GB2278843A - Anti-cancer compounds - Google Patents
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- GB2278843A GB2278843A GB9410907A GB9410907A GB2278843A GB 2278843 A GB2278843 A GB 2278843A GB 9410907 A GB9410907 A GB 9410907A GB 9410907 A GB9410907 A GB 9410907A GB 2278843 A GB2278843 A GB 2278843A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/24—Condensed ring systems having three or more rings
- C07H15/252—Naphthacene radicals, e.g. daunomycins, adriamycins
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- 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
- A61K47/54—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 organic compound
- A61K47/55—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 organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
Abstract
Novel compounds comprising an antioestrogenic moiety linked to an antitumour moiety by means of a spacer group are characterised in that the spacer group is a minimum of 10 atoms in length excluding any atoms from the antioestrogenic moiety or the antitumour moiety which contribute to the linkage. The antitumour moiety has increased specificity. Compounds which are biological breakdown products thereof, including at least part of the spacer group, are also novel.
Description
ANT1NCER R COMPOUNDS FIELD OF THE INVENTION
This invention relates to novel compounds and their use as chemotherapeutic agents.
BACKGROUND TO THE INVENTION
The number of chemotherapy agents of use in the treatment of malignant tumours has expanded rapidly. The diversity of tumours has led to the development of a wide range of chemotherapeutic agents of varying specificity. For example many agents designed to be active at steroid hormone receptors are cytotoxic to cells not bearing those receptors. Similarly antitumour agents such as anthracyclines are often active at non-tumourous cells. It is the lack of specificity of many such agents that leads to the undesirable side effects associated with chemotherapy. These side effects often result in a patient taking a combination of chemotherapeutic agents together with other drugs to minimise the side effects. There is therefore a need for more specific chemotherapeuti c agents.
SUMMARY OF INVENTION
The present invention provides novel compounds of use as chemotherapeutic agents that seek to overcome the drawbacks of prior art chemotherapeutic agents, and provide an improved specificity of chemotherapeutic activity.
Accordingly, the invention relates to compounds comprising an antioestrogenic moiety linked to an anti tumour moiety by means of a spacer group, characterised in that the spacer group is a minimum of 10 atoms in length excluding any atoms from the anti-oestrogenic moiety or the anti tumour moiety which contribute to the linkage.
The term "antioestrogenic moiety" is used herein to refer to a chemical that has activity at an oestrogen receptor that results in an inhibition of protein biosynthesis.
The term "anti tumour agent" is used herein to refer to a chemical having direct or indirect antimitotic activity, other than by activity at an oestrogen receptor. Such an agent may be an alkylating agent, an intercalating agent or an antimetabolite, e.g. an antifolate, etc. A wide range of such agents is discussed in general in The Oxford Textbook of Medicine, 2nd
Edition, pages 4.131 to 4.139 (Oxford University Press).
More particularly the invention relates to compounds wherein the antioestrogenic moiety is selected from the group consisting of compounds of general formula (I)
wherein R1 is hydrogen or hydroxy, R2 is hydrogen or C14 alkyl,
R3 is unsubstituted aliphatic C14 alkyl or aliphatic C14 alkyl substituted by one or more halogen, nitro, amino, aldehyde, keto, hydrazino or alcohol groups, R4 is halogeno and R5 and R6 are hydrogen or aliphatic C14 alkyl, R5 and R6 being the same or different; or a 2,3-diphenylindole, 2-benzoyl-3-phenylthiophene, an acetoxy-substituted triarylethene, a 1,2-diphenylethane or a 2-phenylindene, and the anti tumour moiety is selected from the group consisting of anthracycline drugs such as the compounds doxorubicin, daunorubicin, epirubicin, idarubicin and also mitoxantrone and the compounds methotrexate, aclacinomycin A,
DUP-941, cyclophosphamide and ifosphamide.
It has been found that when an antloestrogenic agent is linked to an anti tumour agent by means of a spacer, there is an increase in the specificity of the anti tumour moiety. The length of the spacer is important in contributing to this effect. It is this observation that lies at the heart of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Preferred compounds of the present invention are those wherein the antloestrogenic agent is tamoxifen, 2-methyl-4-hydroxytamoxi fen, 4-lodotamoxifen, 4-fl uorotamoxi fen or 4-hydroxytamoxifen and the antitumour agent is doxorubicin, methotrexate or mitoxantrone. Most preferably the antloestrogenic moiety is tamoxifen or 4-hydroxytamoxifen.
Any chemical group can be used as a spacer group to link the antloestrogenic and anti tumour agents. The length of the spacer group must be a minimum of 10 atoms in length. This excludes any atoms contributing to the linkage which derive from the anti tumour moiety or the antioestrogenic moiety. The maximum length of the spacer group is not as important. The length of the spacer is preferably a maxlmum of 22 or 24 atoms in length and more preferably of between 12 and 18 atoms in length, preferably between 13 or 16 atoms in length and more preferably 14 atoms in length.The spacer group may be made up from a spacer moiety and separate linker moieties at either end of the spacer molety that chemically link the spacer moiety to the antloestrogenic and antitumour moieties. In this case, where there are lanker moietles, the number of atoms in the linker moiety or moieties contributes to the number of atoms when calculating the length of the spacer group.Suitable spacer moieties include Michael Addition spacers, for example, glutaraldehyde amidinium, hydrocarbons, oligopeptides and polypeptides including peptide polymers, oligosaccharides and polysaccharides, for example, dextran or glycogen, polymers such as polyalkylcyanoacrylates and proteins such as bovine or human albumin, lmmunoglobulins, monoclonal antibodies, ferritin, catalase or superoxide dismutase.
Sultable linker moieties include the following chemical groups: dlazonium, hydrazone, hemiacetal, hemiketal, acetal, ketal, hemisuccinate, carboxymethylamine, oxazol idine, sulphydryl, cystamine, carbon-ester, phosphate-ester, thioester, thloether, imine (Schiff base), ether or N-hydroxy succinamide ester. The lanker molety can be the same or different at either end of the spacer. Preferably, however, the spacer group does not require separate lanker moieties and links directly to the active moieties, for example being a dicarboxylic acid, a polyawino acid or a polysaccharide. Preferred examples of a dicarboxylic acid are those of the formula HOOC(CH2)nCOOH where n is from 8 to 20 (providing a spacer group from 10 to 22 atoms), preferably 10-16 and more preferably 12, of a polyamino acid is polyglutamic acid or polylysine and of a polysaccharide is dextran.
Preferably the spacer group makes an ester or amide bond linkage with the antloestrogenic and anti tumour agent. Most preferable is a spacer group that is a dicarboxylic acid of the formula HOOC(CH2)nCOOH where n is 8 or 12. An example of a preferred compound of the invention is that of formula (11)
In this compound the antloestrogenic moiety is 4-hydroxytamoxifen and the anti tumour agent is doxorubicin. The spacer group is a dicarboxylic acid of the formula HOOC(CH2)12COOH making an ester bond linkage with the 4-hydroxytamoxifen and an amide bond linkage with doxorubicin.
A further aspect of the present invention includes compounds that are the biological breakdown products of compounds comprising an antioestrogenic moiety linked to an antitumour moiety, with the proviso that the biological breakdown product includes at least part of the spacer group.
The term "biological breakdown product" is used herein to refer to any product that may result from the hydrolysis, spontaneous or enzymatic breakdown of a compound of the present invention.
Thus, the present invention further relates to compounds comprising an antioestrogenic moiety or an antitumour moiety linked to all or part of a spacer group.
By way of example with reference to the compound of formula (it), biological breakdown products may include the compounds of formulae (III) and (IV)
All the compounds of the present invention are believed to be novel.
The invention further includes the use of these compounds in therapy, particularly in the treatment of a cancer.
The compounds of the present invention may be formulated with a physiologically acceptable diluent or carrier for use as pharmaceuticals for both veterinary, for example in mammals, and particularly human use by a variety of methods. For instance, they may be applied as a composition incorporating a liquid diluent or carrier, for example an aqueous or oily solution, suspension or emulsion, which may often be employed in injectable form for parenteral administration and therefore may conveniently be sterile and pyrogen free. Oral administration may also be used and although compositions for this purpose may incorporate a liquid diluent or carrier, it is more usual to use a solid, for example a conventional solid carrier material such as starch, lactose, dextrin or magnesium stearate.Such solid compositions may take the form of powders but are more conveniently of a formed type, for example as tablets, cachets, or capsules (including spansules). Alternative, more specialized types of formulation include liposomes and nanoparticles.
Other types of administration than by injection or through the oral route which are of use in both human and veterinary contexts include the use of suppositories or pessaries. Another form of pharmaceutical composition is one for buccal or nasal administration. Other formulations for topical administration include lotions, ointments, creams, gels and sprays.
Compositions may be formulated in unit dosage form, 1.e. in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
Whilst the dosage of the compound used will vary according to the activity of the particular compound and the condition being treated, it may be stated by way of guidance that a dosage selected in the range from 10 to 500 mg/kg per body weight per day.
All the antioestrogenic and anti tumour moieties of use in forming the compounds of the present invention must possess an accessible group capable of linking to the spacer group, e.g. the hydroxy group of 4-hydroxytamoxifen, the amino group of the amino sugar daunosamine of doxorubicin. For the antioestrogen moiety tamoxifen and others like it not having an immediately accessible group for linkage to a spacer, the skilled man will be capable of identifying a "non-essential" side group, i.e. a side chain that may be replaced without detracting from the biological activity of the moiety concerned, for example the ethyl group of tamoxifen, that can be modified to provide an accessible group by replacement with an aminomethyl group providing the possibility of an amide linkage bond between tamoxifen and a spacer.
The invention will now be demonstrated in the following
Examples.
ABBREVIATIONS USED
DoxoTam n : A compound according to the invention comprising
doxorubicin (Doxo) and 4-hydroxytamoxifen (Tam) linked by an HOOC(CH2)nCOOH spacer group, where n is 2, 8 or 12.
Tam n : A 4-hydroxytamoxifen derivative linked to
HOOC(CH2)nCOOH where n is 2, 8 or 12.
Doxorester n : A doxorubicin derivative linked to HOOC(CH2)nCOOH where n is 2, 8 or 12.
EXAMPLE 1
Svnthesis of Tamoxifen and Doxorubicin esters
I. SYNTHESIS OF TAMOXIFEN ESTERS
Z-4-Hydroxytamoxifen was prepared as described in McCague R.
(1986) J. Chem. Res. (S) 58-59 and J. Chem. Res. (M) 0771-0793.
(a) Svnthesis of Z-4-Hvdroxvtamoxifen Succinovl Ester (Tam 2)
Z-4-Hydroxytamoxifen (5 mg, 12.9 pmol) was dissolved in pyridine (10 ml) and succinyl chloride (4 p1, 37.9 pmol) was added and the mixture was stirred at room temperature for 2 hours. The pyridine was evaporated using compressed air and the residual solid redissolved in phosphate buffer pH 7.0. The compound was separated by column chromatography (2.5 cm x 15 cm, silica gel column, eluted with 25% ethyl acetate in hexane, triethylamine, glacial acetic acid (100:5:5)), the resulting white solid (approximately 3 mg, 6.16 pmol, 47.8%) had an Rf of 0.78 slightly less than Z-4-hydroxytamoxifen (0.83) using normal phase TLC with a mobile phase of acetoneltriethylamine (20:1).
Analysis: Mpt : decomposition at approximately 1850C.
IR : 3300-3800 cm-l (COOH), 1660 cm-l (C=0), 1020-1340 cm-l (ethoxy), 3000-3100 cm-l (C-H, aromatic), 1600 cm-l (C=C, aromatic).
1H NMR (CDC13) : (270 mHz) 0.97 (3H, t, CH2CH3), 1.13 (EtOH), 2.50 (2H, q, CH2CH3), 2.74 (2H, t, OCH2CH2N), 2.82 (6H, s, NMe2), 2.88 (2H, d, CH2CH2COOH), 2.94 (2H, d, CH2CH2COOH), 3.01 (EtOH), 3.88 (2H, q, OCH2CH2N), 6.78 (2H, d, meta to OCH2CH2N), 7.00 (2H, d, ortho to OCH2CH2N), 7.07-7.59 (remaining ArH), 10.10 (1H, s, CH2CH2COOH, D20 exchangeable).
Mass Spectrum: m/e (relative intensity) 474 (40, C30H30N03), 72 (15, C4H1ON), 58 (51, C3H8N).
Elemental Analysis: Found C, 71.45%; H, 8.06%; N, 4.90%, calculated for C30H31N05 C, 76.59X; H, 6.60X; N, 2.982.
(b) Svnthesis of Z-4-Hvdroxvtamoxifen Sebacovl Ester (Tam 8)
Z-4-Hydroxytamoxifen (50 mg, 129 pmol) was dissolved in pyridine (10 ml) and sebacoyl chloride (30 p1, 141 pmol) added.
The mixture was stirred at room temperature for 8 hours, then the pyridine was removed using compressed air. The residual solid was redissolved In 25X ethyl acetate in hexane and separated using normal phase column chromatography (2.5 cm x 15 cm, silica gel for flash chromatography, eluted with 25% ethyl acetate in hexane, triethylamine, glacial acetic acid (100:5:5)). The ester (30 mg, 52.7 pmol, 40.81 has a retention factor (Rf 0.10) less than Z-4-hydroxytamoxifen (Rf 0.44) and so eluted after the parent compound.
Analysis: Mpt : 46-480C.
IR : 3300-3800 cm-l (COOH), 1660 cm-l (C.O), 1020-1340 cm-l (ethoxy).
1H NMR (CDC13) : (270 mHz) 0.98 (3H, t, CH2CH3), 1.21 (EtOH), 2.17 (6H, s, NMe2), 2.28 (2H, s, (CH2)8), 2.52 (2H, q, CH2CH3), 2.73 (2H, t, OCH2CH2N), 2.49-2.73 (m, (CH2)8), 3.87 (2H, q,
OCH2CH2N), 6.75 (2H, d, meta to OCH2CH2N), 6.97 (2H, d, ortho to
OCH2CH2N), 7.16-7.57 (remaining ArH), 9.50 (1H, s, CH2CH2COOH,
D20 exchangeable).
Mass Spectrum: m/e (relative intensity) 554 (1, M), 496 (1, C33H36N04), 387 (1, C26H29N02), 58 (105, C3H6N).
Elemental Analysis: Found C, 86.64%; H, 10.50Z; N, 5.05X,
calculated for C36H43N05 C, 77.98%; H, 7.76Z; N, 2.53X.
(c) Svnthesis of Z-4-Hvdroxvtamoxifen Dodecanediovl Ester (Tam 12)
Z-4-Hydroxytamoxifen (50 mg, 129 pmol) was dissolved in pyridine (10 ml) and dodecanedioyl chloride (30 p1, 120 pmol) added. The mixture was stirred at room temperature for 8 hours.
The pyridine was then removed using compressed air. The residual solid was redissolved in 25% ethyl acetate in hexane and separated using normal phase column chromatography (2.5 cm x 15 cm, silica gel for flash chromatography, eluted with 25% ethyl acetate in hexane, triethylamine, glacial acetic acid (100:5:5)). The ester (35 mg, 55.8 mol, 48.8X) has a retention factor (Rf 0.12) less than Z-4-hydroxytamoxifen (Rf 0.44) and so eluted after the parent compound.
Analysis: Mpt : 49-510C.
IR : 3300-3800 cm-l (COOH), 1660 cm-l (C-O), 1020-1340 cm-l (ethoxy).
1H NMR (CDC13) : (270 mHz) 0.97 (3H, t, CH2CH3), 1.12 (EtOH), 2.23 (6H, s, NMe2), 2.33 (2H, s, (CH2)12), 2.48 (2H, t, CH2CH), 2.73 (2H, t, OCH2CH2N), 2.50-2.84 (2H, m, (CH2)12), 3.87 (2H, q,
OCH2CH2N), 6.72 (2H, d, meta to OCH2CH2N), 6.99 (2H, d, ortho to
OCH2CH2N), 7.06-7.57 (remaining ArH), 7.88 (1H, s, CH2CH2COOH,
D20 exchangeable).
Mass Spectrum: m/e (relative intensity) 628 (1, M), 570 (1, C33H37N05), 387 (1, C26H29N02), 58 (105, C3H6N).
Elemental Analysis: Found C, 72.40Z; H, 9.32%; N, 3.84%, calculated for C40H53N05 C, 76.40%; H, 8.40X; N, 2.30X.
11. SYNTHESIS OF DOXORUBICIN ESTERS (a) Svnthesis of Doxorubicin Succinovl Ester (Doxorester 2) Doxorubicln HC1 (8 mg, 12.8 pmol) was dissolved in dichloromethane (10 ml). Succinyl chloride (5 p1, 47.35 pmol) was added with l-ethyl-3-(3'-dimethylaminopropyl) carbodilmide (ECDI) (14 mg, 70 pmol). The mixture was stirred at room temperature for 4 hours. Any unreacted doxorubicin was removed by extraction with phosphate buffer, pH 5.0 (at which pH the unreacted doxorubicin will be ionized). The dichloromethane was evaporated to give a red solid which was then recrystallised from chloroform.The compound (5 mg, 8.5 pmol, 66.7X) has an Rf of 0.93 compared to an Rf of 0.46 for doxorubicin using normal phase
TLC with a mobile phase of chloroformlmethanolltriethylamine (70:10:1).
Analysis: Mpt : 210-2120C.
IR : 3000 cm-l (alkyl), 1700-1750 cm-l (C=O),
3300-3800 cm-l (COOH).
1H NMR (CDCl3) : (270 mHz) 1.12 (3H, d, CH3, C5'), 1.91 (2H, s,
C2'), 2.20 (2H, s, C9), 2.67 (2H, s, C7), 1.80/2.40 (2H, d, (CH2)2), 3.20 (1H, s, CH, C3'), 3.40 (1H, s, CH, C4'), 3.70 (3H, s, CH30), 3.82 (1H, q, CH, C5'), 4.26 (2H, s, C14), 4.54 (1H, s, C10), 4.99 (1H, s, Cl'), 7.23 (1H, t, C3), 7.26 (2H, d, C2, C4), 13.08/13.85 (2H, s, OH, (C6, Cll) D20 exchangeable), 9.37 (1H, S, COOH, D20 exchangeable).
Mass Spectrum: m/e (relative intensity) 627 (1, M), 158 (19, C6H8N04), 129 (35, C5H7N03), 115 (51, C4H5N03), 101 (5, C4H503).
Elemental Analysis: Found C, 60.21X; H, 4.80%; N, 2.45%, calculated for C31H31NO14 C, 59.33%; H, 4.94%; N, 2.23.
(b) Synthesis of Doxorubicin Sebacoyl Ester (Doxorester 8)
Doxorubicin HC1 (8 mg, 12.8 pmol) was dissolved in dichloromethane (10 ml). Sebacoyl chloride (5 %l, 229 pmol) was added with l-ethyl-3-(3'-dimethylaminopropyl) carbodiimide (ECDI) (14 mg, 70 pmol). The mixture was stirred at room temperature for 4 hours. Any unreacted doxorubicin was removed by extraction with phosphate buffer, pH 5.0 (at which the unreacted doxorubicin will be ionised). The dichloromethane was evaporated to give a red solid, which was recrystallised from chloroform. The compound (4 mg, 5.52 pmol, 43.1%) has an Rf of 0.64 compared to an Rf of 0.46 for doxorubicin using normal phase TLC with a mobile phase of chloroformlmethanolltriethylamine (70:10:1).
Analysis: Mpt : 214-2150C.
IR : 3000 cm- (alkyl), 1700-1750 cm- (C=O),
3300-3800 cm1 (COOH).
1H NMR (CDCl3) : (270 mHz) 1.18 (3H, d, CH3, CS'), 1.61 (2H, s, C2'), 2.17 (2H, s, C9), 2.56 (2H, s, C7), 2.10-2.49 (2H, m, (CH2)8), 3.23 (1H, s, CH, C3'), 3.49 (1H, s, CH, C4'), 3.66 (3H, s, CH30), 3.85 (1H, q, CH, C5'), 4.11 (2H, s, C14), 4.81 (1H, s, C10), 5.07 (1H, s, C1'), 6.85 (1H, t, C3), 7.26 (2H, d, C2, C4), 13.85/13.08 (2H, s, OH, (C6, Cll) D20 exchangeable), 9.19 (1H, s, COOH, D20 exchangeable).
Mass Spectrum: m/e (relative intensity) 711-(1, M), 158 (24, C6H8N04), 129 (43, C5H7N03), 115 (43, C4H5N03), 101 (9, C4H503).
Elemental Analysis: Found C, 62.31Z; H, 6.21Z; N, 1.82%, calculated for C37H43N014 C, 62.45%; H, 6.05X; N, 1.97%.
(c) Svnthesis of Doxorubicin Dodecanedioyl Ester (Doxorester 12)
Doxorubicin HC1 (8 mg, 12.8 pmol) was dissolved in dichloromethane (10 ml). Dodecanedloyl chloride (5 rl, 200 pmol) was added with l-ethyl-3-(3'-dimethylaminopropyl) carbodilmide (ECDI) (14 mg, 70 pmol). The mixture was stirred at room temperature for 4 hours. Any unreacted doxorubicin was removed by extraction with phosphate buffer, pH 5.0. The dichloromethane was evaporated to give a red solid, which was recrystallised from chloroform. The compound (6 mg, 7.68 pmol, 60.0X) has an Rf of 0.63 compared to an Rf of 0.46 for doxorubicin using normal phase TLC with a mobile phase of chloroformlmethanolltriethylamine (70:10:1).
Analysis: Mpt : 230-235 C.
IR : 3000 cm-l (alkyl), 1700-1750 cm-l (C.O), 3300-3800 cml (COOH).
1H NMR (CDC13) : (270 mHz) 1.18 (3H, d, CH3, C5'), 1.61 (2H, s, C2'), 2.20 (2H, s, C9), 2.57 (2H, s, C9), 1.87-3.17 (2H, m, (CH2)12), 3.33 (1H, s, CH, C3'), 3.49 (1H, s, CH, C4'), 3.66 (3H, s, CH30), 3.79 (1H, q, CH, C5'), 4.10 (2H, s, C14), 4.97 (1H, s, ClO), 5.25 (1H, s, C1'), 6.83 (1H, t, C3), 7.26 (2H, d, C2, C4), 13.85/13.08 (2H, s, OH, (C6, Cll) D20 exchangeable), 9.20 (1H, s, COOH, D20 exchangeable).
Mass Spectrum: m/e (relative intensity) 767 (1, M), 158 (24, C6H8N04), 129 (43, C5H7N03), 115 (43, C4H5N03), 101 (9, C4H503).
Elemental Analysis: Found C, 63.71%; H, 6.53X; N, 1.65Z, calculated for C41H51N014 C, 64.15%; H, 5.34%; N, 2.58%.
EXAMPLE 2
Svnthesis of Z-4-Hvdroxvtamoxifen - Doxorubicin Prodruas I. SYNTHESIS OF Z-4-HYDROXYTAMOXIFEN SUCCINOYL ESTER
DOXORUBICIN PRODRUG (DoXOTAM 2)
Z-4-Hydroxytamoxifen succinoyl ester (Tam 2, 5 mg, 10.2 pmol) was dissolved in phosphate buffer pH 7.0 (10 ml). Doxorubicin
HC1 (5 mg, 8 pmol) in 3 ml distilled water was added with l-ethyl-3- (3'-dimethylaminopropyl)carbodiimide (ECDI) (14 mg, 70 pmol). The mixture was stirred at room temperature for 4 hours.The product was removed by extraction from phosphate buffer, pH 5.0 (at which unreacted doxorubicin will be lonised) into dichloromethane which was evaporated to give a pink solid (4 mg, 3.95 pmol, 49.4Z) which was recrystallised from methanol. HPLC studies using 35% acetonitrile, 0.02M NaH2P04, 0.05% triethylamine, pH 4.0 on a C18 reverse phase column (10 cm) showed a peak with a retention time of about 2.30 minutes. After acid hydrolysis of the product (gentle heat at pH 4.0 for 10 minutes) there was a peak at approximately 6.69 minutes corresponding to doxorubicin. Normal phase TLC with a mobile phase of acetoneltriethylamine (20:1) also showed a component in the acid hydrolysed product corresponding to 4-hydroxytamoxifen.
Analysis: Mpt : 162-1650C.
IR : 1612 1, 1587 cm-l 1282 cm-, 1204 cm-l 1010 cm-, 990 cm- (Doxorubicin).
1H NMR (CDC13) : (270 mHz) 0.89 (3H, t,m CH2CH3), 1.15 (3H. d. CH3. C5'), 1.25 (s, EtOH), 1.91 (2H. s. C2'), 2.03 (6H, s, NMe2), 2.20 (2H. s. C9), 1.80 (2H, d, CH2CH2), 2.22 (2H, d, CH2CH2), 2.50 (2H, q, CH2CH3), 2.74 (2H, t, OCH2CH2N), 2.67 (2H. s. C7), 3.32 (1H. s. CH. 3'), 3.34 (1H. s.
CH. 4'), 3.80 (2H, q, OCH2CH2N), 3.60 (3H. s. CH30), 3.82 (1H. C5'), 4.24 (2H. C14), 4.39 (1H. C10), 4.99 (1H. s. C1'), 6.89 (2H, d, meta to OCH2CH2N), 7.18-7.49 remaining H,
(underlining indicates signals due to doxorubicin).
Mass Spectrum: m/e (relative intensity) 629 (C31H35N013,51), 570 (C28H30NO12,14), 558 (C34H42N205,23), 542 (C27H30N011,2), 430 (C28H32NO2,34), 402 (C26H28N02,22), 356 (ClgHl607,5), 282 (ClgH24NO,3) Elemental Analysis: Found C, 66.40%; H, 5.46X; N, 2.52X, calculated for C57H60N2 15 C, 67.502; H, 5.92%; N, 2.76%.
II. SYNTHESIS OF Z-4-HYDROXYTAMOXIFEN SEBACOYL ESTER
DOXORUBICIN PRODRUG (DOXOTAM 8)
Z-4-Hydroxytamoxifen sebacoyl ester (Tam 8, 5 mg, 4.6 pmol) was dissolved in dichloromethane (10 ml). Doxorubicin HC1 (5 mg, 8 pmol) was added with N,N' dicyclohexylcarbodiimide (10 mg, 48 pmol). The mixture was stirred at room temperature for 4 hours. Unreacted doxorubicin was removed by extraction with pH 5.0 phosphate buffer and the product recrystallised from methanol to give a red solid. TLC studies using normal phase silica gel plastic backed plates with a mobile phase of chloroform:methanol:triethylamine (70:10:1) showed a component (4 mg, 3.65 pmol, 45.65%) (Rf 0.65) significantly different from doxorubicin (Rf 0.46).
Analysis: Mpt : 181-1840C.
IR : 1612 cm-, 1587 cm-, 1282 cm- 1204 cm-
1010 cm-, 990 cm- (Doxorubicin).
1H NMR (CDC13) : (270 mHz) 0.91 (3H, t, CH2CH3), 1.17 (3H. d. CH3. C5'), 1.67 (2H. s. C2'), 1.90 (6H, s, NMe2), 2.05 (2H. s. C9'), 2.17 (2H, s, (CH2)8), 2.49 (2H. s. C7), 2.29-2.81 (2H, m, (CH2)8), 2.78 (2H, t, OCH2CH2N), 2.51 (2H, t,
CH2CH3), 3.20 (1H. s. C3'), 3.76 (1H. q. C5'), 3.54 (1H. s. C4'), 3.84 (1H. q. C5'), 3.85 (2H, q, OCH2CH2N), 4.19 (2H. s. C14), 4.53 (1H. s. ClO), 5.63 (1H. s. Cl'), 6.69 (2H, d, meta to 0CH2CH2N), 6.78 (1H. t. C3), 6.82-7.31 remaining H,
(underlining indicates signals due to doxorubicin).
Mass Spectrum: m/e (relative intensity) 1040 (C6oH66N015,67), 697 (C36H43N013,33).
Elemental Analysis: Found C, 68.10X; H, 6.41%; N, 2.21%,
calculated for C63H72N2015 C, 68.90%; H, 6.56Z; N, 2.55%.
III. SYNTHESIS OF Z-4-HYDROXYTAMOXIFEN DODECANEDIOYL ESTER
DOXORUBICIN PRODRUG (DOXOTAM 12)
Z-4-Hydroxytamoxifen dodecanedloyl ester (Tam 12, 5 mg, 4.3 pmol) was dissolved in dichloromethane (10 ml). Doxorubicin
HCl (5 mg, 8 pmol) was added with N,N' dicyclohexylcarbodiimide (10 mg, 48 pmol). The mixture was stirred at room temperature for 4 hours.
Unreacted doxorubicin was removed by extraction with pH 5.0 phosphate buffer and the product recrystallised from methanol to give a red solid. TLC studies using normal phase silica gel plastic backed plates with a mobile phase of chloroform:methanol: triethylamine (70:10:1) showed a component (3 mg, 2.60 pmol, 32.5%) (Rf 0.56) significantly different from doxorubicin (Rf 0.46).
Analysis: Mpt : 203-204 C.
IR : 1612 cm-, 1587 cm-, 1282 cm-, 1204 cm'l 1010 cm'l, 990 cm-l (Doxorubicin).
1H NMR (CDC13) : (270 mHz) 0.91 (3H, t, CH2CH3), 1.22 (3H. d. C5'), 1.59 (2H. s. C2'), 2.22 (2H. s. C9), 2.29 (6H, s, NMe2), 2.36 (2H, s, (CH2)12), 2.45 (2H, t, CH2CH3), 2.56 (2H. s. C7), 2.65 (2H, t, OCH2CH2N), 1.90-2.86 (2H, m, (CH2)12), 3.19 (1H. s. C3'), 3.46 (1H. s. C4'), 3.61(3H.s.
CH3O), 3.75 (1H. g. C5'), 3.93 (2H, q, OCH2CH2N), 4.08 (2H. s.
C14), 4.88 (1H. s. C10), 5.29 (1H, s. Cl'), 6.58 (2H, d, meta to
OCH2CH2N), 6.76 (1H. t. C3), 6.85-7.26 remaining H,
(underlining indicates signals due to doxorubicin).
Mass Spectrum: m/e (relative intensity) 1098 (C64H76N015,33), 755 (C40H53N013,43).
Elemental Analysis: Found C, 70.51Z; H, 7.29%; N, 3.51X, calculated for C67H80N2015 C, 69.70Z; H, 6.93%; N, 2.43X.
EXAMPLE 3
In vitro testing The compounds prepared in Examples 1 and 2 above were tested in Th vitro culture assays on an oestrogen receptor positive cell line MCF-7 (Soule, H.D. et al. (1973) J. Natl. Cancer Inst., 51, 1409-1416) and an oestrogen receptor negative cell line HS05787 (Hackett, A.a. it al. (1977) J. Natl. Cancer Inst., 58, 1795-1806). These were performed alongside controls of doxorubicin HC1 and 4-hydroxytamoxifen alone and in combination.
The cell lines MCF-7 and HS-0578T were routinely maintained as monolayer cultures in RPMI 1640 (Gibco) cell culture medium supplemented with foetal calf serum (10%), sodium pyruvate (1 mM) penicillinlstreptomycin (50 I.U. ml-l of each), and buffered with
HEPES (25 mM). The cells were incubated at 370C in an atmosphere of 95X airy5% carbon dioxide.
Cvtotoxicitv studies
Cells were harvested from exponentially growing cultures via trypsinisation, counted with a haemocytometer, and diluted to produce appropriate suspensions. An aliquot of these cells (5 x 103 cells) were plated into each of 96 wells of a microtitre plate and incubated for 24 hours to ensure logarithmic growth.
The cells were exposed for 96 hours to drug concentrations in the range 0.002 to 25.000 pM, and cytotoxicity was assessed by means of an MTT assay (jabbar, S.A.B. it al., Br. J. Cancer, 60, 523-528). The results were expressed as IC50 values with respect to untreated control cells.
RESULTS
Table 1 below shows the IC50 (pM) values obtained for the compounds tested
Table 1 IC5O pM ER+ ER- MCF-7 HS0578T
Doxorubicin 0.173 0.220
4-Hydroxytamoxifen 0.218 NA
DoxoTam 12 1.760 > 25
DoxoTam 8 17.88 NA
DoxoTam 2 0.252 0.365
Tam 12 NA NA
Tam 8 NA NA
Tam 2 3.532 NA
Doxo 12 0.136 0.198
Doxo 8 0.303 0.370
Doxo 2 0.224 0.188
NA = Not Applicable
These results show that the prodrugs doxotam 12 and doxotam 8 were selectively toxic to the ER+ cells whereas these prodrugs were inactive against the ER negative cell lines tested. Doxotam 2, on the other hand was unselective in its toxicity to both the MCF-7 and HS0578T cell lines, hence supporting the invention that the linker must be at a minimum length.
EXAMPLE 4
In vivo activity of Doxotam 12 in female nude Balb/C mice implanted with MCF-7 tumour cells.
Studies on the in vivo effects of Doxotam 12 have initially shown that this prodrug is remarkably non-toxic to rodents. The
LD50 value for doxorubicin is 10 mg kg-l by intravenous route in mice, hence initial toxicity studies with Balb/C nude mice (mean weight 30g, three mice per dose level) used escalating doses of
Doxotam 12 by intravenous route up to a maximum dose of 20 mg kg-l (since this contains the equivalent of 10 mg kg-1 doxurubicin).
The mice suffered no apparent toxic effects from the highest dose administered, hence a new escalating regime was used up to 400 mg kg-l were well tolerated by the mice.
In a parallel study, attempts were made to implant MCF-7 breast tumour spheroid cells by subcutaneous route into the right flank of immunodeficient female Balb/C nude mice. Initial attempts to implant tumour were unsuccessful, and it was suspected that these oestrogen receptor (ER) positive cells require oestrogen supplemention for growth. When subcutaneous oestradiol pellets were used, MCF-7 tumour growth in Balb/C mice was achieved. This enabled a study on the effects of Doxotam 12 on MCF-7 tumour bearing mice to be made.
Eleven Balb/C (30g) female nude mice were subcutaneously implanted with pellets containing 17ss-oestradiol (0.72 mg,
Innovative Research of America). A suspension containing spheroidal clumps of cultured MCF-7 cells (106 ml-l ) were innoculated into the right flank of these mice. The mice were left for a sufficient time period (38 days) for a tumour mass to be detected. The tumour mass was assessed by weekly caliper measurements, and the values for the relative tumour volume recorded (compared to volume at first detection). The mice were divided into two groups: in the first group six mice were used as controls, and were intravenously administered vla the tail vein with the solvent used for prodrug solution (10X dimethyl sulphoxide in 0.97e NaCl); in the second group five animals were treated with prodrug in solvent, and were administered with a single dose of 20 mg kg- Doxotam 12 by the intravenous route in the tail vein. Both groups were then monitored for relative tumour volume for up to 65 days, after which the mice were killed owing to the distress caused to the mice by the tumour.The results are shown in Table 2 below
Relative tumour voles for implanted MCF-7 cells In female Balb/C nude 1ce TABLE 2
Day Solvent Control Doxotaml2 (n.6) 20ma/ka i.v. (n.5) 0 1.00 1.00
7 1.70+0.18 1.610.54 14 2.04*0.57 1.47*1.34
21 3.58+1.49 2.23+1.44 31 6.42*2.80 3.89*3.13 38 8.91+3.76 4.753.48
44 10.80*3.65 6.314.93
51 13.6014.77 9.19*5.50 58 18.20*5.57 11.206.37
65 24.80*9.82 13.40*7.43 These data show that the implanted tumours from the control group undergo an exponential, though relatively slow growth during the course of the experimental period. The drug treated group show an initial reduction in relative tumour volume, then exponential growth from 14 days onward. However there is no significant difference between pairs of equivalent points for these curves at the dosage level used.
These data indicate that Doxotam 12 has tumoricidal activity at 20 mg kg- via the intravenous route. These initial studies have shown that this tumoricidal activity has been achieved with little if any toxicity to the mice.
Claims (11)
1. Compounds comprising an antioestrogenic moiety linked to an anti tumour moiety by means of a spacer group characterised in that the spacer group is a minimum of 10 atoms in length excluding any atoms from the antioestrogenic moiety or the anti tumour moiety which contribute to the linkage.
2. Compounds as claimed in claim 1 in which the spacer group is from 10 to 22 or 24 atoms in length.
3. Compounds as claimed in claim 1 in which the spacer group is from 12 to 18 atoms in length.
4. Compounds as claimed in claim 1 in which the spacer group is 14 atoms in length.
5. Compounds as claimed in claims 1, 2, 3 or 4 wherein the spacer group is a dicarboxylic acid, a peptide or a polysaccharride.
6. Compounds as claimed in claim 1 or 2 wherein the spacer group is a dicarboxylic acid of the formula HOOC(CH2)nCOOH where n is from 8 to 20.
7. Compounds as claimed in any of claims 1 to 6, wherein the spacer group makes an ester or amide bond linkage with the antioestrogenic and anti tumour moieties.
8. Compounds as claimed in claim 1, wherein the antioestrogenic moiety is selected from the group consisting of compounds of general formula (I)
wherein R1 is hydrogen or hydroxy, R2 is hydrogen or C14 alkyl, R3 is unsubstituted aliphatic C14 alkyl or aliphatic C14 alkyl substituted by one or more halogen, nitro, amino, aldehyde, keto, hydrazino or alcohol groups, R5 and R6 being the same or different, or a 2,3-diphenylindole, 2-benzoyl-3-phenylthiophene, an acetoxy-substituted triarylethen, a 1,2-diphenylethane or a 2-phenylindene and the anti tumour moiety is selected from the group consisting of compounds doxorubicin, methotrexate, mitoxantrone, danurobicin, epirubicin, idarubicin, acracinomycin A, DUP-941, cyclophosphamide or fosphamide.
9. Compounds as claimed in claim 8, wherein the antioestrogenic moiety is tamoxifen or 4-hydroxytamoxifen.
10. Compounds as claimed in any preceding claim that are of the formula (II)
11. Compounds that are a biological breakdown product of any of the compounds defined in any of claims 1 to 7, with the proviso that the breakdown product includes at least part of the spacer group.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB939311719A GB9311719D0 (en) | 1993-06-07 | 1993-06-07 | Anti-cancer compounds |
| GB939311717A GB9311717D0 (en) | 1993-06-07 | 1993-06-07 | Anti-cancer compounds |
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| Publication Number | Publication Date |
|---|---|
| GB9410907D0 GB9410907D0 (en) | 1994-07-20 |
| GB2278843A true GB2278843A (en) | 1994-12-14 |
Family
ID=26303016
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB9410907A Withdrawn GB2278843A (en) | 1993-06-07 | 1994-06-01 | Anti-cancer compounds |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU6803494A (en) |
| GB (1) | GB2278843A (en) |
| WO (1) | WO1994029327A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0740650A1 (en) * | 1994-01-28 | 1996-11-06 | University Of Kentucky Research Foundation | Codrugs as a method of controlled drug delivery |
| WO2002053091A2 (en) * | 2000-12-29 | 2002-07-11 | Dospharma | Medicinal combination of a biguanine (metformin) and arginine |
| WO2003061626A1 (en) * | 2002-01-18 | 2003-07-31 | Control Delivery Systems, Inc. | Polymeric gel system for the controlled delivery of codrugs |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998051702A1 (en) * | 1997-05-14 | 1998-11-19 | Sloan-Kettering Institute For Cancer Research | Methods and compositions for destruction of selected proteins |
| CN102219812B (en) * | 2011-04-14 | 2014-04-09 | 中国药科大学 | Tumor targeting deoxyglucose composite drug and preparation method thereof |
| EP3353159A4 (en) * | 2015-09-22 | 2019-03-27 | The Regents of The University of California | Modified cytotoxins and their therapeutic use |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2201419A (en) * | 1987-02-24 | 1988-09-01 | Erba Farmitalia | Anthracycline-oestrone derivatives |
| DE3907290A1 (en) * | 1989-03-07 | 1990-09-13 | Gerhard Prof Dr Eisenbrand | STEROID HORMONE RECEPTOR AFFINES ANTITUARY ACTIVE SUBSTANCES |
| US5149794A (en) * | 1990-11-01 | 1992-09-22 | State Of Oregon | Covalent lipid-drug conjugates for drug targeting |
| AU1424492A (en) * | 1991-02-20 | 1992-09-15 | Christopher Capelli | Non-protein intracellular receptor binding conjugates and a method of use thereof |
-
1994
- 1994-06-01 GB GB9410907A patent/GB2278843A/en not_active Withdrawn
- 1994-06-01 WO PCT/GB1994/001185 patent/WO1994029327A1/en active Application Filing
- 1994-06-01 AU AU68034/94A patent/AU6803494A/en not_active Abandoned
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0740650A1 (en) * | 1994-01-28 | 1996-11-06 | University Of Kentucky Research Foundation | Codrugs as a method of controlled drug delivery |
| EP0740650A4 (en) * | 1994-01-28 | 2000-04-19 | Univ Kentucky Res Found | Codrugs as a method of controlled drug delivery |
| WO2002053091A2 (en) * | 2000-12-29 | 2002-07-11 | Dospharma | Medicinal combination of a biguanine (metformin) and arginine |
| WO2002053091A3 (en) * | 2000-12-29 | 2004-06-10 | Dospharma | Medicinal combination of a biguanine (metformin) and arginine |
| WO2003061626A1 (en) * | 2002-01-18 | 2003-07-31 | Control Delivery Systems, Inc. | Polymeric gel system for the controlled delivery of codrugs |
| AU2003205278B2 (en) * | 2002-01-18 | 2008-08-07 | Psivida Us Inc. | Polymeric gel system for the controlled delivery of codrugs |
Also Published As
| Publication number | Publication date |
|---|---|
| AU6803494A (en) | 1995-01-03 |
| WO1994029327A1 (en) | 1994-12-22 |
| GB9410907D0 (en) | 1994-07-20 |
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