WO1996024598A1 - [4-hexadecyl-3-methoxy-butyl] phosphonic acid and its protein conjugates useful as anti-cancer agents - Google Patents

[4-hexadecyl-3-methoxy-butyl] phosphonic acid and its protein conjugates useful as anti-cancer agents Download PDF

Info

Publication number
WO1996024598A1
WO1996024598A1 PCT/CA1995/000068 CA9500068W WO9624598A1 WO 1996024598 A1 WO1996024598 A1 WO 1996024598A1 CA 9500068 W CA9500068 W CA 9500068W WO 9624598 A1 WO9624598 A1 WO 9624598A1
Authority
WO
WIPO (PCT)
Prior art keywords
pharmaceutically acceptable
carbon atoms
chain containing
aliphatic chain
atom
Prior art date
Application number
PCT/CA1995/000068
Other languages
French (fr)
Inventor
Hassan Salari
Original Assignee
The University Of British Columbia
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of British Columbia filed Critical The University Of British Columbia
Priority to PCT/CA1995/000068 priority Critical patent/WO1996024598A1/en
Priority to AU15734/95A priority patent/AU1573495A/en
Publication of WO1996024598A1 publication Critical patent/WO1996024598A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3808—Acyclic saturated acids which can have further substituents on alkyl
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment

Definitions

  • This invention pertains to the synthesis of [4- hexadecyl-3-methoxy-butyl] phosphonic acid and its protein conjugates, and the use of these compounds as anti-cancer agents.
  • European Patent No. P0230 575A2 discloses a group of glycerophospholipid compounds having an alkyl chain of C2-C22 and a methoxy group at the sn-2 position and a phosphocholine at the sn-3 position. These compounds are stated to be useful as anti-cancer agents.
  • the present invention provides for an anti- cancer compound of the general formula:
  • R 2 is OX or NHR
  • T is an oxygen or sulphur atom
  • R is an aliphatic chain containing 12 to 20 carbon atoms and R 2 is a protein moiety, or OX where X is H, or a pharmaceutically accept ⁇ able cation.
  • the compound includes either of the opposite stereochemical configurations [ (R) or (S) ] , or a mixture of stereoisomers.
  • R is an aliphatic chain containing 12 to 20 carbon atoms and R 2 is a protein moiety, or OX where X is H, or a pharmaceutically acceptable cation.
  • the compound includes either of the opposite stereochemical configurations [ (R) or (S) ] , or a mixture of stereoisomers.
  • R is an aliphatic chain containing 12 to 20 carbon atoms and R 2 is a protein moiety, or OX where X is H, or a pharmaceutically acceptable cation.
  • the compound includes either of the opposite stereochemical configurations [ (R) or (S) ] , or a mixture of stereoisomers.
  • a method of treating cancer, lung cancer, colo ⁇ rectal cancer, leukemia, lymphoma or melanoma in a mammal afflicted with cancer, lung cancer, colorectal cancer, leukemia, lymphoma or melanoma, comprising treating the afflicted mammal with a therapeutic amount of a phosphonic compound of the following general formula:
  • T is either an oxygen or sulphur atom
  • R 1 is an aliphatic chain containing 12 to 20 carbon atoms
  • R 2 is a protein moiety, or OX where X is H, or a pharmaceutically acceptable cation.
  • the compound includes either of the opposite stereochemical configurations [ (R) or (S)], or a mixture of stereoisomers.
  • R 1 is an aliphatic chain containing 12 to 20 carbon atoms and R 2 is a protein moiety, or OX where X is H, or a pharmaceutically acceptable cation.
  • the compound includes either of the opposite stereochemical configurations [ (R) or (S)], or a mixture of stereoisomers.
  • the phosphonic compound is administered to the afflicted mammal at a dosage of 5 to 50 mg/Kg body weight, and may be adminis- ' tered to the afflicted mammal orally, intravenously, intramuscularly, intradermally, subcutaneously, topically or intravenously in the form of a liposome or other lipid vesicle, with or without a pharmaceutically acceptable carrier.
  • the phos ⁇ phonic compound can be administered directly to the af ⁇ flicted mammal's bone marrow, blood, blood cells, leuko ⁇ cytes, lymphocytes or other such extracorporeal prepara ⁇ tion containing the mammal's diseased cells, with or without a pharmaceutically acceptable carrier.
  • R. is an aliphatic chain containing 12 to 20 carbon atoms and R 2 is a protein moiety, or OX where X is H, or a pharmaceutically acceptable cation.
  • the compound includes either of the opposite stereochemical configurations [ (R) or (S)] , or a mixture of stereoisomers.
  • the phosphonic compound is administered to the afflicted mammal at a dosage of 5 to 50 mg/Kg body weight, and may be adminis ⁇ tered to the afflicted mammal orally, intravenously, intramuscularly, intradermally, subcutaneously, topically or intravenously in the form of a liposome or other lipid vesicle, with or without a pharmaceutically acceptable carrier.
  • the phos ⁇ phonic acid can be administered directly to the afflicted mammal's bone marrow, blood, blood cells, leukocytes, lymphocytes or other such extracorporeal preparation containing the mammal's diseased cells, with or without a pharmaceutically acceptable carrier.
  • These phosphonic compounds are useful as anti- cancer agents since they inhibit the growth of malignant cells.
  • the phosphonic compounds as described above or as claimed include either of the opposite stereochemical con ⁇ figurations [(R) or (S)] or a mixture thereof.
  • the enantiomeric phosphonic acid ( [4-hexadecyl ⁇ oxy-3 (R) -methoxy-butyl] phosphonic acid) can be prepared according to the method above using the corresponding starting material.
  • the phosphonic compounds of the invention, wherein T is sulphur can be synthesized according to the following reaction sequence.
  • the enantiomeric phosphonic acid [4-hexadecyl ⁇ thio-3 (R) -methoxy-butyl] phosphonic acid
  • the enantiomeric phosphonic acid [4-hexadecyl ⁇ thio-3 (R) -methoxy-butyl] phosphonic acid
  • the phosphonic compounds of the invention wherein T is oxygen or sulphur and R 2 is a protein moiety, can be synthesized according to the following reaction sequence. OC 16 H 33 PC 0°C eO H O II
  • the specific protein-conjugate phosphonic com- pound may be selected by use of the appropriate dimethyl phosphonate starting material.
  • An example is provided here for the preparation of a [4-hexadecyloxy-3 (S) -methoxy- butyl] phosphonic-protein conjugate.
  • In vitro tests such as MTT assay, have been conducted to establish that phosphonic acids inhibit the growth of cancer cells and kill them.
  • Tumor cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin (50 units/ml) , streptomycin (50 ⁇ g/ml) and mer- captoethanol (5 ⁇ g/ml) in an atmosphere of 5% C0 2 .
  • the cells were passaged weekly by serial 1/10 to 1/10,000 dilutions.
  • the cell viability and growth were constantly monitored by staining with trypan blue exclusion dye or the incorporation of tritiated thymidine.
  • the MTT assay is performed using 3- [4,5- dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) , a water soluble tetrazolium salt yielding a yellow ⁇ ish solution when prepared in media or salt solutions lacking phenol red. Dissolved MTT is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by active mitochondrial dehydrogenase enzymes of living cells. Dead cells do not cause this conversion.
  • the converted dye can be solubilized with DMSO, and the dissolved material measured spectrophotometrically, yield- ing absorbance as a function of concentration of converted dye.
  • MTT working solution 50 ⁇ L of a 1:5 (v/v) diluted stock solution prepared as per the manufacturer's directions
  • Table 1 shows the effect of 72 hours' exposure to various concentrations of the phosphonic acid of the above formula (compound IV) on the viability of HT-29 cells.
  • Table 2 shows the results of an assay performed to observe the effects of compound IV (phosphonic acid) on H460 (human cell lung cancer) cells.
  • Table 2 shows the effect of 72 hours' exposure to various concentrations of the phosphonic acid of the above formula (compound IV) on the viability of H460 cells.
  • phosphonic acids compounds of the formula shown above are cytotoxic against H460 human colorectal cancer cells and lung cancer cells over 72 hours when administered as the sodium salt . This effect is dose -dependent , and demonstrates the anti-cancer activity of phosphonic acids .
  • Table 3 shows the effect of 72 hours' exposure to various concentrations of the phosphonic acid of the above formula (compound IV) on the viability of metastatic Colon 26 cells.
  • phosphonic acids of the formula shown above are cytotoxic against Colon 26 murine metastatic colon cancer cells over 72 hours when administered as the sodium salt . This effect is dose- dependent , and demonstrates the anti -cancer activity of phosphonic acids .
  • Examples 1 and 2 clearly demonstrate that the phosphonic compounds of the invention have a cytotoxic action against colorectal cancer, and are thus useful as anti-cancer agents.
  • HL-60 tumor cells a human myeloleukemic cell line
  • MTT cell viability assay when the compound is administered as the sodium salt. This effect is indicative of the anti-cancer activity of the above phosphonic acid (compound IV) .
  • Table 4 shows the effect of 72 hours' exposure to various concentrations of the phosphonic acid of the above formula (compound IV) on the viability of HL-60 cells.
  • phosphonic acid compounds of the formula shown above are cytotoxic against HL-60 human myeloleukemic cells over 72 hours when administered as the sodium salt . This effect is dose -dependent , and demonstrates the anti -cancer activity of phosphonic acids .
  • Table 5 shows the effect of 72 hours' exposure to various concentrations of the phosphonic acid of the above formula (compound IV) on the viability of L1210 cells.
  • phosphonic acid compounds of the formula shown above are cytotoxic against L1210 murine lymphoma over 72 hours when administered as the sodium salt. This effect is dose- dependent, and demonstrates the anti-cancer activity of phosphonic acids.
  • (IV) has a cytotoxic effect against RPMI 7951 cells (human melanoma cell line) in the MTT cell viability assay when the compound is administered as the sodium salt. This effect is indicative of the anti-cancer activity of the above phosphonic acid (compound IV) .
  • Table 6 shows the effect of 72 hours' exposure to various concentrations of the phosphonic acid of the above formula (compound IV) on the viability of RPMI 7951 cells.
  • phosphonic acids of the formula shown above are cytotoxic against RPMI 7951 human melanoma cells over 72 hours when adminis ⁇ tered as the sodium salt. This effect is dose-dependent, and demonstrates the anti-cancer activity of phosphonic acids.
  • Example 5 clearly demonstrates that the phos ⁇ phonic compounds of the invention have a cytotoxic action against human melanoma, and are thus useful as anti-cancer agents .
  • compound IV showed to be a potent inhibitor of phosphatidylinositol 3 Kinase (PI 3 Kinase) as shown in Table 7.
  • PI 3 Kinase phosphatidylinositol 3 Kinase
  • the PI j Kinase activity is determined as the incor- poration of P at the 3-position of the inositol ring of phosphatidylinositol (PI) is in the presence of 32 P-ATP.
  • the compounds of the invention are useful as anti-cancer agents, and may be administered safely by either parenteral, oral or topical routes in pharmaceutical preparations such as injections, tablets, capsules, liquid preparations or ointments. These preparations are used by an appropriate route of administration, depending on the specific affliction, patient conditions and other factors. Injections may be given intravenously, intramuscularly, intradermally or subcutaneously. The dose of compound can be selected based on the patient weight, treatment regimen or purpose of administration, generally within the range of 5 to 50 mg/Kg.
  • compositions may be adminis ⁇ tered 1 to 4 times daily, at 2 to 7 day intervals, or as otherwise necessary to maintain a therapeutic level of the compound in body tissues depending on the specific afflic- tion, patient conditions, treatment regimen or purpose of administration.
  • Injections, intravenous infusions and similar preparations are prepared by conventional methods in either aqueous solution or physiological saline containing 20% propylene glycol and a preservative such as 0.5% ascorbic acid, with an upwardly adjusted pH in phosphate buffer.
  • the drug solution is sterilized by passing it through a 22 ⁇ m filter, and distributed into glass vials in approximate ⁇ ly 1 ml aliquots to provide a unit dosage.
  • the aliquots are then lyophilized, and the vials tightly stoppered and capped to maintain sterility.
  • the drug may be reconsti ⁇ tuted in the vial by the addition of physiological saline or aqueous diluent.
  • Unit dosage tablets can be prepared by compressing a mixture of 40 mg of phosphonic acid compound, 200 mg of lactose, and 50 mg AvicelTM into the form of a tablet.
  • a similar drug mixture may also be contained in unit dose within a cellulose-based capsule.
  • An ointment or cream may also be prepared by conventional methods by mixing the phosphonic acid compound in a commercially-available glycerine-based cream. The cream is applied topically directly to the afflicted area.
  • the compounds of the invention may also be administered in the form of a liposome.
  • a mixture of phosphonic acid compound and lecithin is mechanically treated to form a bilayer (one side hydrophilic, the other hydrophobic) which spontaneously forms micelles (lipo- somes) . These may be filtered to obtain liposomes of uniform size (approximately 10 nm) and dose (approximately 50 mg/L) . Liposomes are sterilized by filtration through a 22 ⁇ m filter, and administered as an intravenous sol- ution.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

This invention pertains to the synthesis of [4-hexadecyl-3-methoxy-butyl] phosphonic acid and its protein conjugates, and the use of these compounds as anti-cancer agents. A method of treating cancer in a mammal afflicted with cancer, comprising treating the afflicted mammal with a therapeutic amount of a phosphonic compound of formula (I), wherein T is an oxygen or sulphur atom, R1 is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceutically acceptable cation, and either of the opposite stereochemical configurations [(R) or (S)], or a mixture of stereoisomers thereof, and pharmaceutically acceptable salts thereof.

Description

r4-HEXADECYL-3-METHOXY-BUTY l PHOSPHONIC ACID AND ITS PROTEIN CONJUGATES USEFUL AS ANTI-CANCER AGENTS
FIELD OF THE INVENTION
This invention pertains to the synthesis of [4- hexadecyl-3-methoxy-butyl] phosphonic acid and its protein conjugates, and the use of these compounds as anti-cancer agents.
BACKGROUND OF THE INVENTION
European Patent No. P0230 575A2, dated April 12, 1986, discloses a group of glycerophospholipid compounds having an alkyl chain of C2-C22 and a methoxy group at the sn-2 position and a phosphocholine at the sn-3 position. These compounds are stated to be useful as anti-cancer agents.
U.S. Patent No. 4,408,052, dated February 25, 1981, assigned to Takeda Chemical Industries, Osaka, Japan, claims a group of phospholipid carbamates useful as anti- tumor agents.
Canadian Patent No. 1,248,534, dated January 10, 1989, granted to Takeda Chemical Industries of Japan, protects a group of ketolyso phospholipids, which pur¬ portedly are useful as antitumor agents.
U.S. Patent No. 4,515,722, dated May 7, 1985, granted to Merck Sharp & Dohme, protects a group of phosphatidylinositol analogs which are evidently effective in inhibiting phospholipase C and thereby have utility as anti-inflammatory and analgesic agents.
U.S. Patent No. 5,219,845, dated June 15, 1993, granted to The University of British Columbia, protects a group of substances with a glycerol backbone linked to a phosphorus atom and a polar head group useful as anti- inflammation agents.
None of these patents discloses [4-hexadecyl-3- methoxy-butyl] phosphonic acid useful as an anti-cancer agent.
SUMMARY OF THE INVENTION
The present invention provides for an anti- cancer compound of the general formula:
CH, Ri
CH OCH3
O
II
CH, CH, - P
OX
(I) R2 is OX or NHR
wherein T is an oxygen or sulphur atom, R, is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is H, or a pharmaceutically accept¬ able cation. The compound includes either of the opposite stereochemical configurations [ (R) or (S) ] , or a mixture of stereoisomers. A phosphonic compound of the general formula:
CH,
CH OCH,
CH, CH, P - I OX
(II) wherein R, is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is H, or a pharmaceutically acceptable cation. The compound includes either of the opposite stereochemical configurations [ (R) or (S) ] , or a mixture of stereoisomers.
A phosphonic compound of the general formula;
CH, Ri
CH OCH3
O
II
CH. CH, - P - I OX
(III)
wherein R, is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is H, or a pharmaceutically acceptable cation. The compound includes either of the opposite stereochemical configurations [ (R) or (S) ] , or a mixture of stereoisomers.
A method of treating cancer, lung cancer, colo¬ rectal cancer, leukemia, lymphoma or melanoma in a mammal afflicted with cancer, lung cancer, colorectal cancer, leukemia, lymphoma or melanoma, comprising treating the afflicted mammal with a therapeutic amount of a phosphonic compound of the following general formula:
CH2 T R
CH OCH3
O
CH2 CH2 P R2
I OX
(I) wherein T is either an oxygen or sulphur atom, R1 is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is H, or a pharmaceutically acceptable cation. The compound includes either of the opposite stereochemical configurations [ (R) or (S)], or a mixture of stereoisomers.
A method of treating cancer of lung, colorectal cancer, leukemia, lymphoma or melanoma with a therapeutic amount of a phosphonic compound of the following formula:
CH2 O R
CH OCH3
O
CH2 CH2 P -
I OX
(II)
wherein R1 is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is H, or a pharmaceutically acceptable cation. The compound includes either of the opposite stereochemical configurations [ (R) or (S)], or a mixture of stereoisomers. The phosphonic compound is administered to the afflicted mammal at a dosage of 5 to 50 mg/Kg body weight, and may be adminis- ' tered to the afflicted mammal orally, intravenously, intramuscularly, intradermally, subcutaneously, topically or intravenously in the form of a liposome or other lipid vesicle, with or without a pharmaceutically acceptable carrier. In the case of leukemia or lymphoma, the phos¬ phonic compound can be administered directly to the af¬ flicted mammal's bone marrow, blood, blood cells, leuko¬ cytes, lymphocytes or other such extracorporeal prepara¬ tion containing the mammal's diseased cells, with or without a pharmaceutically acceptable carrier.
A method of treating cancer of lung, colorectal cancer, leukemia, lymphoma or melanoma in a mammal with a therapeutic amount of a phosphonic compound of the follow- ing formula:
CH2 S Ri
CH OCH3
CH, CH, P
I
OX
(III) wherein R., is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is H, or a pharmaceutically acceptable cation. The compound includes either of the opposite stereochemical configurations [ (R) or (S)] , or a mixture of stereoisomers. The phosphonic compound is administered to the afflicted mammal at a dosage of 5 to 50 mg/Kg body weight, and may be adminis¬ tered to the afflicted mammal orally, intravenously, intramuscularly, intradermally, subcutaneously, topically or intravenously in the form of a liposome or other lipid vesicle, with or without a pharmaceutically acceptable carrier. In the case of leukemia or lymphoma, the phos¬ phonic acid can be administered directly to the afflicted mammal's bone marrow, blood, blood cells, leukocytes, lymphocytes or other such extracorporeal preparation containing the mammal's diseased cells, with or without a pharmaceutically acceptable carrier.
These phosphonic compounds are useful as anti- cancer agents since they inhibit the growth of malignant cells.
The phosphonic compounds as described above or as claimed include either of the opposite stereochemical con¬ figurations [(R) or (S)] or a mixture thereof.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
1. Production of the Compounds of the Invention
(a) Synthesis of [4-hexadecyloxy-3-methoxy-butyl] phosphonic acid
The phosphonic compounds of the invention, wherein T is oxygen, can be synthesized according to the following reaction sequence:
Figure imgf000008_0001
K CO, C16H330 \^^< LiCH2P(O)(Q e)2
MeOH BF3. EtO, THF, -78 ° to - 20°C
Figure imgf000009_0001
6
OC16H33
MeO H II
OH
OH
The reaction of 20 mmol of (S) - (+) -glycidly tosylate and 30 mmol of 1-hexadecanol in 50 ml methylene chloride in the presence of catalytic boron trifluoride etherate was carried out. After purification by flash chromatography (elution with 5:1 hexane/ethyl acetate), followed by three recrystallizations from ether-hexane, there was obtained 7.41g (80%) of ring-opened product 4 (1- O-hexadecyl-s/i-glycerol 3-0-p-toluene-sulfonate) .
To a suspension of 15 mmol of the tosylate 4 in 100 ml of dry methanol was added 30 mmol of powdered potassium carbonate at 0°C. The reaction mixture was stirred for 3 hours at 0°C, diluted with 300 ml of ethyl ether, and filtered through a pad of silica gel. The filtrate was concentrated under reduced pressure, and the residue was dissolved in hexane and filtered through a pad of silica gel to give 3.95g (98%) of the epoxide 5 (hexadecyl (S) -2-oxirany1methyl ether) as a white solid, which was used without further purification.
To a solution of 40 mmol of dimethyl methanephos- phonate in 30 ml of dry THF was added dropwise 40 mmol of /i-butyllithium (a 2.5 M solution in hexane), and the reaction was stirred for 30 minutes at -78°C. To this mixture was added dropwise boron trifluoride etherate (40 mmol) , followed by a solution of 20 mmol of the epoxide 5 in 100 ml of THF. The reaction mixture was stirred for 3 hours at -78°C and then warmed to -20°C and stirred for a further 1 hour. The mixture was quenched by the addition of saturated aqueous ammonium chloride solution and was concentrated under reduced pressure. The product from the aqueous residue was extracted with ether, and the combined extracts were washed with brine, dried over sodium sulfate, and concentrated under vacuum. Purification by flash chromatography on silica gel (elution with chloroform- methanol, 25:1) gave 7.54g (89%) of the product 6 (dimethyl 4- (hexadecyloxy) -3 (S) -hydroxybutanephosphonate) as a white solid after lyophilization from hexane.
To a mixture of 5.0 mmol of the hydroxy phos- phonate 6 and llg of silica gel (previously heated at 150°C for 2 hours under high vacuum) was added an ether solution of diazomethane (20 molar equivalents based on substrate) at 0°C. After the mixture had stirred at 0°C for 6 hours, another 20 molar equivalents of diazomethane solution was added, and the mixture was stirred for 24 hours at 0°C. The silica gel was removed from the reaction mixture by filtration and washed with ether. The product was purified by flash column chromatography on silica gel (elution with chloroform-methanol, 50:1) to give 1.94g (88%) of the product 7 (dimethyl 4- (hexadecyloxy) -3 (S) -methoxybutane- phosphonate) as a colourless oil.
To a solution of 0.1 mmol of methoxy phosphonate 7 in 5 ml of methylene chloride was added 2.7 mmol of bromotrimethylsilane. After the mixture was allowed to stand for 2 hours at room temperature, volatile materials were removed under vacuum. The residue was dissolved in THF-water (17 ml, 8:1 by volume), and the mixture was allowed to stand for 2 hours at room temperature. The solvents were removed under vacuum, and the residue was dried by repeated azeotropic distillation with dry 2- propanal under vacuum.
Lyophilization from benzene gave 408 mg (100%) of the phosphonic acid 1 ( [4-hexadecyloxy-3 (S) -methoxy-butyl] phosphonic acid) as a white solid.
The enantiomeric phosphonic acid ( [4-hexadecyl¬ oxy-3 (R) -methoxy-butyl] phosphonic acid) can be prepared according to the method above using the corresponding starting material.
(b) Synthesis of [4-hexadecylthio-3-methoxy-butyl] phosphonic acid
The phosphonic compounds of the invention, wherein T is sulphur, can be synthesized according to the following reaction sequence.
1
Figure imgf000011_0001
8
CH2N2,Si02
Figure imgf000011_0002
Figure imgf000011_0003
11 In situ NaBH4-mediated opening of (S) -glycidol (prepared by asymmetric epoxidation of allyl alcohol) with hexadecyl mercaptan yielded the starting material 8 (1- (hexadecylthio) -jn-glycerol) .
A mixture of 10 mmol of the diol 8, 15 mmol of triphenylphosphine, and 15 mmol of diethyl azodicarboxylate in 50 ml of benzene was refluxed for 24 hours. After removal of the solvent, 50 ml of ether was added, and the precipitate of phosphine oxide was removed by filtration. The filtrate was concentrated under vacuum, and the residue was purified by flash chromatography (elution with 20:1 hexane-ethyl acetate) to give 2.71g (86%) of the product 9 (hexadecyl (S) -2-oxiranylmethyl thioether) as a white solid.
To a solution of 20 mmol of dimethyl methanephos- phonate in 15 ml of dry THF was added dropwise 20 mmol of n-butyllithium (a 2.5 M solution in hexane) . After the reaction mixture was stirred for 30 minutes at -78°C, 20 mmol of boron trifluoride ethereate was added dropwise, followed by a solution of 5.0 mmol of the epoxide 9 in 50 ml of THF. The reaction mixture was stirred for 3 hours at -78°C, warmed to -20°C, stirred for 1 hour, and then quenched by the addition of saturated aqueous ammonium chloride solution. The mixture was concentrated under reduced pressure, extracted with ether, and the combined extracts were washed with brine, dried over sodium sulfate, and concentrated under vacuum. Purification by flash chromatography on silica gel (elution with chloroform- methanol, 25:1) gave 1.98g (90%) of the product 10 (di¬ methyl 4- (hexadecylthio) -3 (S) -hydroxybutanephosphonate) as a white solid after lyophilization from hexane.
To a mixture of 5.0 mmol of the hydroxy phos- ponate 10 and llg of silica gel (previously heated at 150°C for 2 hours under high vacuum) was added an ether solution of diazomethane (20 molar equivalents based on substrate) at 0°C. After the mixture had stirred at 0°C for 6 hours, another 20 molar equivalents of diazomethane solution was added, and the mixture was stirred for 24 hours at 0°C. The silica gel was removed from the reaction mixture by filtration and washed with ether. The product was purified by flash column chromatography on silica gel (elution with chloroform-methanol, 50:1) to give 1.94g (88%) of pure product 11 (dimethyl 4- (hexadecylthio) -3 (S) -methoxybutane- phosphonate) as a colourless oil.
To a solution of 0.1 mmol of methoxy phosphonate 11 in 5 ml of methylene chloride was added 2.7 mmol of bromotrimethylsilane. After the mixture was allowed to stand for 2 hours at room temperature, volatile materials were removed under vacuum. The residue was dissolved in THF-water (17 ml, 8:1 by volume), and the mixture was allowed to stand for 2 hours at room temperature. The solvents were removed under vacuum, and the residue was dried by repeated azeotropic distillation with dry 2- propanol under vacuum. Lyophilization from benzene gave the phosphonic acid 2 ( [4-hexadecylthio-3 (S) -methoxy- butyl] phosphonic acid) as a white solid.
The enantiomeric phosphonic acid ( [4-hexadecyl¬ thio-3 (R) -methoxy-butyl] phosphonic acid) can be prepared according to the method above using the corresponding starting material.
(c) Synthesis of protein-conjugated Phospholipid compounds
The phosphonic compounds of the invention, wherein T is oxygen or sulphur and R2 is a protein moiety, can be synthesized according to the following reaction sequence. OC16H33 PC 0°C eO H O II
P(OMe)2
Figure imgf000014_0001
0^16^33
MeO H O Nal
II
P. acetone reflux
NHR
13 OMe
R
Figure imgf000014_0002
The specific protein-conjugate phosphonic com- pound may be selected by use of the appropriate dimethyl phosphonate starting material. An example is provided here for the preparation of a [4-hexadecyloxy-3 (S) -methoxy- butyl] phosphonic-protein conjugate.
Dimethyl 4- (hexadecyloxy) -3 (S) -methoxybutane phosphonate 7 in benzene was cooled to 0°C (ice-salt bath) and an equimolar amount of PC15 was added so that the temperature did not exceed 10°C. After 1 hour of stirring, the solvent and P0C13 were removed under high vacuum. The resulting acid chloride 12, was used without further purification.
To a solution of acid chloride 12 dissolved in THF was added triethylamine (1.2 equivalents) and the protein moiety (1 to 4 equivalents) . This mixture was allowed to react for up to 14 hours at room temperature, catalysed by DMAP. The solvents were removed under vacuum to yield the phosphonamide 13.
To a solution of the phosphonamide 13 dissolved in acetone was added sodium iodide. The mixture was allowed to reflux for 3.5 hours, permitting the selective monodealkylation of the phosphonamide to yield the protein- conjugated phosphonic salt 3.
2. Biological Activity
In vitro tests, such as MTT assay, have been conducted to establish that phosphonic acids inhibit the growth of cancer cells and kill them. In vitro tests, as well as in vivo testing using animal models of cancer, are useful indicators of the cytotoxic activity of new anti- cancer compounds. While it would be ideal to test new compounds in human beings, such testing is unethical, and thus it is acceptable to extrapolate results of testing new anti-cancer compounds in vitro and in vivo in animal models to the human condition.
Experiments were performed using a number of different tumour cell lines. Tumor cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin (50 units/ml) , streptomycin (50 μg/ml) and mer- captoethanol (5 μg/ml) in an atmosphere of 5% C02. The cells were passaged weekly by serial 1/10 to 1/10,000 dilutions. The cell viability and growth were constantly monitored by staining with trypan blue exclusion dye or the incorporation of tritiated thymidine.
The MTT assay is performed using 3- [4,5- dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) , a water soluble tetrazolium salt yielding a yellow¬ ish solution when prepared in media or salt solutions lacking phenol red. Dissolved MTT is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by active mitochondrial dehydrogenase enzymes of living cells. Dead cells do not cause this conversion. The converted dye can be solubilized with DMSO, and the dissolved material measured spectrophotometrically, yield- ing absorbance as a function of concentration of converted dye. Approximately 2500 cells/well were incubated at 37°C for 24 hours in a 96-well microtiter plate. Various concentrations of the test compound (dissolved in HEPES buffer) or vehicle, diluted in 100 μL of the culture medium, or culture medium alone were added to each well, and the cells incubated for a further 24 to 72 hours. MTT working solution (50 μL of a 1:5 (v/v) diluted stock solution prepared as per the manufacturer's directions) was added to each well and the cells incubated at 37°C for 4 hours. The culture supernatant was aspirated, leaving 10 to 20 μL in the bottom of each well, and 150 μL DMSO was added to solubilize any converted dye. The absorbance in each well was read in a spectrophotometer at 540 nm, and the cell viability in the wells containing the test co - pounds expressed as a percentage of the absorbance in control wells. (a) Activity of phosphonic acids against colorectal cancer
Example 1
Activity of Phosphonic Acids Aσainst
Human Colorectal Cancer Cells
It has been discovered that the phosphonic acid of the following formula:
CH2 O C16H33
CH OCH3
O II CH2 CH2 P OH
<!>H
(IV)
has a cytotoxic effect against HT-29 cells (human color- ectal cancer) in the MTT cell viability assay when the compound is administered either as the sodium salt or the free acid. This effect is indicative of the anti-cancer activity of the above phosphonic acid (compound IV) .
Table 1 shows the effect of 72 hours' exposure to various concentrations of the phosphonic acid of the above formula (compound IV) on the viability of HT-29 cells.
Table 1
MTT Cell Viability Assay of HT-29 Tumor Cells
Exposed to Various Concentrations of
Phosphonic Acid (Compound IV) for 72 Hours
Phosphonic acid Mean (n = 3) Absorbance (% of control) concentration (μM)
0.0 100.00
0.1 99.97
0.5 89.21
1.0 77.29
2.5 62.20
5.0 56.71
10.0 35.50
25.0 12.98
50.0 9.14
Example 2
Table 2 shows the results of an assay performed to observe the effects of compound IV (phosphonic acid) on H460 (human cell lung cancer) cells. Table 2 shows the effect of 72 hours' exposure to various concentrations of the phosphonic acid of the above formula (compound IV) on the viability of H460 cells.
Table 2
MTT Cell Viability Assay of H460 Tumor Cells
Exposed to Various Concentrations of
Phosphonic Acid (Compound IV) for 72 Hours
Phosphonic acid (Na salt) Mean (n = 3) Absorbance concentration (μM) (% of control)
Figure imgf000019_0001
100
0.2 96
0.4 88
0.8 78
1.6 47 3.2 12
6.4 0
12.8 0
As can be seen from Tables 1 and 2 , phosphonic acids compounds of the formula shown above (compound IV) are cytotoxic against H460 human colorectal cancer cells and lung cancer cells over 72 hours when administered as the sodium salt . This effect is dose -dependent , and demonstrates the anti-cancer activity of phosphonic acids .
Example 3
Activity of Phosphonic Acids Against Murine Metastatic Colon Cancer Cells
It has been discovered that the phosponic acid compound of the following formula:
CH2 O C16H33
CH OCH3
I II
CH, CH2 P OH
OH (IV)
has a cytotoxic effect against metastatic Colon 26 cells (murine colon cancer) in the MTT cell viability assay when the compound is administered as the sodium salt. This effect is indicative of the anti-cancer activity of the above phosphonic acid (compound IV) .
Table 3 shows the effect of 72 hours' exposure to various concentrations of the phosphonic acid of the above formula (compound IV) on the viability of metastatic Colon 26 cells.
Table 3
MTT Cell Viability Assay of Metastatic Colon 26 Tumor Cells Exposed to Various Concentrations of Phosphonic Acid (Compound IV) for 72 Hours
Phosphonic acid (Na salt) concentration Mean (n = 3) Absorbance (μM) (% of control)
0.0 100.00
0.1 106.91
0.5 101.20
1.0 105.21
2.5 105.17
5.0 87.32
10.0 61.46 25.0 18.01
50.0 0.46
As can be seen from Table 3 , phosphonic acids of the formula shown above (compound IV) are cytotoxic against Colon 26 murine metastatic colon cancer cells over 72 hours when administered as the sodium salt . This effect is dose- dependent , and demonstrates the anti -cancer activity of phosphonic acids .
Examples 1 and 2 clearly demonstrate that the phosphonic compounds of the invention have a cytotoxic action against colorectal cancer, and are thus useful as anti-cancer agents. (b) Activity of Phosphonic Acids Against Leukemia and Lymphoma
Example 4
Activity of Phosphonic Acids Against a Human Myeloleukemic Cell Line
It has been discovered that the phosphonic acid of the following formula:
Figure imgf000022_0001
O
II
CH, CH, P OH
OH
(IV)
has a cytotoxic effect against HL-60 tumor cells (a human myeloleukemic cell line) in the MTT cell viability assay when the compound is administered as the sodium salt. This effect is indicative of the anti-cancer activity of the above phosphonic acid (compound IV) .
Table 4 shows the effect of 72 hours' exposure to various concentrations of the phosphonic acid of the above formula (compound IV) on the viability of HL-60 cells.
Table 4
MTT Cell Viability Assay of HL-60 Tumor Cells Exposed to Various Concentrations of Phosphonic Acid (Compound IV) for 72 Hours
Phosphonic acid (Na salt) concentration Mean (n = 3) Absorbance (μM) (% of control)
0.0 100
0 1 95
0.2 93
0 4 88
0 8 80
1.6 61
3.2 40
6.4 6
12.8 0
As can be seen from Table 4 , phosphonic acid compounds of the formula shown above (compound IV) are cytotoxic against HL-60 human myeloleukemic cells over 72 hours when administered as the sodium salt . This effect is dose -dependent , and demonstrates the anti -cancer activity of phosphonic acids .
Example 5
Activity of Phosphonic Acids Agsint Mouse Lymphoma
It has been discovered that the phosphonic acid of the following formula:
CH2 O C16H 33
CH OCH3
CH. CH, OH in
(IV)
has a cytotoxic effect against L1210 cells (murine lymphoma) in the MTT cell viability assay when the compound is administered as the sodium salt. This effect is indica¬ tive of the anti-cancer activity of the above phosphonic acid (compound IV) .
Table 5 shows the effect of 72 hours' exposure to various concentrations of the phosphonic acid of the above formula (compound IV) on the viability of L1210 cells.
Table 5
MTT Cell Viability Assay of L1210 Tumor Cells Exposed to Various Concentrations of Phosphonic Acid (Compound IV) for 72 Hours
Phosphonic acid (Na salt) concentration Mean (n = 3) Absorbance (μM) (% of control)
0.0 100
0.1 95
0.2 81
0.4 87
0.8 78
1.6 62
3.2 31 6.4 3
12.8 0
As can be seen from Table 5, phosphonic acid compounds of the formula shown above (compound IV) are cytotoxic against L1210 murine lymphoma over 72 hours when administered as the sodium salt. This effect is dose- dependent, and demonstrates the anti-cancer activity of phosphonic acids.
Examples 4 and 5 clearly demonstrate that the phosphonic compounds of the invention have a cytotoxic action against leukemia and lymphoma, and are thus useful as anti-cancer agents. (c) Activity of Phosphonic Acids Against Melanoma
Example 6
Activity of Phosphonic Acids Against Human Melanoma
It has been discovered that the phosphonic acid compound of the following formula:
CH O C^gH33
CH OCH3
CH, CH, P OH OH
(IV) has a cytotoxic effect against RPMI 7951 cells (human melanoma cell line) in the MTT cell viability assay when the compound is administered as the sodium salt. This effect is indicative of the anti-cancer activity of the above phosphonic acid (compound IV) .
Table 6 shows the effect of 72 hours' exposure to various concentrations of the phosphonic acid of the above formula (compound IV) on the viability of RPMI 7951 cells.
Table 6
MTT Cell Viability Assay of RPMI 7951 Tumor Cells
Exposed to Various Concentrations of
Phosphonic Acid (Compound IV) for 72 Hours
Phosphonic acid (Na salt) concentration Mean (n = 3) Absorbance (μM) (% of control)
0.0 100
0.1 91
0.2 89
0.4 88
0.8 74
1.6 67
3.2 52 6.4 37
12.8 7
As can be seen from Table 6, phosphonic acids of the formula shown above (compound IV) are cytotoxic against RPMI 7951 human melanoma cells over 72 hours when adminis¬ tered as the sodium salt. This effect is dose-dependent, and demonstrates the anti-cancer activity of phosphonic acids.
Example 5 clearly demonstrates that the phos¬ phonic compounds of the invention have a cytotoxic action against human melanoma, and are thus useful as anti-cancer agents .
In addition to the cytotoxicity, compound IV showed to be a potent inhibitor of phosphatidylinositol 3 Kinase (PI3Kinase) as shown in Table 7. Table 7
Effect of Compound IV on the Activity of PI3Kinase as Determined bv Incorporation of 32P to PIP ^2.,
Compound IV concentration(μM) PI3Kinase activity(32P CPm)
Control 4105±205
20 427±28
200 32±7
Inhibitory constants: 1C50 = 10μM; Ki - 2 μM
The PIjKinase activity is determined as the incor- poration of P at the 3-position of the inositol ring of phosphatidylinositol (PI) is in the presence of 32P-ATP.
3. Usage and Dosage
The compounds of the invention are useful as anti-cancer agents, and may be administered safely by either parenteral, oral or topical routes in pharmaceutical preparations such as injections, tablets, capsules, liquid preparations or ointments. These preparations are used by an appropriate route of administration, depending on the specific affliction, patient conditions and other factors. Injections may be given intravenously, intramuscularly, intradermally or subcutaneously. The dose of compound can be selected based on the patient weight, treatment regimen or purpose of administration, generally within the range of 5 to 50 mg/Kg. These compound preparations may be adminis¬ tered 1 to 4 times daily, at 2 to 7 day intervals, or as otherwise necessary to maintain a therapeutic level of the compound in body tissues depending on the specific afflic- tion, patient conditions, treatment regimen or purpose of administration. Injections, intravenous infusions and similar preparations are prepared by conventional methods in either aqueous solution or physiological saline containing 20% propylene glycol and a preservative such as 0.5% ascorbic acid, with an upwardly adjusted pH in phosphate buffer. The drug solution is sterilized by passing it through a 22 μm filter, and distributed into glass vials in approximate¬ ly 1 ml aliquots to provide a unit dosage. The aliquots are then lyophilized, and the vials tightly stoppered and capped to maintain sterility. The drug may be reconsti¬ tuted in the vial by the addition of physiological saline or aqueous diluent.
Tablets are prepared by conventional methods. Unit dosage tablets can be prepared by compressing a mixture of 40 mg of phosphonic acid compound, 200 mg of lactose, and 50 mg Avicel™ into the form of a tablet. A similar drug mixture may also be contained in unit dose within a cellulose-based capsule.
An ointment or cream may also be prepared by conventional methods by mixing the phosphonic acid compound in a commercially-available glycerine-based cream. The cream is applied topically directly to the afflicted area.
The compounds of the invention may also be administered in the form of a liposome. A mixture of phosphonic acid compound and lecithin is mechanically treated to form a bilayer (one side hydrophilic, the other hydrophobic) which spontaneously forms micelles (lipo- somes) . These may be filtered to obtain liposomes of uniform size (approximately 10 nm) and dose (approximately 50 mg/L) . Liposomes are sterilized by filtration through a 22 μm filter, and administered as an intravenous sol- ution. As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.

Claims

WHAT IS CLAIMED IS:
1. A method of treating cancer in a mammal afflicted with cancer, comprising treating the afflicted mammal with a therapeutic amount of a phosphonic compound of the formula:
CH2 T Ri
CH OCH3
I n
CH2 CH2 P R,
I OX
wherein T is an oxygen or sulphur atom, R, is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceuti¬ cally acceptable cation, and either of the opposite stereo- chemical configurations [ (R) or (S)], or a mixture of stereoisomers thereof, and pharmaceutically acceptable salts thereof.
2. A method as claimed in claim 1, wherein T is an oxygen atom, R1 is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
3. A method as claimed in claim 1, wherein T is -an oxygen atom, R1 is an aliphatic chain containing 12 to 20 carbon atoms and 33 hydrogen atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceuti¬ cally acceptable cation, for treatment of colorectal, and lung cancers, or lymphoma, leukemia or melanoma.
4. A method as claimed in claim 1, wherein T is an oxygen atom, R1 is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
5. A method as claimed in claim 1, wherein T is a sulphur atom, R, is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
6. A method as claimed in claim 1, wherein T is a sulphur atom, R., is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms and R2 is OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
7. A method as claimed in claim 1, wherein T is a sulphur atom, R, is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms, R2 is a protein moiety, and X is a hydrogen atom, or a pharmaceutically acceptable cation.
8. A method as claimed in claim 1, wherein the protein moiety is an antibody targeted to a specific antigen or other cellular marker, used in the treatment of cancer.
9. A method as claimed in claim 1, wherein the phosphonic compound, or a pharmaceutically acceptable acid or salt thereof, is administered to the afflicted mammal orally, intravenously, intramuscularly, intradermally, subcutaneously, topically or intravenously in the form of a liposome or other lipid vesicle, with or without a pharmaceutically acceptable carrier.
10. A method as claimed in claim 1, wherein the phosphonic compound is administered to the afflicted mammal at a dosage of 5 to 50 mg/kg body weight.
11. A method of treating colorectal cancer in a mammal afflicted with colorectal cancer, comprising treat¬ ing the afflicted mammal with a therapeutic amount of a phosphonic compound of the formula:
CH2 T R.,
CH OCH3
I o
CH2 CH2 P R2
OX
wherein T is an oxygen or sulphur atom, R,, is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceuti¬ cally acceptable cation, and either of the opposite stereo¬ chemical configurations [ (R) or (S)], or a mixture of stereoisomers thereof, and pharmaceutically acceptable salts thereof.
12. A method as claimed in claim 11, wherein T is an oxygen atom, R1 is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
13. A method as claimed in claim 11, wherein T is an oxygen atom, R1 is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms and R2 is OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
14. A method as claimed in claim 11, wherein T is an oxygen atom, R1 is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms and R2 is a protein moiety, and X is a hydrogen atom, or a pharmaceutically acceptable cation.
15. A method as claimed in claim 11, wherein T is a sulphur atom, R, is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
16. A method as claimed in claim 11, wherein T is a sulphur atom, R, is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms and R2 is OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
17. A method as claimed in claim 11, wherein T is a sulphur atom, R., is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms, R2 is a protein moiety, and X is a hydrogen atom, or a pharmaceutically acceptable cation.
18. A method as claimed in claim 11, wherein the protein moiety is an antibody targeted to a specific antigen or other cellular marker, used in the treatment of colorectal cancer.
19. A method as claimed in claim 11, wherein the phosphonic compound, or a pharmaceutically acceptable acid or salt thereof, is administered to the afflicted mammal orally, intravenously, intramuscularly, intradermally, subcutaneously, topically or intravenously in the form of a liposome or other lipid vesicle, with or without a pharmaceutically acceptable carrier.
20. A method as claimed in claim 11, wherein the phosphonic compound is administered to the afflicted mammal at a dosage of 5 to 50 mg/kg body weight.
21. A method of treating leukemia or lymphoma, melanoma or lung cancer in a mammal afflicted with these cancers, comprising treating the afflicted mammal with a therapeutic amount of a phosphonic compound of the formula: CH,
CH OCH3
O
II
CH, CH, - P -
I OX
wherein T is an oxygen or sulphur atom, R, is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceuti- cally acceptable cation, and either of the opposite stereo¬ chemical configurations [ (R) or (S) ] , or a mixture of stereoisomers thereof, and pharmaceutically acceptable salts thereof.
22. A method as claimed in claim 21, wherein T is an oxygen atom, R, is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
23. A method as claimed in claim 21, wherein T is an oxygen atom, R1 is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms, and R2 is OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
24. A method as claimed in claim 21, wherein T is an oxygen atom, R, is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms, R2 is a protein moiety, and X is a hydrogen atom, or a pharmaceutically acceptable cation.
25. A method as claimed in claim 21, wherein T is a sulphur atom, R1 is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
26. A method as claimed in claim 21, wherein T is a sulphur atom, R, is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms and R2 is OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
27. A method as claimed in claim 21, wherein T is a sulphur atom, R., is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms, R2 is a protein moiety, and X is a hydrogen atom, or a pharmaceutically acceptable cation.
28. A method as claimed in claim 21, wherein the protein moiety is an antibody targeted to a specific antigen or other cellular marker, used in the treatment of leukemia or lymphoma.
29. A method as claimed in claim 21, wherein the phosphonic compound, or a pharmaceutically acceptable acid or salt thereof, is administered to the afflicted mammal orally, intravenously, intramuscularly, intradermally, subcutaneously, topically or intravenously in the form of a liposome or other lipid vesicle, with or without a pharmaceutically acceptable carrier.
30. A method as claimed in claim 21, wherein the phosphonic compound is administered to the afflicted mammal at a dosage of 5 to 50 mg/kg body weight.
31. A method as claimed in claim 21, wherein the phosphonic compound, or a pharmaceutically acceptable acid or salt thereof, is administered directly to the afflicted mammal's bone marrow, blood, blood cells, leukocytes, lymphocytes or other such extracorporeal preparations containing the mammal's diseased cells, with or without a pharmaceutically acceptable carrier.
32. A method of treating melanoma in a mammal af- flicted with melanoma, comprising treating the afflicted mammal with a therapeutic amount of a phosphonic compound of the formula:
CH,
CH OCH3
I II
CH2 CH2 P R.
I OX
wherein T is an oxygen atom, R1 is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceutically acceptable cation, and either of the opposite stereo¬ chemical configurations [ (R) or (S)], or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof.
33. A method as claimed in claim 32, wherein T is an oxygen atom, R,, is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
34. A method as claimed in claim 32, wherein T is an oxygen atom, R., is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms, and R2 is OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
35. A method as claimed in claim 32, wherein T is an oxygen atom, R1 is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms, R2 is a protein moiety, and X is a hydrogen atom, or a pharmaceutically acceptable cation.
36. A method as claimed in claim 32, wherein T is a sulphur atom, R., is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
37. A method as claimed in claim 32, wherein T is a sulphur atom, R, is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms and R2 is OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
38. A method as claimed in claim 32, wherein T is a sulphur atom, R, is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms, R2 is a protein moiety, and X is a hydrogen atom, or a pharmaceutically acceptable cation.
39. A method as claimed in claim 32, wherein the protein moiety is an antibody targeted to a specific antigen or other cellular marker, used in the treatment of melanoma.
40. A method as claimed in claim 32, wherein the phosphonic compound, or a pharmaceutically acceptable acid or salt thereof, is administered to the afflicted mammal orally, intravenously, intramuscularly, intradermally, subcutaneously, topically or intravenously in the form of a liposome or other lipid vesicle, with or without a pharmaceutically acceptable carrier.
41. A method as claimed in claim 32, wherein the phosphonic compound is administered to the afflicted mammal at a dosage of 5 to 50 mg/kg body weight.
42. A method as claimed in claim 1, wherein the phosphonic compound includes either of the opposite stereo¬ chemical configurations [ (R) or (S) ] , or a mixture of stereoisomers.
43. The compound as claimed in claim 1 used as inhibitor of PI3Kinase, and the biological systems that this enzyme is used to prevent a symptom.
44. A phosphonic compound of the formula:
CH, T Ri
CH OCH,
CH, CH, P - I OX
wherein T is an oxygen or sulphur atom, R1 is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceuti¬ cally acceptable cation, and either of the opposite stereo¬ chemical configurations [ (R) or (S) ] , or a mixture of stereoisomers thereof, and pharmaceutically acceptable salts thereof.
45. A compound as claimed in claim 44, wherein T is an oxygen atom, R1 is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceutically acceptable cation.
46. A compound as claimed in claim 44, wherein T is an oxygen atom, R, is an aliphatic chain containing 16 carbon atoms and 33 hydrogen atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceuti- cally acceptable cation, for treatment of colorectal, and lung cancers, or lymphoma, leukemia or melanoma.
47. A compound as claimed in claim 46 wherein R2 is OH and OX is OH.
48. A compound as claimed in claim 44 wherein R is NHR.
49. The use of a compound of the formula:
CH2 T i
CH OCH3 I O
I ii
CH, CH2 P R2
2 I
OX
wherein T is an oxygen or sulphur atom, R1 is an aliphatic chain containing 12 to 20 carbon atoms and R2 is a protein moiety, or OX where X is a hydrogen atom, or a pharmaceuti¬ cally acceptable cation, and either of the opposite stereo¬ chemical configurations [ (R) or (S) ] , or a mixture of stereoisomers thereof, and pharmaceutically acceptable salts thereof, in the treatment of cancer in a mammal afflicted with cancer, comprising administering to the afflicted mammal a therapeutic amount of the compound of the formula.
PCT/CA1995/000068 1995-02-10 1995-02-10 [4-hexadecyl-3-methoxy-butyl] phosphonic acid and its protein conjugates useful as anti-cancer agents WO1996024598A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CA1995/000068 WO1996024598A1 (en) 1995-02-10 1995-02-10 [4-hexadecyl-3-methoxy-butyl] phosphonic acid and its protein conjugates useful as anti-cancer agents
AU15734/95A AU1573495A (en) 1995-02-10 1995-02-10 {4-hexadecyl-3-methoxy-butyl} phosphonic acid and its protienconjugates useful as anti-cancer agents

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CA1995/000068 WO1996024598A1 (en) 1995-02-10 1995-02-10 [4-hexadecyl-3-methoxy-butyl] phosphonic acid and its protein conjugates useful as anti-cancer agents

Publications (1)

Publication Number Publication Date
WO1996024598A1 true WO1996024598A1 (en) 1996-08-15

Family

ID=4173069

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA1995/000068 WO1996024598A1 (en) 1995-02-10 1995-02-10 [4-hexadecyl-3-methoxy-butyl] phosphonic acid and its protein conjugates useful as anti-cancer agents

Country Status (2)

Country Link
AU (1) AU1573495A (en)
WO (1) WO1996024598A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8119126B2 (en) 1998-03-24 2012-02-21 Chugai Seiyaku Kabushiki Kasha Inhibiting vascularization using antibodies to CXCR4 and SDF-1
CN112321446A (en) * 2020-11-25 2021-02-05 华南理工大学 Synthesis method of amide derivative

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0230575A2 (en) * 1985-12-04 1987-08-05 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Medicament active against tumours, containing hexadecylphosphocholine
US5219845A (en) * 1991-04-25 1993-06-15 The University Of British Columbia Phosphonates as anti-inflammation agents

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0230575A2 (en) * 1985-12-04 1987-08-05 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Medicament active against tumours, containing hexadecylphosphocholine
US5219845A (en) * 1991-04-25 1993-06-15 The University Of British Columbia Phosphonates as anti-inflammation agents

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI Z ET AL: "Phosphonate isosteres of phospholipids", TETRAHEDRON LETT. (TELEAY,00404039);93; VOL.34 (22); PP.3539-42, UNIV. RHODE ISLAND;DEP. CHEM.; KINGSTON; 02881; RI; USA (US) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8119126B2 (en) 1998-03-24 2012-02-21 Chugai Seiyaku Kabushiki Kasha Inhibiting vascularization using antibodies to CXCR4 and SDF-1
CN112321446A (en) * 2020-11-25 2021-02-05 华南理工大学 Synthesis method of amide derivative

Also Published As

Publication number Publication date
AU1573495A (en) 1996-08-27

Similar Documents

Publication Publication Date Title
US5776915A (en) Phosphocholines of retinoids
JPH0859677A (en) Alkylphosphocholin derivative and its production
EP1102776A1 (en) Water soluble prodrugs of hindered alcohols or phenols
EP1305322B1 (en) Derivatives of branched-chain lipophilic molecules and uses thereof
US20130190524A1 (en) Cyclitols and their derivatives and their therapeutic applications
AU2001280043A1 (en) Derivatives of branched-chain lipophilic molecules and uses thereof
WO1996024598A1 (en) [4-hexadecyl-3-methoxy-butyl] phosphonic acid and its protein conjugates useful as anti-cancer agents
CA2180754C (en) Heteroaryl-substituted deoxy glycero-phosphoethanolamines
Bittman et al. Isosteric phosphonate analogs of ET-16-OMe. Synthesis and biological evaluation of the enantiomers of 2'-(trimethylammonio) ethyl 4-(hexadecyloxy)-3-methoxybutanephosphonate and 2'-(trimethylammonio) ethyl 4-(hexadecylthio)-3-methoxybutanephosphonate
US4647685A (en) 2-alkoxy-1-((2-trialkylaminoethoxy)phosphinyloxy)-alkenes and alkynes, hydroxy inner salts
US5506217A (en) Phosphonates as anti-cancer agents
EP1578754A2 (en) Antiprotozoal ring-substituted phospholipids
Tsivgoulis et al. A reinvestigation of the synthesis of arsonolipids (2, 3-diacyloxypropylarsonic acids)
FI84832C (en) FOERFARANDE FOER FRAMSTAELLNING AV NYA FOSFO-FOERENINGAR.
FI65261B (en) FOERFARANDE FOER FRAMSTAELLNING AV FARMAKOLOGISKT ANVAENDBART DINATRIUMSALT AV N- (FOSFONOACETYL) -L-ASPARAGINSYRA
US20060116354A1 (en) Antineoplastic ether lipid compounds with modifications at the sn-3 carbon
EP1587513A2 (en) Antineoplastic ether lipid compounds with modifications at the sn-2 carbon
WO2004062596A2 (en) Antineoplastic ether lipid compounds having a four carbon backbone
EP0581793A1 (en) Phosphonates and phosphinates as anti-cancer, -inflammatory, -allergy and -myocarditis agents
EP0171968A1 (en) Use of a phospholipid derivative
US20020173489A1 (en) Compositions containing a mixture of phosphorous compounds and alkylglycerols
EP1583552A2 (en) Antineoplastic ether lipid compounds
WO2004062595A2 (en) Antineoplastic ether lipid compounds with modifications at the sn-1 carbon
EP1421941A1 (en) Phospholipid derivatives having a polyhydroxylated aliphatic carbocycle
CA2438414A1 (en) Enantiomers of unsaturated alkyllysophosphonocholines and use as anti-neoplastics

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU JP KE KG KP KR KZ LK LR LT LU LV MD MG MN MW MX NL NO NZ PL PT RO RU SD SE SI SK TJ TT UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase