Thermosensitive cyclotriphosphazene-platinum complex conjugate, its preparation method and anticancer agent containing the same

The present invention provides a novel thermosensitive cyclotriphosphazene-platinum complex conjugates represented by Formula 1, ##STR1## PA1 wherein m is a repeating unit of poly(alkoxyethylene glycol) selected from the integers 2, 7 and 12; n represents the length of the alkyl chain selected from the integers 0, 1, 2 and 3; x represents the length of the anionic amino acid residue selected from the integers 0 (amino malonic acid derivatives), 1 (aspartic acid derivatives) and 2 (glutamic acid derivatives); A.sub.2 is a bidentate chelating diamine selected from the group consisting of 2,2-dimethyl-1,3-propanediamine (dmpda), trans(.+-.)-1,2-diaminocyclohexane (dach) and 1,1-diaminomethylcyclohexane (dmach).

BACKGROUND OF THE INVENTION
 The present invention relates to a thermosensitive
 cyclotriphosphazene-platinum complex conjugate which can be administered
 systemically or locally and has an excellent anticancer activity, its
 preparation method and an anticancer agent containing the same as an
 active ingredient. More particularly, the compound of the present
 invention is a biodegradable cyclotriphosphazene-platinum complex
 conjugate exhibiting thermosensitivity in the temperature range including
 the body temperature.
 The present inventors have found that the derivatives obtained by
 nucleophilic substitution of chlorine atoms in
 hexachlorocyclotriphosphazene ((NPCl.sub.2).sub.3) with a low or high
 molecular weight hydrophilic poly(alkoxyethylene glycol) and a hydrophobic
 amino acid ester exhibit thermosensitivity (Korean patent application No.
 99-48800). The present inventors have succeeded in the preparation of
 their platinum complexes by introducing the (diamine)platinum(II) ion to
 the hydrolyzed amino acid groups of these trimeric derivatives. To our
 surprise, these novel trimeric platinum complex conjugates also exhibited
 thermosensitivity in a wide temperature range including the body
 temperature as well as high anticancer activity. These new thermosensitive
 platinum complex conjugate anticancer agents showing a controlled release
 property has never been reported. These compounds can be administered
 systemically or locally since the targeted drug delivery is possible using
 their thermosensitivity. We expect, therefore, these new compounds will
 provide a new and significantly improved therapeutic regimen in the
 treatment of solid tumors.
 Thermosensitive polymers in the present invention refer to the polymers
 that can be solubilized in water at low temperatures but precipitates
 above a certain critical temperature due to the rapid decrease of their
 water-solubility. When such a phase transition is reversible, the phase
 transition temperature is called a lower critical solution
 temperature(LCST) or a cloud point. Below LCST, the hydrogen bonding
 between the polymer-water molecules is stronger than the hydrophobic
 interaction between the polymer-polymer molecules. As the temperature
 increases, however, the hydrogen bonding between the polymer-water
 molecules weakens whereas the hydrophobic interaction between the
 polymer-polymer molecules increases resulting in the precipitation of
 polymers in aqueous solution.
 These thermosensitive polymers were widely studied in many fields is
 including mainly drug delivery systems, medical biomaterials, thin films,
 the separation process of biochemical reactions, cosmetics and optics.
 However, most of the conventional thermosensitive organic polymers are
 known to be hydrolytically non-degradable. In recent years, a few
 biodegradable polymers were reported (Jeong, B. et. al., Nature, 388, 860
 (1997); Song, S.-C. et. al., Macromolecules, 32, 2188(1999); Lee, S. B.
 et. al., Macromolecules, 32, 7820 (1999)). Up to date, however, no
 thermosensitive anticancer drug has been reported. Cisplatin, a platinum
 complex, approved as an anticancer agent in 1979 by FDA in the United
 States has been used as one of the most effective chemotherapeutic agents
 to treat a variety of cancers such as testicular, ovarian, bladder, and
 head and neck cancers. Its use is limited, however, due to its high
 toxicity (LD.sub.50 =13 mg/Kg, M. J. Cleare, Biochimie 60, 835(1978)).
 Even though the second-generation anticancer agent, carboplatin exhibits
 much lower toxicity than cisplatin (LD.sub.50 =180 mg/Kg, M. J. Cleare,
 Biochimie 60, 835(1978)), it is not widely used since it is lower in
 anticancer activity and more expensive than cisplatin. Therefore, there is
 a great need for a third-generation anticancer agent that has higher and
 wider anticancer activity with lower toxicity than cisplatin.
 SUMMARY OF THE INVENTION
 Therefore, an object of the present invention is to develop a
 third-generation anticancer agent having a higher anticancer activity and
 lower toxicity than the conventionally used cisplatin.
 To achieve this goal, the present inventors have discovered a novel class
 of the cyclotriphosphazene-platinum complex conjugate anticancer agents
 whose LCST can be designed variably for the desired purpose and controlled
 precisely, by introducing the solubilizing agent and the platinum complex
 into the biodegradable cyclotriphosphazene. These new
 cyclotriphosphazene-platinum complex conjugate anticancer agents of the
 present invention may be administered systemically or locally by using the
 thermosensitive properties of the drugs.
 More particularly, an object of the present invention is to provide
 oligomeric thermosensitive cyclotriphosphazene-platinum complex conjugates
 having a stereo-specific chemical structure and LCST that can be designed
 for the desired purpose, by nucleophilic substitution of the chlorine
 atoms in hexachlorocyclotriphosphazene with a poly(alkoxyethylene glycol)
 and an amino acid ester, followed by hydrolysis of the substituted amino
 acid ester and subsequent reaction with a (diamine)platinum salt and the
 preparation method thereof.
 Another object of the present invention is to provide an anticancer agent
 having the thermosensitive cyclotriphosphazene-platinum complex conjugate
 as an active ingredient.
 DETAILED DESCRIPTION OF THE INVENTION
 To achieve the above-mentioned goals, the present inventors have performed
 the following experiments. Hexachlorocyclotriphosphazene was reacted first
 with a hydrophilic poly(alkoxyethylene glycol) having different molecular
 weights, and then with a variety of hydrophobic amino acid esters to
 obtain a new class of cyclotriphosphazene derivatives with thermosensitive
 properties. The substituted amino acid eaters in the trimers were
 hydrolyzed with alkali, and then reacted with a (diamine)platinum(II) salt
 to obtain a cyclotriphosphazene-platinum complex conjugate formed by
 covalent bonding between the carboxylate groups of the amino acid and the
 platinum(II) cation. Surprisingly, we have found that these trimeric
 platinum conjugates exhibit thermosensitivity in a wide range of
 temperature depending on the kinds of substituents and the platinum
 derivatives. Furthermore, we have found that the LCST of the
 cyclotriphosphazene-platinum complex conjugates can be designed and
 controlled for the desired application purpose since the LCST of these
 cyclotriphosphazene-platinum complex can be varied easily by appropriate
 choice of different poly(alkoxyethylene glycol), amino acid and platinum
 derivatives.
 The preparation method of the novel stereo-specific
 cyclotriphosphazene-platinum complex conjugates represented by Formula 1
 of the present invention is described in detail as follows.
 ##STR2##
 wherein, m is an integer selected from 2, 7 and 12; n is an integer
 selected from 0, 1, 2 and 3; x is an integer selected from 0, 1 and 2; and
 A.sub.2 is a bidentate chelating diamine selected from the group
 consisting of 2,2-dimethyl-1,3-propanediamine (dmpda),
 trans(.+-.)-1,2-diaminocyclohexane (dach) and 1,1-diaminomethylcyclohexane
 (dmach).
 In the above, m selected from the integer of 2, 7, or 12 is a repeating
 unit of poly(alkoxyethylene glycol), n selected from the integer of 0 to 3
 represents the length of the alkyl chain, x selected from the integer of 0
 to 2 represents the length of the anionic amino acid residue.
 The trimeric amino acid ester derivatives represented by Formula 2 was
 prepared by reacting hexachlorocyclotriphosphazene first with hydrophilic
 poly(alkoxyethylene glycol) and then with a variety of hydrophobic amino
 acid esters. By reacting the trimeric derivatives of Formula 2 with 3
 moles of alkali earth metal hydroxide of Formula 3 or with 6 moles of
 alkali metal hydroxide of Formula 4 in methanol or ethanol for 3.about.5
 hours, the intermediate cyclotriphosphazene-alkali earth metal salt of
 Formula 5 or the cyclotriphosphazene-alkali metal salt of Formula 6,
 respectively, were obtained. With this intermediate, 3 moles of
 (diamine)platinum sulfate or nitrate of Formula 7 was reacted in distilled
 water for 3.about.7 hours at 1.about.5.degree. C., and then the reaction
 mixture was freeze-dried.
 ##STR3##
 M(OH).sub.2 Formula 3
EQU M'OH Formula 4
 ##STR4##
 ##STR5##
 A.sub.2 PtL.sub.2 Formula 7
 In Formulas 2-7, m, n, x and A are as defined as in Formula 1; R is
 selected from the group consisting of methyl, ethyl or benzyl groups; M is
 an alkali earth metal ion such as barium or calcium; M' is an alkali metal
 ion such as lithium, sodium or potassium; and L.sub.2 is selected from
 sulfate or nitrate ion.
 The aqueous solution of (diamine)platinum salt of Formula 7 was prepared by
 reacting (diamine)platinum iodide with an acidic salt of silver according
 to the method in the literature (R. C. Harrison, Inorg. Chimica Acta 46,
 L15 (1980)).
 To the above freeze-dried product was added alcohol with stirring and then
 the solution mixture was centrifuged. After the supernatant solution was
 separated and condensed by evaporation at a reduced pressure, an excess
 amount of ether or hexane was added to precipitate the final product. The
 precipitation process using a solvent pair of alcohol/ether or hexane was
 repeated 2.about.3 times and the final precipitate was vacuum-dried. To
 remove small amounts of other isomers in the final product, an appropriate
 LCST lowering compound (NaCl, KCl, CF.sub.3 CH.sub.2 OH, or CH.sub.3
 (CH.sub.2).sub.3 OH) depending on the LCST of the isomeric forms of the
 trimeric derivatives was added to the aqueous solution of the product to
 induce precipitation of the purer product. The supernatant aqueous phase
 was removed by centrifugation to obtain the purified precipitate. This
 purification process was repeated 2.about.3 times and then the final
 solution was freeze-dried to obtain the thermosensitive
 cyclotriphosphazene-platinum complex conjugates of Formula 1.
 The preparation process of the present invention is schematically
 represented in the following Reaction Scheme 1.
 ##STR6##
 The invention will be further illustrated by the following examples, but is
 not limited to the examples given.
 Elemental analysis of carbon, hydrogen, nitrogen in the compounds of the
 present invention was performed in the Special Analysis Center at KIST by
 using carbon, hydrogen and nitrogen analyzer (Perkin Elmer), and
 phosphorous and platinum analysis was performed using Polyscan 61E ICP.
 Hydrogen and phosphorous nuclear magnetic resonance spectra were obtained
 using Varian to Gemini-300, and LCST was measured by using Perkin-Elmer
 Lamda 18 UV/VIS spectrophotometer.