Compound, oleyl-2-pyridinioethyl phosphate having antifungal and antiprotozoal properties

Phospholipids, inclusive of pharmaceutically acceptable salts thereof, of the formula ##STR1## wherein R.sup.1 is a C.sub.8-30 aliphatic hydrocarbon residue, R.sup.2, R.sup.3 and R.sup.4 are independently hydrogen or lower alkyl or ##STR2## represents cyclic ammonio and n is 0 or 1, exhibit inhibitory activity to multiplication of tumor cells and antimycotic (antifungal) and antiprotozoal activities, and are useful for inhibiting multiplication of tumor cells and prolonging the survival time of tumor-bearing warm-blooded animal, for treating or preventing a disease in an animal caused by a mycete (fungus) and for treating or preventing a plant disease.

This invention relates to novel antifungal and antitumor agents. More 
particularly, this invention relates to antifungal and antitumor agents 
containing a phospholipid, inclusive of a pharmaceutically acceptable salt 
thereof, of the formula 
##STR3## 
wherein R.sup.1 is a C.sub.8-30 aliphatic hydrocarbon residue, R.sup.2, 
R.sup.3 and R.sup.4 are independently hydrogen or lower alkyl or 
##STR4## 
represents cyclic ammonio, and n is 0 or 1. 
Referring to the above formula (I), the C.sub.8-30 aliphatic hydrocarbon 
residue represented by R.sup.1 includes straight or branched chain 
saturated or unsaturated aliphatic hydrocarbon residues (e.g. alkyl, 
alkenyl, alkynyl, etc.), which may be substituted or unsubstituted. The 
alkenyl group may be Z- or E-configuration. R.sup.1 may have further one 
or more substituents such as hydroxy, mercapto, amino, oxo, carbamoyl, 
carboxy, halogen, C.sub.3-7 cycloalkyl, C.sub.3-7 cycloalkenyl, aryl (e.g. 
phenoxy, tolyl, phenyl, etc.), etc. As a preferred embodiment of R.sup.1, 
there may be mentioned, for example, C.sub.8-30 alkyl group [e.g. 
n-dodecyl, n-tridecyl, n-tetradecyl, 3,7,11-trimethyldodecyl, 
n-pentadecyl, n-heptadecyl, n-octadecyl, n-eicosyl, n-docosyl, 
3,7-dimethyloctyl, (1-octyl)nonyl 3,7,11,15-tetramethylhexadecyl], among 
which C.sub.10-30 alkyl group is more preferred, C.sub.8-30 alkenyl group 
[e.g. 8-tridecenyl (.DELTA..sup.8), 3,7,11-trimethyl-2,6,10-dodecatrienyl, 
8-tetradecenyl (.DELTA..sup.8), 8,11-tetradecadienyl (.DELTA..sup.8,11), 
8-heptadecenyl (.DELTA..sup.8), 2-octadecenyl, 9-octadecenyl (oleyl), 
9,15-octadecadienyl, 9,12,15-octadecatrienyl, 8,11,14-heptadecatrienyl 
(.DELTA..sup.8,11,14), 8,11-octadecadienyl (.DELTA..sup.8,11), 
4,7,10,13-nonadecatetraenyl (.DELTA..sup.4,7,10,13), phythyl, 
3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl, 
3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenyl, 
12-(2,3-cyclopentenyl)dodecyl, 12-(2,3-cyclopentenyl)-5-dodecenyl, 
11-hydroxy-8-heptadecenyl, 
3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraenyl, 
4,7,10,13-nonadecatetraenyl], C.sub.8-30 alkynyl group [e.g. 
9-octadecynyl, 9,15-octadecadiynyl, heptadecan-8-ynyl,4-decynyl], 
C.sub.8-30 aralkyl [e.g. 15-(4-n-butylphenyl)pentadecyl, 
.omega.-(p-tolyl)heptadecyl, 
6-(4-n-pentylphenyl)hexadecyl,15-phenylpentadecyl], and 
15-(4-n-butylphenoxy)pentadecyl or 6-(4-n-pentylphenoxy)hexadecyl. 
R.sup.2, R.sup.3 and R.sup.4 are independently hydrogen or lower alkyl 
which may be substituted. As the lower alkyl group, there may be 
mentioned, for example, C.sub.1-5 alkyl group (e.g. methyl, ethyl, propyl, 
i-propyl, n-butyl). These groups may further have one or more substituents 
such as hydroxycarbonyl, lower (C.sub.1-3) alkoxycarbonyl, hydroxy, cyano 
or lower (C.sub.1-3) alkoxy. 
As the cyclic ammonio group represented by 
##STR5## 
there may be mentioned, for example, pyridinio, oxazolio, thiazolio, 
pyridazinio, quinolinio or isoquinolinio, and these groups may further 
have one or more substituents such as C.sub.1-4 alkyl (e.g. methyl, 
ethyl), hydroxy, hydroxyethyl, aminoethyl, amino (imino), carbamoyl or 
ureido. The above-mentioned cyclic ammonio group includes cases where any 
two groups of R.sup.2, R.sup.3 and R.sup.4 form a ring together with the 
quaternary nitrogen atom and the remaining one group is C.sub.1-4 alkyl 
group (e.g. methyl, ethyl), for example, N-methylmorpholinio or 
N-methylpiperadinio. 
The compound (I) may be present in, for example, the form of a salt 
representable by the formula 
##STR6## 
wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are as defined above and 
A.sup.- is an anion such as chlorine, bromine or iodine ion or by the 
formula 
##STR7## 
wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are as defined above, and 
M.sup.+ is an alkali metal (e.g. Na, K) ion. The compound (I) can form a 
salt together with an alkaline earth metal (e.g. Ca, Mg) ion. 
Further, this invention relates to novel phospholipids, inclusive of 
pharmaceutically acceptable salts thereof, of the formula 
##STR8## 
wherein R.sub.a.sup.1' is a C.sub.8-30 aliphatic hydrocarbon residue, and 
R.sub.a.sup.2', R.sub.a.sup.3' and R.sub.a.sup.4' are independently 
hydrogen or lower alkyl or 
##STR9## 
represents cyclic ammonio, and of the formula 
##STR10## 
wherein R.sub.b.sup.1' is a C.sub.8-14 or C.sub.18-30 aliphatic 
hydrocarbon residue, and R.sub.b.sup.2', R.sub.b.sup.3' and R.sub.b.sup.4' 
are independently hydrogen or lower alkyl or 
##STR11## 
represents cyclic ammonio. 
Referring to the above formula (I'A), R.sub.a.sup.1', R.sub.a.sup.2', 
R.sub.a.sup.3' and R.sub.a.sup.4' are of the same meaning as R.sup.1, 
R.sup.2, R.sup.3 and R.sup.4 respectively. 
Referring to the formula (I'B), the C.sub.8-14 and C.sub.18-30 aliphatic 
hydrocarbon residues represented by R.sub.b.sup.1' include straight or 
branched chain saturated or unsaturated hydrocarbon residues having 8 to 
14 and 18 to 30 carbon atoms. Practical embodiments of the aliphatic 
hydrocarbon residues are as exemplified in the foregoing and they may 
optionally have substituents as mentioned above. 
R.sub.b.sup.2', R.sub.b.sup.3' and R.sub.b.sup.4' are of the same meaning 
as R.sup.2, R.sup.3 and R.sup.4 respectively. 
The compounds (I'A) and (I'B) may be present in the form of salts 
corresponding to the salt (Ia) or (Ib). 
Among the compounds of the formula (I), those wherein n equals to 1 can be 
produced by the following methods. 
Method A 
A compound of the formula 
EQU R.sup.1 --OH (II) 
wherein R.sup.1 is defined above, is allowed to react with a compound of 
the formula 
##STR12## 
wherein X and Y are respectively halogen (e.g. chlorine, bromine, iodine), 
to give a compound of the formula 
##STR13## 
wherein R.sup.1, X and Y are as defined above, which is then subjected to 
hydrolysis with water to give a compound of the formula 
##STR14## 
wherein R.sup.1 and Y are as defined above. This compound is allowed to 
react with a compound or a salt thereof of the formula 
##STR15## 
wherein each symbol is as defined above, to give the compound (I). 
When two or more of R.sup.2, R.sup.3 and R.sup.4 are hydrogen, the 
following Method B can also be employed. 
Method B 
A compound of the formula 
##STR16## 
wherein X is as defined above, and either one of R' and R" is 
--COOCH.sub.2 C.sub.6 H.sub.5, --COOC.sub.6 H.sub.5, --CHO, --COCF.sub.3, 
--COCH.sub.2 C.sub.6 H.sub.5, --Si(CH.sub.3).sub.3 or --C(C.sub.6 
H.sub.5).sub.3 and the other one is R.sup.2, or R' and R", on cyclization 
with the adjacent nitrogen atom, form succinimido or phthalimido, is 
allowed to react with the compound (II). The reaction product is treated 
with water, then subjected to deprotecting reaction by a per se suitable 
conventional methods to give a compound 
##STR17## 
wherein R.sup.1 and R.sup.2 are as defined above. 
The compounds (I) wherein n equals to 0 and 1 can both be produced also by 
the following method. 
Method C 
A compound of the formula 
##STR18## 
wherein R.sup.1 and n are as defined above, is led to its reactive 
derivative, and then the reactive derivative is allowed to react with a 
compound of the formula 
##STR19## 
wherein R.sup.2, R.sup.3 and R.sup.4 are as defined above and A.sup.- is 
an anion such as chlorine, bromine, iodine or tosyl ion, to give the 
compound (I). 
The material compounds for the above-mentioned reactions can all be easily 
produced by known methods or analogous methods thereof. 
The reaction between (II) and (III) can be conducted in an inert solvent 
(e.g. benzene, toluene, dichloromethane, tetrahydrofuran) in accordance 
with a conventional manner in the presence or absence of a base (e.g. a 
tertiary base such as pyridine, picoline, triethylamine). When the 
reaction is conducted in the presence of a base, the reaction temperature 
ranges from a temperature under ice-cooling to a room temperature. When 
the reaction is conducted in the absence of a base, the reaction 
temperature may be raised by heating in order to accelerate the reaction. 
Hydrolysis of (IV) is conducted by removing the solvent used for producing 
(IV) and adding water or by adding water to the reaction mixture as it is, 
which may, upon necessity, be heated up. For accelerating the hydrolysis, 
an inorganic base such as sodium hydrogen carbonate, sodium carbonate or 
sodium hydroxide may be added in accordance with a conventional manner. 
(V) can be purified by means of chromatography, but it may be used for the 
subsequent reaction without purification. 
The reaction between (V) and (VI) can be conducted in an inert solvent 
(e.g. benzene, toluene, tetrahydrofuran) or by using (VI) itself as the 
solvent. The reaction can be conducted at a temperature ranging from a 
room temperature to the boiling point of the solvent used, but when (VI) 
is a low-boiling point compound (e.g. trimethylamine), the reaction is 
preferably conducted in a sealed vessel. The objective compound (I) can be 
purified by a per se conventional methods such as a silica-gel 
chromatography, recrystallization, or reprecipitation. 
The reaction between (II) and (III') can be conducted in a manner analogous 
to that between (II) and (III). Removal of a protecting group may 
sometimes be accomplished by mere reaction with water in the presence of 
an acid or alkali, but it can be effected also by means of catalytic 
reduction or chemical reaction. For example, benzyloxycarbonyl group or 
trityl group can be removed by a catalytic reduction in a solvent (e.g. 
water, acetic acid, alcohol, tetrahydrofuran and a mixture thereof) in the 
presence of a catalyst (palladium-carbon, Raney nickel, platinum oxide, 
etc.) in accordance with a conventional manner. Succinimido group or 
phthalimido group can be removed by treating with hydrazine. 
The method of leading (VII) to its reactive derivative in Method C can be 
conducted by a per se known method. For example, a method which comprises 
reacting (VII) with phosphorus pentachloride to lead to phosphoric 
chloride, or activating (VII) with a per se known condensing reagent (e.g. 
2,4,6-trimethylbenzenesulfonyl chloride, 8-quinolinesulfonyl chloride, 
2,4,6-isopropylbenzenesulfonyl imidazolide, 2,4,6-trimethylbenzenesulfonyl 
tetrazolide, dicyclohexyl carbodiimide, etc.), then by allowing (VIII) to 
react. 
The compound (I'A) can be produced in accordance with Method C, and the 
compound (I'B) can be produced in accordance with Method A, B or C. 
In addition to direct cytotoxicity to tumor cells, the compounds (I) and 
pharmaceutically acceptable salts thereof have host-mediated antitumor 
activity, though the mechanism has not yet been cleared. The compounds of 
the formula (I) wherein R.sup.1 is a C.sub.14-20 aliphatic hydrocarbon 
residue exhibit especially excellent effects. Specifically, when 
administered to animals bearing spontaneous carcinomas in mice or rats, 
carcinogen-induced solid tumors, MM46 derived from mastrocarcinoma, 
Ehrlich carcinoma, sarcoma 180, etc., or to nude mice implanted with human 
cancer cells, these compounds display life-span prolonging effects. The 
antitumor effect on sarcoma 180 and MM46 are respectively shown in Tables 
1 and 2. 
The antitumor agent of this invention displays excellent life-span 
prolonging effects in warm-blooded animals with a malignant tumor such as 
leukemia or solid tumor (e.g. digestive tract cancer or lung cancer). The 
compound (I) is generally available as a crystalline powder or a powder. 
Since it is sufficiently hydrophilic and lipophilic, the compound can be 
formulated into variety of pharmaceutical compositions such as injections, 
tablets, capsules, solutions, ointments, etc. 
Injectable solutions and solutions for drip infusion, for instance, can be 
prepared in the conventional manner using physiological saline or an 
aqueous vehicle containing glucose and/or other auxiliaries. Tablets, 
capsules, etc. can also be prepared by the established pharmaceutical 
procedures. These preparations may take unit dosage forms for application 
by the routes of administration suited for the purposes e.g. intravenous 
or subcutaneous preparations or preparations for direct injection at an 
effected area in the case of injectable solutions. The dosage of the 
compound (I) for tumor-bearing warm-blooded animals is selected according 
to the clinical condition, route of administration, etc. and may generally 
range from about 0.1 to 100 mg/kg body weight or preferably from about 0.5 
to 30 mg/kg body weight. The frequency of administration may be daily or 
at intervals of 2 to 7 days. For a sustained effective tissue 
concentration, the regimen of 1 to 3 divided doses daily or a drip 
infusion over a protracted time may also be feasible. 
Further, the compound (I) has antimycotic activity. Its antimycotic 
spectrum covers Trichophyton, Aspergillus niger, Penicillium and yeasts 
and, therefore, the compound is of value in the treatment and prevention 
of diseases (e.g. trichophytia) of which these organisms are causative 
agents. 
Such an antimycotic preparation can be produced by the established 
pharmaceutical procedure and while the relative amount of the active 
compound in the preparation is not particularly critical, the amount of 
the compound of this invention may range from about 0.01 to 70 weight % or 
preferably about 0.1 to 5 weight % of the total composition when the 
preparation is intended for the treatment of trichophytia for instance. 
Such an antimycotic preparation can be conveniently applied in the 
conventional manner, e.g. by direct coating or spraying to the affected 
site once to several times daily. 
The compound (I) is also active against phytopathogenic pests, especially 
fungi and, therefore is useful as an agricultural fungicide for combating 
such plant diseases as rice blast, rice Helminthosporium leaf spot, rice 
stem rot, gray mold and cucumber anthracnose. The agricultural fungicides 
can be prepared in the conventional manner. The proper content of the 
active compound is generally about 1-90% for emulsifiable concentrates, 
wettable powders and the like, and about 0.1-10% for oil solutions, dusts 
and the like, and about 5-50% for granular preparations. Emulsifiable 
concentrates, wettable powders and the like are preferably sprayed after 
adequate dilution with water or the like (e.g. 50-5,000-fold dilution). 
These agricultural fungicides can be applied in the conventional manner 
and generally in a proportion of about 10 to 300 g as the active compound 
to each 10 ares of land. The concentration of the active component in such 
fungicidal preparations is about 10 to 1000 ppm. 
The compound (I) of this invention is only sparingly active against 
bacteria in general and yet is active against protozoa (e.g. Tetrahymena), 
which activity in association with the aforesaid antifungal activity 
thereof makes the compound (I) of value as an antifungal/antiprotozoal 
agent for the assay of bacterial ecologies in the soil, activated sludge, 
body fluids, etc. The compounds of the formula (I) wherein R.sup.1 is a 
C.sub.12-16 aliphatic hydrocarbon residue have excellent actions mentioned 
above. Thus, for example, in isolating useful bacteria from the soil, or 
in detecting the activity of bacteria alone to the exclusion of protozoa 
and fungi for operation or analysis of the activated sludge process in 
waste water treatment, selective growth of bacteria is possible without 
allowing fungi and protozoa present in the sample to grow. Specifically, 
the test sample is added to a liquid or solid culture medium, then 0.1 ml 
of an aqueous solution of the compound (I) having a concentration of about 
10 .mu.g/ml to 100 mg/ml is added, and incubation is performed. 
The following production examples, test examples and dosage form examples 
are further illustrative but by no means limitative of this invention.

PRODUCTION EXAMPLE 1 
Tetradecyl 2-trimethylammonioethyl phosphate 
Myristyl alcohol (5 g) and 2-bromoethyl phosphorodichloridate (9.02 g) were 
dissolved in benzene (40 ml). To the solution was added pyridine (2.95 g) 
dropwise. The mixture was stirred at room temperature for four hours. 
Then, the solvent was evaporated off. To the residue was added water, 
which was refluxed for one hour and half. The reaction mixture was cooled 
with ice, to which was added concentrated hydrochloric acid (6 ml). The 
mixture was subjected to extraction with chloroform. The extract was 
washed with water, dried and concentrated to dryness to give tetradecyl 
2-bromoethyl phosphate, to which was added 20% toluene solution (60 ml) of 
trimethylamine. The mixture was left standing for five days, then 
concentrated to dryness. The residue was dissolved in methanol, to which 
was added silver carbonate (8.4 g). The mixture was refluxed for one hour 
and half, followed by filtration when hot. The filtrate was concentrated 
to dryness. The residue was purified by subjecting it to a silica-gel 
chromatography twice. [First: silica-gel, 70 g; eluent, methanol. Second: 
silica-gel, 70 g; eluent, chloroform-methanol-water=65:25:4 (by volume)]. 
The fractions containing the object compound were collected and 
reprecipitated from chloroform-acetone to give white powder (3.67 g). 
IR(KBr)cm.sup.-1 : 3400, 2915, 2850, 1660, 1490, 1465, 1240, 1075, 970. 
TLC: Rf=0.1 (silica-gel, CHCl.sub.3 --MeOH--H.sub.2 O, 65:25:4 (by 
volume)). 
PRODUCTION EXAMPLE 2 
Tetradecyl 2-pyridinioethyl phosphate 
An analogous reaction to Production Example 1 was conducted by employing 
myristyl alcohol (5 g) and 2-bromoethyl phosphorodichloridate (9.02 g) to 
yield an intermediate compound, tetradecyl 2-bromoethyl phosphate. To this 
compound was added pyridine (25 ml), and the mixture was refluxed for one 
hour and half. The reaction mixture was left standing at room temperature 
overnight, followed by concentration to dryness. The residue was dissolved 
in methanol, to which is added silver carbonate (8.4 g). The mixture was 
refluxed for one hour and half, followed by filtration when hot. The 
filtrate was concentrated to dryness. The residue was purified subjecting 
it to a silica-gel chromatography twice. [First: silica-gel, 75 g; eluent, 
methanol. Second: silica-gel, 70 g; eluent, 
chloroform-methanol-water=65:25:4 (by volume)]. The fractions containing 
the object compound were collected and reprecipitated from chloroform and 
acetone to give the end-product as white powder. The yield was 3.65 g. 
IR(KBr)cm.sup.-1 : 3400, 3050, 2920, 2850, 1630, 1490, 1235, 1080, 1049, 
918, 778. 
TLC: Rf=0.15 (silica-gel, chloroform-methanol-water, 65:25:4 (by volume)). 
Elemental Analysis: C.sub.21 H.sub.38 NO.sub.4 P.multidot.0.75H.sub.2 O. 
Calcd.: C, 61.07; H, 9.64; N, 3.39; P, 7.50. Found: C, 60.91; H, 9.66; N, 
3.68; P, 7.52. 
PRODUCTION EXAMPLE 3 
Oleyl 2-trimethylammonioethyl phosphate 
An analogous reaction to Production Example 1 was conducted by employing 
oleyl alcohol (4.8 g) and 2-bromoethyl phosphorodichloridate (6.9 g) to 
yield 2.98 g of the object compound as hygroscopic white powder. 
IR (liq) cm.sup.-1 : 3400, 2920, 2850, 1650, 1460, 1220, 1080, 963. 
TLC: Rf=0.1 (silica-gel, chloroform-methanol-water, 65:25:4 (by volume)). 
Elemental Analysis: C.sub.23 H.sub.48 NO.sub.4 P.multidot.H.sub.2 O. 
Calcd.: C, 61.17; H, 11.16; N, 3.10; P, 6.86. Found: C, 61.31; H, 11.19; 
N, 2.93; P, 7.26. 
PRODUCTION EXAMPLE 4 
Oleyl 2-pyridinioethyl phosphate 
An analogous reaction to Production Example 2 was conducted by employing 
oleyl alcohol (4.8 g) and 2-bromoethyl phosphorodichloridate (6.9 g) to 
yield 2.58 g of the object product as white powder. 
IR(KBr)cm.sup.-1 : 3400, 2925, 2850, 1631, 1490, 1235, 1074, 780. 
NMR(CDCl.sub.3).delta.: 0.90(3H), 1.26(22H), 1.87-2.20(4H), 3.48-4.63(6H), 
4.93-5.50(4H, m), 7.96-8.65(3H, m), 9.51(2H, d, J=6 Hz). 
Elemental Analysis: C.sub.25 H.sub.44 NO.sub.4 P.multidot.1.5H.sub.2 O. 
Calcd.: C, 62.48; H, 9.86; N, 2.91; P, 6.45. Found: C, 62.18; H, 9.82; N, 
2.72; P, 6.55. 
PRODUCTION EXAMPLE 5 
Docosyl trimethylammonioethyl phosphate 
1-Docosanol (3.26 g) and 2-bromoethyl phosphorodichloridate (2.53 g) were 
dissolved in a mixture of dichloromethane (6 ml) and carbon tetrachloride 
(3 ml), and the solution was stirred overnight. The reaction solution was 
refluxed for two hours, then the solvent was evaporated off. To the 
residue was added water (20 ml), which was refluxed for one hour. The 
reaction mixture, after cooling, was subjected to extraction with ether 
(70 ml). To the extract were added a given amount of BaSO.sub.4 and water 
(2 ml). The mixture was stirred sufficiently, and the precipitating powder 
was collected by filtration. The power was dissolved in mixture of 3% 
hydrochloric acid (50 ml) and ether (50 ml) under vigorous stirring. The 
ether layer was taken, and the ether was evaporated off under reduced 
pressure. To the residue was added toluene (60 ml) containing 
trimethylamine (12 g). The mixture was heated at 60.degree. C. in an 
autoclave for 48 hours. The reaction solution was concentrated to dryness 
under reduced pressure. The residue was dissolved in methanol (50 ml). To 
the solution was added silver carbonate (4.0 g), and the mixture was 
refluxed. The insolubles were removed by filtration. The filtrate was 
concentrated to dryness under reduced pressure. The residue was purified 
by means of chromatography using silica-gel (15 g). The product was 
subjected to re-precipitation from chloroform-acetone to give 1.30 g of 
the object compound as white powder. 
IR(film)cm.sup.-1 : 3400, 2910, 2845, 1650, 1460, 1220, 1080, 1050, 960. 
NMR(CDCl.sub.3).delta.: 0.67-1.67(43H), 3.33(9H), 3.57-4.67(6H, m). 
Elemental Analysis: C.sub.27 H.sub.58 NO.sub.4 P.multidot.4H.sub.2 O. 
Calcd.: C, 57.61; H, 11.80; N, 2.49. Found: C, 57.62; H, 11.60; N, 2.61. 
PRODUCTION EXAMPLE 6 
Stearyl 2-trimethylammonioethyl phosphate 
An analogous reaction to Production Example 1 was conducted by employing 
stearylalcohol (2.71 g) and 2-bromoethyl phosphorodichloridate (3.63 g) to 
give 1.32 g of the object compound as white powder. 
IR(KBr)cm.sup.-1 : 2920, 2850, 1230, 1080. 
NMR(CDCl.sub.3).delta.: 0.9(3H), 1.25(32H), 3.25(9H, s), 3.5-4.5 (6H, 
broad). 
Elemental Analysis: C.sub.23 H.sub.50 NO.sub.4 P.multidot.1.5H.sub.2 O. 
Calcd.: C, 59.71; H, 11.55; N, 3.03; P, 6.69. Found: C, 59.93; H, 11.44; 
N, 3.02; P, 6.51. 
PRODUCTION EXAMPLE 7 
3,7,11,15-Tetramethylhexadecyl 2-trimethylammonioethyl phosphate 
Dihydrophytol (0.60 g) and bromoethyl phosphorodichloridate (0.83 g) were 
dissolved in dry benzene (10 ml). To the solution was added dropwise dry 
pyridine (0.2 ml) under stirring. The mixture was stirred at room 
temperature for three hours, then cooled with ice, to which was added 2 ml 
of water. The mixture was again stirred vigorously at room temperature for 
two hours. The solvent was evaporated off, and the residue was dissolved 
in ether. The ether solution was washed with water, and then concentrated 
to dryness. The residue was dissolved in toluene (10 ml) containing 
trimethylamine (2 g), and the solution was left standing for five days. 
The reaction solution was concentrated under reduced pressure. The residue 
was subjected to a silica-gel chromatography (eluent, methanol) to yield 
0.404 g (43%) of the object compound as colorless solid matter. 
IR(film)cm.sup.-1 : 3350, 2850, 1460, 1380, 1225, 1080, 965. 
NMR(90 MHz, CDCl.sub.3).delta.: 0.82, 0.87, 0.89(s, 15H), 1.20(m, 22H), 
1.5(m, 2H), 3.38(s, 9H), 3.6-4.4(m, 6H). 
Elemental Analysis: C.sub.25 H.sub.54 NO.sub.4 P.multidot.0.5H.sub.2 O. 
Calcd.: C, 63.53; H, 11.73; N, 2.96; P, 6.55. Found: C, 63.54; H, 12.07; 
N, 2.96; P, 6.31. 
TLC Rf=0.26 (silica-gel, CHCl.sub.3 --MeOH--H.sub.2 O, 65:25:4 (by 
volume)). 
PRODUCTION EXAMPLE 8 
(i) Cetylphosphonic acid 
To ethyl phosphite (1.6 g) dissolved in 5 ml of tetrahydrofuran was added 
sodium (180 mg), and the mixture was dissolved. To this solution was added 
cetyl tosylate (514 mg), and the mixture was refluxed for ten hours. The 
reaction solution was concentrated to dryness under reduced pressure. To 
the residue was added water, which was acidified with concentrated 
hydrochloric acid, followed by extraction with ether and dried on Na.sub.2 
SO.sub.4. From the extract was removed the solvent by evaporation, and the 
residue was subjected to a silica-gel (7 g) chromatography. Fractions 
eluted with chloroform were collected and concentrated to dryness. To the 
residue was added concentrated hydrochloric acid (3 ml), which was 
refluxed for 18 hours. The resulting precipitates were collected by 
filtration, followed by recrystallization from n-hexane to give 240 mg 
(60%) of colorless needles. 
IR(KBr)cm.sup.-1 : 2920(CH), 2850(CH), 1100(P.dbd.O). 
Elemental Analysis: C.sub.16 H.sub.35 O.sub.3 P. Calcd.: C, 62.71; H, 
11.51; P, 10.11. Found: C, 63.13; H, 11.76; P, 9.99. 
(ii) 2-Trimethylammonioethyl cetyl phosphonate 
Cetyl phosphonate (153 mg) and choline tosylate (0.5 g) are added to 4 ml 
of pyridine. The mixture was dissolved by heating at 50.degree. C. To this 
solution was added trichloroacetonitrile (2 ml). The mixture was heated at 
50.degree. C. for 50 hours. One half volume of pyridine was evaporated off 
under reduced pressure. To the residue was added under stirring 
acetonitrile (20 ml). The resulting precipitates were collected to yield 
colorless powder (160 mg). The powder was dissolved in a mixture of 
tetrahydrofuran and water (7:3). The solution was poured onto a mixed 
resin (3 ml) of IRA410-Dowex 50W (2:1). The fractional eluate was 
concentrated to dryness under reduced pressure. The residue was subjected 
to a silica-gel (10 g) column chromatography using 
chloroform-methanol-water [65:25:4 (by volume)] as the eluent. The 
fractional eluates were collected and concentrated to dryness. The residue 
was recrystallized from chloroform-acetone to give 120 g (61%) of 
colorless needles. 
IR(KBr)cm.sup.-1 : 2910(CH), 2850(CH), 1465(CH.sub.2), 1200(P.dbd.O), 
1190(P.dbd.O), 1077, 1045, 960. 
PRODUCTION EXAMPLE 9 
3,7,11-Trimethyldodecyl 2-trimethylammonioethyl phosphate 
In 10 ml of benzene was dissolved 1.0 g (4.38 mmol) of 
3,7,11-trimethyl-1-dodecanol. To the solution, while cooling with ice, 
were added 1.80 g (4.38 mmol.times.1.7) of 2-bromoethyl 
phosphorodichloridate and 0.59 g (4.38 mmol.times.1.7) of pyridine. The 
mixture was stirred for 2.5 hours at room temperature, to which was added 
2 ml of water, followed by vigorous stirring for further 2.5 hours. The 
reaction solution was concentrated, and the residue was subjected 
extraction with ether. The ether extract was washed with water, then the 
solvent was evaporated off. The residue was dried and dissolved in 10 ml 
of toluene containing 2 g of trimethylamine, then the solution was left 
standing for four days. The reaction was concentrated to dryness. The 
residue was subjected to a silica-gel chromatography [first eluent: 
methanol, second eluent: chloroform-methanol-water (65:25:4 by volume)] to 
give 0.57 g (34%) of the object compound as colorless solid. 
TLC Rf=0.2(CHCl.sub.3 --MeOH--H.sub.2 O 65:25:4 (by volume)). 
IR(film) cm.sup.-1 : 3390, 2955, 2870, 1460, 1380, 1230, 1085, 970. 
NMR(90 MHz, CDCl.sub.3).delta.: 0.82, 0.89(s, 12H), 1.0-1.7(m, 18H), 
3.40(s, 9H), 3.6-4.1(br. 4H), 4.1-4.5(br. 2H). 
Elemental Analysis: C.sub.20 H.sub.44 NO.sub.4 P.multidot.1.5H.sub.2 O. 
Calcd.: C, 57.12; H, 11.26; N, 3.33; P, 7.36. Found: C, 56.99; H, 11.20; 
N, 3.59; P, 7.34. 
PRODUCTION EXAMPLE 10 
(E)-2-Octadecenyl 2-trimethylammonioethyl phosphate 
(E)-2-Octadecen-1-ol (1.34 g, 5 mmol) and 2-bromoethyl 
phosphorodichloridate (1.57 g, 6.5 mmol) were dissolved in benzene (25 
ml). To the solution was added a solution of pyridine (514 mg, 6.5 mmol) 
in benzene (2.5 ml) dropwise. The mixture was stirred at room temperature 
for four hours. Then, the solvent was evaporated off. To the residue was 
added water (50 ml), which was refluxed for one hour and half. After 
cooling, the mixture was extracted with chloroform. The extract was dried 
and concentrated to dryness to give (E)-2-octadecenyl 2-bromoethyl 
phosphate, to which was added 20% toluene solution (40 ml) of 
trimethylamine. The mixture was left standing for 66 hours, then 
concentrated to dryness. The residue was subjected to a silica-gel column 
chromatography using methanol as an eluent to give 0.5 g (23%) of the 
object compound as colorless powder. 
IR(KBr)cm.sup.-1 : 2920, 2850, 1472, 1240, 1100, 1025, 971. 
NMR(90 MHz, CDCl.sub.3).delta.: 0.87(3H), 1.24(26H), 1.82-2.12(2H), 
3.33(9H, N.sup.+ Me.sub.3), 3.65-3.80(2H), 4.05-4.43(4H), 5.45-5.70(2H). 
Elemental Analysis: C.sub.23 H.sub.48 NO.sub.4 P.multidot.0.75H.sub.2 O. 
Calcd.: C, 61.78; H, 11.16; N, 3.13; P, 6.93. Found: C, 61.77; H, 11.75; 
N, 3.11; P, 6.94. 
PRODUCTION EXAMPLE 11 
(1-Octyl)nonyl 2-trimethylammonioethyl phosphate 
9-Heptadecanol (2.56 g, 10 mmol) and bromoethyl phosphorodichloridate (4.11 
g, 17 mmol) were dissolved in dry benzene (20 ml). To the solution was 
added dropwise dry pyridine (1.34 g, 17 mmol) under stirring. The mixture 
was stirred at room temperature for three hours, then to which was added 
water. The mixture was again stirred for thirty minutes at 80.degree. C. 
The solvent was evaporated off, and the residue was dissolved in ether. The 
ether solution was washed with water, and then concentrated to dryness. 
The residue was dissolved in toluene (25 ml) containing trimethylamine (5 
g), and the solution was left standing for three days. 
The reaction solution was concentrated under reduced pressure. The residue 
was subjected to a silica-gel chromatography [eluent: 
chloroform-methanol-water (65:25:4 by volume)] to yield 0.76 g of the 
object compound as colorless solid matter. 
IR(KBr)cm.sup.-1 : 2925, 2860, 1465, 1380, 1230, 1085, 970, 760. 
NMR(90 MHz, CDCl.sub.3).delta.: 0.87(t, 6H), 1.24(m, 28H), 3.37(s, 9H), 
3.79(br, 2H), 4.0-4.4(br, 3H). 
Elemental Analysis: C.sub.22 H.sub.48 NO.sub.4 P.multidot.0.5H.sub.2 O. 
Calcd.: C, 61.37; H, 11.47; N, 3.25; P, 7.19. Found: C, 61.59; H, 11.68; 
N, 3.37; P, 7.26. 
TEST EXAMPLE 1 
Effects against sarcoma 180 
To a group of ICR mice was administered intraperitoneally 1 mg of the test 
compound dissolved in 0.2 ml of physiological saline per mouse. Four days 
later, 1.times.10.sup.5 cells of sarcoma 180 per mouse were administered 
intraperitoneally. The survival rates (T/C) of the test group to the 
control group administered with only physiological saline were assayed. 
The results are as shown in Table 1. 
TABLE 1 
______________________________________ 
Effects against sarcoma 180 
Test Compound Survival Rate (T/C) 
______________________________________ 
Production Example 1 
217 
Production Example 2 
152 
Production Example 3 
302 
Production Example 4 
308 
Production Example 5 
167 
Production Example 6 
200 
Production Example 11 
282 
______________________________________ 
TEST EXAMPLE 2 
Effects against MM46 
To each of C3H/He mice group was transplanted intraperitoneally 
1.times.10.sup.4 cells of MM46 carcinoma. Four times from five days to two 
days before the transplantation and four times from two days to five days 
after the transplantation, eight times in total, 250 .mu.g of the test 
compound dissolved in 0.2 ml of physiological saline was administered 
intraperitoneally. The number of mice surviving on the 24th day after 
transplantation of the carcinoma cells was compared with that of the 
control group administered with saline. The results are as shown in Table 
2. 
TABLE 2 
______________________________________ 
Effects against MM46 
Test Compound Surviving mice/Test mice 
______________________________________ 
Physiological saline 
0/5 
(control) 
Production Example 3 
3/5 
Production Example 4 
2/5 
______________________________________ 
TEST EXAMPLE 3 
The antifungal and antiprotozoal activities of the compounds of this 
invention are as shown in Tables 3 and 4 respectively. 
The antimycotic (antifungal) activity values as shown in Table 3 were 
assayed on various organisms including phytopathogenic microorganisms 
using 1% glucose-bouillon agar medium, and the minimum inhibitory 
concentration (MIC) was determined by the serial dilution method. 
The antiprotozoal activity values as shown in Table 4 were assayed using 
Tetrahymena pyriformis W strain as the test organism and an assay medium 
composed of 20 g of tryptose peptone (manufactured by Difco), 1 g of yeast 
extract, 2 g of glucose, 1000 ml of distilled water and 10 ml of 1M 
phosphate buffer (pH 7.0). The incubation was continued at 28.degree. C. 
for 44 to 48 hours and the minimum inhibitory concentration (MIC) of the 
compounds of this invention was determined by the broth dilution method. 
TABLE 3 
______________________________________ 
Antifungal Activity [MIC (.mu.g/ml)] 
Pro- 
duc- 
tion 
Exam- 
ple Test Organism 
No. 1 2 3 4 5 6 7 8 9 
______________________________________ 
1 12.5 6.25 12.5 3.12 12.5 25 25 6.25 3.12 
2 25 12.5 50 3.12 25 25 50 12.5 3.12 
3 12.5 25 6.25 1.56 12.5 12.5 3.12 1.56 50 
4 50 25 25 3.12 50 25 6.25 6.25 50 
6 25 25 12.5 6.25 50 50 6.25 3.12 100 
7 -- -- -- 1.56 -- 6.25 25 ND 100 
______________________________________ 
______________________________________ 
Test Organism 
______________________________________ 
1. Aspergillus niger IFO-6341 
2. Penicillium citrinum 
IFO-6352 
3. Saccharomyces cerevisiae 
IFO-0209 
4. Pyricularia oryzae IFO-5279 
5. Helminthosporium oryzae 
IFO-7503 
6. Gibberella fujiknroi 
7. Botrytis cinerea IFO-5365 
8. Helminthosporium sigmoideum 
IFO-4867 
9. Colletctrichum lagenarium 
IFO-6207 
______________________________________ 
TABLE 4 
______________________________________ 
MIC (.mu.g/ml) against Tetrahymena pyriformis W strain 
Production Example No. 
MIC 
______________________________________ 
1 2 
2 .gtoreq.4 
3 0.4 
4 2.about.4 
5 &gt;4 
6 .ltoreq.1 
7 4 
8 2 
10 .ltoreq.1 
11 .ltoreq.1 
______________________________________ 
DOSAGE FORM EXAMPLE 1 
Oleyl 2-trimethylammonioethyl phosphate (80 g) is dissolved in 1 liter of 
distilled water, the solution is passed through a sterilization filter, 
poured into 1,000 vials (1 ml per vial) and lyophilized, and the vials are 
tightly stoppered. 
Separately, a solution containing xylitol or mannitol (100 g in 2 liters) 
in distilled water for injection is poured into 1,000 ampules for 
injectable solution (2 ml per ampule) in an aseptic manner, and the 
ampules are sealed by fusing. 
For administration, the powder in one vial is dissolved in the 
above-mentioned xylitol (or mannitol) solution in one ampule. 
DOSAGE FORM EXAMPLE 2 
Tablets, each weighing 370 mg and having a diameter of 9.5 mm, are prepared 
in a conventional manner by mixing the ingredients: 
______________________________________ 
(1) Oleyl 2-pyridinioethyl phosphate 
100 mg per tablet 
(2) Lactose 200 mg per tablet 
(3) Corn starch 51 mg per tablet 
(4) Hydroxypropylcellulose 
9 mg per tablet 
______________________________________ 
followed by granulation, addition of corn starch (8 mg per tablet) and 
magnesium stearate (2 mg per tablet) and tableting. 
DOSAGE FORM EXAMPLE 3 
Tablets containing tetradecyl 2-trimethylammonioethyl phosphate are 
prepared in the same manner as in Dosage Form Example 2, and coated with a 
solution of hydroxypropylmethylcellulose phthalate (14 mg per tablet) and 
castor oil (1 mg per tablet) in an acetone-ethanol (4:6) mixture, the 
concentration of the solutes being 7%. Thus are obtained enteric coated 
tablets.