CA1114400A - Antiviral amine and amidine derivatives of glycerol and propanediols - Google Patents

Abstract

Abstract of the Disclosure Novel amine and amidine derivatives of di-O-(n-higher alkyl and alkenyl)-glycerols and -propanediols, and their pharmaceutically acceptable acid addition salts, are useful for combating viral infectins in mammals. Of particular interest in 1,3-di-O-(n-hexadecyl)-2-0-(3-aminopropyl)-glycerol, and its pharmaceutically acceptable acid addition salts.

Description

P.C. 5860 I .

A~TIVIRAL AMI~E AND AI~IDINE DERIVATIVES
OF GLYCEROL AND PROPANEDIOLS
' Virus inf~ections which attack mammals, including man, , are normally contagious afflictions which are capable of .
causing great human suffering and economic loss. .Unfortunately, the discovery o~ antiviral compounds is far more complicated and difflcult than the discovery.of antibacterial and anti- ¦
fungal agents~ This is due, in part, to the close structural 10 . similarity o~ viruses and the structure of certain essential cellular components such as ribonucleic and deoxyribonucleic acids. Nevertheless, numerous nonviral "antiviral agents", . i.e substances "which can produce either a protective or therapeutic e~fect ~o ~he clear detectable advantage of the : -!i virus infected host, or any material that can significantly ~, enhance an~ibody ormation, impro~e antibody activity7 i~ . 1, ! lmprove non-speci~ic resistance, s?eed convalescence or depress .
symptomsn.lHerrman et~ al., Proc. Soc. Exptl. Biol. Med., 103, . ,j 625 ~19~0], have ~ieen described in the llteraturec The list of ' i : i ~ :' 'i, : ~ ~ : -1~14~r~

reported antiviral agents includes, to name a few, interferon and synthetic materials such as amantadine hydrochloride, pyrimidines~J
biguanides, guanidine, pteridines and methisazone. Because of j the rather.narrow range of viral infections that can be treated by each of the antiviral agents commercially available at the present time, new synthetic antiviral agents are always welcomed as potentially valuable additions to the armamentarium of medical techno~ogy~
1' The cells of mammals produce, in response to virus infection,~
j. a substance which enables cells to resist the multiplication of , a variety of viruses. The viral-resisting or viral-interfering substances are referred to as "interferons". The interferons are glycoproteins which may differ in their physico-chemical properties, but all exhibit the same biological properties; namely~, they inhibit a wide range of unrelated viruses, have no toxic or other deleterious effects on cells, and are species-specific ¦
tLocxart~ Frontiers of Biology, Vol. 2, "Interferons", edited by Finter, W. B. Saunders Co., Philadephia, 1966, pages 19-20).
! No practical, economical method has;yet been deveioped for . .
;~20~ the preparatlon of exogenous interferon for routine clinical use I against ~iral infections. An alternEtive approach to producing interferonhas, therefore, been pursued, which comprises~adminis-~ ering to: the animal to be protected or treated a non-viral : :, sub:stanc~ which ;timulates- or lnduces- production of interferon.
25; ~ ' in the cells.~ The intereron producod in this fashion is i referred to as "endogenous" interferon.
~ U.S. Patont~-No. 2~,738,:351 disclosos that compounds of the ; : ~:1 general formula~

~ ~ ::CH-Z-ALK-B

-Y-CH2~

wherein each of Rl and R2 may be alkyl, aralkyl, aryl, cyclo-alkyl, nitro-substituted aryl, halogen-substituted aryl, alkyl-substituted aryl, or alkoxy-substituted aryl, each of X, Y and Z may be oxygen, sulfur or sulfonyl, ALK is straight or branched alkylene of from one to six carbon at.oms, and B may be di(lower)-alkylamino, piperidino, morpholino, pyrrolidino, (lower alkyl)-pyrrolidino, N'-alkyl-piperazino or pipecolino, are local anesthetic agents. Additionally, the discussion of alternate synthetic routes (see Col. 1, 11. 57-70, of said patent) dis-closes intermediates of the above formula wherein B is aminoand (lower alkyl)amino. However, none of the compounds specific-ally enumerated in the disclosure of said patent contain an alkyl Rl or R2 larger than n-pentyl. Furthermore, in none of these compounds are both Rl and R2 alkyl and both X and Y
oxygen.
Insecticidal and miticidal compounds of the formula 1 f 2 fH - (CH2)q~A

~ .
wherein Rl and R2 may each be, inter alia, lower alkylthio; q is 0 to 5; and A may be, inter alia, l-piperidino or di(lower ~20 alkyl)amino are dissolved in Japanese Patent J7-6042-177.
It has now been discovered that certain novel amine and amidine derivatives of di-O-(n-higher alkyl and alkenyl)-glycerols and -propanediols are capable of combating viral infections in mamma:Ls. The novel compounds of this invention are those of the foxmulae ~ ;

: ~ : : -:
~ .

::
~ ~ 3 :; : : : ,~ :

R -O-CH

fH-O-Y-NHR3 or fH~-O-Y-NHR3 Rl-O-CH II

and the pharmaceutically acceptable acid addition salts thereof, wherein Rl and R2 are each selected from the group consisting of normal alkyl of from 14 to 18 carbon atoms and n-octadec-9-enyl, Y is selected from the group consisting of ' ' ' ' OH
' ; ~ -CH2-( 'H-CH2-;

and ~herein the left bond~is connected to O; and~R3 is selected from~the group~consLsting~of~hydrogen and alkyl of from 2 to 4 l0~ carbon atoms ~
The invention~dlsclosed~herein comprises the novel antiviral;compound8~of formulae I~;~and~II and thus makes~avail- -;
abl~novel ph-rmac_utlcal~compo~iLions~containing an antlvirally effective amount of a compound of formulae I or II as the essential active ingredient in a pharmaceutically acceptable carrier, a novel method of prophylactically controlling a viral infection in a mammal which comprises administering an amount effective to prophylactically control said viral infection of a compound of formulae I or II, and a novel method of inducing the production of interferon in a mammal which comprises admin-istering an amount effective to induce the production of inter-feron of a compound of formulae I or II.
The compounds of this invention exhibit antiviral activity against a wide variety of viruses ln vivo in mammals and in vitro in mammalian tissue culture. At least a substantial portion of this activity results from the ability of said com-pounds to induce the production of interferon in the cells, i~e. endogenous interferon.
By "pharmaceutically acceptable" acid addition salts is meant those salts which are non-toxic at the dosages administered. The pharmaceutically acceptable acid addition salts which may be employed include such water-soluble and water-insoluble salts as the hydrochloride, hydrobromide, , .
phosphate, nitrate, sulfate, acetate, hexafluorophosphate, citrate, gluconate, benzoate, propionate, butyrate, sulfo-salicylate, maleate, laurate, malate, fumarate, succinate, oxalate, tartrate, amsonate (4,4'-diaminostilbene-2,2'-disulfonate), pamoate (l,l~-methylene-bis-2-hydroxy-3-naphtho-ate), stearate, 3-hydroxy-2-naphthoate, _-toluenesulfonate, methanesulfonate, lactate/ and suramin salts~
One preferred group of the compounds of formulae I
and II consists of the hydrochloride salts of the bases of formulae I and II.

:A : -5-Another preferred group of the compounds of formulae I and II consists of those wherein Rl and R2 are each normal alkyl of from 14 to 18 carbon atoms.
Another preferred group of the compounds of formulae I and II consists of those wherein Rl and R2 are each normal alkyl of from 14 to 18 carbon atoms and contain the same number of carbon atoms.
Another preferred group of the compounds of formulae I and II consists of those wherein Rl and R2 are each n-hexadecyl. ~-Another preferred group of the compounds of this in- ~ -vention consists of those of formula I.
Another preferred group of the compounds of this invention consists of those of formula II. :
One preferred group of the compounds of formulae I
and II consists of those wherein R3 is hydrogen.
The compounds of formulae I and II above may be prepared from the appropriate 1,2-di-O-(n= higher alkyl or alkenyl)-glycerol and l,3-di-O-(n-higher alkyl or alkenyl)-glycerol starting materials by methods familiar to those skilled in the art. Thus, according to the invention, the compounds of formulae I and II are prepared by a process which comprises the steps of (a) reactlng a compound of the formula Rl-O-IH2 TH-O-YI X ' or ' ~ fH2 R -O-CH XI

.
-~ 6 wherein Y' is selected from the group consisting of -CH -CH / ~ -CH -- ~ CH CH2;

with, when R3 is alkyl, a compound of the formula R3NH2; or with, when R3 is hydrogen, an azide salt, followed by reduction of the resulting azido derivative; and (b) if desired, con-verting the resulting compound of formula I or II to a pharmaceutically acceptable acid addition salt thereof.
Acid addition salts of the bases of formulae I and II
may be prepared by conventional procedures such as by mixing the amine or amidine compound in a suitable solvent with the required acid and recovering the salt by evaporation or by precipitation upon adding a non-solvent for the salt. Hydro-chloride salts may readily be prepared by passing hydrogen chloride through a solution of the amine or amidine compound in an organic solvent. As can be seen by reference to the examples herein, many of the isolated hydrochloride or dihydro-. , .
chloride salts of the bases of formulae I and II tend to contain ~; a significant water content. Whether this observed "trapped"
water is randomly occluded during crystallization, or corres-.
ponds to formation of true molecular hydrates, or results Erom the occurrence of some other phenomenon, is not known. In any event, the salts contalning~"trapped" water may be efficaciously formulated and administered~without preliminary dehydration.

.
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~ -7-- The 1,2-di-0-(n-higher alkyl)-glycerol start1ng materials may be prepared by the method of Kates, M. et al., Etiochemistry,

2, 394 (1963). The 1,3-di-0-(n-higher alkyl)-glycerol starting materials may be prepared by the method of Damico, R., et al., J. Iipid Res., 8, 63 (1967). The 1,2- and 1,3-di-0-(n-higher alkenyl)-glycerol starting materials may be prepared by the method of Bauman, W.J. and Mangold, H.K., J. Org. Chem., 31, 4g8 ' (19663.
~i The antiviral activity of the compounds of this invention ' was determined by the use o~ two independent procedures. In '1 the first, the test compound is administered to mice by the : - .
" intraperitoneal route eighteen to twenty-four hours prior to challenging them with a lethal dose of encephalomyocarditis t~C) lll ~irus. Survival data are taken during the ten da~s after i challenge and compàred with the data for unprotected animals.
The procedure in which the drug is given eighteen to twenty-four hours before, and at a dLstinctly diffèrent site from, virus ! injection is designed to eliminate ~ocal effects between drug and virus and identify only those compounds which produce a ~ystemic antiviral responseL
n the second procedure, monolayers of human nasal polyp , cells grown on microtiter plates are treated with the test compound about eighteen hours before treatment with a lethat dose ¦
of vesicular stomatitis virus (VSV). The test compound is washed !
25~ ~ away from the~monolayers~beore virus ~reatment. Culture fluid ; extracted from t:he plates~ after a post challenge incubation period :A
,, ~

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!l 4~

is titrated for the amount of infectious virus present in microti ter plates of L-929 mouse fibroblasts. Comparison is made with the virus yield data for culture fluid extracted ~rom unprotected polyp cells.
Additionally, many of the compounds of this invention were tested for their ability to enhance the known antiviral activity of polyinosinic:polycytidylic acid. Finally, certain of the compounds were also tested for their ability to induce circulatin interferon in mice a~ter parenteral administration! using the procedure described by Hoffman, W.W., et al., Antimicrobial Agents and Chemothera~y~ 3, 498-501 (1973).
-Parenteral, topical or intranasal administration of the above-described amines and amidines to a mammal be~ore exposure of the mammal to an infectious virus provides rapid resistance to the virus. Preferably, administration should take place from about two days to~about one day before exposure to the virus, although this will vary somewhat with the particular animal speci ~s and the particular in~ectious virus.
~hén the materi~als of this invention are administered, they 20~ are most~easily~and economically used in a dispersed form~ln~an acceptable carrier.~ ¦~When~it is said~that this material is dispersed, it means~that the partioles may be molecular in size and held~in~true solution in a sultable solvent or that the ~partlcles~may be~collo1dal in slze and dispersed~through a 11guid ~ ' ,~ :

I ~ : :

~ ` ~ 9 1 ;

phase in the form of a suspension or an emulsion. The term "dispersed" als~ means that the particles may ~e mixed with and spread throughout a solid carrier so that the mixture is in the form of a powder or dust. This term is also meant to encompass S l mixtures which are suitable for use as sprays, including sol-utions, suspensions or emulsions of the agents of this invention.
When aaministered parenterally (subcutaneously, intra-muscularly, intraperitoneally) the materials of this invention are used at a level of from about 1 mg.~kg. of body weight to j about 250 mg./kg. body weight. The favored range is from about 5 mg./kg. to about 100 mg./kg. of body weight, and the preferred range from about S mg. to about 50 mg.~kg. of body weight. The dosage, o~ course, is dependent upon the mammal being treated I' and the particular amine or amidine compound involved and is to be determined ~y the individual responsible for its administratiol.
, Generally, small doses will be administered initially with graduaI
increase in dosage until the optimal dosage level is determined , . .
for the particular subject under treatment.
. .
Vehicles suitable for parenteral injection may be either 2~a: ~ aqueous such as water, isstonic saline, isotonic dextrose, Rlnger' s j solution, or non-aqueous such as fatty oils of vegetable~origin cottonseed! peanut oil, corn, sesamej and other non-aqueous ¦ ~ ~ehicles which will not interfere with the effica~y of the prepara_ tion and are non-toxic in the volume or proportion used (glycerol, ~ ethanol, propylen~e glyool, sorbitol). Additionally, compositions¦
;~ ~ suitable~for exte~poraneous prepar~tion of solutions prior to administration may~advantageously be made. Such compositions may ; ~ include liquid dlluents, for example, propylene glycol, diethyl carbonate, glycerol, sorbitol.

~; " ~ : . . .

~ ~ ~, : . / 0 In practicing the intranasal route of administration o~
this invention any practical method can be used to contact the antiviral agent with the respiratoxy tract of the-mammal.
Effective me~hods include administration of the agent by intra-nasal or nasopharyngeal drops and by inhalation as delivered by anebulizer or an aerosol. Such methods of administration are of pxac~ical importance because they provide an easy, safe and efficient method of practicing this invention. For intranasal ~ aaministration o~ the agent, usually in an acceptable carrier, a li' concentration of agent between 1.0 mg./ml. and 100 mg./ml. is j satisfactory~ Concentrations in the range of about 30 to 50 , mg./ml. allow administration of a convenient volume of material.
,¦ For topical application the antiviral agents are most I, conveniently used in an acceptable carrier to pexmit ease and , control of application and better absorption. Here also concen-trations in the range of from about 1.0 mg./ml. to about 250 mg.~ml. are satisfactory. In general, in the above two methods of administration a dose within the range of about l.0 mg./kg. to about 250 mg./kg. of body weight and, preferably, from about 5.0 , mg./kg. t~ about 50 mg.~kg. of body weight will be administered~
The compounds employed in this inveneion may be employed alone, i~e~, without other medicinals, as mixtures of more than .
one ~f the herein-described compounds, or in combination with other medic;nal agents, such as analgesics, anesthetics, ant~-septics, decongestants, antbiotics, vaccines, buffe~in~ agentsand inorganic salts~, eo afford desirable pharmacological prop-erties. Furthert they may be administered in combination with , hyaluronidase to avoid or, at least, to minimize local irritation !
and ko increase the rate of absorption of the compound. Hyal-~ uronidase levels of at least about 150 (U.S.P.) units are " effective in this respect although higher or lower le~els can; I -of cours~, be used.

.
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Those materials of this invention which are water-insoluble, including those which are of low and/or dif~icult solubility in water, are, for optimum results, administered in formulations, e.~., suspensions, emulsions, which permit formation of particle ; sizes of less than about 20~. The particle sizes of the form-ulations influence their biological activity apparently through -better absor~tion of ~he active ma~erials. In ~ormulating these materials various surace active agents and protective colloids ~ are used. Suitable surface active ayents are the partial esters ~ of common fatty acids, such as lauric, oleic, stearic, with ', hexitol anhydrides deri~ed from sorbitol, and the polyoxyethylene ~derivatives of such ester products. Such products are sold under the trademarks "Spans" and ~Tweens," respectively, and are avail-; able from ICI United States Inc., Wilmington, Del. Cellulose ' ethers, especially cellulose methyl ether (Methocel, availablefrom the Dow Chemical Co., Midland, Mich.) are highly efficient as protective colloids for use in emulsions containing the ~; .
ma~erials o~ this invention.
' The water-soluble materials described herein are adminis-tered for optimum results in aqueous solution. Typically they are administered in phosphate bufered saline. The water-insoluble i- :
compounds are administered in formulations of the type described above or in various other formulations as previously note~.
j Dimethylsul~oxide serves as a suitable vehicle for water-insoluble compounds. A representative formulation for such compounds ; comprisas formulating 25 to 100 mg. of the chosen drug as an emul-... ~ .
' sion by melting and mixing with equal parts of poly-sorbate 80 and 1',' ' I :

A 1, . 1, ,1 . . .

.,: , . I

~L$14~rJ`~ 11 glycerin to which hot (80C.) water is added under vigorous mix-. ing. Sodium chloride is added in a concentrated solution to a final concentration of 0.14 M and sodium phosphate, pH 7, is added to a final concentration of 0.01 M to give, for example, the fol-¦
1'~ lowing representative composition:
.,, , . .

~ Drug mg /ml.
'. Polysorbate 80 50.0 ', Glycerin 50.0 10 Ji Sodium Phosphate Monobasic Hydrous 1.4 ! Sodium Chloride 7.9 ,~~ Water 842.0 1001.3 , In certain instances, as where clumping of the drug 1 particles occurs, sonication is employed to provide a homogenous ' system.
i, The following examples illustrate the invention but are not !
: to be construed as limiting the same.
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1,3-Di-0-(n-hexadecyl)-2-0-(3-aminopropyl)-glycerol Hydrochloride A. 1,3-Di-0-(n-hexadecyl)-~-0- ~ l)-q~ycerol ! A mixture of 1,3-di-0-(n-hexadecyl)-glyceroi (80 g., 148 mmoles), acryloni~rile (1.49 kg., 28.1 mmoles) and aqueous 2N .
sodium hydroxide (1.2 1.) was heated to 50C. Tetrabutylammonium ., hydroxide (19~2 g-. of 40 wt. % a~ueous solution, 29.15 mmoles) was sl~owly added, causing the temperature of the exothermic ` reac~ion mixture to rise t~ about 80 to 90~C. The reaction - ¦
~. mixture was then s~irred for 20 minutes without any external . heating, followed by cooling to 20C. and addition of water . (1.0 1~. A.solid material, a mixture o~ unreacted and i cyanoethylated 1,3-di-0-(n-hexadecyll-glycerol, was isolated and treated again with ~resh acrylonitrile ~1.49 kg., 28.1 mmoles), aqueous 2N sodium hydroxide ~1.2 1.) and tetrabutylammon~um hydroxide (19.~ g. of 40 wt. ~ aqueous solution, 29.15 mmoles) for 2~ minute~ with stirring at 50C.~ followed ~ cooling and addition of water (1.0 1.). The resulting 1,3-di-O~(n-hexadecyl)-:! _ 2-0-(2-cyanoeth~ glycerol solids were filtered, washed con-secutive~y with water, methanol and acetonitrile, and dried . ~82 g., 93% yield, m.p. 45-46C., ir (C~C13) 2250 cm 1, n.m.r.
(CDC13) ~ 3~92 ~t~ 2~ NCCH~CH20-) t 3r33~3~67 (m, 9, C~C15H31]2)~ 2n ~2 (t~ 2, NCC ~ CH2O-) and 0 75-1 58 tm~, 62, aliphat:ic protons)l.
. : ' " .

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., ! ' B. Title Compound A mixture of 1,3-di~0-(n-hexadecyl)-2-0-(2-cyanoethyl)-glycerol ~20.5 g~, 34.5 mmoles)~ tetrahydrofuran (200 mlO), ethanol (10 ml~) and Raney nickel catalyst (3 g.) was saturated S with ammonia gas at 0 to 5C. and then hydrogenated (~0 psi~ in a Paar hydrogenator for 3 hours at room temperature. The mixture '~ was then filtered, the catalyst washed with tetrahydrofuran , (50 ml~.), and the total filtrate evaporated in vacuo to an oil.
'i This procedure was repeated three more times with fresh reactants , and catalyst to yield a total of 77 g. of oil, The oil was '~ dissolved in ether ~500 ml.) and the solution washed with 2 ~t. %
aqueous ammonium hydroxide solution (500 ml.), ~ried (MgSO4)r filtered and evaporated in_vacuo to yield a solid. The solid was ', dissolved in methanol (300 ml.) and the. solution satura*ed with hydrogen chloride gas and then evaporated in vacuo to a solid.
This solid was cry tallized from ethyl acetate to yield the named !
~: , product wi*h a slight impurity t63 g., 72% yield, m.p. 69-70~C.), ¦
: ~ and then recrystallized twice from isopropanol:acetonitrLle tl:l, 800 ml.~)lg7.5 g., 54% yield, m.p..58-59C., n.m.r, tCDC13) ~; ", ~ ~ H2NCN2CH2CH2 O~ 3 . 55 (m, 9, -OCH 1CH OC~2C

3.24 (t,~2,~H2NCH2CH2CH20-), 2.04 tm, 2, H2NCH2C~ 2 , 0.90-1.32 (m, 62, aliphatic protons), elemental analysis : ~ calculated: 72.04g C; 12 . 73% H; 2.21% N; found: 71.80% C,~12.41%
'' H; 2 ~ 30~ N~ a ~ ~ ~
.

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) EXAMPLE'; 2-7 In like manner to that desc:ribed.in Example 1 the following compounds were prepared by usin~ the appropriate 1,3- or 1,2-di-O-(n-higher alkyl)-glycerol as starting material:
.1 , Rl_o_lH2 IH-O(CH233NH2 ,i, ~ R2 CH2 .
., f H2 - O ( CH2 ) 3NH2 ~ Rl-O-~H II
10 . R2-0- H
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1,3-Di~0-(n-hexadecyl)-2-0-(2-aminoethyl)-glycerol Hydrochloride A. 1,3-di-0-(n-hexadecyl)-2-0-(?-carboxyethyl)-glycerol A mixture of 1,~-di-0-(n-hexadecyl)-2 0 (2-cyanoethyl)-: glycerol (4O8 g., 8.1 mmoles), concentrated hydrochloric acid ; (50 ml.3 and formic acid (50 ml.) was stirred for 16 hours at reflux, then cooled and extracted with ether (3 X 100 ml.)~ The ~; combined ether extract was washed with water (200 ml.), dried i ' tMgSO4), filtered and evaporated in vacuo to yield 1,3-di-O-'. (n-hexadecyl~-2-0-(2-carboxyethyl)-glycerol solids (4.5 g.), , which were puri~ied by silica gel chromatography (elution with toluene:ethanol)13~5 y.9 71% yield, m.p. 43-45C., ir (CHC13) ' 1740 cm 1, n.m.r. (CDC13) S 3.93 (t, J = 6 Hz,'2, -OCH2CH2COOH) and 2.6~ (t, J = 6 Hz, 2, -OCH2CH2COO~.]. :
.
: 15 '.` B. Title Compound 1,3-Di-0-(n-~exadecyl)-2-0-(2-carboxyethyl)-glycerol 53,5 g~
~.7 mmoles) was dissolved in a mixture of benzene t55 ml.) and concentrated sul~uric acid (5.89 g.). Hydrazoic acid (6.34 ml. of - , ~ 4.65,wt. % benzene solution, 6.0 mmoles) was then added dropwise ;. ~ -and the resulti~g mixture stirred for 2 hours a~ room temperature.
~: ?l Thin layer chromatography (TLC) analysis showed about 50~ reaction '~ o~ the 2-carboxyethyl compound. Additional hydrazoic acid ; ~6.34 ml. of 4.65 wt. % benzene solution, 6.0 mmoles) was ~dded dropwise;and the reaction,mixture stirred for another 16 hours ~;25 at 40C. ~LC cmalysis now shswed that the reaction was essentially ' ; j : : : , complete. Watex (SO ml.) and aqueous 2N sodium hydroxide were , then addèd~and the resulting mixtuxe extracted with ether ~ ~9 ~s~
i, ~ , ~ - j .; ~

j. ~
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(3 X 200 ml.). The combined ether extract was dried (Na2S04), filtered, saturated with hydrogen chloride gas and evaporated in vacuo to yield a solid. The solid was purified by silica gel ! chromatography (elution with chloroform:methanol) and recrystal- ;
lized from hot ethyl acetate ~570 mg., 16% yield, m.p. 79-80C., ` n.m.r. (CDC13j ~ 3.9~ ~m, 2, -OC~2CH2NH2) and 3.22 (m, 2, -OCH2CH~NH2)l elemental analysis calculated: 71.62% C;
; , ,i 12.67% H; 2.Z6~ ~; found: 70.90% C; 12.19% H; 2.05~ N~.
' EXAMPLE 9 : .
10 "1,3-Di-0-(n-hexadëcyl)-2-D- .
,~3-ethylaminoprop~l)~ylycerol Hydrochloride 'j A. 1,3-Di-0-( -hexadecyl)-2-0-(3-acet~mldo~ropyl)-glycerol 1,3-Di-0-(n-hexadecyl~-2-0-t3-aminoprspyl)-glycerol hydrochloride (1.0 g., 1.6 mmoles) was added to a mixture of ; potassium carbonate (830 mg., 6.0 mmoles) and benzbne ~75 ml.).
,. , . .", . . .
'J Acetyl chlorid~ ~150 mg., 1.9 mmoles) was then added and the resulting mixture stirred for one hour at reflux. Additional acetyl chloride (1~0 mg., 1.9 moles) was added and the reaction , mixture stirred for another hour at reflux~ TLC analysis showed ' that the reaction was essentially complete. The xeaction mixture was cooled, water ~75 ml.) added, and th xe~ulting '~ mixture extracted with ether ~3 X 100 ml.~. The com~ined ether extract was~drie~ (MgSO~), filtered and evaporated _n_vacuo to yield ; the named compound t800 mg~, 79% yield, m.p. 53-54C., ir ~HC13~ 1 3400 and 1670 cm 1, n.m.r. (CDC13) ~ 1.97 (s, 3, -NHCOC~3)~.
. . .

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B. Title Compound 1,3-Di-O~(n hexadecyl)-2-0-(3-acetamidopropyl)-glycerol (700 mg., 1.1 mmoles) was dissolved in ether (100 ml.) and treated .
with lithium aluminum hydride t500 mg., 13 mmoles) Water (100 ml.) was then added and the mixture extracted with ether ~2 X 100 ml.). The combined ethe:r extract was dried (MgS04~, filtered, treated with hydrogen chloride gas and evaporated `, in vacuo to a sol~d, which was recrystallized from hot ethyI
, acetate ~470 mg., 66% yield, m.p. 61-62C.; n.m.r. (CDC13)~ 1.47 (t, 3, -NHCH2CH3), el~mental analysis calculated: 72,51% C;
1~.78% ~; 2.11% N; found: 72.47% C; 12.56% H; 2.03% Nl.
, EX~MPLE 10 :: 1,3-Di-0-(n-hexadecyl)-2-0-. (3-isopropylaminopropy1~-gl~cerol~Hydrochloride .
, 1,3-Di-0-(n-hexadecyl)-2-0-(3-aminopropyl)-glycerol ! ~ydrochloride (700 m~., 1.1 mmoles) was dissolved in a solu~ion . of acetic acid (1.05 ml.3, sodium acetate ~350 mg., ~.3 mmole~) and acetone (1.3 ml.). Sodium borohydride (1.2S g., 33 mmoles) was added in small portions u~ti} TLC analysis showed that all the : . ' ~ 3-aminopropyl compound~had been consume~. The reaction mixture was then treated with aqueaus 2N sodium~hydroxide ~20 ml.) and : ~ water (20:ml.), and extracted with ether (3 X 40 ml.). ~he ~
~ .
: ~ combined ether extract was~dried (MgS04), fiItered, treated with hydrogen ~hloride gas, and then evaporated in vacuo to a~
solid, which wa~3 recrystallized ~rom hot ethyl a~eta~e 1~10 mg., solid contained about 1/2 mole H20 per mole~named product, 28~
yield,~ m.p.~72-'73C., n.m.r.~(CDC13) ~1.42 (d, 6,~-NHCH~CH3]2), elemental analylsis calcu1ated: 71.~82~ C;~12.79% H; 2.04% N;
,' fOUnd 71~92% ~; 12L46% H; 1~94% N]. .

,~ d~ I ~

i.

EXA*IPLE 11 1,2-Di-0-(n-hexadecyl)-3-0-(2-isopropylaminoethy~?-~lycerol Hydroi_hloride A. 1,2-Di-O-~n-hexadecyl)-3-0-all~ lycerol 5 '~ Sodium hydride (1.78 g. of 50 wt. ~ dispersion in mineral oil, 37 mmoles) was added at 60C. ko a solution of 1,2-di-0-~I tn-hexadecyl)-glycerol (10 g., 18.5 mmoles) in N,N-dimethyl-formamide (100 ml.), and the resulting solution stirred for 20 ~! minutes a~ 60C. Allyl bxomide ~4.47 g., 37 mmoles) was then ' added dropwise and the resulting mixture stirred for 3 ho~rs , at 90C., cooled, cautiosly diluted with water (200 ml.) to quench¦
the reaction, and extracted with ether t3 X 150 ml.~. The combined !` .
, ether extract was w~shed with saturat~d aqueous sodium chloride ,, solution, dried (MgSO4), filtered and evaporated in vacu~ to ~ ~n oil, which was purified by silica gel chromatoqraphy (elution , with benzene~ ~10 g., 93% yield, oil, n.m.r. (CDC13)~ 5.66-6.16 ~m, 1, -OCH2CH=CH2), 5.25 (d of doublets, 2, -OCH2CH=C~ ) and , 4.03 ~d~ 2,-OC~I~OEI=CH2)~
: ~S B. 1~2-D1-O- (n-hexadecyl)-3-0-formylmethyl-~lycerol j, Osmium tetroxide (90 mg.,.354 mmoles~ was added to a solution o~ di-0-(n-hexadecyl)-3-0-aIlyl-glycerol (4.5 g., i 7~75 mmoles) in tetrahydrofuran:water ~3 1, 120 ml.), and the ~ ~ resulting solution stirred for 5 minutes at room tempera~ure~
; ~, Sodium ~eriodate ~9 g., 42 mmoles) wa then added and the reactio~
'' solution stirred or 16 hours at room temperature under nitrogen.
" The reaction solution was then diluted with water ~150 ml.) and extracted with ether ~2 X 150 ml.). The combined ether i ,' extract was washed with water ;~}50 ml.), dried (MgSO4) and I j e~aporated n~vacuo to an oil, which was purified by silica gel ~ cbromatography ~elution with benzene:ethyl acetate)/ 12.6 y., il 57 yield, waxy solid, ir (CHC13) 1735 cm 1, ~m.r. ~CDC13)~ 9.381 (t, J = 1 H~ OCH2C~0) and 4.07 (d, J = 1 Hz, 2, OCH~CHO)I. ¦

~ ~ .
, i' . C. Title Compound . Sodium cyanoborohydride (0.1 g., 1.6 mmoles~ was added to a solution of 1,2-di-0-(n-hexadecyl)-3~0-formylmethyl-glycerol ,. ~1.5 g~, 2.6 mmoles) and isopropylamine (0.89 g., 15 mmoles) , in methanol:te~rahydro~uran (1:1, 50 ml. ), and the mixture stirred i' for two hours a~ room temperature. The pH was then adjusted . to 6 with 5N methanolic hydrochlori~ acid t additional sodium yano~orohydride (0.1 g., 1.6 mmoles) added, and the re~ction mixture then stirred for another 60 hours at roo~ temperature, .
il filtexed, treated with aqueou~ 3N sodium hydroxide (10 ml.) and ii saturated aqueous sodium chloride solution (200 ml.), and extracte ¦! with ether~2 X l5D ml.). The combined ether extract was driea (MgSO4), ~ilterea and evaporated in vacuo to an oily solid, which . .
. was purified by silica gel chromatography (elution with benzene: .
~15 j ethanol) and dissol~^ed in methanol. The solution was treated with hydrogen.chloride gas and evaporated in vacuo to yield a solid, '~ which was recrystalli~ed from ethyl acetate [400 mg., solid . contained about 1/4 mole H20 per mole named product, 23~ y~eid, 11 m.p. 71 72C~, n.m.r ~CDC13) ~ 1.42 (d, J = 6 Hz, ~, -N~CH~CH312), , elemental analysis c~lculated. 72.02% C; 12.76~ ~; 2.10% ~;
: 1. found: 71.89~ C; 12.34% H; 2.09% Nl. .
, EX~PL~ 12 :
~'~ 1,2-Di-0-(n-hexadecyl)-3-0-[2-(2-hydroxyethylamino)-i ethyl]-~y~erol Hydrochloride _ 25 ~ In like manner to that desoribed in Example 11 the named i~ compound was prepared by reacting 2-hydroxyethylamine with 1 f 2-di-0-(n-hexadecyl~-3-0-formylmethyl-glycerol ~solid contained ~: , about 1~2~ le ~2 per mole named product, m.p. 125-126C., ~" elemental analys:ls calculated: 69.54~ C; 12.42% H; 2~07~o N;
~ ound: 69.62~ C; 12.08% H; 2.29% N].
` 1! ` .
` ` 11 ` ' ~

, i, EXAMPLE 13 ; 1,3-Di-0-ln-hexadecyl)-2-0-(4-aminobutyl)-~lyCerol Hydrochloride ' A. 1,3-Di-0-(n-hexadecyl)-2-0-(3-hydroxyp~y~L~ycerol Borane methyl sulfide (B~S) complex (6.5 ml., 68.5 mmoles) was added at 0 to 5C. to a solution of 1,3-di-0-(n-hexadecyl)- j 2-0-allyl-glycerol (10.82 g., 18.6 mmoles, prepared as in i' Example llA) in hexane tl90 ml. ), and the resulting solution ~) stirre,d for 3 hours at room temperature. The reaction solution jl was then cooled again to 0 to 5C. and ethanol ~17.3 ml.) added ,I dropwise to decompose residual ~MS. The reactio~ solution was then treated with a~ueous 3N sodium hydroxide (13 ml.) and !i 30 wt, % aqueous hydrogen peroxide (11 ml.), stirred for 16 hours j at reflux, cooled, and poured into ice water containing sodium ¦~ bisulfite. The ice water solution was stirred until it gave " a negati~e starch-iodide test for peroxides, and then extracted !~ with ether (3 X 200 m~ ). The combined ether extract was washed with water (200 ml.), washed with sàturated aqueous sodium l~, chloride solution ~200 ml.~, dried (MgSO~), filtered and Ij evaporated in_vacuo. The resulting product was puri~ied by ', silica gel ohromatography (elution with benzene:ethanol) ~5 g., - 45~ yield, m.p. 29C., n.m.r~ (CDC13)~3.80 (t, J = 5 Hz, 2, -OCH2CH2CH2OH) and 3.75 ~t, J = 5 Hz, ~, -OCH2CH2CH2OH)].
'~ B. 1,3-Di-O-(n-hexadecyl)-2-0-[3-(p-tosyloxy)propyl]-~lycero 1 j 1,3-Di-0-~n-hexadecyl)-2~0-t3-hydryoxypropyl)-glycerol ' ~8.0 g., 13.4 mmoles) was added at 10C. to a solution of ~-i toluenesul~onyl chloride (5~25 g., 27.5 mmoles) and pyridine (10 m 1.
j in methylene chloride (200 ml.),~and the mixture stirred for 6Q
hours at room temperat~ure. Water (200 m~.) was then added, the i' I' .
1~
.

., . ..
, methylene chloride and aqueous phases separated, and the latter ¦, extracted with methylene chloride (2 X 150 ml.). The three . methylene chloride layers were combined, washed with water , (2 X 150 ml ~ dried (MgS04)~ filtered and evaporated in vacuo.
The resulting ~osylate was purified.by silica gel chromatography ,l (elution with benzene) 13.0 g.0 10% yield, oil, ix ~CHC13) 1130 , and 1350 cm 1, n.m.r. (CDC13) ~ 7.53 ~q, 4, protons on phenyl ,1 ring), 4.15 ~t, 2, -S03CH2CH2CH20-), 3.63 (t, 2, -S03CH2CH2CH20-), .
~! 3-42 ~m~ 9, -OCH[CH2OCH2~1,H31]2), 2.45 (s, 3~ Ar-CH~), l.go ¦l (m, 2, -S03CH2C~ CH20-) and 0.90-1.50 (m, 62, aliphatic protons)]
,j C. 1~3-Di-o~(n-hexadecyl)-2-o-(3-cyanopropyl)-glycerol Il 1,3-Di-0-~n-hexadecyl)-2-0-[3~ t~syloxyJpropyl]-glycerol i'~ (3.0 g., 4.0 mmoles) was aissolved in a solution of sodium , cyanide tO.5 g.y 10 mmoles) in N,N-dimethylformamide (50 ml.), / and the resulting solu~ion stirred for 16 hours at 80DC., cooled, .
! diluted with water (lOOjml.) and extracted with ether t3 X 100 ml.~.
The combined ether extract was washëd consecutively with lN
ii hydrochloric acid (3 X 75 ml.), saturated aqueous sodium bicar~onate solution (3 X 75 ml.), water (75 ml.3 ana saturated ~ aqueous sodium chIoride solution (75 ml.), then dried (MgS~4), filtered and evaporated in vacuo to yield a waxy ~olid that was used in the next step wi~hout further puri~icat~on t2Oo g., 1l 83~ yield, ir `(C~C13~ 2250 cm 1].

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,I D. Title Compound Lithium aluminum hydride (800 mg., 21 mmolPs) was added to . a solution of 1,3 di-0 (n-hexadecyl)-2-0-(3-._yanopropyl)-glycerol . t2.~ g., 3.3 mmoles) in ether (100 ml~), and the mixture stir~ed , for 60 hours at room temperature. Enough water to quench thë
,~ reaction was added cautiously, followed by an additional 100 ml.
~l of water. The resulting mixture was stirred for another hour at Ii room temperature and the~ extracted with ether (3 X 100 ml ) ! The combined ether extract was washed with saturated aqueous 1~ sodium chloride solution ~3 X 75 ml.), dried ~MgSO4), filtered . .
Ij and evaporated in va-_uo to an oil, which was purified by silica 1, gel chromatography (elution with benzene:ethanol) and then ii dissolved in ethanol. The solution was trea~ed with hydrogen 1~ chloride gas and then evaporated in vacuo to y1eld a solid, which .
~ was recrystallized.. from ethyl acetate 1444 mg., 21~ yield, m.p.
61.5-63.5''C., n.m.r. ~CDC13) ~ 3.67 (t, 2, -OCH2CH~CH2C~aNH2), ii ~5 tm~ 9, OCHlCH?OCH2C15~31121~ 3-10 ~t, ~, -O~2c~2cH2cH2~H2)~
¦¦ 1.50-2.00 (~, 4, -OCH2C~ CH2CH2~H2~ and 0.80-l.S0 (m, 62, ali~hati .¦~ protons), elemental analysis calculated: 72.23~ C; 12.74% H;
il ~.16% N; ~ouna: 72.53~ C; 12.42e ~; 2.10% N~.

I! EXAMPLE I4 1~ 1,2-Di-0-(n-hexadecyl)-3-0- .
'l(3-aminomethylbenz~i)-glycerol ~ydrochloride .
¦A. 1,2 Di-0-(n-hexadec~ 3-0-(3-c~nobenzyl)-~lycer~l :25 ~l, Sodium hydride (1.056 g. of 50 wt. ~ mineral oil-dispersion, I
22 mmoles) was added to~a solution of 1,2-di-0 (n-hexadecyl)- .
'".glycerol (9.73 g., 18 mmoles) in tetrahydrofuran (150 ml-.) and the resultin~ solu~on stirred for 20 minutes at room ~', temperature under nitrogen. m-Cyanobenzyl bromide (4.0 g., 20 i~ ~moles) was added and the reaction mixture stirred overnight at 'i'j ;' , , , , 11 ~.
.
, . . .

room tempera~ure under nitrogen. Water (200 ml.~ was then added cautiously and the resul~ing mixture extracted with ethyl acetate (3 X 150 ml.)~ The combined ethyl acetate extract was dried (MgSO4), filtered and evaporated in vacuo to an oil s I! (12 ~- ), which was purified by si~ica gel chromatography i1 (elution with benzene:hexane) ~8.0 g., 68% yield, oil, - '! ir (CHC13) 2230 cm~
il B. Title.Compound .

ii A solution of 1 t 2-di-0-(n-hexadecyl)-3-0-(3-cyanobenzyl)-o i! glycerol (1.0 g., 1.5 mmoles) in ether (10 ml.) was slowly added ' under ni~royen to a suspension of lithium aluminum hydride ~0,057 g., 1.5 mmoles) in ether ~0 ml.), and the resulf-ing mixture stirred for one hour at refluX under nitrogen and I~ then cooled. Water ~50 ml.) was added cautiously and the mixture !i extracted with ethër ~3 X 50 ml.). The comhined ether extract '~ was dried (~gSO4), filtered and evaporated in vacuo to an oil, '' which was purified by silica g~l chromatography (elution with il benzene:ethanol) and then dissol~ed in ethyl acetate. The solution was treated with hydrogen chloride gas and then ev~porated in va uo to yield a solid, which was recrystallized , from ethyl acetate ~220 mg., 21% yi~ld, m.p. 88-90C., elemental ,' analysis calculate~: 74.14~ C; 11~87% H; 2.01% N; ~ound:
74.35% C; 11.54% H; 2.15% Nl. ~ - .
.
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!, ExAMPLES 15-?6 , In like manner to that descxibed in Example 14 the foilowing I
i: compounds were prepared by using the appropriate 1,3- or 1,2-di- I
, O-(n-higher alkyl or alkenyl)-glycerol and cya~obenzyl bromide i' as starting materials: .

i! Rl- O--CH2 'C~I2NH2 3 CH-O~CH
2 C~2 ' . . .
3 ~CH2NH2 -O-CH ~ II .

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E:lemental Ana7 ysis Exampl~ Calcuiated (%) Found ~%) :, Num~er C: H N C ~ N
~ _ _ _ _ _ '' 15 7~.14 11.87 2.01 73.89 11.43 1.99 . 16 73.20 11.85 1.98 73~17 11.53 2.2~
. 17 72 . 2~ 11 . 831 . 9672 . 52 11 . 46 1 . ~0 , 18 72 . 63 11 . 482. 1772 . 62 11~ 81 2. 43 , 19 . 74 . 1~ 11 . 872 . 0173 . 9~il . 25~ 2 . 02 ,t 20 73.14 11.65 2.19 72.86 11.~4 2.11 10 !¦ 21 74 . 99 12 . 061. 8674 . 97 1~. 7~ 3 22- 74 . 50 11 . 571 . 85~4 . 4011 . 08 . 2. 08 j 23 73. 14 11 ,. 652 . 1972 . 8411. 30 ~ . 26 ~1 24 74 .14 11. 87? O 0174 . 33 11~ 55 2 .15 ,. 25 74 . 99 12 . 061. ~67J, . 50. 11 . 30 1 . gl ,' 26 74.06 11.54 1.83 74.00 10.99 1.93 - ... .
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, . 1,2-Di-0-(n-hexadecyl)-3-0-(4-aminomethy ~ lycerol Hydrochloride I A. 1,-2-Di-0-(n-hexadecyl)--3-0-tp-tosyl)-glycerol j; . In like manner to that desl~ribed in Example 13B the named .' compound was prepared by reactillg 1,2-di-0-(n-he*adecyl)-glycerol j~ with ~-tol,uenesulfonyl chloride~. Purification was accomplished .
~¦ by recrystallization from ethyl acetate [m.p. 53-55C., ir Il (CHC13) 1360 and 1180 ~m 11. .
!~ B. 1,2-Di-0-(n-_exadecyl)-3-0-(4 ~ nophenyl)-gl~cerol , A mixture of 1,2-di-0-(n-hexadecyl)-3-0-(p-tosyl)-glycerol (1.4 g., 2.0 mmoles), sodium 4-cyanophenolate ~0.5 g., 3.5 mmoles~
and xylene (100 mlO) was stirred for 16 hours at reflux. Since ! the reaction was no~ yet complete the xylene was xemoved by ; distillation and replaced by N,N-dimethylformamlde ~100 ml.), and the resulting solution stirred for another 16 hours a~ 150C. .
' The reaction solution was then coolëd, diluted with water (100 ml ) .. and extracted with ether (2 X 100 ml.). 'I'he combined ether .
, extra~t was washed consecutively with 3N hydxochloric acid j ~100 ml.), 10 wt. % aqueou~ sodiùm bicarbonate solution ~100 ml.) and water ~100 ml.), dried (MgS04), filtered and evaporated . ¦
~I sn vacuo to an o~l, which was puri~ied b~. silica gel chromatography '¦ telution with ~enzene~) ~0.65 g., 50% yield, m.p. 53-55C., ir CXC13) 2210 c~-1].
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C. Title_Comp und 1,2-Di-0-(n-hexadecyl)-3-0-(4-cyanophenol)-glycerol -tO.60 g., 0.93 mmole) was added to a suspension of lithium ~1 aluminum hydride (0~3 g.~ 7.9 ~noles) in ether ~25 ml.), and ~! the resulting mix ure stirred for 30 minutes at room temperature;
Wa~er ~25 ml.) was then added czlutiously, the ether and aqueous ~, phase$ separa~ed, and the latter extracted with ether (3 X 25 ml.
il and ethyl acetate ~25 ml.). The five organic extracts were .
Ii combined, dried (MgS04), iltered and evapoxated i~ vacuo to an ii -oil, which wa dissolved in ether. The solution was treated jj With hydrogen chloride gas, causing precipitation of a solid ."-10.41 g., 64% yield, m.p. 110-112C~, n~m.r. .~CDC13) ~ 4.02 ~ s, 2, -eH2NH2j, elemental analysis calculated- 73.91% C:
t 11.81~ ~; 2.05% N, found: ?3.62~ c; 11.71% ~; 2.14~ N].
!,` EXAMPLES 28-30 ~i In like manner to that des~ribed in Example 27 B-C the '~ following compouDds were prepared~rom ~he appropriate tosylate .
" tprepared as in Example 27A) and sodium cyanophenolate: .
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i EX~MPLE 31 .1,2-Di--0-(n-hexadecyl)-3-0-!i[4-(3-aminoprop~ henyl]-~lycerol ~ydrochloride In like manner to that described in Example 27 the named . compound was prepared by using isodium 4-(2-cyanoethyl)phenoiate in place of sodium 4-phenolate ~m.p~ 153-1S5C., elemental il analysis calcul~ted: 74.374 C; 11.91~ H; 1.97~ N; found:
¦l 74.13~ C; 11.44% H; 2.08% N).
. ¦i EXAMPLE 32 . .
j ~ 1,2-Di(n-hexadecyloxy)-3-~3-aminometh~lbenzylamino)-propane Dihydrochloride I ¦ 1,2-Di-0-~n-hexadecyl3-3-0~ tosyl)-glycerol (3.48 g., '! 5~0 mmoles) was added to a solution of m-x~lylenediamine to-68 g, i, 5.0 mmoles) in N,N-dimethylformamide ~20 mI.). ~he resulting lS i~ mixture was stirred ~or one hour at 90~C. and then poured into j ice water (150 ml.), causing the formation o~ solids which were isolated by filtra~ion, purified b~ silica gel chromatography ;~
telution with benzene:ethanol) and ~hen dissolved in ethyl . acetat~. The solution was treated with hydrogen chloride gas ,~ and then evaporated in vacuo to yield a solid, which was re- :
crystallized from et~yl acetate tO.29 g., 8~ yield, m.p. 78-80C.
n.m.r. (CDC13) ~ 4.24 (s, 2, Ar-CH2NH-) and 4.37 ~s, 2, Ar-C~ NH2),.elemental analysis calcula~ed 70.55~ C;
11.570 H; 3.83~ N; found: 70.64% C; 11.29% H; 3,62~ N].

. ~, J ~ : .
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!
il ! Ex~IP~E 33 !
" 1,2-Di-O-(n-hexadecyl~-3-0-(3-isopropylamino-,. 2-hydroxyprop~l?-glycerol Hydrochloride i A. 1, 2-Di_O- (n-hexadecyl)-3-0=(2,3-epoxyp o~E~ ycerol i~ A solution of 1,2-di-0-(n-hexadecyl)-3-allyl-glycerol t5.8 g., 10.0 mmoles) and m-chloroperbenzoic acid ~1.86 g., j' 10.8 mmoles) in benzene t50 ml.) was stirred at reflux for 16 I~ hours ~he reaction mixture was then coolea, treated with - ¦1, satura~ed aqueous sodium bisulfite solution tlO ml.) and l~ saturated aqueous sodium bicarbonate ~olution (50 ml.3, and , extracted with ether (3 X 50 ml.~. The combined ether extract ¦? was washed with water (100 ml.), washed with saturated aqueous Il~ sodium chloride solution (100 ml.) t dried (MgS04), filtered and '~ evaporated in vacuo ~o an oil (4 . 9 g., 82~ yield, olefinic I, protons absent by n.m.r. analysis), wh;ch was purified by silica !~ gel chromatography telution with benzene:ethyl acetate) (4.2 g., ;.
i, 7Q% yieid, oil-solidified on standing)~
~! B. Title Compound ¦, A s~lution o~ 1,2-di-0-(n-hexadecyl)-3-0-(2,3-epoxypropyl)-' glyoerol (2.0 g., 3.35 mmoles) ln isopxopylamine (40 ml~ was ~, ., .
'~ hea~ed in a stain~ess ~teel bomb or 16 hours at 10~C., cooled, ; l~ concentrated in vacuo and dis~olved in ether (100 ml.). The ' ether soIution wa~ washed with.lN hydrochloric acid (100 ml.), ' dried (MgS04), filtered, treated with charcoal, filtered again, I; and then cooled by immersion of the ~la5k in a Dry Ice-acetone : 1~ bath, causing fonmat~on o~ a precipitate. The precipi~ate was .
isolated ~y filtration (1.3 g.) and:puri~ied by silica gel ! chromatography ~elution with benzene:ethanol) [720 mg., solid ¦ contained about 1/2 molie H20 per mole named prod~lct, 31~ yield,-' m.p. 55-57C~, n.m.r~ tcDcl3)~ 1.45 ~d, 6, -NHCH[C 3I2), elementa~
analysis calculated: 70.19% C;.12.50% ~; 2.00% N; found: 70.10% ¦
j; C; 12.19% H; 1.87% ~

; ~j ' .~3, ~f jl ; EXAMPLES 34-37 ' In like manner to that described in ~xample 33B the ~ollowing compounds were Drepared by reacting the appropriate 1. 2,3-epoxide ~prepared as in Example 33A) and alkylamine:

~' .. n-hexadecyl-0-1H2 ~H

I! CH-O-CH2 HCH2NHR3 li ' n-hexadecyl-O-CH2 ' 'I ' ' ~ fH2-o-cE~2~HcH2N~R3 .
Ij n-hexadecyl-O- 3 ~ II
n-hexadecyl-O-CH2 ii . -:
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1,2-Di-O~(n-hexadecyl)--3-0-(3-amin~-2-hydroxy~ropyl ? -qlYcerol H~drochloride A . 1, 2-Di-O- ( n-hexadecvl)-3-0=(3-azido-2-hydroxYpropyl)-ylycerol A solution of sodium azide (0.5 g., 7.7 mmoles) in water ~5 ml.) was added to a refluxing solution of 1,2-di-0-(n-hexadecyl)-3-0-(2 J 3-epoxypropyl)-glycerol (3.3 g., 5.5 mmoles) in 1,4-dioxane (100 ml.1, and the resulting solution stirred at reflux for 16 hours. Since the reaction was not yet complete, additional sodium azide (0.5 g., 7.7 mmoles) was added and the reaction stirred at reflux for another 16 hours. The reaction solution was then cooled, concentrated in vacuo, diluted with water (100 ml.) and extracted with ether (3 X 100 ml.). The combined ether extract was washed with water (100 ml.), dried (MgSO4), filtered and evaporated in vacuo to an oil which solidi~ied on standing [2.2 g., 62% yield, ir (CHC13) 2105 cm ].
B. Title Compound I Lithium aluminum hydride (300 m~, 7.9 mmoles) was added -. .
¦ to a solution of 1,2-di 0-(n-hexadecyl) 3-0-l3-a~ido-2-hydroxy-¦ propyl)-glycerol (2.2 g., 3.4 mmoles) in ether (100 ml.), and the resulting mixture stirred for one hour at room temperature.
Ethanol (5 ml.) and water ~200 ml.) were added to guench the . reaction, and the mixture then extracted with ether (2 X 100 ml.) .
: 25 The combined ether:extract was dried (MgSO4), filtered and e~aporated in vacuo. The resulting product was purified by silica gel chromatography (elution with benzene:ethanol) and then converted to the hydrochloride salt [800 mg., solid ~ . .
'. ~,,~,1 . - -~

~ k L~

contained about 2 moles H20 per mole named product, 34% yield, m.p. 149-150C., n.m.r. (CDC13) 4.00-4.35 (m, 1, -OCH2CHOCHCH2NH2), 3.33-3.73 (m, 11, C15H31CH2CH2CH[CH2C15H31]
CH2OCH2-), 3.03-3.25 (m, 2, -OCE~2CHOHCH~NH2) and 0.87-1.67 (m, 62, aliphatic protons), elemental analysis calculated:
66.48~ C; 12.33% H; 2.04~ N; found: 66.68% C; 11.85~ H; 2.02% N] .
XAMPLE: 39 1,3-Di-0-(n-hexadecyl)-2-0-(3-amino-2-hydroxy~roEyl)-glycerol In like manner to that described in Example 38 the named compound was prepared from 1,3-di-0-(n-hexadecyl)-2-0-(2,3-epoxypropyl)-~lycerol (prepared as in Example 33A) (free base, m.p. 61-63C., elemental analysis calculated: 74.33% C, 12.96%
H; 2.28~ N; found: 74.49% C; 13.10% H; 2.12% N).

1,2-Di-0-(n-hexadecyl)-3-0-(2-aminoprop~ll-glycerol Hydrochloride A. 1,2-Di-0-(n-hexadecyl)-3-0-[2-(p-tosyloxy)propyll-~lycerol In like manner to that described in Example 13A and B, 1,2-di-0-(n-hexadecyl)-3-0-allyl-glycerol was reacted with BMS, and the resulting 2-hydroxypropyl and 3-hydxoxypropyl compounds converted to their corresponding tosylates. A separation was not attempted at this stage; the mixture of tosylates was used dlrectly in the next step.

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B. 1,?-Di-O- (n-hexadecyl)-3-0-(2-azidopropyl~-glycerol The resulting mixture of tosylates (3.0 g.~ 4.0 mmoles) . was dissolved in N,N-dimethylacetamide (50 ml.) and treated with a solution of sodium azide (0.326 g., 5.0 mmoles) in water (5 ml.) ; for 16 hours at 90C. The reaction solution was then cooled, ~ diluted with water (200 ml.), and extracted with ether (2x150 ml.) .
i~ The combined ether extract was washed with water, dried (Mg504~, , filtered, and evaporated i= vacuo to an oil 12 g~, 81% yield;
Il ir (CHC13) 2100 cm 1], a mixture of the 2-azidopropyl and 3- .
~ azidopropyl compounds, which was used without further purification ¦. in the next step. . .
!l C. Title Compound . . .
The resulting mixture of azides (2 g., 3;2 mmoles) was dissolved in ether (lOC ml.), treated with lithium aluminum 15 ~j hy~rid~ ~0.4 g., 10.5 mmoles), and allowed to stix for 2 hours " at room temperature. Excess hydride was destroyed by cau~ious , addition of ethanol (10 mlO) and water ~150 ml.), and the mixture ~` then extracted with ether 12xlO0 ml.~. The com~ined ether extrac~
; 1i~ was dried (MgS04), filtered, and concentrated in vacuo to an oil ~ 8 g.), whi~h was purified by silica gel chromatography~(elutio~
with benzene:ethanol) and then converted to the hydrochloride sal~
by ~issolution and treatment wit~ hydrogen chloride gas. The .
i,' sal~ was recrys~allized from ethyl acetate (0.21 g., solid con~aineo ~ about 1/2 1e H~O per mole ~amed produ~t, 10% yield, m.p. 56-58Cj., ' elemental asalysi6 calculated: 71.03% C; 12.70~ H; 2.18% N;
, found: 71.11% C; 12.91% H; 2.16~ N).

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~ EXAMPLE_41 .
: 1,2-Di-0-(n-octadecyl)-3-0-(2-aminopropyl)-glyCerOl Hydrochloride In like manner to that described in Example 40A)1,2-di-0- ¦.
(n-octadecyl)-3-0-(2-hydroxypropyl)-glycerol was prepared from j 1,2-di-~-~n-octadecyl)-3-0-allyl-glycerol. The named compound ji was prepared rom 1,2-di-~-(n-octadecyl)-3-0-(2-hydroxypropyl)-1~ glycer,ol in like manner to that described in Example 40 B-C (soli !i contained about 1 mole H20 per mole named product, m.p~ 65-67C., i, elemental analysis calculated: 71;19% C; 12.B0% H; 1.98% N;
10 ~ found: 71~12~ C; 12.52% H; 1.92% ~). . .

~ _ ' 1,2-Di(n-hexadç~loxy)-3 aminopropane Hydrochloride . j !1 In like manner to that des~ribed in Example 40B, 1,2-di-0- .

¦' ~n-hexadecyl)-3-0-t~-tosyl)-glycerol was converted to 1,2-di-ii (n-hexadecyloxy)-3-azidopropane. This intermediate was converted ,, to the ti~-le compound in like manner to that described in Example ,~ 40C (m.p~ 78-80C., elemental analysis cals~ulated: 72.93% C; .

.94% H; 2.43% N; foun~ 73.08% C; 13.08% ~; 2.65% N) ~ } ' ' ' .
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1,3-Di(n-hexadecyloxy ! -2-aminopr~ne Hydrochloride In like manner to that described in Example 42 the named compound was prepared from 1,3-di-0-(n-hexadecyl~ 2 0-(p-tosyl)-¦ glycerol (prepared as in Example 27A) (m~p.58-60C., elemental analysis calculated: 72.93% C; 12.94% H; 2.43~ N; found:
72.65% C; 13.02~ H; 2.59% N).

l 1~2-Di(n~hexadecyloxy)-4-aminobutane Hydrochloride ¦ In like manner to that described in Example 42-the named compound was prepared by using sodium cyanide in place of sodium azide (m.p. 86-37C., elemental'analysis calculated: 73.25% C;
12.97% H; 2.37% N; found: 73.52% C; 12.64% H; 2.50% N).

1~ 1,3-Di(n-hexadecyloxy)-2-(3-aminopropylamino)propane Dihydrochloride A. 1,3'-Di(n-hexade~yloxy~-2-(2-cyanoethylamino)propane A mixture of 1,3-di(n-hexadecyloxy)-2-aminopropane (500 mg., 0.93 mmoles), acrylonitrile (75 ml.) and 2 wt. % aqueous sodium hydroxide solution (75 ml~) was heated to 60C. Tetrabutyl ammonium hydroxide ~1 ml. of 40 wt. % aqueous solution) was then added and the resul'ting mixture stirred for 15 minutes at 90C.
The reaction mixture was then cooled~ causing precipitation of solids, which were isolated by filtration and found (TLC) to ?5 ` contain a large ~uantity of unreacted starting material. Using fresh acrylonitrile and aqueous sodium hydroxide solution in each cycle, the solids were treated two more times by the above procedure.

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~; The third cycle solid product w,as purified by silica gel ` chromatography (elu~ion with toluene:ethyl acetate) ~200 mg., t 36~ yield, m.p. 45-46C.~ ir (C~C13) ~250 cm 1, n.m.r. (CDC13) ~ ¦
, 3.07 (t, 2, -NHCH2CH2CN) and 2.!;3 (t, 2, -NHCH~C~2CN)3.
5 . B... Title Compound .¦
,, A mixture of 1,3-ditn-hexadecyloxy)-2-(2-cyanoethylamino3-!' propane t200 mg., 0.34 mmoles1, tetrahydrofuran (10 ml.), ethanol (20 mi.) and Raney nickel catalyst (0.2 y.) was saturated with ~ ' ammonia gas and then hydrogenated (50 psi) for about 4 hours at .10 ,~' room tempera~ure. The reaction mixture. was ~hen filtered and " evaporated in ~acuo to an oil, which was purified by silica gel chromatography (elution with toluene:ethyl.acetate:.ethanol:
methanol) and then dissolved in ethyl acetate. The solution was , treated with hydrogen chloride gas, causing precipitation o~
~' solids tlO mg., solid contained about 2.5 moles H20 per mole ~; named product, 4% yield, m.p. 235-2.36C., elemental analysis calculated: 63~65% C; 12.51~ ~; 3.90~ N; found: 63.60% C;
11.84~ H; 3.75% N]. .
~i EXAMPLE 46 : 20 ,. 1,2-Di-O-(n hexadecyl)-3-0-(4-amidinophen~ lycerol Hydrochloride A solution of 1,2-di-O-(n-hexadecyl)-300-t4-cyanophenyl)-g~ycerol ~3.5 g., 5.45 mmoles), ethanol (10 ml.) and 1,4_ . dioxane tlOO ml.) was saturated with hydrogen chloride gas a~ .
2S ~ 0C~, and allowled to rea~t for 16 hour~ at ambient temperature.
. The reaction so.lution was then evaporated in vacuo to an oil, the oil dissolved in ethanol (100 ml.), and the resulting solution saturated with ammonia gas, stirred for 3 hours at reflux, dilute ,, c '' ~ !i ~l with water (lS0 ml.), evaporated in vacuo to remove the majority . of the ethanol, and extracted with chloroform (3 X 150 ml.). The !
, combined chloroform extract was dried (MgS04), filtered and evaporated n vacuo to yield a solid, which was purified by ~ silica gel chromatography (elution with benzene:ethanol) and Ij then dissolved in ethyl acetate. The solution was treated with il hydrogen chloride gas and then evapora~ed in vacuo to yield a I¦ solid; which was recrystallized from ethyl aceta~e [1.0 g., 26~
¦I yield, m.p. 220-222Co ~ ir (CHC13) 1670 cm 1, elemental analysis ~, calculated: 72.53~ C; 11.45% H; 4.03~ N; found: 72.67% C;
, 11.38% H; 4.12% N3.
' EXAMPLE 47 . . .
;, 1,2-Di-0-~n-hexadecyl)-3 0--'. (3-amidinobenzyl? ~lycerol Hydrochloriae :
, .The named compound was prepared from 1,2-di 0-(n-hexadecyl)-j. 3-0-(3-cyanobenzyl)-glycerol in like manner to that described . in Example 46 [solid contained about 2 moles H20 per mole named .~ product, 20~ yield, m.p. 155-157C., ir (CHC13) 1670 cm 1, elemental analysis calculated. 69n27~ C; 11.48% H; 3.76% N;
~0 ~I found: 69.11~ C; 10.63~ H; 3.83% N].

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, EXAMPLE 48 1,2-Di-O-(n-hexadecyl)-3-0-~3-(1-hydroxy-2-t-butylaminoethyl)-benzyl]-~lycerol Hydrochloride ; A. 1,2~Di-O-(n-hexadecy~-3-0-(3-formylbenzyl)-~lycer ~ A solution o 1,2-di-0-(n-hexadecyl)-3-0-(3-cyanobenzyl~-glycerol (5.0 g., 7.6 mmoles) and diisobutylaluminum hydride i (1.17 g., 8.2 mmoles) in benzene (25 ml.) was stirred ~or 16 hours at ambient ~emperature. The reactlon mixture was txeated i, with methanol (4.~2 ml.) and water t2.5 ml.) and stirred to l~ decompose unreacted hydride, and then filtered and extracted !~ with benzene (3x25 ml.). The combined benzene extract was dried ~ a2S04), ~iltered and evaporated in vacuo to an oil, which was !~ purified by silica gel chromatography (elution with benzene) i! [2~0 g., 40% yield, oil, ir (C~C13~ 1700 cm , n.m.r. ICDC13) S
', 10.1 (s~ ArCHO)].

, B. 1,2-Di-O-(n-hexadecyl~-3-0-13-~1,2-epoxyethyl)-benzyl]-glycerol _ _ _ _ _ _ _ suspe~sion of sodium hydride` (3.23 g~ of a 57 wt. % disper-sion in mineral oil, ~7 mmoles) in ~imethylsulfoxide (117 ml.) w~s ~0 ' heated under a nitrogen atmosphere at 70 to 75C. until hydrogen -i! evolution stopped 145 min.). Tetrahydxofuran (88 ml.) was added j and the mixture cooled to O to 5C. Trimethylsul~onium iodide ' (13.~7 g., 67 mmoles) was then added in portions, fbllowed by ,' rapid addition of a solution of 1,2-di-0-(n-hexadecylj-3-0-(3-',' ormylbenzyl3-glycerol ~7.0 g., 10.6 mmoles) in tetrahydrofuran (58 ml.); The ~resulting mixture was stirred for 16 hours at I, room temperature, poured into water (200 ml.) and extracted !: with ether (3xlE~O ml.~. The combined ether extract was washed ~- with water t2xlOO ml.? and saturated aqueous sodium chloride 1; solution tlOO ml.), dried ~(MgS04), filtered and evaporated in vacu~
to ~n oil (7.0 g., 98% yield), which was sufficiently pure to be u6ed in~the next step.

i~l11.4~?., ~' C. Title Compound i - . I
A mixture of t-~utylamine (30 ml.~ and 1,2-di-0-(n-hexadecyl~
. 3-0-13-~1,2-epoxyethyl)-ben2yl]-lglycerol (2.0 y., 3Ømmoles) 1. was heated for 9 hours at 100C. in a steel bomb. The reaction S ~' mixture was cooled, t-~utylamine removed by evaporation in va~uo,-¦
and the resulting oil purified b~y silica gel chromatography (elution with benzene ethanol~ and then dissolved. The solution ~! was saturated with hydrogen chloride gas and then evaporated . ~! in vacu~ to yield a solid, which was recrystallized from ethyl ! acetate 1630 mg., solid contained about 1 mole X20 per mole named .
I, product, 27% yie~d, m.p. 49-51C., n.m.r. (CDC13) ~ 1.47 !, ~s, 9, -C[CH.~3), ele~en~al analysis calculated- 71.99~ C;
11.83~ H; 1.75% N; found- 71.B6% C; 11.30~ H, 1.69% N~.

~5 !~ 1,3-Di-0-(n-hexadecyl)-2~0-~3-tl-hydroxy-2- .
~, t-butylaminoethy~-benzyl]-glycerol Hydrochlor de !- In like manner to that descri~ed in Example 48 A-B, 1,3-di-~ 0-(n-hexadecyl)-2-0-(3-cya~obenzyl)-glycerol (prepared as in !~ Example 14A)- was converted to 1,3-di-0-(n~hexadecyl)-2-0-13-(1,2-'. epoxyethyl)-benzyll-glycerol. The title compouna was prepared ', by reacting said epoxy compound ~ith t-butylamine in like manner l~ to that described in Example 48C (solid contained about 1 mole 2 per mole named product, m.p. 43-45C., elemental analysis l~ calculated: 71.99% C; 11.83% H; 1.75% N; found: 72.06% C;
1, 11.43% ~ }.71% N~.

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;1,2-Di-0-[n-hexadecyl)-3-0-[3-(1-hydroxy-2-;iso~ropylaminoethyl?-benzyl]-glycerol Hydrochloride In like manner to that descr.ibed in Example 48C the named . compound was prepared by using isopropylamine in place of t-.' butylamine ~solid contained about 3~4 mole EI20 per mole named ' product, m.p. 53-55C., elemental analysis calculated: 72.17%
, C; 11.79~ H; 1.79% N, found: ?2.11~ C; 11.55% H; 1.92% N).
~ EXAMPLE 51 ~ 2,3-Di(n-hexadecyloxy)propyl]-4-aminomethyl-4~ .
,~henyl~iperidine Dihydrochloride A. ~-12,3-Di[n-hexadec~loxy)propyl]-4-cyano-4-~heny~piperiai le jt A mixture of 1,2-di-0-(n-hexadecyl)-3-0-(~-tosyl)-~lycexol `. (6.96 g., la mmoles), 4-cyano-4-phenylpiperidine hydrochloride ., ~2.23 g., 10 mmoles), triethylamine 12 ml.) and N,N-dimethyl- .
,~ ~ormamide (4~ ml.) was stirred for 16 hours at 95 to lOO~C.
~ The reaction mixture W2S then cooled, diluted with water ~200 ml.~
and ex racted with ethyl acetate (3xlS0 ml.). The combined ethyl ¦
j. ac~tate extract was dried (MgSO4), filtered and evaporated l~ in vacuo to an oil 16 g.), which was purified b~ column chroma~
: ,, tography (elution with benzene:ethyl acetate~ loil, ir ~CHC13) : 1 2220 om~l~i.
.

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i, j B. Title Compound A solution of 1-[2,3-di(n-hexadecyloxy)propyl]-4-cyano-4-phenyl-piperidine (2.5 g., 3.6 mmoles) in ether (100 ml.) was treated with lithium aluminum hydride (0.4 g., 10.5 mmoles), and the resulting mixture stirred for 4 hours at room temperature. The reaction mixture was treated cautious-ly with water ~100 ml.) and extracted with ether ~3xlO0 ml.). The combined ether extract was dried (MgS04), filtered and evaporated in vacuo to an oil, which was purified by silica gel chromatography (elution with benzene:
ethanol) and then dissolved. The solution was treated with hydrogen chlor-ide gas and then evaporated in v~acuo to yield a solid, which was recrystal-lized from ethyl acetate (1.1 g., solid contained about 3/4 mole H20 per mole named product, 40% yield, m.p. 132-134C., elemental analysis calculat-ed: 70.60% C; 11.53% H; 3.50% N; found: 70.74% C; 11.34% H; 3.40% N).

In like manner to that described in Example 51 the following com-pounds were prepared from the appropriate 1,2-di-0-(n-alkyl or alkenyl)-3-0-~p-tosyl)-glycerol ~prepared as in Example 27A):

H21-N ~ 2 H2 phenyl .

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.: EXAMPLE 56 ~ ri-o- (n-hexadecyl)-3-0-~2-[di(2-j: hydroxyeth~l)amino~,ethyl)-ql~cerol Hydrochloride In like m~nner to that described in Example 11 the named compound was pr,epared by reacting 1,2-di-0-(n-hexadecyl)-3-0- .
formylmethyl-glycerol with di(2-hydroxyethyl)amine (solid contained about 1/4 mole H20 per mole named product, m.p.
194-195'"C., elemental analysis calculated: 6~.06% C; 12.20~. H;
I' 1.96% N; found: 69.12% C; 11,.76% ~; 1.90% ~)O
1i ' . EX~MELE 57 In Vivn-Activity of 1,3-Di-0-tn-hexadecyl)-2-0~
(3-aminopropxl)-yl~cerol Hydrochloride Against EMC Virus , Formulation as an emulsion was accomplished by mel~ing !1 and mixing equal parts of the named compound, polysorbate 80, l' and glycerin, and then dispersing the mix~ure. in hot water under I vigorous mixing. The formulation was then adjusted to final -" concentrations of 0.14M sodium chloride and 0 01M s~dium .
phosphate, pH 7. Further diluti,~ns were made with 0 .14M sodium , chloride -O~OlM sodium phosphate, p~ 7 buffer solutio~.
j, Three groups of ten female albino mice (20-25 g. body Il weight) were given 0.5 ml. intrapeeritoneal injections containing .
` dosage levels of 1.5! 5 and 15 mg. of the named ~ompound~kg.
,' body weight, respectively. A fourth control group of ten I~ m~ce was-given no such injection. ~Eighteen to twenty-~our ~25 i~ hours later all four groups ware challenged with 0.2 ml.
subcutaneous injection containing ~0 times the I.D50, the dosage " level causing a 50~ death rate in unprotec~ed mice in ten days, of encephalomyocarditis (EMC) virus.' Survival d, a were recorded. .
over the next~ten days and the relative survival (Sr) calculated:

'Dosa~e Leve3. of Named Compound C~r (avera~e of seven experiments) 15 mg./kg. : 61 ! 5 45 .
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" Antiviral activity is expressed as the relative survival.~; (Sr) in experimental groups compared to the contr~l on the tenth day after challenge. Sr is defined by the formula . Sx ~ > . _xi ~~ ~ ..
'` Sr ~i=l to 10 i=l to 10 X 100 I? _ _ - , . .
100 ~ 100 -~, ,. .ei I! iCl to 10 ?~ wherei,n Sr = relative survival ~ !l Sx = percent survival after ten days in experimenta 1 ?, Xi = number of survivors on khe ith day in ¦¦ . experimental group ,, ei = number of survivors on the ith day in control i~ group .

ii EXAMPLES SB-86 ; In like manner to that described in Example 57 the n vivo .
i activity agains~ EMC ~irus was determined for the compounds lis~ed below.
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li EXAMPLE 87 Reduc~ion of Virus Yield on Human Polyp Cells In Vitro by 1 3-' Di-O-(n-hexadecyl)-2-o-(3-aminopropyl-)-glycero r ~-y~ L~e ,; Growth medium was prepared by supplementing Eagle's minimum essential medium (100 ml.) with lOOX concentrated antibiotic-antimycotic solution (2 ml.), 200 mM glutamine solution tl ml.~, i,lOOX concentra~ed nonessential am.ino acids solution (1 ml.), 7~ 100 mM sodium pyruvate solukion (1 ml- ) t and hPat-inactiYated , , fetal calf serum (10%j. Each well of 9Ç-well microtiter 10 . i~plates was seeded with about 50,000 human nasal polyp cells suspended in 0.2 ml. growth mediu~. ..~he plates were then ,jincubated for 8-10 days at 37C. in a 5~ rO2 atmosphere to .
establish monolayers of cells.
Ii At the end o~ the 8-10 day cell gr~wth period co~fluent 1S ! monolayers on the plates were wàshed four times with phosphate ! buffered saline and immediat~ly afterward treated with 0.2 ml. .
per well of maintenance medium containing 10, 5~0~ lrO~ 0~5r i'0.1 and Of~g./ml. of the named compound, respectively.
,IThe maintenance medium was identical to ~he growkh medium des-i~cribed above except that the level of said fetal cal serum was ~2%. The plates were incuba~ed for another 18 hours at 37C., ':and the monolayers then washed ~our times with phospha~e buffered !j saline to remove the named compound, challenged with a composition ~,containing about lOOD times the TCID5~ . the dosage level I causing a 50% inlection rate in unpr~tected cultures, of ,vesicular-stomatitis virus (VSV) ox a two hour (37C.) ads~rption.
'period, washed ~our times with phosphate buffered saline to remove . ,,, .

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. unadsorbed virus particles, and refed with 0.2 ml. per well of ; said maintenance medium. The plates were then incubated for 7 hours at 37C., and the culture ~luid from 5-8 replicate cells , harvested from each plate~ stored fro2en in test tubes, and then S 'j titrated for the amount of infectious virus present in micr~titer ¦
I plates of L-92g mouse fibro~las~.s~ The ~-929 mouse cultures ¦, were scored microscopically and ,malyzed ab~ut three to four I! days later, with the ~ollowing percentage decreases in virus ¦¦ yield ~with respect to the contxol) determined for the five !~ concentratio~s.of named compound tested: .
~,Percenta~e Reduction of ~irus Yield ¦iConcentratlon ~g.~ml.)~~of Named Compound ,i10 5-0 1-0 ' 0.5 ~.1 !1~4~ 90% 84% 75% ~68~ .
~¦~XAMPLES 88-121 ~! In like manner to th~t described in Example 87 the reduction . of ~irus yield o~ human polyp cells in vitro was determined fo~ .
' the compounds listed below. .

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~1 E u~l ~ a a Ii o~O
" I,~ V~ V '~ . .

~ u~ + ~ !
v~

~. ~ ,,1 ~ `1, + ~ + ~.

,,' "' ~.` '' ' ,. ... .... .
. , ' '. `.., V .

a E ~ 1 5 v `. - . . ~ .
,' '~1 ' ' - J,~ "

t t!~ t!~ t~ t~ 3 ~ ~ _ I' ~ Z trl t~ I t.~ t~ _l " ~ ~

4~o ~bility of 1,3-Di-0-~n~hexadecyl)-2-0-(3-aminopropyl)-glycerol _ H~rochloride to Induce Circulatin~ In~erferon._ _ ' A mixture of equal weights of the named compound, po~.ysorbate, s 80 and glycerol was ~used and then homogenized in hot 0.14M
~'s~dium chloride containing O.OlM sodium phosphate, pH 7 (PBS)~
,~The resulting oil-in-water emulsion was readily dilu~ed with PBS
for administration.
jl Female Swiss mice (2Q-25 g. body weight) were injected .
ji (0O5 ml., intraperitoneal) with ~. ~uantity of the above diluted ~emulsion containing 25 mg. of the named compou~d/kg. body weight.
! Eigh~, twelve, sixteen and kwenty hours after injecti~n samples of plasma were withdrawn from four mice and popled. Serial ',dilutions in h-i5 (Leib~vitz3 medium containing 5~ fetal ~alf 1~ , serum were incubated in microtiter plates overnight at 37C.
! on confluent monolàyers of L-929 mousa fibroblasts. The mono-' layers were then washed with protein-~ree medlum, challenged ¦'with 10 times the TCID50, i.e. the dosage level causing ~ 50%.
,'in~ection rate in .unpr~ ected cultures, o~ vesicular stomatitis virus 5VSV~ for a one hour (37C.) adsorption period, washed, ,~retxea~ed wi~h ~-15 medium oontainin~ 5% fetal calf serum and ,i,then incubated again for 48 hours at 37C. The L-929 cultures were ~hen scored micro~copically for viral cytopathology and ,analyzed, with the plasma interfero~ level, the recipro~al of ~he plasma dilution conferring 50% protection to the L-g29 m~nolayers~l idetermined. ¦.
'~ A second experiment followed the above procedure, except ,that the mice wer~e inje~ted with 10 mg. of the named ~ompound/kg.
'body weight and slamples of peritoneal wash were taken from four ¦.
,.~mice and pooled at six, nine, twelve, fifteen and eîghteen hour~
~l l !l , ! /

V~$~
, I .

a~ter`injection. The ~amples were taken by exposing the peritoneal membrane, injecting 1 ml. o~ Hank's balanced salt solution containing 100 penicillixl units/ml. and 100 ~g.
streptomycin/ml. in~o th~ peritoneal cavity, briefly massaging ~the abdomen, and then aspirating the periton~al wash.
The following data were obtaiined ~rom these two experiments: , I, In~erferon Leve~s (unitsfml.) , Source,of Tlme ~hrs.r a~ter Injection . " Interferon 6 8 9 ~ = 16 18 2~
I. Plasma - 34- 67 - 52 40 ,'Peritoneal ~wash .. 16 - 768 320 448 - 448 - .

j~ EX~MPLES 123 129 . ~n like manner to that described in Example ~22 the ability 15 ,, tD induce circulating in~erferon was determLned for the compounds .
,'listed below.
~ ~" ' . '~., ' '. i ,' ' .
,1 . .
.
" \ ' .
~~
.
., .
,1, . ' .

' ~

- ! I

1, o ~ ~J ;~, 1~ .4 ~ U~ N
,. ~ ~ u)~
U~ ~ ~ ~ It O~
:1 ~ ~_~ u~
.,~ C ~ , I
;! t~ u~

,' ~ ~
_ ~I ~ ~0 ~ ~ ~ C~ll D3 1~ ~
il , a~ ~ , ~Dl ~ 0~ ~ ~ ~ U.
a) ~ ~1 ~
i ~ 'E~ ~ ~v~ r .
. . .
. . . ..
. . .
~, ,, , . .
.' ~ l ~ ~ u~ ~ ~o ,, ! ~
1. _ H ~ t ' r-- O ~ U~ O .
~la s'l ~~ .D ~t ~r ~ O
~U . ~ 1 1~ . ,. ~. ' .
lî ,_ ~ ~ ~ ~ , -! ~ ~ _I ~1 1~ '1 ~ ~ ~1 ~ t ,~
O Vl ~ . .
! -~ ~ ,, a) ~ C~ co o ~, ~ , S ~ , ~r ~ ~ ~I N r~ l r~ ~ m tH E l ~ ~ . .
a t ~
tO h fi ~
,; ~x ~ ~
~ I s.s U ~ ' ' ¦ ~ ~i .t ~ ~ u7 O o D
t . U~ U~ E3 . O X . . O O ~ .
.

.. O
t~
; ' Q) Q) ~ r~ t ' I
H H '~1 ~i r-i . .
Ei R ~ ~ . .
;, ~ F ~ ~ ~ ~ 3`3 ; X :5 ~ 1~ R
. .1~1 Z . ~

~j , _~ I

Enhancement of Polyinosinic-Polycytidylic Acid [Poly (I:C)]-induced Cellular Resistance to V.i.ral Infection by 1~3-Di-0-~n-hexadecyl)-2-0-(3-aminopropyl)-glycerol Hydrochloride

5 ; Growth medium was pxepared by supplemen~ing Eagle's minimum essential medium (100 ml.) wi~h lOOX concentrated antibiotic- l . antim~cstic solution (2 ml.), 200 mM glutamine solutlon ~1 ml.),ald ,j heat-inactivated fetal calf sexum (5%~. Mouse L-9~9 ~ibroblasts ;, were s.uspended in grow~h medium, and each well of 96-well microtiter ¦'plates was seeded wi h 0.2 ml. of said suspension containing 2~,000 ¦, to 30,000 cells. The plates were incubated for 2 to 4 days at 37~
in a 5% C02 atmosphere to establish monolayers o~ cells. ~he , plates were washed four times with phosphate buffered saline im~ediately prior to treatment. . .
i .
', Poly (I:C) was prepared at concentrations of 5.0, 1.0, 0.2 .
and 0.04~g./ml. in the medium described above minus calf serum.
i, 0.1 ml. of each dilution was combined in a checkerboard arrange- ¦
ment on the L-929 cell monolayers wi~h 0.1 ml. dilutions contai~ing . 29.0, 4.0, 0.8, 0.16 and 0~032~g. of the named compouna per ml.
2 0 . said serum-free medium. Control wells were expo~ed to either pol~
'. (I:C) or the named compound alone. The plates were i~cubat d ` for 6 hours at 37C. in a 5% CO2 atmosphere, washed four times ',with phospha~e buffered saline, and reed with 0.~ ml. per weil ,' growth medium containing 2% fetal calf serum. After 18 more :j hours of incubation, the plates were scored for toxicity and then j challenged with 0.1 ml. per well of a vesi~ular stomatitis virus (VSV) suspension containing lO to 30 times the TCID50 (tissue ¦ .
culture infective dose causing a ~0~ infection rate)O The '~'' ' ' i 1., , .~ ~ .

~,i'l '' ~

\

rr~
.;

plates were incubated for ano~her 3 to 4 days and then scored microscopically for cytopathogenic effect (CPE). Cells protected from ~irus in~ection were free of CPE. The minim~n protective dose (ME~D) of poly (I:C) alone wa.~ noted, and the amount of S . enhanced or augmented ankiviral acti~ity caused by comkination with the named compound recorded :Eor 4ach dilutivn level of said . named compound.
~, .
!l~ Enhancement of Pol (~:C1-induced .
1! Cellular Resï ~e to Viral Infection `Concëntrat1on (~./ml.) of Named Compound .'2~.0 4.0 0.8 ~.16 0.~32 .
.,125X 125X 125X 5X. C5X.
, Note: comkining poly(I:C~ with named compound pro~vides same antiviral effect as increasing poly ~I:C) concentration indicated .
number o~ times. .... . .

` In like manner to that described in Example 130 the enhance-ment of poly~I:C)-induced cellular resistance to viral infection .
w.s determ~n Eor the compcunds listed below.

'' I
, . . .
I '~ .
,~ .

L~

j. . I

'I

+1 +1 +1 +1 1 rl +1 1 1 +1 ~ I + + + + ~ t + + + +, , +

11 ~W ~ I + ~ + ~ ~ ~ I t ~
1~1 . . ' ., ', ' .

~ ~ ~ rl ~l _l ~ _I N t~ ~ N

:

E~ t~ ~ ~ ~ ~ ~" ~ ~ ~ ~ ~ ~

' ~

!

+~ 1+11+l+l1+++

Il o C o . . .

C~ J4~ ~ + I + ~ ~ + * t ~ + ~ +

. C '`11 ' \ ' ' ';

0 .1 ! .
II.~D ~ ~ w i c4 o~ O ~ o ~ ' .

1,~ ' ' ';1 ' 1 ' !l ~ æ ~ ri ~
il ' P~ 1i G~

, .

A~¦ +l +l +l l +l ~ l Il ~ .~ q.l + ~ + ~ ~ .

~ 1 + + + + + +

~ co L ;~

A

.~,' ~ D ~ .
1' ~ z " , ,~ I a .

.; 1, ''

Claims (7)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for preparing a compound of the formula I
or II

and the pharmaceutically acceptable acid addition salts thereof, wherein R1 and R2 are each selected from the group consisting of normal alkyl of from 14 to 18 carbon atoms and n-octadec-9-enyl, Y is selected from the group consisting of and wherein the left bond is connected to O; and R3 is selected from the group consisting of hydrogen and alkyl of from 2 to 4 carbon atoms, which comprises the steps of (a) reacting a compound of the formula X

or XI

wherein Y' is selected from the group consisting of and with, when R3 is alkyl, a compound of the formula R3NH2; or with, when R3 is hydrogen, an azide salt, followed by reduction of the resulting azido derivative; and (b) if desired, con-verting the resulting compound of formula I or II to a pharm-aceutically acceptable acid addition salt thereof.

2. A process according to claim 1 wherein R1 and R2 are each normal alkyl of from 14 to 18 carbon atoms.

3. A process according to claim 2 wherein R1 and R2 are each n-hexadecyl.

4. A process according to claim 1 wherein a compound of formula (X) is reacted with said compound of the formula R3NH2 or with said azide salt.

5. A process according to claim 1 wherein a compound of formula (XI) is reacted with said compound of the formula R3NH2 or with said azide salt.

6. A process according to claim 1, 4 or 5 wherein Y' is and said compound of formula (X) or (XI) is reacted with an azide salt followed by reduction of the resulting azido derivative.

7. A compound of the formula (I) or (II) as defined in claim 1, or a pharmaceutically acceptable acid addition salt thereof, when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.