 1.6 0.631 -- -- -- -- 
18 &gt;100 .about.100 
-- -- -- -- 
__________________________________________________________________________ 
.sup.a +40% 
.sup.b +21% 
.sup.c "--" indicates the compound was not tested.