CA1169433A - Method for preparing an antihypertensive agent comprising 1-0-alkyl-2-acetoyl-sn-glycero-3- phosphocholine - Google Patents

Abstract

METHOD FOR PREPARING AN ANTIHYPERTENSIVE AGENT

ABSTRACT OF THE DISCLOSURE
The composition of this invention is I-O-alkyl-2-acetoyl-sn-glycero-3-phosphocholine, having the ionic structural formula:

Description

9~33 . .

METHOD FOR PREPARING AN ANTIHYPERTENSIVE AGENT
COMPRISING l-o-ALKyL-2-AcEToyL~lsN-GLycERo-3-pHospHocHoLINE

Background of the InYention Field of the Invention The invention relates to the control of hypertension (high blood pressure) in human or veterinary medicine by the administration of pharmaceutical agents having hypotensive activlty.
.
Description of the Pr~or Art In descr1bing the work of others herein we do not admi~ thct such work is actually prior art under 35 USC 102~or 35 U~C 103 or thit the work was actually prior in ~ime to the making of the invent~on described and claimed herein. We reserve the right to establish a date of conception or reduction to practi~ce prior to the effeçtive date of any publ k ation, pctene, or work herein described.
Muirhead et al in "Reversal of Hypertens~on by Transplants and Lipid Extrac~s of Cultured Renomedullary Interst~tial Cells,"
Laboratory Investigation Yol. 35, No. 2, pp~ 162-172 (1977) describe the antihypertensive activity of lipids of un~dentified chemical struc- -ture extracted from rencl medulla tlssue. The extracted material was ` subieçted to reduction with NaAlH2~0CH2CH~OCH3)2, acetic acld treatment ,. .

~69433 and lipophilic chromatography on Sephadex (registered trademark). In some instances the material was subjected to chromatography on Florisil (registered trademark) and acetylation with acetic anhydride prior to Sephadex chrolnatography. The treated material demonstrated hypo-tensive activity in rats and rabbits. The active agents in the material were not identified or separated from other components. A compound isolated from bovine brain, which in some respects resembled lysophosphati-dylcholine was shown to depress blood pressure in Tsukatani et al, Chemical Pharmaceutical Bulletin (Tokyo) vol. 24 p p. 2294-1200 (1976).
Statement of the Objects ~ . .
It is an object of this invention to provide a highly effective antihypertensive composition for use in human and veterinary medicine.
It is a further object to provide a method for preparing the anti-hypertensive agent from readily available commerclal materials.
It is a further object to provide a method for reducing blood pressure in warm-blooded animals, including humans.
Other objects, advantages, and novel features of the invention will become apparent to those skilled in the art upon examination of the following detailed description and accompanying drawings.
Summary o~ the Invention The composition of this invention is 1~0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine, having the ionic structural formula:
H~,C-O-CH2-R
O
CH3C!O-CH

H2C_o-lp-ocH2cH2N(cH3~3

- 2 -~ ~69433 wherein R is saturated alkyl having 9-21 carbon atoms, or salts or hydrates of the composition. Preferably R has 13-19 carbon atoms and most preferably R has 15 carbon atoms. The composition of thiS inven-tion iS useful for reducing hypertension in warm~blooded animals, including humans, when administered either orally or by innoculation, e.g., intravenous injection.

The invention also includes a method for preparing an antihypertensive agent comprising l-0-alkyl-2-acetoyl-sn-glycero~3-phosphocholine comprising the steps of (a) reacting a l-0-alkyl glycerol, said alkyl having 10-22 carbon atoms, with a benzyl halide to produce a first reaction product having the structural formula H2C-O-alkyl HO-C-H

H2C-O-CH2-c6H5 (b) reacting said first reaction produc~ With an acetylating agent to produce a second reaction product having the structural formula H2C-O-alkyl CH3-~-0-CH

H2C-O-CH2-c6H6 (c) reacting the second reaction product with hydrogen to produce d 1-0-alkyl-2-acetoyl-sn-glycerol, (d) react1ng said 1-0-alkyl-2-acetoyl-sn-glycerol with a 25 phosphating agent to produce a thlrd reaction product having the struc-tural formula H~C-O-alkyl CH3!C-O~CH

OH

- 3 ~ 1~9433 (e) react1ng sald thlrd reactlon product with chollne-p-toluene sulfonate to produce a fourth reaction product mlxture contalning l-0-alkyl-2-acetoyl-sn-glycero-3-phosphochollne, sald alkyl belng saturated havlng 10-22 carbon atoms, and (f) recoverlng 1-0-alkyl-2-acetoyl-sn glycero-3-phosphocholine from said reactlon mixture.
Brief Descrtptlon of the Vlews of the Drawlng Fig. l is a data traclng illustrattng the mean arterial pressure response of a hypertensive rat to intravenous injectlon of the agent of thts invention.
Flg. 2 is a data traclng lllustrating the dose responses of mean arterial pressure o~ a hypertenslve rat to the agent of this invent10n.
Fig. 3 is a htstogram illustrating the duratlon of mean arterial pressure reductlon in hypertensive rats from the lntravenous and oral adminlstratlon of the agent of this invention.
Fig. 4 is a graph showlng the prolonged effect of mean arterlal pressure reductlon for hypertensive rats from the intravenous and oral admlnistratlon of the agent of this invention.
Detalled_Descr1ption of the Invention This 1nvention ls based upon the discovery that phosphollptds having the structure ln lonlc form:

1~ 1 .

H2 C-0-P-0CH2CH2N(cH3)3 f;
- 3a -~ 169~33 demonstrate profound hypotensive activity in warm-blooded animals. The R substituent of the phospholipid of this invention is straight chain or branched saturated alkyl having 9-21 carbon atoms. The above phospholipid is designated 1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine, in accordance with stereospecific numbering asdescribed in "The Nomenclature of Lipids," IUPAC-IUB Commission on Biochemical Nomenclature~ Lipids, ~ol. 12, No. 6, pp. 455-468 (1977).
1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholines exist as amorphous gummy solids at room temperature and are generally light yellow in color. The 1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine com-positions can also exist as a salt or as a hydrate. In the salt form, a cation would be associated with the negatively charged si-te of the structure and an anion would be associated with the positively charged site. As a hydrate, a hydrogen atom would be associated with tile nega-tive site and a hydroxyl group with the positive site. The com-position, its salt and its hydrates are soluble in aqueous and organic solutions. The compositions can be readily synthesized in a highly pure form from lipids occurring in nature, and from commercially available materials.
For pharmaceutical applications the phospholipid should be admi-nistered as a composition of matter consisting essentially of the 1-0 alkyl-2-acetoyl-sn-glycero-3-phosphocholines, and which can include phar-maceutical carriers. By "consisting essentially of" it is meant free from impurities or other components in amounts sufficient to materially degrade the antihypertensive activity of the phospholipid. The anti-hypertensive agent of this invention consists essentially of material having the ionic structural formula:

~ ~69~33 CH3!~-0-C-H

, H2C-O-IlOCH2CH2N(CH3~3 o-wherein R is saturated alkyl having 9-21 carbon atoms, or salts or hydrates thereof, in combination with a pharmacologically compatible carrier~ Pharmacologically compatible carriers are liquid or solid substances which do not interfere with the anti-hypertensive activity of the phospholipid. Examples of such carriers for intravenous or oral administration are water, saline or other aqueous solution, emulsions, ethanol ~olu~ion~, a1bumin solutions, emulsions with propylene glycol or glycerine and liposomal preparations. For oral administration the phospholipids can be administered in solid form in capsules or in tab-lets. Conventional solid pharmaceutical carriers such as starch-lactose mixtures and Avicel [registered trademark), a microcrys~alline cellu-lose, can be used if desired. The phosphollpids of this invention can be administered singly or as a mixture of alkyl homologues, where R in the l-O-CH2-R group can vary from 9-21, preferably 13-19 carbon atoms.
It is important that the composition be substantially free from toxic imputities which can cause undesirable side effects. Phosphocho-line lipids having a lyso group (-OH) at position 2 are known to cause hemolysis. Consequently, the compositlon of matter of this invention consist;ng essentially of 1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine should be substantially free of 2-lyso-sn-glycero-3-phosphocholines such ~ ~69~33 as 1-0-alkyl-2-lyso-sn glycero-3-phosphocholines having the ionic struc-tural formula:
H2C-O-Ctl2-R

H0-~CH

H2 C-0- 1 0cH2cH2N(cH3)3 O~
wherein R' is an aliphatic hydrocarbon group, or salts or hydrates thereof.
tO In its method of use aspects this ;nvention comprises depressing blood pressure in a warm-blooded animal, including human be;ngs, by administering to the animal an amount of the 1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholines effective to reduce the blood pressure of the animal. ~ased on tçsts with hypertensive rats the ef~ective dosage of 1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine to provide an antihy-pertensive effect lasting up to about 48 hours is about 0.025-0.060 milligram per kilogram body weight for intravenous administration and about 0.040-0.20 milligram pèr kilogram body weight for oral admi-nistration. Effects of shorter duration can be achieved with smaller dosages.
1-0-alkyl-2-acyl-sn glycero-3-phosphocholines having 18-carbon acyl groups at the sn-2 position have shown to be ineffective for reducing blood pressure, however compositions having shorter sn-2 acyl groups, such as 3-5 carbon atoms can be expected to exhibit hypotensive activity. Such 1-0-alkyl-2-acyl-sn-glycero-3-phosphocholines demonstrating hypotensive activity are contemplated as equ;valents to ~ 9 ~ 3 ~

1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholines in the compositions and methods of this invention.
The phospholipids of this invention can be prepared ~rom naturally occurring lipids such as occur in heart tissue or they can be prepared from commercially available sources. In one synthesis method, a lipid composition of the ionic formula H2-C-O-Rl 1l +
H2C-0-IP-OcH2-cH2-N(cH3)3 O
wherein R1, is a saturated or unsaturated aliphatic hydrocarbon radical having 10-22 carbon atoms, preferably 14-20, and most preferably 16 carbon atoms, and R2 is an acyl group, is saponified by reaction with a base such as sodium or potassium hydroxide to form the 2-lyso phospholipid: -Hf-O-R

I O
H2C-0-IP-OcH2-cH2-N(cH3)3 O : .
The 2-lyso-phospholipid can be hydrogenated to saturate the R1 group if necessary. The hydrogenation step can alterna-tlvely be carried out before saponification. The 1-0-alkyl-2-lyso-sn-glycero-3-phosphocholine is then acetylated by reacting with an acetylating agent, such as acetic acid, acetyl chloride, or acetic anhydride to ~ ~69~33 produce l-o-alkyl-2-acetsyl-srl-glycero-3-phosphocholine~ which is reco-vered from the reaction mixture, e.g., by thin layer chromatography.
The antihypertensive agent can also be prepared from a racemic mixture of ()-alkyl-phospholipid stereoisomers by reacting a first mix-ture containing 1-0-alkyl-2-acyl-sn-glycero-3-phosphocholine and 3-0-alkyl-2-acyl-sn-glycero-1-phosphocholine with a lipase such as phospho-lipase A2 which is speci-fic for deacylating the sn-2 position of the natural (l-0-alkyl) isomer to provide a second mixture containing 1-0-alkyl-2-lyso-sn-glycero-3-phosphocholine. The 1-0-alkyl-2-lyso-sn-glycero-3-phosphocholine is then reacted with an acetylating agent, su~h as acetic anhydride, acetic acid, or acetyl chloride to produce 1~0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine, which is separated, e.g., by thin layer chromatography, from unreacted material.
The antihypertensive agent of this invention can be prepared from a l-0-alkyl glycerol by reacting the alkyl glycerol with a benzyl halide such as benzyl chloride to produce a 1-0-alkyl-2-lyso~3-benzyl composition having the structural formula : H0-C-H

H 1 0 CH2-C6~I5 This product is reacted with an acetyla-ting agent such as acetic anhydride, acetyl chloride~ or acetic acid to acetylate the sn-2 posi-tion, producing a product having the formula

4 3 3 H~C-0-R

This product is reacted with hydrogen, in the presence of a hydrogena-tion catalyst if desired, to produce 1-0-alkyl-2-acetoyl-sn-glycerol which is reacted with a phosphating agent such as POC13 to produce a reaction product having the structural formula I O

H2C-O-~-OH
H
This product is reacted with choline-p-toluene sulfonate, CH3-C4H~-S03_ CH2CH2N(CH3)3, to produce 1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine which can be recovered from the reaction mixture using the chromatographic methods described.
: The following examples illustrate the laboratory scale preparation 20 and testi~g o~ phospholipids according to this invention. The synthe-tic methods can readily be amplified to large scale, for example, by using high pressure liq ld chromatography p~ocedures..

' ~:
~' :
_ g `~

1 ~69~33 Example I
Preparation of 1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine from Heart Tissue (a) Extraction of Lipids One hundred grams of fresh beef heart (obtained from a slaughterhouse) was minced, 500 ml of chloroform was added, and the mixture was homogenized in a Polytron blender. Methanol (500 ml) was added and the mixture again homogenized in the blender. The homogenate was centr;fuged at 1850 rpm for 10 minutes in a refrigerated centrifuge to pellet proteins. To the solvent extract (750 ml) was added 390 ml water with vigorous stirring. This mixture was transferred into 40 ml centrifuge tubes and centrifuged at 1850 rpm for 10 minutes, whereupon the solution separated into a water-methanol phase and a chloroform phase which contained most of the dissolved lipids. The chloroform phases were pooled and transferred to a rotary evaporator to remove the solvent. The lipid residue was dissolved in chloroform for chroma-tographic analysis.
(b~ Isolation of 1,2-diradyl-sn-gl~cero-3-phosphocholine The lipid resldue contained a mixture of compounds of the formula:

O~

1 o H2C-O-~-OCH2CH2N(cH3)3 ~ 1~9~3~ -where R1 is generally a mixture of alk-1-enyl, alkyl, and acyl groups and R2 is an aliphatic group. Approximately 50 mole percent of the lipid fraction was l-alk-l-enyl-2-acyl-sn-glycero-3-phosphocholine.
The lipid fraction was separated by thin layer chromatography by banding the lipid solution onto an 8" x 8" glass TLC plate coated with silica gel HR (MC/B Manufacturing Chemists, Inc., Cincinnati, Ohio) and employing a developing solvent of chloroform:methanol:glacial acetic acid:water (50:25:8:4 respect;vely by volume). A phosphatidylcholine-containing standard (isolated from rat liver) was used. Similar chro-matographic results can be obtained using egy phosphatidylcholine. Thephospholipid fraction had an Rf (migration distance relative to the solvent front) about the same as the pho~phatidylcholine standard. With a developing solvent of chloroform:methanol:glacial acetic acid:water (50:25:8:3) the phospholipid fraction has an average Rf of 0.25, ranging from 0.22-0.28, on the silica gel. As is customary in the art, iodine staining was used to identify the segregated lipids in one track of the TLC plate. The portion of the silica gel plate containing the unstained phospholipid fraction was scraped away. The phospholipids were eluted from the loaded silica gel by washing with a solution pre-pared from 3.5 ml H20, 6.5 ml methanol containing 2Z acetic acid, and 7ml chloroform. The eluate formed two phases, the lipids distributing to the chloroform phase.
(c) Hydrogenation of Carbon-Carbon Double ~onds The isolated phospholipid fraction from step (b) was dried with N2 and 3 ml ethanol, containing about 25 mg Adam's catalyst (PtO2 H20~, was added. Hydragen was gently sparged through the mixture for about 20 ~;9~33 seconds and the sample was capped and shaken. The hydrogen sparging was repeated twice more. Chloroform (3 ml) was added and the mixture was centrifuged to separate the catalyst. Thè supernatant was removed, the catalyst was rinsed with 2:1 chloroform:methanol, and the super-natants were combined. The supernatants were dried with N2 and theresidue was dissolved in 6 ml of 2:1 chloroform-methanol. After hydro-genation, a portion of the product was tested by exposure to HCL. No released aldehydes were detected, indicating that no plasmalogens (alk 1-e~yl groups) existed in the hydrogenated lipid.
(d) Replacement of Acyl Group at Position 2 with An Acetoyl Group A~ter hydrogenation, the product was mildly saponified to remove acyl groups by adding 0.6 ml of 0.33 N potassium hydroxide in methanol to the sample dissovled in 1.2 ml o~ chloroform, followed by mild shakiny for 20 mlnutes at room temperature. The saponification mixture was cooled at 0C, and 0.5 ml of 6N HCl, 0.8 ml chloroform, 1.2 ml of methanol, and 1.5 ml of water were added to neutralize the potassium hydroxide. The mixture of solvents formed two phases, with the chloro-form phase containing the deacylated lipid product. After centrifu~a-tion at 1500 rpm for 10 minutes the lower chloroform layer was removed and the upper layer was extracted twice more with chloroform. The three chloroform extracts were combined and evapora~ed ~o dryness. The products, which are 2-lyso-phospholipids, were separated from fatty acids by preparatiYe thin layer chromatography using the same type of silica gel plat~s and developing solvent as in step (b) with the phosphatidylcholine (from rat liver) standard. The lyso-phospholipid product has about the same Rf as lyso-phosphatidylcholine and was ?

~ 3 ~

recovered from the plate and eluted frorn the silica gel as in step (b).
With a developing solvent of chlorofornl:methanol:glacial acetic acid:water (50:25:~:3) the lyso-phospholipid product has a rather sharp Rf of 0.11 on the silica gel. The lyso-phospholipid is 1-0-alkyl-2-lyso-sn-glycero-3-phosphocholine having the formula:
H~C-0-CH2-R

O
H2 C-O-Ifl-O-cH2-cH2-N(cH3)3 , O-The lyso-phospholipid was acetylated by heating the samples in 2.5 ml of acetic anhydride:pyridine (4:1, vol./vol.) -for 45 minutes at 65-70C. Heating at 100C for one hour is now preferred. Excess acetic anhydride and pyridine were removed at a temperature of 60-70C with a stream of N2. The acetylated compounds were isolated from lyso-compounds by preparative thin layer chromatography using the same type of silica gel plates and developing solvent as described in step (b).
The standard was the rat iiver phosphatidylcholine. The 1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine product had a slightly lower Rf than the phosphatidylcholine standard and the 2-lyso-phospholipid had an Rf lower than the-2-acetoyl prod~ct. The 1 0 alkyl-2-acetoyl-sn-glycero-3-phosphocholine product was extracted from the silica gel as described in step (b), and, af~er evaporation of solvents, is ready for use. With a developing solvent of chloroform:methanol:glacial acetic acid:water (50:25:8:3) the 1-0-alkyl-2-acekoyl-sn-glycero-3-phosphocholine has an Rf of about 0.11 +.01. The nuclear magnetic . .

~ ~9433 resonance spectrum for this composition showed positions of the proton signals for the acetoyl and the three N-terminal methyl groups which agree with the spectra described for triacetin and phosphatidylcholine by Chapman et al., J. Biol. Chem. 241, pp. 5044-5052 (1966). It has been determined that better recovery is obtained when the saponifica-tion is performed before the hydrogenation step due to the solubility of the lyso-compound in the hydrogenation solvent. Acetic acid (10%), rather than HCl, should then be used for the neutralization step.
The length of the alkyl chain in the l-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine was determined by reduction of the composition with NaAlH2(0CH20CH3)2 to form alkylglycerols which were treated with 1 ml acetone containing 0.5 ~1 of 70~ HC104 acid for 5 minutes at room temperature to prepare isopropylidine derivatives. Analysis of the isopropylidenes by gas liquid chromatography showed the alkyl chain lengths, which correspond to R~1 in the structural formula of the 1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine, as se~ forth in the table.
TABLE
Car~ons Double Bonds Branching Weight Percent 14 - 0 yes ~ 1.6 14 0 no 1.3 0 yes 9.4 0 no ; 3.9 16 0 yes 2.4 16 0 no 65.7 17 0 yes 1.4 18 0 no 8.8 19 0 no ~ 1.2 0 yes .

~ 3 3 Example II
.

Antihypertensive Activity Tests The 1^0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine of Example I was tested for antihypertensive activity at Baptist Memorial Hospital, Memphis, Tennessee, under the direction of Drs. E. Eric Muirhead and Lawrence W. Byers by administration to members of a strain of hypertensive rats having one kidney. The rats had a clip reducing the flow of blood through the renal artery of the kidney. Each rat had a mean arterial pressure of 170-190 mm Hg. prior to administration, as -~
obtained by averaging arterial pressure for the preceeding three days.
The compound was administered in saline solution and in a saline solu-tion containing albumin, both intravenously and by mouth. For the intravenous injection, a catheter was implanted ln the inferior vena cava. For adminl~tration by mouth, the composition was passed through ~15 a tube directly into the stomach. Of the rats receiving the com-position by mouth, one rat had a single dose of 13 micrograms and two rats each recelved a single dose of 20 micrograms. ~Seven rats received multiple doses of 40-80 micrograms each.~ O~the rats receiving the composition intravenously, single doses~of 1.0-6.4 micrograms were~
given, or multiple doses, each amounting~to 4-6 micrograms, were given for a total~dDse of 12-24 micrograms.~ ~ ~
The acute effect of the intravenous~doses was eval~uated by a bolus injection while the arterial pressure was continuously recorded.
The prolonged depressor eff~ect was evaluated by introducing the com-pound, either intravenously or by mouth, at a slow rate w;th thearterial pressure not being allowed to decrease below ~0-100 mm Hg.
~ , ?

1 169~33 Two sets of controls were employed. A group of seven hypertensive rats were injected with the vehicle (0.4-0.5 ml of 0.9% saline~.
Another group of 16 hypertensive animals were injected with the acyl analogue of the alkyl ether lipid ~an acyl group replacing the alkyl ether linkage at position 1~, which showed no antihypertensive acti-vity. Similar tests were run showing that a non-acetylated 1-0-alkyl phospholipid (-OH at position 2) was inactive as a blood pressure depressor agent.
Fig. 1 illustrates the acute depressor effect elicited by a single bolus intravenous dose of 6 micrograms of the composition of Example I.
Within about 2 seconds, the arterial pressure receded from 185 to 50-70 mm. Hg, remaining maximally depressed for about 5 minutes, thereafter gradually increasing, but remaining 25 mm Hg below the original level after one hour.
Fig. 2 illustrates the dose response of the acute depressor effect of the compo3ition of Example 1 when 63, 126, and 189 nanogr~ms were injected as a bolus. The immediate depressor effect amounted to -60, -85, and -95 mm Hg, respectively.
Fig. 3 summarizes the prolonged depressor effect of the composi-tion of Example I when administered intravenously and by mouth bymultiple doses totaliny about 20-80 micrograms orally or 12-24 micrograms intravenously.
Fig. 4 demonstrates change in wean arterial pressure over time for the animals receiving multiple doses of the composition of Example I.
The controls had no change in mean arterial pressure.

~xampl è ~ ~ ~ I
Preparation of 1-0-alkyl-2-acetoyl- _-glycero-3-pllosplloclloline from 1-0-alkyl-2-acyl-phosplloliyids ; A racemic mixture (a powder) of the stereoisomers 1-0-hexadecyl-2-octadecenoyl-sn-glycero-3~phosphocholine and 3-0-hexadecyl-2-octadecenoyl-sn-glycero-1-phosphocholine, was obtained from R. Berchtold, Biochemisches Labor., Bern, Switzerland. The racemic mixture (7-10 mg) was dissolved in 3 ml of diethyl ether, to which was added 0.4 ml borate buffer (0.1 M, pH 7~0) containing CaCl2 (2.5 x 10-3 M). To this mixture was added 4 mg of phospholipase A2, obtained from Ophiophagus hannah venom (Ross Allan Reptile Institute, Silver Springs, Fla.). The mixture was shaken in a vortex mixer for two hours, then evaporated to dryness using Nz. The residue was extracted three times with chloroform-methanol (2:1) using 3.3 ml each time. The phospholipase A2 is specific for the natural isomer (l-O-alkyl-), and converts it to the 2-lyso- composition.
The extracts were pooled, evaporated to dryness, and the 2-lyso-compositlon was isolated by TLC as described in Example I. Two ml acetic anhydride and 0.5 ml pyridine were added to the isolated 2-lyso com-position and the solutions heated to 100C for 1 hour to acetylate the 2-position of the natural isomer. The sample was blown to dryness~with N2 and redissolved in ch1oroform-methanol~(2:1), The solutlon was banded ~on 4" of an 8" by 8" plate coated with silica gel HR. The resulting 1-0-hexadecyl-Z-acetoyl-sn-glycero-3-phosphocholine was separated~by thin layer chromatography using the developing solvent and standard phosphati-dylcholine of Example I. ~The 1-0-hexadecyl-2-acetoyl-sn-glycero-3-phosphocholine had an R~ slightly lower than the standard phosphati-dylcholine. The unreacted 3-0-hexadecyl-2-octadecenoyl-sn-glycero-1-;~ phosphocholine and fatty acids had Rf values the same as and higher,, ~ , : ' ' :~ 169`4~3 respectively, than the standard. The 1-0-hexadecyl-2-acetoyl-sn-glycero-3-phosphocholine had the formula H21C-O-C16~33 S CH3-1Cl-o-C-H

H2-C-0-~!OCH2CH2~(CH3~3 with no branching in the -C16H33 chain. Tests for hypotensive activity were performed on hypertensive rats as in Example II, and the composition showed essentially the same antihypertensiYe activity. The stereoisomer 3-0-hexadecyl-2-acetoyl-sn-glycero-1-phosphocholine was also prepared.
The isomer showed only slight hypotensive activity which possibly was due to the presence of small amounts of the 1-0-alkyl composition.
Example IY
Preparation of 1-alkyl-2-acetoyl-sn-glycero-3-phosphocholine from a 1-alkyl-glycerol Chimyl alcohol (predominately 1-hexadecylglycerol~ having the for-mula H2-C-0(CH2~15CH3 l ' .

(available from commercial sources) is benzylated according to the proce-dure of Sowden~ et al., J. hm. Chem. Soc. _3 p. 3244 (1941) by refluxing with stoichiometric benzyl chloride in diethyl ether containing powdered sodium for about 70 hours in the absence of moisture. The precipitated , i .. . .

16~33 NaCl is filtered and washed with diethyl ether. The diethyl ether solu-tions are evaporated under vacuum leaving the benzyl derivative having the formula H~C-O(C~2)15C~3 HO-C-H

H2C-O-CH2-C6~5 The benzyl derivatiYe is acetylated by reaction with acetic anhydride as described in Example I, step (d3, to provide a composition of the formula H2C-O-(CH2)15CH3 O I .
CH3-CI-O-lCH

The benzyl group is removed by dissolving the acetylated material in an organic solvent such as n-hexane and hydrogenating the sample in a Parr hydrogenator at 20 psig H2 at room temperature for 5 hoursS using palladium black catalyst, ~o result in the production of 1-hexadecyl-2-acetoyl-sn-glycerol, H~C-O-(C~2~15CH3 ~ I

H -OH
Choline toluene sulfonate is prepared according to the procedure of Brokerhoff et al. Lipids Vol. 14~ p. 88, (1978) by neutralizing an aqueous solution of choline hydroxide with p-toluene sulfonic acid.

19 `

1 ~)9 ~ 3 ~ J

The water is removed by repeated evaporation with toluene, and the pro-duct is crystallized as the salt form from acetone and stored in air-tight bottles.
The 1--hexadecyl-2-acetoyl-sn-glycerol is dissolved in ethanol-free .
chloroform containing an equal molar amount of dry quinoline and an equal molar amount of POCl3. The mixture is heated to about 45C for 30 minutes whereupon the 3-OH position is phosphated to produce H2~C (CH2)15 3 lo C~2lcl0f~

H2C-O-~OH
OH
After cooling, twice the stoichiometric amount of choline toluene sulfonate is added, stirring for 5 hours at room temperature. A few milliliters of water is added and stirred for an additional 30 minutes.
The-mixture is then extracted with several 50 ml portions of chlorofor~
and the extracts are washed successively with water, 3% aqueous Na2C03, '~ HCl and water. If an emulsion forms, sufficient wash material is added to break the emulsion. The chloroform extrac~ is dried over Na2S04 and the chloroform is evaporated. The resulting solid material is 1-hexadecyl-2-acetoyl-sn-glycero-3-phosphocholine, which can be purified by thin layer chromatography as~described in Example I, sec-tion (d).

The foregoing descriptions of the compositions and methods of this invention have been presented for purposes of illustration and descrip-tion and are not intended to be exhaustive or to limit the invention to the precise forms disclosed. The examples were chosen and described in S order to best expla;n the princ;ples of the invent;on and their prac-tical application to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modifica-tions as are suited to the particular use contemplated. It is intended that the scope of the invention be def;ned by the cla;ms appended .
hereto.

Claims (4)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for preparing an antihypertensive agent comprising 1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine comprising the steps of (a) reacting a 1-0-alkyl glycerol, said alkyl having 10-22 carbon atoms, with a benzyl halide to produce a first reaction product having the structural formula (b) reacting said first reaction product with an acetylating agent to produce a second reaction product having the structural for-mula (c) reacting the second reaction product with hydrogen to produce a 1-0-alkyl-2-acetoyl-sn-glycerol, (d) reacting said 1-0-alkyl-2-acetoyl-sn-glycerol with a phosphating agent to produce a third reaction product having the struc-tural formula (e) reacting said third reaction product with choline-p-toluene sulfonate to produce a fourth reaction product mixture containing 1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine, said alkyl being saturated having 10-22 carbon atoms, and (f) recovering l-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine from said reaction mixture.

2. The method of claim 1 wherein the alkyl group in said 1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine contains 14-20 carbon atoms.

3. The method of claim 1 wherein the alkyl group in said 1-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine contains 16 carbon atoms.

4. l-0-alkyl-2-acetoyl-sn-glycero-3-phosphocholine compound pro-duced by the process of claim 1, claim 2 or claim 3.