WO2004062596A2 - Antineoplastic ether lipid compounds having a four carbon backbone - Google Patents

Abstract

Ether lipid compounds of formula (I), pharmaceutically-acceptable salts, prodrugs or isomers thereof are provided, where 5 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 having a Four Carbon Backbone

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention provides novel ether lipid compounds having a four

carbon backbone, 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:

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

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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.

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Bittman, R. in Phospholipids Handbook: Chemical Preparation of

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

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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) , l-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 protem 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 ether lipid compounds having a four carbon backbone 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:

Formula (I)

R¹ is selected from the group consisting of alkyl, alkenyl and alkynyl. R² is selected from the group consisting of:

/ \

— N 0 -O-S— R5 > - OR. -OR⁴ and H.

\ /

0

X¹ is selected from the group consisting of

O O

I I I I

— P — an d P O-

I _ I _

O O X² is selected from the group consisting of:

— CH₂(CH₂)_mNO₂, -CH₂(CH₂)_mNT(R⁴)₃, -CH₂(CH₂)_mC(R⁴)₃, and — CH₂(CH₂)_mP⁺(R⁴)₃.

R⁴ is selected from the group consisting of C,_.3 alkyl and H. R⁵ is selected from the group consisting of C,_.3 alkyl. m is 0, or an integer from 1 to 10.

When R¹ is — C_IgH₃₇ and R² is — OMe, then X² is not R— CH₂(CH₂)_mN⁺(R⁴)₃ Preferably, R¹ is represented by Y Y², wherein: Y¹ is (CH₂)n₁(CH=CH)n₂(CH₂)n₃(CH=CH)n₄(CH₂)n₅(CH=CH)n₆(CH₂)n₇

(CH=CH)n₈(CH₂)n₉, the sum of n_!+2n₂+n₃+2n₄ is equal to n₅+2n₆+n₇+2n_s+n₉ is an integer of from 3 to 23, n_t is zero or an interger of from 1 to 22, n₃ is zero or an integer of from 1 to 19, n₅ is zero or an integer of from 1 to 16, n₇ is zero or an integer of from zero to 16, n₉ is zero or an integer of from 1 to 10, and each of n₂, n₄, n₆ and n₈ is independently zero or 1; and

Y₂ is — CH₃ or— CO₂H. Preferably, R¹ is selected from the group consisting of — Cι₈H₃₇ and — C₁₆H₃₃. More preferably, R¹ is — C₁₈H₃₇. Preferably, R³ is hydrogen. Preferably, R² is — OMe or selected from the group consisting of:

O

—N O , — O-S— _R5 and -N(R⁴)₂. ^ ' O

More preferably, R² is — OSO₂Me.

Preferably, X¹ is

Preferably, X² is — CH₂(CH₂)_mN⁺(CH₃)₃, more preferably where m is an

integer from 1-7.

PPrreeffeerraabbllyy,, X X² iiss „ .._CCHH₂J(CCHH₂)-—N N⁺⁺ > while R is CH₃.

Preferably, X² is: — CH₂(CH₂)C(CH₃)₃, -CH₂(CH₂)P⁺(CH₃)₃₅

H₃C -CH₂(CH₂)-N^| ' (more preferably while R⁴ is CH₃),

(more preferably while R⁴ is CH₃), or

-CH₂CH₂NO₂.

Preferably, X¹ is while X² is

-CH₂(CH₂)_mN⁺(CH₃)₃, most preferably while m is an integer from 1-7 and/or R² is -OMe.

Preferably, R² is selected from:

O

/ \

— N O —O-S— _R5 and ~N(R⁴)₂,

\ / II O

more preferably, while R⁴ is CH₃ and R⁵ is CH₃. Preferred ether lipids according to Formula (I) include:

CH₃

) :

and

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 ofthe 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 ofthe 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 ofthe 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-mefhyl--.tz-

glycero-3-phosρhocholine (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 scheme for the synthesis of compounds of the invention.

FIG. 3 depicts growth inhibitory effects of new ether lipids against normal human (WI-38) and murine (NIH-3T3) fibroblast cell lines and the human colon tumor cell line HT29. L-ET-18-OCH₃ and D-ET-18-OCH₃ are shown for comparison. The values used are the larger ofthe numbers when repeat experiments were performed.

FIG. 4A-4I depicts GI₅₀ values for compounds sent for testing at NCI's Drug Discovery Program for screening against numerous human tumor cell lines (renal, ovarian, colon, CNS, non-small cell lung, leukemia, breast, melanoma and prostate.)

FIG. 5 depicts the in vivo efficacy of new ether lipids against B16/F10

melanoma in mice. D-EL and L-EL are the D and L isomers of ET-18-OCH₃.

DETAILED DESCRIPTION OF THE INVENTION

As 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 compounds of

formula (I), having a four carbon backbone. 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 "sn-l, " "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, gly colic acid,

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

tartaric 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, piper izine, piper idine, 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 rales 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 fungoides, 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).

A general route to the synthesis of the compounds of formula (I) is shown

in Figure 2. As shown in Figure 2, to a stirred solution of the alcohol, e.g. 1-

octadecanol and (S)-4-bromo-l,2-epoxybutane (I) in anhydrous CH₂C1₂, was added

BF₃ etherate. Other alcohols may be used in place of 1 -octadecanol, depending

upon what substitution is desired at the sn-l position. The reaction mixture was

stirred for about 18 hr or until the reaction was complete under nitrogen

atmosphere. After the reaction is complete, the solvent is removed under reduced

pressure, the resulting white crude solid is purified via column chromatography or

other suitable means to yield the alcohol (II).

To the alcohol (II) in anhydrous CH₂C1₂, was added 2,6-ditertbutyl-4-

methyl-pyridine. After 5 min of stirring, methyl triflate was added and the reaction

was continued at reflux at 40° C for 16 hr. Reaction was monitored by TLC

After the reaction was complete, the solvent was removed under reduced pressure.

The resulting residue is taken up in ethyl acetate and washed with 2M HCl (2 x

100 ml), 5% NaHCO₃, and dried over Na₂SO₄. Solvent filtration and removal under reduced pressure resulted in a white solid, which is purified by column

chromatography or any other suitable technique to yield methyl ether (III).

It should be noted that the alcohol of compound (II) may be further

derivatized using any suitable technique to provide compounds of formula (I) with

various substituents at the sn-2 position. Alternatively, the alcohol group may be

converted into a leaving group, and subsequently reacted with various nucleophiles

for different substitution at the sn-2 position.

A solution of compound (III) and excess of tris(trimethylsilyl) phosphite is

heated at 125° C for 24 hr to yield IV. The unreacted phosphite and

bromotrimethylsilane are removed, e.g. via vacuum distillation at ≤ 90° C. After

the distillation, the crude bis-silylphosphite IV was cooled to room temperature

and was subjected to hydrolysis in THF:H₂O (9: 1) at room temperature for about

12 hr or until the reaction is complete to yield V. The crude waxy white product

was dried and used in the next step without further purification.

Compound V may then be derivatized with a variety of headgroups to yield

compounds of formula (I). For instance, in a typical procedure,

trichloroacetomtrile, and 2- trimethylammoethanol tosylate are added to compound

V in anhydrous pyridine.

The solution is heated to about 50° C for 48 hrs under N₂ atmosphere or

until the reaction is complete. Solvent is then removed under reduced pressure. The product may be isolated and purified using standard techniques known in the

art.

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.

Formulation 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/tablet)

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 carboxymethyl 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 mixmre 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

Xanthan 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 Quantitv

Active Ingredient MO 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 une 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, 17fh 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, tumor icidal 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-l 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 mmor 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 mimmized 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-l 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 alk l-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 hexadecylphosphocholine (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

DMF = dimethylformamidf

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 = optical density

q = quartet

s = singlet

sept = septuplet

t = triplet

THF = tetrahydrofuran

tic = thin layer chron

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, Ronkohkoma, 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 Per kin-Elmer 1600 FT spectrophotometer. Optical rotations were

measured on a IASCO 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

Routes to the Synthesis of the Ether Lipids

1 -O-Octadecyl-2-hydroxy-4-bromobutane (II)

To a stirred solution of 1-octadecanol (36.0 g, 0.133 mol) and (S)-4-bromo-l,2-

epoxybutane (I) (20.0 g , 0.133 mol) in 500 ml anhydrous CH₂C1₂, was added BF₃

etherate (5ml, 0.005 mol), and the reaction mixture was stirred for 18 hr under

nitrogen atmosphere. After 18 hr, the solvent was removed under reduced pressure,

the resulting white crude solid was purified via column chromatography by elution

with 4% EtOAc in hexane to give white solid; 28 g in 50% yield; I 0.5

(hexane:ethyl acetate, 9: 1); Η NMR (300 MHz, CDC1₃) δ (ppm) 4.0-3.9 (m, IH), 3.6-3.3 (m, 6H), 2.1-1.8 (m, 2H), 1.6-1.9 (br s, IH), 1.6-1.5(m, 2H), 1.4-1.2 (br

s, 30H), 0.9-0.8 (t, 3H) [α]²⁵ _D = - 9.7° (c 6.5, CHC1₃).

l-O-Octadecyl-2-O-methyl-4-bromobutane (III)

To l-O-Octadecyl-2-hydroxy-4-bromobutane (II) (2.0 g, 4.75 mmol) in 40 ml of

anhydrous CH₂C1₂, was added 2,6-ditertbutyl-4-methyl-pyridine (4.9 g, 24 mmol).

After 5 min of stirring, methyl triflate (3 ml, 26.6 mmol) was added and the reaction

was continued at reflux at 40° C for 16 hr. TLC was checked and solvent was

removed under reduced pressure. The resulting residue was taken up in ethyl acetate

and washed with 2M HCl (2 x 100 ml), 5 % NaHCO₃, and dried over Na^. Solvent

filtration and removal under reduced pressure resulted in a white solid, which was

purified on a silica gel column by elution with 2% EtOAc in hexane, yielding 1.4 g

(68%) as flaky powder; IL 0.9 (hexane:ethyl acetate, 15:85); ^lR NMR (300 MHz,

CDC1₃) δ (ppm) 3.6-3.4 (m, 10H), 2.2-2.0 (m, 2H), 1.7-1.6 (m, 2H), 1.4-1.2 (br s,

30H), 0.9-0.8 (t, 3H); [ ] ²⁵ _D + 7.15° (c 2.0, CHC1₃).

l-O-Octadecyl-2-O-methyl-butyl-4-phosphonic acid (V)

A solution of compound (III) (1 g, 2.3 mmol) and excess of tris(trimethylsilyl)

phosphite (5 ml) was heated at 125° C for 24 hr to yield IV. The unreacted phosphite

and bromotrimethylsilane were removed via vacuum distillation at ≤ 90° C. After the

distillation, the crude bis-silylphosphite IV was cooled to room temperature and was subjected to hydrolysis in THF:H₂O (20 ml, 9:1) at room temperature for 12 hr to

yield V. The crude waxy white product was dried and used in the next step without

further purification; I 0.7 (chloroform:methanol:water, 60:40:5); ^!H NMR (300

MHz, CDC1₃) δ 3.5-3.4 (m, 8H), 2.0-1.7 (m, 4H), 1.6-1.5 (m, 2H), 1.4-1.2 (m,

30H), 0.9 (t, 3H); [ ] ²⁵ _D = -2.4° (c 0.575, CHC1₃); [α]²⁵ _D = -6.81° (c 0.74,

CHCl₃:MeOH, 1: 1).

2'-Trimethylaminoethyl-l-O-octadecyl-2-O-methylbutane-4-phosphonate (1)

To a solution of l-O-Οctadecyl-2-O-methyl-butyl-4-phosphonic acid (V) (100 mg, 0.2

mmol) in anhydrous pyridine (15 ml), was added (2 ml, 20 mmol) of

trichloroacetomtrile, and 2- trimethylaminoethanol tosylate (900 mg, 3 mmol).

Solution was heated at 50° C for 48 hrs under N₂ atmosphere. Solvent was removed

under reduced pressure. Dark solid was dissolved in 9:1 THF:H₂O and passed

through a TMD-8 ion-exchange resin column using the same e ients. Solvent

removal under reduced pressure left a residue, which was purified via column

chromatography by elution with gradients of CHC1₃, MeOH and H₂O. Compound 1

was lyophilized from cyclohexane to give 39 mg (38%), as white flaky solid; R_/0.4

(chloroform:methanol:water, 60:40:5); [ ] = + 1.23° (cθ.25, CHCl₃:MeOH, 1:1).

Η NMR (300 MHz, CDC1₃) δ (ppm) 4.4-4.3 (m, 2H), 3.9-3.8 (m, 2H), 3.5-3.3 (m,

17H), 1.8-1.6 (m, 6H), 1.4-1.3 (m, 30H), 0.9-0.8 (t, 3H); calculated for C₂₈H₆₀O₅NP

m/z 522.4 (M.H⁺). Compounds 2-7 were prepared from the phosphonic acid (V) by the same

procedure as described for 1.

3'-Trimethylaminopropyl-l-O-octadecyl-2-O-methylbutane-4-phosphonate (2): 45 mg

(42%) flaky white powder after lyophilization from cyclohexane; I 0.4

(chloroform:methanol:water, 60:40:5); [α]²⁵ _D = + 1.57° (c 1.2, CHCl₃:MeOH,

1:1)Η NMR (300 MHz, CDC1₃) δ (ppm) 4.0-3.9 (m, 2H), 3.8-3.7 (m, 2H), 3.5-3.3

(m, 17H), 2.2-2.0 (t, 2H), 1.9-1.5 (m, 6H), 1.4-1.2 (m, 30H), 0.9-0.8 (t, 3H);

calculated for C₂₉H₆₂O₅NP m/z 536.4 (M.H⁺).

4'-Trimethylaminobutyl-l-O-oct.adecyl-2-O-methy1butane-4-phosphonate (3): 47 mg

(39%) after lyophilization from cyclohexane; IL 0.4 (chloroform:methanol:water,

60:40:5); [α]²⁵ _D = + 2.76° (c 0.25, CHCl₃:MeOH, 1:1); Η NMR (300 MHz,

CHC1₃) δ (ppm) 4.0-3.9 (m, 2H), 3.8-3.6 (m, 2H), 3.5-3.2 (m,17H), 1.9-1.7 (m,

4H), 1.7-1.5 (m, 4H), 1.4-1.2 (m, 30H), 0.9-0.8 (t, 3H); calculated for C₃₀H₆₄O₅NP

m/z 550.4 (M.H⁺).

4^,-N_TN-Dimethylpiperidyl-l-O-octadecyl-2-O-methylbutane-4-phosphate (4): 46 mg

(37%) as white flaky powder; IL 0.4 (chloroform: methanol: water, 60:40:5); [α]²⁵ _D

= -0.25° (c 0.2 ,1: 1 MeOH:CHCl₃); Η NMR (300 MHz, CDC1₃) δ (ppm) 4.6-4.5 (m, IH), 3.8-3.6 (m, 4H), 3.4-3.2 (m, 14H), 2.3-2.1 (m, 4H), 1.8-1.5 (m, 6H), 1.4-

1.2 (m, 30H), 0.9-0.8 (t, 3H); calculated for C₃₀H₆₂O₅NP m/z 548.4 (M.H⁺).

3'-Dimethylbutyl-l-O-octadecyl-2-O-methylbutane-4-phosphonate (5): 60 mg (44%)

as white powder; Pγ0.7 (chloroform: methanol: water, 60:40:5); ^JH NMR (300 MHz,

CDC1₃) δ (ppm) 4.0-3.9 (m, 2H), 3.5-3.3 (m, 8H), 2.7-2.4 (br s, IH), 1.8-1.5 (m,

8H), 1.4-1.2 (m, 30H), 0.9-0.8 (t, 12H); calculated for C₂₉H₆₀O₅NP m/z 521.4

(M.H⁺).

2 ' -Trimethylphosphoniumethyl- 1 -O-octadecy 1 -2-O-methylbutane-4-phosphonate (6) :

20 mg (16%) as white powder after lyophilization from cyclohexane; I 0.4

(chloroform:methanol:water, 60:40:5); Η NMR (300 MHz, CDC1₃) δ (ppm) 4.4-4.2

(m, 2H), 3.5-3.4 (m, 8H), 2.8-2.7 (m, 2H), 2.2-2.1 (d, 9H), 1.9-1.6 (m, 6H), 1.4-

1.2 (m, 30H), 0.9-0.8 (t, 3H); calculated for C₂₈H₆₀O₅P₂ m/z 539.4 (M.H⁺).

2'-Cyclopentanyl-2'-trimethylaminoethyl-4-O-octadecyl-2-O-methylbutane-4-

phosphate- .7>: 45 mg (30%); I 0.5 (chloroform: methanol: water, 60:40:5); [ ] ²⁵ _D

=. +1.78° (c 0.25 , CHCl₃:MeOH, 1:1); Η NMR (300 MHz, CDC1₃) δ (ppm) 3.5-

3.4 (m, 17H), 2.6-2.1 (m, 14H), 1.4-1.2 (m, 30H), 0.9-0.8 (t, 3H); calculated for

C₃₁H₆₄ONP m/z 576.5 (M.H⁺). 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.

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 mmor 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, stodies 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 Screening of New Ether Lipid (NEL) Derivatives

The following protocol measures platelet aggregation in whole blood

utilizing 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-18-OCH₃, but any species, including

human may be substituted. 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.

Collection of Blood Sample for in vitro Platelet Aggregation Testing:

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 solution : blood). One (2.0 mL) Vacutainer tube 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.

Procedure for TLC ELL- 12 dilutions: Test samples were provided from the

Molecular Mechanisms Group either as powder or solutions. Cloudy solutions

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

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

blood (1:40)). 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 — lmL 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 temperatore 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 citrated 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 ran. 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.

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).

Results are shown in Table 1.

Table 1: Platelet aggregation of New Ether Lipid Analogs.

Compound Relative Platelet Aggregation (Ohms) Platelet aggregation

25 /.M JOO μM 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(S) 13 13.5 8 6 3 + + +

3(S) 0 0 0 0 0.5

4(S) 0 0 0 0 0 -

5(S) 0 0 0 nd nd -

6(S) 12 13 19 8 nd + + +

7(S) 8 >4 >4 nd 0 0

Platelet aggregation in dog whole blood was measured in Ohms. *A11 compounds were diluted from saline except 17-21 which, because of poor solubility, were given in DMSO (5S was given in 6% ethanol). Consequently, we believe much of the response noted for 17-21 was a DMSO response since DMSO alone evoked values similar to those recorded. For all experiments, 0.1_/-.M PAF and 100 μM ET-I8-OCH₃ were included as positive controls.

The compounds that elicited a clear platelet aggregation response were IS,

6S and 7S. It should be noted that all of these compounds, S isomers with

phosphonate linkages, had structural orientations consistent with the "L" isomer of

ET-18-OCH₃ (except 4), but only compounds IS, 6S and 7S evoked a platelet

aggregation response. Compound 1 was the structure most similar to ET-18-OCH₃

in that the only difference was that this compound had a phosphonate (methylene

substitution for the phosphate oxygen) linkage in the headgroup. For compounds

6S and 7S, 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. When the number of methylenes in the headgroup was increased

(i.e., compound 3S) or the positive charge was removed (compound 5S), no aggregation was noted indicating that those changes sufficiently altered binding to

the PAF receptor.

II. In Vitro Hemolytic Activity Assessment

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 tobe 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 was

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

culture tobe. 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 l mL of lOO uM + 1 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 temperatore.

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 tabes using

positive displacement pipet. 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. H10 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

The test sample dilutions are shown below:

Shown in Table 2 are the hemolytic activities for the ether lipids. H₅₀ values for washed RBCs of 9-21 μM were reported for all compounds while a value of 28 μM was observed for 13R. Compound 13R also exhibited the highest H₁₀ value in whole blood (Table 2).

Table 2. Hemolytic Activity of New Ether Lipids.

Washed Hitman RBCs Whole Human Blood

Free Compound H₁₀(μM) H₅₀(μM) H₁₀(μM) H₅₀(μM)

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

3S 8 11 600 >2000 4S 7 9 450 >2000

Liposome Systems L-ELL12 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 = dioleoylphosphatidylethanolan-ine-glutaric acid (glutaric acid is covalently attached via the headgroup nitrogen ); CHS = cholesteryl-hemmisuccinate.

TTT. Tn Vitro Growth Inhibition fG ₀.

A. Cell line maintenance

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

GI₅₀ studies: MCF-7: human breast mmor, 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, TJ-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 made in-house and the compounds were

dissolved 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. 100 μl aliquots 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 as 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 with minor modifications. 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 lOOμl 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-UN) 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 as 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 drug concentration, dose response curves were generated and the GI-₅₀

values were calculated. The GI-₅₀ values for each experiment were calculated

using data obtained from three duplicate wells on two separate plates. The mean

GI-₅₀'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.

Results

For growth inhibitory evaluation, five human tumor cell lines (U937;

HT29; A549; MCF7; MCF7/ADR) and two normal fibroblast cell lines (NIH-3T3,

murine; WI-38, human) were used. For comparison, the activity of free L-ET-18-

OCH₃ and D-ET-18-OCH₃ were examined.

Table 3: Growth Inhibition of Tumor/Normal Cells by New Ether Lipids.

GI50 (μM)

Compound Tumor Cell Lines Normal Cell

Line. 3*

MCF7/

TJ937 HT29 A; 49 MCF7 A B. NTH- T 3T3

L-ET-I8-OCH₃ 1.0-1.5 5.5, 6.0 6.5-9.1 9.7-18.6 25.7- 46.6 10-12.8 >40

D-ET-18-OCH, 1.4 5.1 8.0 14.6 25.1 41.4 10-13.5

1(S)* nd 3.0 6.3 26 39.9 - —

3(S) nd <4 6.4, 9.1 19.6, 26.3, 51.8 8.8 22.9 34.5

4(S^,> nd 5.5 7.4 -40 >40 42, 5.3

67.8 5(S) nd 27.8 21.4 31.6 >40

6(S)* nd 5.0 5.6 31.2 34.7

7(S)* nd 5.7 6.2 19.6 29.7

*A human umbilical vein endothelial cell (H-UVEC) line was also examined but it appeared to be very sensitive to ether lipids (for example, the GI₅₀of L- and D- ET-18-OCH₃ were 1.2 and 1.4 μM) and was therefore not included for brevity.

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, NIH-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. With the exception of compounds IS, 7S, and 7S, which aggregated platelets, all ofthe remaining compounds showed relatively good activity with the order of sensitivity the same as that listed above for ET-18-OCH₃. TN. Measurement of DFNDase Activity

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 ofthe invention. At treatment time cell density was 5 l0⁵ cells per mL. Cells were incubated with the ether lipid compounds ofthe 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 DENDase 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 DENDase 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- DEND-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 DENDase activity were calculated after subtraction of background fluorescence of substrate incubated without proteins, and were expressed as percent of control (DENDase activity in untreated cells). Average of DENDase activity, calculated from few independent experiments, was used to calculate percent of L-ether lipid-induced DENDase activity. First, we examined various conditions to compare free ET-18-OCH₃ and

ELL12 because in our studies we were limited by material availability and could only examine free compounds. As shown in Figure 7, 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 ΝELs in Jurkat T Cells at 6 Hours

% L-ET-1 - CTL (DENDase, 6hrs.

[ether lipid], μM

Lipid 1Ω 2Q 3D Ν

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

D-ET-18-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

3S 27.0 ± 3.4 31.5 + 4.3 32.6 + 4.1 4

4S 60.0 + 9.1 58.7 ± 9.3 65.6 ± 10.3 4

Ν is the number replicate experiments.

As shown in Table 7, all ofthe new ether lipids exhibited DENDase activity that was less than both isomers of ET-18-OCH₃. Interestingly, compound 3S exhibited the lowest DENDase activity but was active in vivo against B16/F 10 (recall Figure 3). To further assess 3S cytotoxicity, 3S was compared to ET-18- OCH₃ in terms of Caspase 3, 8, and 9 processing in Jurkat T cells at 5 hours and caspase 3 and 8 at 48 hrs. In all cases compound 3S showed significantly less activity than ET-18-OCH₃. Perhaps this difference in apparent activities is due to an isolated poor activity of 3S against the Jurkat cell line as compared to ET-18-OCH₃. On the other hand, the B16/F10 cell line might be overly or specifically sensitive to this compound. In support of this latter notion, the in vitro GI50 values for several compounds, including ET-18-OCH₃, were very high against B16/F 10 (data not shown) while the value for compound 3S as the lowest: 21 μM for 3S and 51μM for

ET-18-OCH₃. This might suggest an inherent specific sensitivity of the B16/F10 cell line to compound 3S.

N. In Nivo Toxicity and Therapeutic Methods A. Toxicity. Intravenous (xl) or Oral (xl)

CDF1 mice (3/group) were administered a single intravenous or oral dose ofthe 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:.

Bl 6/F10 Marine Melanoma ( iv. / iv. )

Female C57/BL6 mice (5/group) were inoculated iv. with 5 x IO⁴ cells in 0.2 mL 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, as well as 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 ( ip. / iv. )

Female CDF1 mice (7-8/group) were inoculated ip. with 1 x IO⁵ P388 cells in 0.5 mL PBS (day 0). Treatments were administered iv. on days 1, 3, 5, 7, & 9 post inoculation 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 ether lipid compounds to be tested, along with ELL- 12 (L), or Control or on days 1 - 8 post inoculation with 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.

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 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.

DU145 Human Prostate (sc. / iv. )

Male SCID mice (5/group) were inoculated sc. with 2 x 10^δ 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 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 SCID 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 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).

Results

Ten compounds were also sent to NCI's Drug Discovery Program for screening against numerous human tumor cell lines (9 panels total: renal ovarian, colon, CNS, non-small cell lung, leukemia, breast, melanoma, and prostate). The data are shown in Figures 2a-i which also include data regarding D- and L- ET-18- OCH₃ (listed as L-EL and D-EL in the figures).

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 ofthe 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 ofthe appended claims are intended to be included therein. All ofthe 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 alkyl, alkenyl and alkynyl;

R² is selected from the group consisting of

R5 > -N(R⁴)₂, -OR⁴ and H;

X is selected from the group consisting of

O O

I I I I

— P — an d P- -o-

O O

X² is selected from the group consisting of:

-CH₂(CH₂)_mNO₂, -CH₂(CH₂)J^(R⁴)₃, -CH₂(CH₂)_mC(R⁴)₃, and -CH₂(CH₂)_mP⁺(R⁴)₃;

R⁴ is selected from the group consisting of C_t.3 alkyl and H;

R⁵ is selected from the group consisting of C,_₃ alkyl;

and m is 0, or an integer from 1 to 10,

with the proviso that when R¹ is — C₁₈H₃₇ and R² is — OMe, then X² is not R-CH₂(CH₂)_mr^(R⁴)₃.

2. An ether lipid of Claim 1 , wherein R¹ is represented by Y¹ Y², wherein:

Y¹ is (CH₂)n₁(CH=CH)n₂(CH₂)n₃(CH=CH)n₄(CH₂)n₅(CH=CH)n₆(CH₂)n₇ (CH=CH)n_g(CH₂)n₉, the sum of n_!+2n₂+n₃+2n₄ is equal to n₅+2n₆+n₇+2n₈+n₉ is an integer of from 3 to 23, n_} is zero or an interger of from 1 to 22, n₃ is zero or an integer of from 1 to 19, n₅ is zero or an integer of from 1 to 16, n₇ is zero or an integer of from zero to 16, n₉ is zero or an integer of from 1 to 10, and each of n₂, n₄, n₆ and n₈ is independently zero or 1 ; and

Y₂ is — CH₃ or— CO₂H.

3. An ether lipid of Claim 2, wherein R¹ is selected from the group consisting of — C₁₈H₃₇ and — Cι₆H₃₃.

4. An ether lipid of Claim 2, wherein R¹ is — C₁₈H₃₇.

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

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

7. An ether lipid of Claim 4, wherein R² is selected from the group consisting of:

8. An ether lipid of Claim 7, wherein R² is — OSO₂Me.

9. An ether lipid of Claim 4, wherein X is — & — o-

0_

10. An ether lipid of Claim 9, wherein X² is — CH₂(CH₂)_raN⁺(CH₃)₃.

11. An ether lipid of Claim 10, wherein m is an integer from 1 -7.

12. An ether lipid of Claim 9, wherein X is „_CH /_CH )_N⁺ \

13. An ether lipid of Claim 9, wherein X² is and R⁴ is CH₃

14. An ether lipid of Claim 9, wherein X rl^z . is — CH₂(CH₂)C(CH₃)₃.

15. An ether lipid of Claim 9, wherein X² is — CH₂(CH₂)P⁺(CH₃)₃.

16. An ether

17. An ether lipid of Claim 4, wherein X is

18. An ether lipid of Claim 9, wherein X² is

19. An ether lipid of Claim 9, wherein X² is — CH₂CH₂NO₂

20. An ether lipid of Claim 4, wherein X¹ is

21. An ether lipid of Claim 20, wherein X² is — CH₂(CH₂)_raN⁺(CH₃)₃

22. An ether lipid of Claim 21, wherein m is an integer from 1-7.

23. An ether lipid of Claim 20, wherein R² is — OMe.

24. An ether lipid of Claim 20, wherein R² is selected from the group consisting of

O

-IM O — 0-4— PS ^{md "N}(^R4)₂' O

and wherein R⁴ is CH, and R⁵ is CH,

25. An ether lipid of Claim 1 , selected from the group consisting of:

26. An ether lipid of Claim 1 , selected from the group consisting of:

CH₃

27. An ether lipid of Claim 1, having the chemical formula:

28. An ether lipid of Claim 1 , having the chemical formula:

29. An ether lipid of Claim 1 , having the chemical formula:

30. An ether lipid of Claim 1 , wherein the ether lipid is optically active.

31. An ether lipid of Claim 1 , wherein the ether lipid is the D enantiomer.

32. An ether lipid of Claim 25, wherein the ether lipid is the D enantiomer.

33. An ether lipid of Claim 26, wherein the ether lipid is the D enantiomer.

34. An ether lipid of Claim 27, wherein the ether lipid is the D enantiomer.

35. An ether lipid of Claim 28, wherein the ether lipid is the D enantiomer.

36. An ether lipid of Claim 29, wherein the ether lipid is the D enantiomer.

37. 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.

38. 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 phannaceutically acceptable salt, isomer or prodrug thereof.

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

40. A method of treating a mammal afflicted with a cancer which comprises administering to the mammal a therapeutically effective amount ofthe pharmaceutical composition of Claim 37 comprising from about 0.1 mg of the ether lipid per kg ofthe body weight ofthe mammal to about 1000 mg per kg.

41. A method of Claim 40, 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.

42. The method of Claim 40, comprising administering to the mammal an additional biologically active agent.

43. The method of Claim 42, wherein the additional biologically active agent is selected from the group consisting of antineoplastic agents, antimicrobial agents, and hematopoietic cell growth stimulating agents.