WO2004062595A2 - Antineoplastic ether lipid compounds with modifications at the sn-1 carbon - Google Patents

Abstract

Ether lipid compounds of formula (I): pharmaceutically-acceptable salts, prodrugs or isomers thereof are provided, where the variables are as defined herein. The compounds of the invention have antineoplastic activity, and accordingly have utility in treating cancer and related diseases. Enantiomers of these compounds, pharmaceutical compositions, and methods for treating cancer with the pharmaceutical compositions are also provided.

Description

Antineoplastic Ether Lipid Compounds with Modifications at the sn-1 Carbon

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention provides novel ether lipid compounds with

modifications at the sn-1 carbon, pharmaceutically-acceptable salts, prodrugs or

isomers thereof, as well as pharmaceutical compositions, and methods for treating

cancer.

References

The following publications, patents and patent applications are cited in this

application as superscript numbers:

¹ Berdel, W.E. , Br. J. Cancer. 64:208-211 (1991).

² Lohmeyer, M. and Bittman, R., Drugs of the Future. 19:1021-1037

(1994).

³ Berdel, W.E. and Munder, P.G. in Platelet Activating Factor and

Related Lipid Mediators, (F. Snyder, Ed.), pp. 449-468, Plenum Press,

New York, NY (1987).

⁴ Houlihan, W.J., Lohmeyer, M., Workman, P. and Cheon, S.H. , Med

Res. Rev.. 15:157-223 (1995).

⁵ Principe, P. and Braquet, P., Rev. Oncol. Hematol.. 18: 155-178 (1995). Berdel, W. E., Bausert, W.R.E., Fink, U. , Rastetter, J. and Munder, P.

G., Anticancer Res. 1. 345-352 (1981).

Bittman, R. in Phospholipids Handbook: Chemical Preparation of

Glycerolipids, (Cevc, G., Ed.), Marcel Dekker, Inc. , New York, NY,

pp. 141-232 (1993), and references therein.

Berdel, W.E., Andreesen, R., and Munder, P.G. in Phospholipids and

Cellular Regulation, Vol 2., (Kuo. J_τ Ed).. CRC Press: Boca Raton, FL,

pp. 41-73 (1985).

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Wieder, T., Orfanos, C.E. and Geilen, C.G., J. Biol. Chem., 273:

11025-11031 (1998). Bittman, R. , and Arthur, G. in Liposomes: Rational Design, (A.S.

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Arthur, G. and Bittman, R., Biochim. Biophys. A eta. 1390:85-102

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3583-3592 (1999).

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All of the above publications, patents and patent applications are herein

incorporated by reference in their entirety to the same extent as if each individual

publication, patent or patent application was specifically and individually indicated

to be incorporated by reference in its entirety.

State of the Art

Alkyllysophospholipids (ALPs) and alkylphosphocholines (APCs) represent

subclasses of potential antitumor agents collectively known as antitumor ether

lipids (AELs). They do not interact with cellular DNA and are therefore not

mutagenic.^1"3 The antitumor activities of these compounds, which are based on lysophosphatidylcholine, are now well established; the prototype of the

alkyllysophospholipids (ALPs) , 1 -O-octadecyl-2-O-methyl-glycerophosphocholine

(ET-I8-OCH₃), and other ether-linked phosphocholine analogues are in clinical

trials.²' ^4"7 Compound ET-18-OCH₃, a subclass of alkyl lysophospholipids (ALPs),

is known for its anti-cancer activities against breast (MCF-7), Lewis lung (A549),

ovarian (Ovcar-3) cell lines.²'⁴ Structurally similar, the platelet activating factor

(PAF) differs from ET-18-OCH₃ merely in an ester linkage at the sn-2 position of

the glycerol-backbone. Both PAF and ET-18-OCH₃ are known to inhibit protein

kinase C activity and phosphatidylcholine choline biosynthesis.^{3, 31, 32}

ALPs also appear to inhibit the proliferation of tumor cells without

affecting the growth of normal cells.⁸ While the mechanism of inhibition of cell

proliferation has yet to be resolved, various hypotheses have been proposed. In

some cells, ALPs and APCs appear to induce apoptosis as a consequence of

inhibition of phosphatidylcholine synthesis.^9"11 Other theories for the mechanism

of action include activation of the stress activated protein kinase pathways,^12"13

drug-induced increase in cellular ceramide levels,¹⁴ nutrient starvation, inhibition

of transacylase activity, enhanced lipid peroxidation, inhibition of cellular

signaling pathways^15"16 and/or activation of tumoricidal macrophages.^17"18

Other studies have revealed that ALPs affect the activity of a large number

of signaling molecules including protein kinase C (PKC), phosphatidylinositol 3-

kinase, phosphatidylinositol-specific phospholipase C, and diacylglycerol kinase.¹⁹' ²⁰' ¹⁶ Recently another signaling molecule, Raf-1, was added to the list with the

demonstration that ET-18-OCH₃ decreased the levels of Raf-1 associating with the

cell membrane in growth-factor stimulated MCF-7 cells which consequently led to

decreased activation of MAP kinase,²¹ a crucial enzyme required in initiating cell

proliferation.²² It was suggested that Raf-1 is a primary target of ALPs in cells.

The large number of molecules affected by ALPs has complicated the task of

separating their primary site(s) of action from secondary events.

The finding that the glycerol-based ether lipids possess anti-neoplastic

activities, has led investigators to explore isosteric and isoformic analogs of

ET-I8-OCH₃ especially in areas of synthesis, biological and biophysical

properties.^6"7 Compound ET-18-OCH₃, a subclass of alkyl lysophospholipids

(ALPs), is known for its anti-cancer activities against breast (MCF-7), Lewis lung

(A549), ovarian (Ovcar-3) cell lines.²'⁴ ET-18-OCH₃ formulated in liposomes

(ELL- 12), is currently being evaluated in Phase I clinical trial.^29"30

Despite the progress that has been made in understanding the underlying

mechanisms of antitumor ether lipids, there remains a need to develop novel

compounds and compositions for the treatment of disease. Ideally, the treatment

methods would advantageously be based on ether lipids that are capable of acting

as anti-neoplastic agents. SUMMARY OF THE INVENTION

The invention is directed to the discovery of a class of anti-tumor ether

lipid compounds having anti-neoplastic activity. Preferably, the invention

provides bioactive either lipid compound with modifications at the sn-1 carbon or

pharmaceutically-acceptable salts, prodrugs or isomers thereof. The invention also

relates to pharmaceutical compositions comprising these compounds, and methods

for treating cancer.

In one embodiment, the invention relates to an ether lipid having formula (I), or a pharmaceutically acceptable salt, isomer or prodrug thereof:

Forrnula (I)

R¹ is selected from the group consisting of — CH₂CH₂(OCH₂CH₂)_rnO~CH₃ and -(CH₂)_nCH=CH(CH₂)_pCH₃.

R² is selected from the group consisting of —OR³ and

R >3 a „n--d A r R>4 are each independently selected from the group consisting of C alkyl and H; and n, m and p are each independently an integer from 1 to 10.

Preferably, n, m and p are each independently an integer from 1 to 5, more preferably n, m and p are each independently an integer from 1 to 3.

Preferably, R² is selected from the group consisting of — OMe and

— OSO₂Me, or the group consisting of — OEt and — OSO₂Et.

Preferred compounds of Formula (I) include:

Preferably, the compound of Formula (I) is optically active, more preferably, the compound of Formula (I) is the D enantiomer. In a preferred embodiment, the compounds according to the invention will

not aggregate platelets (i.e. , mimic PAF). The chemical structure of PAF (platelet

aggregation factor) is shown in Figure 1. In an embodiment of the invention, the

antitumor ether lipid compounds will avoid PAF recognition while maintaining or

enhancing activity and selectivity. However, in those cases where a platelet

aggregation response to the antitumor ether lipid compounds is observed, co-

administration with a PAF antagonist may be used to block such a response. In

yet another embodiment of the invention, the D isomer is used in order to avoid a

platelet aggregation response.

In a further embodiment of the invention, the antineoplastic ether lipid

compounds will (1) inhibit growth of tumor cells, and (2) inhibit growth of

normal cell lines as compared to tumor cells. Further, it is also preferred that the

compounds of the invention will not aggregate platelets, will not lyse red blood

cells and have desirable pharmacokinetic properties.

Additionally, the invention relates to pharmaceutical compositions

comprising a pharmaceutically acceptable carrier and a pharmaceutically effective

amount of a compound of formula (I). The pharmaceutical compositions may

comprise (a) a liposome, emulsion or mixed miscelle carrier and (b) a pharmaceutically effective amount of compound of formula (I) or a

pharmaceutically acceptable salt, isomer or prodrug thereof. The invention further relates to a liposome comprising a compound of formula (I) or a pharmaceutically acceptable salt, isomer or prodrug thereof.

These pharmaceutical compositions can be used in methods for treating a

mammal afflicted with a cancer, comprising administering to the mammal a

therapeutically effective amount of the pharmaceutical composition. Typical

dosages range from about 0.1 to about 1000 mg of the compound of formula (I)

per kg of the body weight of the mammal per day.

The type of cancer to be treated may be selected from the group consisting

of, but not limited to: lung cancers, brain cancers, colon cancers, ovarian cancers, breast cancers, leukemias, lymphomas, sarcomas, and carcinomas.

The treatment methods according to the invention may also include

administering to the mammal an additional biologically active agent. Any suitable

biologically active agent may be used in combination with the ether lipids of the

invention. In a preferred embodiment, the additional biologically active agent may

be selected from the group consisting of antineoplastic agents, antimicrobial

agents, and hematopoietic cell growth stimulating agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of l-O-octadecanol-2-O-methyl-OT-

glycero-3-phosphocholine (ET-18-OCH₃) (left), PAF (center) and lyso-PC (right).

PAF differs in structure in that the methoxy (-OCH₃) is replaced with an acetyl (-OCOCH₃) group; i.e. , the ether linkage at sn-2 is replaced with an ester linkage.

For lyso-PC, the sn-1 linkage is an ester and a hydroxyl group resides at the sn-2

position.

FIG. 2 depicts a general scheme for the synthesis of compounds of the

invention, comprising (a) protecting the sn-3 alcohol, (b) ring opening of the

epoxide with an alcohol, (c) derivatizing the sn-2 alcohol group, (d) deprotecting

the sn-3 alcohol group, (e) reacting the sn-3 alcohol with phosphorus oxy chloride,

and (f) reacting the phosphate with a choline salt/pyridine, followed by water to

give a compound of formula (I).

DETAILED DESCRIPTION OF THE INVENTION

As stated above, this invention relates to novel ether lipid compounds,

pharmaceutically-acceptable salts, prodrugs, or isomers thereof, which have utility

as anti-neoplastic agents. In particular, the invention relates to ether lipid

compounds of formula (I), having modifications at the sn-1 carbon. However,

prior to describing this invention in further detail, the following terms will first be

defined. Definitions

The term "alkyl" refers to saturated aliphatic groups including

straight-chain, branched-chain, cyclic groups, and combinations thereof. The

alkyl groups preferably have between 1 to 20 carbon atoms.

The term "alkenyl" refers to unsaturated aliphatic groups including

straight-chain, branched-chain, cyclic groups, and combinations thereof, having at

least one double bond and having the number of carbon atoms specified. The

alkenyl groups preferably have between 1 to 20 carbon atoms.

The term "cyclic alkyl" or "cycloalkyl" refers to alkyl group forming an

aliphatic ring. Preferred cyclic alkyl groups have about 3 carbon atoms.

The term "direct link" as used herein refers to a bond directly linking the

substituents on each side of the direct link.

The ether lipids of the invention have a 3 carbon alcohol, glycerol, as the

backbone. With the 3 carbons of glycerol, positions are designated as

stereospecific numbers, sn, to distinguish location. The designations ".STΪ-I , " "sn-

2," and "sn-3" identify glycerol carbons 1, 2, and 3, respectively. The glycerol

carbons are labeled below for formula (I):

Formula (I)

"Pharmaceutically acceptable salt" refers to pharmaceutically acceptable

salts that are derived from a variety of organic and inorganic counter ions well

known in the art and include, by way of example only, sodium, potassium,

calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the

molecule contains a basic functionality, salts of organic or inorganic acids, such as

hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the

like. Examples of pharmaceutically acceptable acid addition salts includes salts

which retain the biological effectiveness and properties of the free bases and which

are not biologically or otherwise undesirable, formed with inorganic acids such as

hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid

and the like, and organic acids such as acetic acid, propionic acid, glycolic acid,

pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,

tartar ic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,

methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid

and the like. Examples of pharmaceutically acceptable base addition salts include

those salts derived from inorganic bases such as sodium, potassium, lithium,

ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum

bases, and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from pharmaceutically acceptable

organic nontoxic bases include salts of primary, secondary, and tertiary amines,

substituted amines including naturally occurring substituted amines, cyclic amines

and basic ion exchange resins, such as isopropylamine, trimethylamine,

diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol,

trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,

hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine,

theobromine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine resins

and the like. Particularly preferred organic nontoxic bases are isopropylamine,

diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline, and

caffeine.

"Prodrug" means any compound which releases an active parent drug

according to formulas (I) in vivo when such prodrug is administered to a

mammalian subject. Prodrugs of a compound may be prepared by modifying

functional groups present in the compound in such a way that the modifications

may be cleaved in vivo to release the parent compound. Prodrugs include

compounds of formula (I) wherein a hydroxy, amino, or sulfhydryl group is

bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl,

amino, or sulfhydryl group, respectively. Examples of prodrugs include, but are

not limited to esters (e.g. , acetate, formate, and benzoate derivatives), carbamates

(e.g., N,N-dimethylamino-carbonyl), and the like. "Isomers" are compounds that have the same molecular formula but differ

in the nature or sequence of bonding of their atoms or the arrangement of their

atoms in space. Isomers that differ in the arrangement of their atoms in space are

termed "stereoisomers. " Stereoisomers that are not mirror images of one another

are termed "diastereomers" and those that are non-superimposable mirror images

of each other are termed "enantiomers. " An enantiomer can be characterized by

the absolute configuration of its asymmetric center and is described by the R- and

S- sequencing rules of Cahn and Prelog, or by the manner in which the molecule

rotates the plane of polarized light and designated as dextrorotatory or levorotatory

(i.e. , as (+) or (-)-isomers respectively). A chiral compound can exist as either

individual enantiomer or as a mixture thereof. A mixture containing equal

proportions of the enantiomers is called a "racemic mixture".

"Treating" or "treatment" of a disease includes:

(1) preventing the disease, i.e. causing the clinical symptoms of the disease

not to develop in a mammal that may be exposed to or predisposed to the

disease but does not yet experience or display symptoms of the disease,

(2) inhibiting the disease, i.e. , arresting or reducing the development of the

disease or its clinical symptoms, or

(3) relieving the disease, i.e. , causing regression of the disease or its clinical

symptoms. A "therapeutically effective amount" means the amount of a compound

that, when administered to a mammal for treating a disease, is sufficient to effect

such treatment for the disease. The "therapeutically effective amount" will vary

depending on the compound, the disease and its severity and the age, weight, etc. ,

of the mammal to be treated.

A "pharmaceutically acceptable carrier" means an carrier that is useful in

preparing a pharmaceutical composition that is generally safe, non-toxic and

neither biologically nor otherwise undesirable, and includes a pharmaceutically

acceptable excipient that is acceptable for veterinary use or human pharmaceutical

use. A "pharmaceutically acceptable excipient" as used in the specification and

claims includes both one and more than one such excipient. Some examples of

suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches,

gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,

microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup,

and methyl cellulose. The formulations can additionally include: lubricating

agents such as talc, magnesium stearate, and mineral oil; wetting agents;

emulsifying and suspending agents; preserving agents such as methyl- and

propylhydroxy-benzoates; sweetening agents; and flavoring agents. The

compositions of the invention can be formulated so as to provide quick, sustained

or delayed release of the active ingredient after administration to the patient by

employing procedures known in the art. " Cancer" refers to a group of diseases characterized by uncontrolled

growth and spread of abnormal cells, often resulting in the formation of a

non-structured mass or tumor. Illustrative tumors include carcinomas, sarcomas

and melanomas, such as basal cell carcinoma, squamous cell carcinoma,

melanoma, soft tissue sarcoma, solar keratoses, Kaposi's sarcoma, cutaneous

malignant lymphoma, Bowen's disease, Wilm's tumor, hepatomas, colorectals

cancer, brain tumors, mycosis ftmgoides, Hodgkin's lymphoma, polycythemia

vera, chronic granulocytic leukemia, lymphomas, oat cell sarcoma, and the like.

Tumors may also include benign growths such as condylomata acuminata (genital

warts) and moles and common warts.

An "anti-neoplastic agent" is a pharmaceutical which inhibits or causes the

death of cancer or tumor cells.

An "antimicrobial agent" is a substance that either destroys or inhibits the

growth of a microorganism at concentrations tolerated by the infected host.

A "hematopoietic cell growth stimulating agent" is one that stimulates

blood cell growth and development, i.e. of red blood cells, leukocytes, and

platelets. Such agents are well known in the art. For example, in order to

increase infection-fighting white blood cell production, recombinant

granulocyte-colony stimulating factor may be used to stimulate the growth of neutrophils. Another example of a hematopoietic cell growth stimulating agent is

recombinant granulocyte macrophage-colony stimulating factor, which increases production of neutrophils, as well as other infection-fighting white blood cells,

granulocytes and monocytes, and macrophages. Another hematopoietic agent is

recombinant stem cell factor, which regulates and stimulates the bone marrow,

specifically to produce stem cells.

Compound Preparation

The compounds of formula (I) can also be prepared via several divergent

synthetic routes with the particular route selected relative to the ease of compound

preparation, the commercial availability of starting materials, and the like. For

instance, the compounds of formula (I) may be synthesized and tested using the

methods ememplified in the examples and the instant specification. Such methods

may be further adapted to produce analogs, derivatives and variants within the

scope of formula (I).

Figure 2 shows the steps and reagents required for the synthesis of ether

lipid compounds of formula (I) with substitution at sn-2 carbon product in only a

few steps in good yields. The starting material shown in the scheme may be

optically active (R)-glycidol or (S)-glycidol, depending upon which isomer is

desired.

In step (a), the alcohol group is protected with a suitable protecting group.

Suitable protecting groups are described, for example, in Protective Groups in

Organic Synthesis, Third Edition (Peter G. M. Wuts and Theodora Greene, Editors), Wiley, John & Sons, Incorporated (1999), which is hereby incorporated

by reference in its entirety. In a preferred embodiment, the protecting group is a

benzyl group.

In a preferred embodiment, O-Benzyl glycidyl ether is used as the starting

material. O-Benzyl glycidyl ether has been used to design and synthesize a class

of novel ether lipids with (R)- and (S) -configurations. This procedure can also be

applied to prepare the phospholipids in multi gram quantities, providing an access

to scale up a wide variety of glycero-based lipids in efficient manners. This route

provides a significant improvement over the conventional method, which involves

eight steps from L-serine as a precursor, for the preparation of 2-aza

phosphocholines.²⁸ This is a simple and short procedure to synthesize (R)- and (S)

ether lipids with substitution sn-2 carbon, starting from the commercially available

O-benzylglycidyl ether precursor with high percentage enantiomeric excess.

In step (b), the epoxide is subjected to ring-opening conditions with an

alcohol. As shown in the scheme, this reaction may be carried out by reacting a

suitable nucleophile in CH₂C1₂ with

at room temperature until the

reaction is complete. Preferred nucleophiles include alcohols, particularly, HO-CH₂CH₂(OCH₂CH₂)_mO-CH₃, HO-(CH₂)_nCH=CH(CH₂)_pCH₃ (cis double bond) and HO-(CH₂)_nCH=CH(CH₂)_pCH₃ (trans double bond).

In step (c), the alcohol moiety may be converted to a -OR³ or -O-SO₂-R⁴

group. For example, in order to covert the alcohol to a -OR³ group, where R³ is a C_M alkyl, the alcohol may be refluxed with 2,6-di-t-butyl-4-methylpyridine with a

suitable alkyling agent in anhydrous methylene chloride at around 60 °C until the

reaction is complete. Examples of suitable alkylating agents include methyl

trifluoromethanesulfonate (methyl triflate) or ethyl trifluoromethanesulfonate (ethyl

triflate).

The alcohol may alternatively be converted to a -O-SO₂-R⁴ group, where

R⁴ is hydrogen or a C₁ alkyl with a suitable sulfonating agent. Examples of

suitable sulfonating agents include methanesulfonyl chloride (mesyl chloride) or

ethanesulfonyl chloride.

In step (d), the protecting group at the sn-3 position may be removed using

suitable deprotecting conditions. For instance, if the protecting group is a benzyl

group, it may be removed by using H₂ gas and 5 % Pd on carbon until the reaction

is complete.

In step (e), the alcohol at the sn-3 position is reacted with POCl₃ and Et₃N.

The reaction is stirred at 0 °C to room temperature for about 3-4 hours or until the

reaction is complete. Next, choline tosylate/pyridine is added and the reaction

proceeds at room temperature until reaction is complete, followed by quenching

with water and stirring at room temperature for about 1 hour.

If an alcohol group is desired at the sn-2 position, the above synthesis may

be modified by protecting the alcohol at the sn-2 position after step (b). This

protecting group selected should be stable under the reaction conditions of steps (d), (e) and (f). The protecting group of the sn-2 alcohol can then be removed at

the end of the synthesis to yield an alcohol.

The desired product is then isolated and purified using any suitable

technique known in the art. For instance, many of the compounds can be easily

purified by column chromatography.

Pharmaceutical Formulations

When employed as pharmaceuticals, the compounds of formula (I) are

usually administered in the form of pharmaceutical compositions. These

compounds can be administered by a variety of routes including oral, rectal,

transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These

compounds are effective as both injectable and oral compositions. Such

compositions are prepared in a manner well known in the pharmaceutical art and

comprise at least one active compound.

This invention also includes pharmaceutical compositions which contain, as

the active ingredient, one or more of the compounds of formula (I) above

associated with pharmaceutically acceptable carriers. In making the compositions

of this invention, the active ingredient is usually mixed with an excipient, diluted

by an excipient or enclosed within such a carrier which can be in the form of a

capsule, sachet, paper or other container. When the excipient serves as a diluent,

it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of

tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,

solutions, syrups, aerosols (as a solid or in a liquid medium), ointments

containing, for example, up to 10% by weight of the active compound, soft and

hard gelatin capsules, suppositories, sterile injectable solutions, and sterile

packaged powders.

In preparing a formulation, it may be necessary to mill the active

compound to provide the appropriate particle size prior to combining with the

other ingredients. If the active compound is substantially insoluble, it ordinarily is

milled to a particle size of less than 200 mesh. If the active compound is

substantially water soluble, the particle size is normally adjusted by milling to

provide a substantially uniform distribution in the formulation, e.g. about 40

mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,

sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth,

gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,

cellulose, sterile water, syrup, and methyl cellulose. The formulations can

additionally include: lubricating agents such as talc, magnesium stearate, and

mineral oil; wetting agents; emulsifying and suspending agents; preserving agents

such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring

agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to

the patient by employing procedures known in the art.

The compositions are preferably formulated in a unit dosage form, each

dosage containing from about 5 to about 100 mg, more usually about 10 to about

30 mg, of the active ingredient. The term "unit dosage forms" refers to physically

discrete units suitable as unitary dosages for human subjects and other mammals,

each unit containing a predetermined quantity of active material calculated to

produce the desired therapeutic effect, in association with a suitable

pharmaceutical excipient. Preferably, the compound of formula (I) above is

employed at no more than about 20 weight percent of the pharmaceutical

composition, more preferably no more than about 15 weight percent, with the

balance being pharmaceutically inert carrier(s).

The active compound is effective over a wide dosage range and is generally

administered in a pharmaceutically effective amount. It will be understood,

however, that the amount of the compound actually administered will be

determined by a physician, in the light of the relevant circumstances, including the

condition to be treated, the chosen route of administration, the actual compound

administered, the age, weight, and response of the individual patient, the severity

of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal active

ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present

invention. When referring to these preformulation compositions as homogeneous,

it is meant that the active ingredient is dispersed evenly throughout the

composition so that the composition may be readily subdivided into equally

effective unit dosage forms such as tablets, pills and capsules. This solid

preformulation is then subdivided into unit dosage forms of the type described

above containing from, for example, 0.1 to about 500 mg of the active ingredient

of the present invention.

The tablets or pills of the present invention may be coated or otherwise

compounded to provide a dosage form affording the advantage of prolonged

action. For example, the tablet or pill can comprise an inner dosage and an outer

dosage component, the latter being in the form of an envelope over the former.

The two components can be separated by an enteric layer which serves to resist

disintegration in the stomach and permit the inner component to pass intact into the

duodenum or to be delayed in release. A variety of materials can be used for such

enteric layers or coatings, such materials including a number of polymeric acids

and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and

cellulose acetate.

The liquid forms in which the novel compositions of the present invention

may be incorporated for administration orally or by injection include aqueous

solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil,

or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and

suspensions in pharmaceutically acceptable, aqueous or organic solvents, or

mixtures thereof, and powders. The liquid or solid compositions may contain

suitable pharmaceutically acceptable excipients as described supra. Preferably the

compositions are administered by the oral or nasal respiratory route for local or

systemic effect. Compositions in preferably pharmaceutically acceptable solvents

may be nebulized by use of inert gases. Nebulized solutions may be inhaled

directly from the nebulizing device or the nebulizing device may be attached to a

face mask tent, or intermittent positive pressure breathing machine. Solution,

suspension, or powder compositions may be administered, preferably orally or

nasally, from devices which deliver the formulation in an appropriate manner.

The following formulation examples illustrate representative pharmaceutical

compositions of the present invention.

For ulation Example 1

Hard gelatin capsules containing the following ingredients are prepared:

Quantity

Ingredient (mg/capsule)

Active Ingredient 30.0

Starch 305.0

Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in 340

mg quantities. Formulation Example 2

A tablet formula is prepared using the ingredients below:

Quantity

Ingredient (mg/tablet)

Active Ingredient 25.0

Cellulose, microcrystalline 200.0

Colloidal silicon dioxide 10.0

Stearic acid 5.0

The components are blended and compressed to form tablets, each

weighing 240 mg. Formulation Example 3

A dry powder inhaler formulation is prepared containing the following

components:

Ingredient Weight %

Active Ingredient 5

Lactose 95

The active ingredient is mixed with the lactose and the mixture is added to

a dry powder inhaling appliance.

Formulation Example 4

Tablets, each containing 30 mg of active ingredient, are prepared as

follows: Quantity

Ingredient mg/tablef)

Active Ingredient 30.0 mg

Starch 45.0 mg

Microcrystalline cellulose 35.0 mg

Polyvinylpyrrolidone

(as 10% solution in sterile water) 4.0 mg

Sodium carboxymefhyl starch 4.5 mg

Magnesium stearate 0.5 mg

Talc - 1.0 mg

Total 120 mg The active ingredient, starch and cellulose are passed through a No. 20

mesh U.S. sieve and mixed thoroughly. The solution of poly vinylpyrrolidone is

mixed with the resultant powders, which are then passed through a 16 mesh U.S.

sieve. The granules so produced are dried at 50° to 60° C and passed through a 16

mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and

talc, previously passed through a No. 30 mesh U.S. sieve, are then added to the

granules which, after mixing, are compressed on a tablet machine to yield tablets

each weighing 120 mg.

Formulation Example 5

Capsules, each containing 40 mg of medicament are made as follows:

Quantity

Ingredient (mg/capsule)

Active Ingredient 40.0 mg

Starch 109.0 mg

Magnesium stearate 1.0 mg

Total 150.0 mg

The active ingredient, starch, and magnesium stearate are blended, passed

through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 150 mg

quantities. Formulation Example 6

Suppositories, each containing 25 mg of active ingredient are made as

follows:

Ingredient Amount

Active Ingredient 25 mg

Saturated fatty acid glycerides 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve and

suspended in the saturated fatty acid glycerides previously melted using the

minimum heat necessary. The mixture is then poured into a suppository mold of

nominal 2.0 g capacity and allowed to cool.

Formulation Example 7

Suspensions, each containing 50 mg of medicament per 5.0 mL dose are

made as follows:

Ingredient Amount

Active Ingredient 50.0 mg

Xanfhan gum 4.0 mg

Sodium carboxymethyl cellulose (11 %)

Microcrystalline cellulose (89%) 50.0 mg

Sucrose 1.75 g

Sodium benzoate 10.0 mg

Flavor and Color q.v.

Purified water to 5.0 mL

The active ingredient, sucrose and xanthan gum are blended, passed through

a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of the

microcrystalline cellulose and sodium carboxymethyl cellulose in water. The

sodium benzoate, flavor, and color are diluted with some of the water and added

with stirring. Sufficient water is then added to produce the required volume. Formulation Example 8

Quantity

Ingredient (mg/capsule)

Active Ingredient 15.0 mg

Starch 407.0 mg

Magnesium stearate 3.0 mg

Total 425.0 mg

The active ingredient, starch, and magnesium stearate are blended, passed

through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 425.0

mg quantities.

Formulation Example 9

A subcutaneous formulation may be prepared as follows:

Ingredient Quantity

Active Ingredient 5.0 mg

Corn Oil 1.0 mL

Formulation Example 10

A topical formulation may be prepared as follows:

Ingredient Quantity

Active Ingredient 1-10 g

Emulsifying Wax 30 g

Liquid Paraffin 20 g

White Soft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin and

emulsifying wax are incorporated and stirred until dissolved. The active

ingredient is added and stirring is continued until dispersed. The mixture is then

cooled until solid.

Another preferred formulation employed in the methods of the present

invention employs transdermal delivery devices ("patches"). Such transdermal

patches may be used to provide continuous or discontinuous infusion of the

compounds of the present invention in controlled amounts. The construction and

use of transdermal patches for the delivery of pharmaceutical agents is well known

in the art. See, e.g., U.S. Patent 5,023,252, issued June 11, 1991, herein

incorporated by reference in its entirety. Such patches may be constructed for

continuous, pulsatile, or on demand delivery of pharmaceutical agents.

Frequently, it will be desirable or necessary to introduce the pharmaceutical

composition to the brain, either directly or indirectly. Direct techniques usually involve placement of a drug delivery catheter into the host's ventricular system to

bypass the blood-brain barrier. One such implantable delivery system used for the

transport of biological factors to specific anatomical regions of the body is

described in U.S. Patent 5,011,472 which is herein incorporated by reference in its

entirety.

Indirect techniques, which are generally preferred, usually involve

formulating the compositions to provide for drug latentiation by the conversion of

hydrophilic drugs into lipid-soluble drugs. Latentiation is generally achieved

through blocking of the hydroxy, carbonyl, sulfate, and primary amine groups

present on the drug to render the drug more lipid soluble and amenable to

transportation across the blood-brain barrier. Alternatively, the delivery of

hydrophilic drugs may be enhanced by intra-arterial infusion of hypertonic

solutions which can transiently open the blood-brain barrier.

Other suitable formulations for use in the present invention can be found in

Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia,

PA, 17th ed. (1985), which is hereby incorporated by reference in its entirety.

Utility

The compounds and pharmaceutical compositions of the invention are useful

as anti-neoplastic agents, and accordingly, have utility in treating cancer in

mammals including humans. As noted above, the compounds described herein are suitable for use in a

variety of drug delivery systems described above. Additionally, in order to

enhance the in vivo serum half-life of the administered compound, the compounds

may be encapsulated, introduced into the lumen of liposomes, prepared as a

colloid, or other conventional techniques may be employed which provide an

extended serum half-life of the compounds.

The amount of compound administered to the patient will vary depending

upon what is being administered, the purpose of the administration, such as

prophylaxis or therapy, the state of the patient, the manner of administration, and

the like. In therapeutic applications, compositions are administered to a patient

already suffering from cancer in an amount sufficient to at least partially arrest

further onset of the symptoms of the disease and its complications. An amount

adequate to accomplish this is defined as "therapeutically effective dose."

Amounts effective for this use will depend on the judgment of the attending

clinician depending upon factors such as the degree or severity of cancer in the

patient, the age, weight and general condition of the patient, and the like.

Preferably, for use as therapeutics, the compounds described herein are

administered at dosages ranging from about 0.1 to about 500 mg/kg/day.

In prophylactic applications, compositions are administered to a patient at

risk of developing cancer (determined for example by genetic screening or familial

trait) in an amount sufficient to inhibit the onset of symptoms of the disease. An amount adequate to accomplish this is defined as "prophylactically effective dose. "

Amounts effective for this use will depend on the judgment of the attending

clinician depending upon factors such as the age, weight and general condition of

the patient, and the like. Preferably, for use as prophylactics, the compounds

described herein are administered at dosages ranging from about 0.1 to about 500

mg/kg/day.

The compounds of the invention may also be used in combination therapy

with one or more additional biologically active agents. Virtually any suitable

biologically active agent may be administered together with the ether lipids of the

present invention. Such agents include but are not limited to antibacterial agents,

antiviral agents, anti-fungal agents, anti-parasitic agents, tumoricidal agents,

anti-metabolites, polypeptides, peptides, proteins, toxins, enzymes, hormones,

neurotransmitters, glycoproteins, lipoproteins, immunoglobulins,

immunomodulators, vasodilators, dyes, radiolabels, radio-opaque compounds,

fluorescent compounds, receptor binding molecules, anti-inflammatories,

antiglaucomic agents, mydriatic compounds, local anesthetics, narcotics, vitamins,

nucleic acids, polynucleotides, etc. The entrapment of two or more compounds

simultaneously may be especially desirable where such compounds produce

complementary or synergistic effects. In particular, such biologically active agents

include, but are not limited to, antineoplastic agents, antimicrobial agents, and

hematopoietic cell growth stimulating agents. For instance, in a recent study of ET-18-OCH₃ and a liposomal incorporated

ET-18-OCH₃, it was found that apoptosis is triggered by this ether lipid by

induction of caspase activation through the release of cytochrome c in a Bcl-XL -

sensitive manner but independently of the CD95 (APO-1/Fas) ligand/receptor

system.^27"28 CD95 is a surface membrane molecule involved in cell activation and

apoptosis. It is expressed by a variety of hematopoietic cells, such as

CD34+/CD38+ stem cells, myeloid cells and lymphocytes. Accordingly, the

compounds according to the invention particularly when formulated in a liposome,

may be used as an adjunct for the treatment of tumors in combination to

myelosuppressive chemo-therapeutic drugs and/or those that use the

CD95-ligand/receptor system to trigger apoptosis.

As noted above, the compounds administered to a patient are in the form of

pharmaceutical compositions described above. These compositions may be

sterilized by conventional sterilization techniques, or may be sterile filtered. When

aqueous solutions are employed, these may be packaged for use as is, or

lyophilized, the lyophilized preparation being combined with a sterile aqueous

carrier prior to administration. The pH of the compound preparations typically

will be between 3 and 11, more preferably from 5-9 and most preferably from 7

and 8. It will be understood that use of certain of the foregoing excipients,

carriers, or stabilizers will result in the formation of pharmaceutical salts. As mentioned above, in a preferred embodiment, the compounds according

to the invention will not aggregate platelets. With respect to avoiding platelet

aggregation, various structural modifications to PAF have been studied, which

provide guidelines for modifications that can be made to the antineoplastic ether

lipids. For instance, PAF activity requires an ether linkage at the sn-1 position.

Interestingly, unlike PAF, compounds having a sulfonate or sulfamoyl linkage at

the sn-2 position, may not be susceptible to PLA₂ hydrolysis.³³ When the ether

linkage is replaced with an ester linkage, the compound becomes susceptible to

PLA inactivation, and no platelet aggregation is observed. It is thus likely that

such compounds may survive enzymatic hydrolysis conditions in aiding prolong

circulation and perhaps could yield potent and selective anti-neoplastic effects.

Further, the D isomer generally elicits no platelet aggregation. Additionally,

when the acetyl group occupying the sn-2 position in PAF is replaced with another

group, PAF activity may be decreased. In this regard, it was found that although

replacement with propionyl doesn't decrease the activity, for every additional

methylene unit added, the activity drops 10 fold compared to PAF. Additionally,

when the acetyl group occupying the sn-2 position in PAF is replaced with a

hydroxyl group (as in lyso PC shown in Figure 1), there is no PAF activity.

While not wishing to be bound by theory, the lack of PAF activity may be due to

the susceptibility of the hydroxyl group to acylation. Finally, when the choline headgroup in PAF is replaced with another moiety, the platelet aggregation effect

is diminished or non-existent.

The L isomer of ET-18-OCH₃ elicits a platelet aggregation response in dog

whole blood, most likely due to its structural similarity to PAF. This response

likely reflects an inherent promiscuity in the PAF receptor for dogs. This

response can be blocked by PAF antagonists. Although no platelet aggregation

has been observed using human blood from healthy volunteers, an aggregation

event has been noted in platelet rich plasma (PRP) processed from the blood of

healthy individuals, and in the whole blood of a few cancer patients. The

physiochemical changes responsible for this have not yet been defined.

One way to avoid any potential hematological problems with ET-18-OCH₃ or

other ether lipids has been to replace the L isomer with the D isomer, which

doesn't elicit an aggregation response in dog whole blood. Particularly in cases

where the D isomer exhibits roughly the same activity as the L isomer in terms of

cytotoxicity and hemolytic activity, the D isomer is a likely candidate if a

replacement is desired.

In a preferred embodiment of the invention, one or more of these factors are

taken into account in order to produce a compound that does not exhibit a platelet

aggregation effect.

In a further embodiment of the invention, the compounds will also not lyse

red blood cells. If however, the compounds do lyse red blood cells, it is often possible to use a liposome carrier to minimize this effect. For instance, although

ET-18-OCH₃ has exhibited antitumor activity in several animal tumor models,⁸ its

clinical use has been restricted by systemic toxic effects, e.g. hemolysis. In this

regard, a stable liposomal system was developed that would incorporate ET-18-

OCH₃ into the bilayer such that its release (exchange out) would be reduced.

Using -molecular monolayer studies of shape complementarity and formulation

optimization, a lipid system which attained ideal packing between the host lipids

and ET-18-OCH₃, resulting in a minimized hemolytic activity was determined.

This a system, known as ELL-12, is now being evaluated in clinical trials,²³

Using such system, hemolysis has not presented a problem at doses exceeding

those previously tried for the free compound.

In yet another embodiment of the invention, the antineoplastic ether lipids

have desirable pharmacokinetic properties. For instance, it may be desirable to

use a compound that is resistant to "rapid metabolism. " While not wishing to be

limited by theory, lyso PC as shown in Figure 1, is thought to be short lived

because (1) the ester linkage is susceptible to phospholipase cleavage to produce

GPC and (2) lyso PC's free hydroxyl is susceptible to acyltransferases. In

contrast, ET-18-OCH₃ is thought to be resistant to the hydrolysis by

membrane-associated phospholipases Al and A2 (PLAl and PLA2) due to its ether

linkages with the sn-1 C 18 chain and sn-2 methyl group. Further, the choline and phosphocholine moieties are known targets for

phospholipases C and D hydrolysis, which yields alkyl-glycerol and

phosphocholine or phosphatidic acid and choline, respectively. One recent

investigation, has shown that ET-18-OCH₃ at and above its cytotoxic

concentrations did not inhibit phosphocholine-specific phospholipase C and

phospholipase D, suggesting that ET-18-OCH₃ is not their primary target and

could survive in biological membranes.²⁴ However, other studies revealed that

ET-18-OCH₃ hexadecylphosphocholme (HPC) can be metabolized by PL-D, thus

making an argument to replace the choline moiety to avoid PC specific PL-D

hydrolysis.^25"26

Likewise, phosphonocholine ET-18-OCH₃ analogs having a methylene

residue instead of oxygen between the phosphorus and the glycerol moiety, could

significantly help in providing less susceptibility to PL-C Furthermore,

modifying the headgroups with entities bulkier than choline may reduce

susceptibility to choline-specific PL-D as well. This inaccessibility to

phospholipases may allow these compounds to behave as long-acting

anti-neoplastic agents.

In an embodiment of the invention, one or more of these factors are taken

into account to produce novel ether lipid compounds that are stable to potential

phospholipase degradation. Specific embodiments of the invention will now be described through

examples. The following synthetic and biological examples are offered to

illustrate this invention and are not to be construed in any way as limiting the

scope of this invention.

EXAMPLES

In the examples below, the following abbreviations have the following

meanings. If an abbreviation is not defined, it has its generally accepted meaning.

bd = broad doublet

bs = broad singlet

c = concentration

d = doublet

dd = doublet of doublets

ddd = doublet of doublets of doublets

DMF = dimethylformamide

DMSO = dimethyl sulfoxide

g — grams

hept. = heptuplet

J = coupling constant

m = multiplet M molar

max — maximum

mg = milligram

min. = minutes

mL = milliliter

mM = millimolar

mmol = millimole

N = normal

ng = nanogram

nm = nanometers

OD or o.d. = optical density

q = quartet

s = singlet

sept = septuplet

t = triplet

THF = tetrahydrofuran

tic = thin layer chromatography

μL = microliter

The antibodies were obtained from the following vendors: Transduction

Laboratories, Lexington, KY (Raf-1, PKB/AKT); New England Biolabs Inc, Beverly, MA (phospho-MAP kinase and phospho- PKB/AKT); Santa Cruz Inc,

Santa Cruz, CA (ERK-1, ERK-2); fetal bovine serum (FBS) from Hyaclone

(Logan, UT).

Additionally, the term "Aldrich" indicates that the compound or reagent used

in the following procedures is commercially available from Aldrich Chemical

Company, Inc. , 1001 West Saint Paul Avenue, Milwaukee, WI 53233 USA; the

term "Fluka" indicates the compound or reagent is commercially available from

Fluka Chemical Corp., 980 South 2nd Street, Ronkonkoma, NY 11779 USA; the

term "Lancaster" indicates the compound or reagent is commercially available

from Lancaster Synthesis, Inc., P.O. Box 100, Windham, NH 03087 USA; and

the term "Sigma" indicates the compound or reagent is commercially available

from Sigma, P.O. Box 14508, St. Louis, MO 63178 USA.

Unless otherwise stated, all temperatures are in degrees Celsius.

NMR spectra were recorded on an IBM-Bruker 200-MHZ or a Bruker 400-

MHZ Spectrometer with Me₄Si as internal standard. Infrared spectra were

recorded on a Perkin-Elmer 1600 FT spectrophotometer. Optical rotations were

measured on a JASCO Model DIP- 140 digital polarimeter using a 1-dm cell.

Methylene chloride and pyridine were distilled from calcium hydride and barium

oxide, respectively. Chloroform was distilled from P₂O₅. All other synthetic

reagents were used as received unless otherwise stated. In these synthetic methods, the starting materials can contain a chiral center

and, when a racemic starting material is employed, the resulting product is a

mixture of R,S enantiomers. Alternatively, a chiral isomer of the starting material

can be employed and, if the reaction protocol employed does not racemize this

starting material, a chiral product is obtained. Such reaction protocols can involve

inversion of the chiral center during synthesis. Alternatively, chiral products can

be obtained via purification techniques which separates enantiomers from a R, S

mixture to provide for one or the other stereoisomer. Such techniques are well

known in the art.

PART I: PREPARATION OF COMPOUNDS

The compounds of the invention were synthesized with high percentage

enantiomeric excess starting from the commercially available optically active O-

benzylglycidyl ether precursor.

Routes to Synthesis of Ether Lipids

A

CH₃S0₂CI

(OCH₂CH₂)_n)OCH₃ H=CH(CH₂)₇CH₃ (trans) H=CH(CH₂)₇CH₃ (cis)

(R)-O-Benzyl glycidyl ether A was subjected to the oxirane ring opening by

the appropriate alcohol in presence of catalytic amounts of boron triflate etherate

to yield B. Compound B was then reacted with methanesulfonyl chloride in

methylene chloride and pyridine to yield C. Cleavage of the benzyl group of

compound C was carried out by hydrogenolysis on 5 % Pd on carbon under hydrogen gas to yield compound D. Finally, compound D was phosphorylated

with phosphorus oxychloride and friemylamine at ambient temperature followed by

coupling with choline tosylate/pyridine and then hydrolysis to yield compounds 1,

2 and 3.

PART II: METHODS FOR EVALUATION OF COMPOUNDS

The ether lipid compounds according to the invention may be screened by

any acceptable method(s) used in the field. For example, the ether lipid

compounds may be examined with respect to the ability of the new compounds (1)

to aggregate platelets (i.e. , mimic PAF), a specific toxicity to avoid or minimize,

(2) to lyse red blood cells, a non-specific toxicity for which a liposome carrier may

be needed, (3) to inhibit growth of tumor cells as a measure of activity, and (4) to

inhibit growth of normal cell lines as compared to tumor cells, a measure of

selectivity. Representative screening assays are contained in the following

description.

After using these screening assays, candidates deemed suitable for in vivo

testing were then synthesized in large scale quantities and some of this material

was sent to the NCI's Developmental Therapeutics Program for a battery of

growth inhibitory studies against various human tumor cell lines (60 cell lines, 9

different panels). The remainder of the material was used to assess in vivo

efficacy using murine tumor models. Additionally, studies were conducted on the lead candidates to discern

mechanism of action with particular emphasis on these agents apoptotic ability as

measured by caspase 3 activity. The details (materials and methods) for these

various assays are described further in the following discussion.

I. Platelet Aggregation Assay

Platelet aggregation in whole blood is measured using a whole blood

aggregometer from Chronolog Corp. The species most often used is dog, since

this species has consistently shown a strong platelet aggregation response in whole

blood to L-ET-I8-OCH₃. However, any species, including human may be used.

Briefly, whole citrated blood is diluted 1:2 with sterile saline and placed in a

warm chamber with a mini stir bar. An electronic probe, measuring electrical

resistance, is inserted in the sample. The aggregometer is calibrated and the

baseline is observed to detect any spontaneous aggregation. The test sample is

added and allowed to run for at least 6 minutes. If the sample is an agonist the

platelets will start to aggregate and stick to the electronic probe causing resistance

across the electrodes to increase. This resistance, in ohms, is measured 6 minutes

post addition of sample. The test samples are run at 25, 100, 200, 400 and 800

uM and compared to 100 uM L-ET-18-OCH₃. A. Collection of Blood Sample for in vitro Platelet Aggregation Testing

Platelet aggregation was assessed using dog whole blood, a system found to

be highly sensitive as it has been demonstrated that the L-isomer of ET-18-OCH₃

invokes aggregation at relatively modest concentrations (but not the D-isomer).

Venous blood is collected in 4.5 mL Vacutainer tubes containing 0.129 M

sodium citrate using a 21G needle or larger. Blood is immediately mixed by gentle

inversion 15-20 times and kept at room temperature. Dilution ratio is 1 to 9 (3.8%

citrate solutiomblood). One Vacutainer tube (2.0 mL) containing EDTA is also

collected for platelet counts. Complete blood counts (CBC) are measured on the

CDC Technologies Hemavet 1500 to ensure that the test subject's platelets are

within normal range. Any vials of hemolytic blood or blood containing any clots

should be discarded. Platelet aggregation testing must be completed within 3

hours of blood collection. After this time the ability of platelets to aggregate

decreases.

B. Procedure for dilutions

Test samples are provided either as powder or solutions. Cloudy solutions

were warmed to —50° C to dissolve any particles. Dilutions were prepared in PBS

or saline at 40X concentration. (25 uL test sample are added to 1000 uL diluted

blood (1:40)). All compounds were diluted in saline. However, if samples were poorly

soluble, they may be diluted in DMSO. It should be noted that since DMSO itself

may elicit a response, a control for DMSO should also be used.

C Procedure for in vitro Platelet Aggregation Test using CHRONO-LOG

Aggregometer Model 560-CA-

The following protocol was used:

1. Turn on aggregometer, aggrolink, monitor, computer and printer at least

15 minutes before testing to warm aggregometer to 37 °C

2. Double click on "AGGLINK" in Windows.

3. Set stir speed to 1000 for whole blood.

4. Set up small beaker with deionized water to clean impedance probes after

each sample. Set up 2 plastic cuvettes with — ImL of saline to store probes in

warming chambers between tests. Set up 1 small plastic cuvette or test tube with

— 2 mL of saline to clean pipet after addition of test articles.

5. Click on "aggregometer"

Click on "test procedure" and set parameters

Procedure Name (Ether Lipid platelet aggregation test)

Channels = 4

Duration = 6:00 (min: sec)

Reagent (test article or L-EL control) Concentration (25-800 uM)

Stirrer = 1000

Gain = 20/5 (20 ohms = 5 blocks)

Enter OK to exit

6. Click on "aggregometer"

Click on "run test" and set parameters

Enter Subject Information

Last Name = WB 1:2

First Name: Subject

ID#= time

Hospital = N/A

Test Procedure (Edit if necessary)

Enter OK to exit

Make sure aggregometer temperature reads 37 °C before testing.

7. Place 1 mL plastic cuvettes into warming wells.

8. Add 1 disposable siliconized stir bar to each cuvette.

9. Add 500 uL saline to 2 cuvettes

10. Add 500 uL whole titrated blood to the two saline containing cuvettes.

M-1000 positive displacement pipet is recommended to measure blood volume.

11. Warm diluted blood 5 min at 37° C in warming wells. 12. Transfer diluted blood samples (in duplicate) to aggregation chambers

and insert impedance probes. Close doors.

13. Calibrate each chamber (This must be done for each test run). Zero

channels to baseline with zero knob. Hold in calibration button and adjust gain to

50%. Observe steady baseline for 1 minute. Recalibrate if necessary.

14. Open chamber door and add 25 uL test sample to each cuvette

15. Rinse capillary pipet piston with saline after each use.

16. Allow test to run at least 6 minutes.

17. Click "aggregometer" and then click on "stop test"

18. Click "Edit"

19. Click "set start & stop time. " Select channel 1 and hold down both

mouse buttons while moving vertical start line to 3-5 seconds prior to sample

injection (the stop time will automatically move to 6 min after start time).

20. Click done. Repeat step 15 and 16 to select channel 3.

21. Click "Edit".

22. Click "compute slope & amplitude" and then check that both channels

are set for 6 min run. Click OK. The aggregometer automatically calculates the

ohms amplitude.

23. Click "file" and click "Save"

24. Click "File"

25. Click "close" 26. Remove impedance probe and place in beaker of deionized water

27. Gently remove any aggregated platelets from probe and place into warm

saline prior to next test.

28. Discard test sample in biological waste.

29. Print out files Platelet Aggregation.

P. Results

Table 1: Platelet aggregation of New Ether Lipid Analogs.

Compound Relativ e Platelet A ggregation (Ohms) Platelet aggregation

25 μM 100 .M 200 μM 400 μM 800 μM

L-ET-I8-OCH₃ 13 14 12 nd nd + + +

D-ET-I8-OCH₃ 0 0 0 nd nd -

1(R) 0 0 0 0 0 -

KS) 0 0 0 0 0 -

Platelet aggregation in dog whole blood was measured in Ohms. For all experiments, O.lμM PAF and 100 μM ET-18-OCH₃ were included as positive controls.

As shown by the data in Table 1, neither compound 1(R) nor 1(S) exhibited

a platelet aggregation response.

While not wishing to be bound by theory, steric hindrance at the sn-2

position may be the reason why the sulfonate compounds 1(R) and 1(S) do not

evoke a platelet aggregation.

Other testing showed the substitution of phosphorus for nitrogen or the

introduction of a ring system in the headgroup chain did not prevent recognition

of these compounds by the PAF receptor; in both instances the number of

methylenes in the headgroup chain were 2. It was also found that when the number of methylenes in the headgroup was

increased or the positive charge was removed, no aggregation was noted indicating

that those changes sufficiently altered binding to the PAF receptor.

II. In Vitro Hemolytic Activity Assay

The hemolytic activity of the various compounds was compared to ET-18-

OCH₃. A compound that is more hemolytic than ET-18-OCH₃ might require a liposome to minimize such side effects, while a compound that is equal or less hemolytic may not require a liposomal carrier (at least under the conditions established here for this screening study).

A. Hemolysis Assay with Washed Human Red Blood Cells

Venous blood was collected in 10 mL Vacutainer tubes containing EDTA

using a 21G needle or larger. Blood was immediately mixed by gentle inversion

15-20 times and kept at room temperature. The blood was transferred from 1

EDTA tube to a 50 mL conical tissue culture tube and the volume was brought up

to 50 mL with PBS.

The blood was centrifuged for 10 minutes at 1500 RPM. The supernatant

was removed and the blood was resuspended up to 50 mL with PBS. Next the

blood was centrifuged for 10 minutes at 1500 RPM. The supernatant was removed, and 2.0 mL of packed red blood cells were

carefully transferred, using positive displacement pipet, into a fresh conical tissue

culture tube. Next, 48 mL PBS was added to achieve a 4% washed RBC solution.

Next, 25, 50, 100 and 200 uM stock solutions of test sample in phosphate

buffered saline (PBS) as follows:

Stock Solution Test Sample PBS

200 uM 200 uL of 20 mM + 19.8 mL

100 uM 5 mL of 200 uM + 5 mL

50 uM I mL of 100 uM + I mL

25 uM 500 uL of 50 uM + 500 uL

Next, 0.5 mL of 4% washed RBC was added to 0.5 mL test sample dilutions

(in duplicate). The final concentration of test sample was 50% of working stock

solution.

The samples were capped or sealed with Parafilm and the samples were

gently mixed. The blood was incubated at 37 °C in gentle rotating water bath for

30 minutes. The samples were centrifuged for 10 minutes at 1500 RPM. Next,

200 uL of supernatant was transferred to a cuvette and 1 mL deionized water was

added. Absorbance was measured at 550 nm vs. a water blank. Next, H₁₀ and H₅₀

were determined by graphing "Absorbance" vs. "Test Sample Concentration. " B. Hemolysis Assay with Whole Human Blood

Venous blood was collected in 10 mL Vacutainer tubes containing EDTA

using a 21G needle or larger. The blood was immediately mixed by gentle

inversion 15-20 times and kept at room temperature.

Stock solutions of 20 mM test sample in phosphate buffered saline (PBS)

were prepared as follows:

Working Stock Test Sample PBS

20,000 uM 100 uL of 20,000 uM

10,000 uM 100 uL of 20,000 uM + 100 uL

5,000 uM 100 uL of 10,000 uM + 100 uL

2,500 uM 100 uL of 5,000 uM + 100 uL

1,000 uM 100 uL of 2,500 uM + 100 uL

500 uM 100 uL of 1,000 uM + 100 uL

Then 270 uL of whole blood was aliquotted in duplicate mini test tubes using

positive displacement pipette. Next, 30 uL of each working stock solution, in

duplicate, was added to the whole blood. Next, 30 uL of PBS was added for

background control. The samples were capped or sealed with Parafilm and gently

mixed. The blood was incubated at 37° C in gentle rotating water bath for 30

minutes. Final concentration of test sample was 10% of working stock solution. Total hemolysis samples of 1 % and 10% whole blood samples in deionized

water were prepared as follows:

1: 100= 10 uL whole blood + 990 uL deionized water

1: 10= 100 uL whole blood + 900 uL deionized water

The samples were freeze thawed 3X in liquid nitrogen then water bath. The

samples were then centrifuged 10 minutes at 1500 RPM. Next, 100 uL of

supernatant was transferred to a cuvette and 1 mL deionized water was added.

The absorbance was read at 550 nm vs. water blank.

H_j0 and H₅₀ were calculated by graphing Percent Total Hemolysis vs. Test

Sample Concentration. The Percent Total Hemolysis = (average o.d of test

sample)/(average o.d. of total hemolysis sample) X 100

Hemolytic activities for L-ET-18-OCH₃ and the corresponding liposomal

formulation, L-ELL-12 are shown in Table 2. As shown by the data in Table 2,

inclusion of compounds in liposomes dramatically decreased hemolytic activity.

Table 2. Hemolytic Activity

Washed Human RBCs Whole Human Blood H,„ (βM) H_m (μM. H,_n (μ ) H^ faM)

Free Compound

L-ET-I8-OCH₃ 11, 10.5 17, 15 600, 550, 700 2000, > 2000

Liposome Systems L-ELL-12 350 >2000 > 2000 > >2000

H₁₀ and H₅₀ values are the concentrations at which the ether lipids produce 10% or 50% hemolysis, respectively. D-ET-18-OCH₃ historically produced the same values as the L isomer and is not shown here. Some experiments were repeated thus the additional entries. For the new liposome formulations, all liposomes were extruded to approximately 100 nm in mean diameter. Choi = cholesterol; DOPC = dioleoylphosphatidylcholine; DOPE-GA = dioleoylphosphatidylemanolarnine- glutaric acid (glutaric acid is covalently attached via the headgroup nitrogen ); CHS = cholesteryl- hernmisuccinate.

TTI. In Vitro Growth Inhibition (GT_r0) Assay

A. Cell line maintenance

The following cell lines were selected from the cell bank for screening and

GI₅₀ studies: MCF-7: human breast tumor, MCF-7/ADR: MCF-7 adriamycin

resistant subline, HT-29: human colon carcinoma, A-549: human non-small cell

lung cancer, NIH-3T3: mouse swiss embryo fibroblast and WI-38: human lung

fibroblast, SKMEL-28: human melanoma, Lewis Lung: mouse lung carcinoma,

DU-145: prostate carcinoma, B16F10: mouse melanoma, L1210: murine

lymphocytic leukemia, P-388: murine leukemia, U-937: human histolytic

lymphoma. Except HT-29, WI-38, NIH-3T3 and Lewis Lung which were

obtained from the American Type Culture Collection (Rockville, MD) all the other

cell lines were obtained from National Cancer Institute - Frederick Research

Facility (Frederick , MD). All the cell lines were grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS) except WI-38 and DU-145 which were

grown in EMEM + 10% FBS at 37°C, 5% CO₂ and 100% humidity and NIH-

3T3, Lewis Lung, B16F10 and L1210 which were grown in DMEM containing

10% FBS (10% HS for L1210). All the adherent cell lines were detached from the

culture flasks by addition of 2-3 ml of 0,05% trypsin-EDTA. Thereafter, trypsin

was inactivated by addition of lOmL of 10% serum-containing RPMI-1640

medium. Cells were separated into a single-cell suspension by gentle pipetting

action. Depending on the cell type, 3,000 to 10,000 cells were plated onto 96-well

plates a day prior to the drug treatment, in a volume of 100 μl per well.

B. Drug Treatment

The test compounds were dissolved in PBS or saline at a stock concentration

of approximately 20 mM, which is 400 times the desired final maximum test

concentration. The stock solutions were then diluted with complete medium to

twice the desired final concentration. A 100 μl aliquot of each dilution was then

added to the designated wells. After 3 days of incubation, cell growth was

determined.

C Sulforhodamine B (SRB) assay

The SRB assay was performed with minor modifications to the method

described by Monks, A., Scudiero, D., Skehan, P., Shoemaker, R., Paull, K., Vistica, D.,Hose, C, Langley ., Cronise,P., and Vaigro-Wolff, A., "Feasibility

of a high-flux anticancer drug screen using a diverse panel of cultured human

tumor cell lines, " J Natl Cancer Inst. 83: 757-766 (1991), which is hereby

incorporated by reference in its entirety. Following drug treatment, cells were

fixed with 50μl of cold 50% (wt/vol) trichloroacetic acid (TCA) for 60 minutes at

4°C. The supernatant was discarded, and the plates were washed six times with

deionized water and then air dried. The precipitate was stained with 100/χl SRB

solution (0.4% wt/vol in 1 % acetic acid) for 10 minutes at room temperature, and

free SRB was removed by washing three times with 1 % acetic acid, and the plates

were then air dried. Bound SRB was solubilized with Tris buffer (lOmM), and the

ODs were read using an automated plate reader (Bio-Rad, Model 3550-UV) at

490nm. Background values were subtracted from the test data, and the data was

calculated as a % of control. The GI₅₀ represents the concentration of test agent

resulting in 50% of net growth compared to that of the untreated samples. In this

assay, ODs were also taken at time 0 ( the time the drugs were added ) If the ODs

of the tested samples were less than that of time 0, cell death had occurred.

Percentage growth was calculated by the method described by Peters, A.C,

Ahmad, I., Janoff, A.S., Pushkareva, M. Y., and Mayhew, E. "Growth Inhibitory

effects of liposome-associated l-o-octadecyl-2-o-methyl-sn-glycero-3-

phosphocholine, " Lipids. 32:1045-1054 (1997). The raw optical density data was

imported into an Excel spreadsheet to determine dose responses. Percentage growth was calculated as follows: (T-T₀)/(C- T₀)x 100 where (T)=mean optical

density of treated wells at a given drug concentration, (T₀)=mean optical density

of time zero wells, and (C)=mean optical density of control wells, or if T < T₀

where cell killing has occurred, then percent death can be calculated as follows:

(T- T₀)/ (T₀) x 100. By varying drag concentration, dose response curves were

generated and the GI₅₀ values were calculated. The GI₅₀ values for each experiment

were calculated using data obtained from three duplicate wells on two separate

plates. The mean GI₅₀'s from each independent experiment.

D. Cell Growth Assay

To determine the growth inhibition in the suspension cell lines, cell numbers

were directly counted instead of using the SRB assay which determines the total

cell protein. One day prior to the drug treatment, 40,000 cells per well were

seeded into 24-well plates in a volume of 0.5mL. Stock solutions were diluted

with complete medium to twice the desired final concentrations, and then 0.5mL

aliquots of each dilution were added to the designated wells. After 3 days

incubation, cell growth was determined by counting cell number using a coulter

counter (Z-M, coulter). Cell counts were also taken at time 0 and subtracted from

the test results to give net growth. The GI-50 represents the concentration of test

agent resulting in 50 % of net growth compared to that of the untreated control

samples. E. Results

For growth inhibitory evaluation, five human tumor cell lines were used

(U937; HT29; A549; MCF7; MCF7/ADR) and two normal fibroblast cell lines

(NIH-3T3, murine; WI-38, human). For comparison, the activity of free L-ET-

18-OCH₃ and D-ET-18-OCH₃ was examined.

Table 3: Growth Inliibition of Tumor/Normal Cells

As shown in Table 3, both L and D isomers of ET-18-OCH₃ gave essentially identical results with the order of sensitivity for the cells lines being U937>HT29>A549>MCF7>MCF7/ADR, H-3T3 (normal cell line). WI-38 cells were moderately sensitive to both ether lipids with GI50 values of 10-13 μM, which was 3-4 times lower than that for the NIH-3T3 cells at 41-47 μM.

TV. Measurement of DEVDase Activity

In a recent study, it was found that ELL- 12 triggers apoptosis by induction of caspase activation through the release of cytochrome c in a Bcl-xL - sensitive manner but independently of the CD95 (APO- 1/Fas) ligand/receptor system.²⁷ To determine whether the new ether lipids produce their growth inhibitory effects via the same apoptotic mechanism as ELL-12, and to correlate these results with activity, DEVDase activity, which is a specific correlate of Caspase 3 activity, was examined.

Suspension cells were seeded at density 3.2xl0⁵ cells per mL in RPMI-1640 medium (Bio-Whittaker) supplemented with 10% heat inactivated FBS (Bio- Whittaker). Cells were pre-incubated overnight prior to treatment with the ether lipid compounds of the invention. At treatment time cell density was 5xl0⁵ cells per mL. Cells were incubated with the ether lipid compounds of the invention for various periods of time, usually 6 hours. Cells were collected, washed with IxDPBS and resuspended in 110 μl of Buffer A (10 mM HEPES-KOH, pH 7.4, 2 mM EDTA, 0.1 % CHAPS, 5 mM DTT, 1:100 dilution of protease inhibitors cocktail (Sigma)-DTT and protease inhibitors should be added just before use). After 10 min incubation on ice in order to lyse cells, samples were frozen on dry ice/ethanol and kept at -20°C until analysis.

At the time of analysis of DEVDase activity frozen pellets were kept on ice until thawed and after vigorous vortexing samples were centrifuged at 14,000 rpm for 6 min. Supernatant was transferred into another tube and 2 μl, in triplicates, were used for protein measurement using Bradford reagent (Bio-Rad).

Measurement of DEVDase activity was carried out in 100 μl volume, where 10 μg of protein was delivered in 50 μl of Buffer A and 40 μM of substrate Ac- DEVD-AMC was also delivered in 50 μl of Buffer A. All measurements were done in triplicates. Reaction was carried out for 1 hour and generation of fluorescent product of reaction (aminomethylcoumarin) was measured by reading fluorescence at l_em 460 nm (l_ex 360 nm). Changes in DEVDase activity were calculated after subtraction of background fluorescence of substrate incubated without proteins, and were expressed as percent of control (DEVDase activity in untreated cells). Average of DEVDase activity, calculated from few independent experiments, was used to calculate percent of L-ether lipid-induced DEVDase activity.

First, various conditions to compare free ET-18-OCH₃ and ELL-12 were examined. As shown in Figure 4, there is indeed a difference between liposomal compound and free drug. This difference is likely due to availability and, consistent with this notion, subsequent experiments showed that longer incubation times caused the differences to diminish (data not shown). However, the 6 hour time frame made for convenient testing and was used for comparative studies.

Table 4. DEVDase Activity of NELs in Jurkat T Cells at 6 Hours

% L-ET180CH3 (DEVDase, 6hrs) Tether lipidl. μM

Lipid 1Q 2Q 2Q N

L-ET-I8-OCH₃ 100 ± 8.8 100 ± 10.4 100 ± 9.3 7

D-ET-I8-OCH₃ 79.3 + 9.7 89.2 ± 12.2 92.1 ± 11.8 4

L-ELL-12 66.1 ± 8.1 78.5 + 10.8 71.3 ± 9.4 4

D-ELL-12 56.5 ± 6.8 78.2 ± 10.5 85.5 + 10.4 4

N is the number of replicate experiments. V. In Vivo Toxicity and Therapeutic Methods

A. Toxicity

Intravenous (xl) or Oral (xl)

CDF1 mice (3 /group) were administered a single intravenous or oral dose of the ether lipid compounds to be tested. Mortality was recorded daily and body weights were recorded at least twice weekly for an observation period of 30 days.

B. Therapeutic

B 1 /F10 Murine Melanoma (iv, / iv.)

Female C57/BL6 mice (5/group) were inoculated iv. with 5 x 10⁴ cells in 0.2 L PBS (day 0). On days 10, 12, 14, 16, & 18 post-tumor inoculation, mice were treated iv. with the ether lipid compounds to be tested, along with ELL- 12 (L), D- EL, L-EL or Control (0.9% NaCl). Mice were sacrificed by carbon dioxide inhalation on day 22, lungs were excised, inflated and fixed with 10% Formalin. Lungs were counted "blind" for tumor nodules using a magnifier. The mean number of nodules per treatment group was determined. P388 Murine Leukemia (ipJ iv.)

Female CDF1 mice (7-8/group) were inoculated ip. with 1 x 10⁵ P388 cells in 0.5 mL PBS (day 0). Treatments were administered iv. on days 1, 3, 5, 7, & 9 post inoculation with with the ether lipid compounds to be tested, along with ELL- 12 (L), L-EL, or Control. Mice were checked daily for mortality and the percent of survival was determined.

P388 Murine Leukemia (ip./ ip.)

Female CDF1 mice (4-8/group) were inoculated ip. with 1 x 10⁵ P388 cells in 0.5 mL PBS (day 0). Treatments were administered ip. on days 1 - 10 post inoculation with with the ether lipid compounds to be tested, along with ELL- 12 (L), or Control or on days 1 - 8 post inoculation withNEL-AlS, -A3S, -A3R, -A5S, - A7R, -A9S, -A21S, -A26R, D-EL, L-EL or Control (NaCl). Mice were checked daily for mortality and the percent survival was determined.

LI 210 Murine Leukemia (ip./ iv.)

Female DBA/2 mice (3-5/group) were inoculated ip. with 1 x 10⁵ cells in 0.5 mL PBS (day 0). Treatments were administered iv. on days 1, 3, 5, 7, & 9 post inoculation with with the ether lipid compounds to be tested, along with D-EL, L-EL or Control (NaCl). Mice were checked daily for mortality and the percent survival was determined. DTT145 Human Prostate (so. / iv.)

Male SOD mice (5/group) were inoculated sc. with 2 x 10⁶ cells in O.lmL PBS (day 0) and the tumors were allowed to reach a volume of -250 mm³ at the start of treatment. Treatments were administered iv. on days 27, 28, 29, 30, & 31 with with the ether lipid compounds to be tested, along with ELL- 12 (D), ELL- 12 (L), L- EL, or Control (NaCl). Tumors were measured with calipers and tumor volume

(mm³) was calculated as (Length x (Width)² x p).

MX-1 Human Mammary (sc. / iv.)

Female SCJD mice (5/group) were inoculated sc. with 10 mg/O.lmL tumor mince (day 0), and the tumors were allowed to reach a volume of ~200mm³ at the start of treatment. Treatments were administered iv. on days 13, 15, 17, 19, & 21 with the ether lipid compounds to be tested, along with ELL- 12 (D), ELL- 12 (L), L- EL, or Control (NaCl). Tumors were measured with calipers and tumor volume

(mm³) was calculated as (Length x (Width)² x p).

The invention has been described with reference to specific embodiments. Substitutions, omissions, additions and deletions may be made without departing from the spirit and scope of the invention defined in the appended claims. From the foregoing description, various modifications and changes in the composition and method will occur to those skilled in the art. All such modifications coming within the scope of the appended claims are intended to be included therein. All of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

The claimed invention is:

1. An ether lipid having formula (I), or a pharmaceutically acceptable salt, isomer or prodrug thereof:

Formula (I) wherein:

R¹ is selected from the group consisting of ~CH₂CH₂(OCH₂CH₂)_mO—CH₃ and --(CH₂)_nCH=CH(CH₂)_pCH₃;

R² is selected from the group consisting of —OR³ and

R³ and R⁴ are each independently selected from the group consisting of C ₄ alkyl and H; and

n, m and p are each independently an integer from 1 to 10.

2. An ether lipid of Claim 1 , wherein n, m and p are each independently an integer from 1 to 5.

3. An ether lipid of Claim 2, wherein n, m and p are each independently an integer from 1 to 3.

4. An ether lipid of Claim 1 , wherein:

R² is selected from the group consisting of — OMe and — OSO₂Me.

5. An ether lipid of Claim 4, wherein R² is — OSO₂Me.

6. An ether lipid of Claim 4, wherein R² is — OMe.

7. An ether lipid of Claim 1, wherein:

R² is selected from the group consisting of — OEt and — OSO₂Et.

8. An ether lipid of Claim 1 , wherein the ether lipid is selected from the group consisting of:

C^

9. An ether lipid of Claim 8, wherein the ether lipid is:

10. An ether lipid of Claim 8, wherein the ether lipid is:

11. An ether lipid of Claim 8, wherein the ether lipid is:

12. An ether lipid of Claim 8, wherein the ether lipid is optically active.

13. An ether lipid of Claim 9, wherein the ether lipid is the D enantiomer.

14. An ether lipid of Claim 10, wherein the ether lipid is the D enantiomer.

15. An ether lipid of Claim 11 , wherein the ether lipid is the D enantiomer.

16. A pharmaceutical composition comprising a pharmaceutically effective amount of an ether lipid of Claim 1 or a pharmaceutically acceptable salt, isomer or prodrug thereof, and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition comprising:

(a) a liposome, emulsion or mixed miscelle carrier and

(b) a pharmaceutically effective amount of an ether lipid of Claim 1 or a pharmaceutically acceptable salt, isomer or prodrug thereof.

18. A liposome comprising an ether lipid of Claim 1 or a pharmaceutically acceptable salt, isomer or prodrug thereof.

19. A method of treating a mammal afflicted with a cancer which comprises administering to the mammal a therapeutically effective amount of the pharmaceutical composition of Claim 16 comprising from about 0.1 mg of the ether lipid per kg of the body weight of the mammal to about 1000 mg per

kg-

20. A method of Claim 19, wherein the cancer is selected from the group consisting of lung cancers, brain cancers, colon cancers, ovarian cancers, breast cancers, leukemias, lymphomas, sarcomas and carcinomas.

21. The method of Claim 19, comprising administering to the mammal an additional biologically active agent.

2. The method of Claim 21, wherein the additional biologically active agent is selected from the group consisting of antineoplastic agents, antimicrobial agents, and hematopoietic cell growth stimulating agents.