US20010018531A1 - Water soluble analogs and prodrugs of paclitaxel - Google Patents
Water soluble analogs and prodrugs of paclitaxel Download PDFInfo
- Publication number
- US20010018531A1 US20010018531A1 US09/788,344 US78834401A US2001018531A1 US 20010018531 A1 US20010018531 A1 US 20010018531A1 US 78834401 A US78834401 A US 78834401A US 2001018531 A1 US2001018531 A1 US 2001018531A1
- Authority
- US
- United States
- Prior art keywords
- paclitaxel
- analogs
- prodrugs
- malyl
- mmol
- Prior art date
- Legal status (The legal status 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 status listed.)
- Granted
Links
- 0 CCC(C(*)C(C(C)(C(CC1OC[C@]11*)OC)C1([C@@]1(CC2)OC(*)=O)C=*)=O)=C(C)[C@@](*C([C@@](C(*(C=C)C(C)=O)C(C3)C3=C)OC(CC3OC(C)(C)OC3=O)=O)=O)CC12OC Chemical compound CCC(C(*)C(C(C)(C(CC1OC[C@]11*)OC)C1([C@@]1(CC2)OC(*)=O)C=*)=O)=C(C)[C@@](*C([C@@](C(*(C=C)C(C)=O)C(C3)C3=C)OC(CC3OC(C)(C)OC3=O)=O)=O)CC12OC 0.000 description 3
- MOYLYSBOUVNTEB-UHFFFAOYSA-N [H]N(C(=O)C1=CC=CC=C1)C(C1=CC=CC=C1)C(OC)C(=O)OC1CC2(O)C(OC(=O)C3=CC=CC=C3)C3([H])C4(O[Ac])COC4CC(OC)C3(C)C(=O)C(O[Ac])C(=C1C)C2(C)C Chemical compound [H]N(C(=O)C1=CC=CC=C1)C(C1=CC=CC=C1)C(OC)C(=O)OC1CC2(O)C(OC(=O)C3=CC=CC=C3)C3([H])C4(O[Ac])COC4CC(OC)C3(C)C(=O)C(O[Ac])C(=C1C)C2(C)C MOYLYSBOUVNTEB-UHFFFAOYSA-N 0.000 description 2
- SLVNANFPVQYFNQ-UHFFFAOYSA-N [H]N(C(=O)C1=CC=CC=C1)C(C1=CC=CC=C1)C(O)C(=O)OC1CC2(O)C(OC(=O)C3=CC=CC=C3)C3([H])C4(O[Ac])COC4CC(O)C3(C)C(=O)C(O[Ac])C(=C1C)C2(C)C Chemical compound [H]N(C(=O)C1=CC=CC=C1)C(C1=CC=CC=C1)C(O)C(=O)OC1CC2(O)C(OC(=O)C3=CC=CC=C3)C3([H])C4(O[Ac])COC4CC(O)C3(C)C(=O)C(O[Ac])C(=C1C)C2(C)C SLVNANFPVQYFNQ-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
- C07D305/14—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the invention relates to water soluble prodrugs in which the solubulizing groups are non toxic acids which are attached to paclitaxel as an ester functionality to the C2′- and/or C7-hydroxyl position.
- These prodrugs are stable in aqueous solution but are readily hydrolyzed at physiological conditions to the parent drug.
- Paclitaxel (1) is a natural diterpenoid, isolated from the Pacific yew tree ( Taxus brevifolia ) . Paclitaxel has been approved for treatment of patients with advanced ovarian cancer or breast cancer.
- paclitaxel Although paclitaxel has demonstrated to be an unique antitumor agent, it has several disadvantages.
- One of the major problems is its poor solubility in water, which makes formulation difficult in relation to the intravenous administration. Due to this poor solubility, paclitaxel is formulated, using a 1:1 mixture of cremophor EL (a polyethoxylated castor oil) and ethanol (Rowinsky, E. K. et al. J. Natl. Cancer Inst. 1990, 82, 1247). This mixture is diluted with 5% dextrose in water or saline prior to lengthy infusion.
- Prodrug strategies consist of temporary modification of the physiochemical properties of a compound through chemical derivatization. Such temporary chemical modification is usually designed to alter aqueous solubility and biodistribution while the pharmacological properties of the parent drug remain intact.
- Prodrugs can be designed to be converted in a predictable way, in vivo, to the active drug by either an enzymatic mechanism or by hydrolysis initiated under physiological pH conditions.
- Chemical stability is critical to the formulation and storage of any water soluble analog/prodrug of paclitaxel, since partial degradation to the poorly soluble parent drug is likely to lead to precipitation of paclitaxel.
- the enzymatic stability (in rat, human plasma or in vivo) is important in relation to the degradation of the prodrugs to paclitaxel or to an active metabolite of paclitaxel.
- a further object of this invention is to produce water soluble analogs of paclitaxel which possess (in vitro or in vivo) antitumor activity in the same extent as paclitaxel.
- FIG. 1 illustrates the protection of one of the carboxylic acids together with the ⁇ -hydroxyl group of malic acid ( 2 ) as an isopropylidene functionality, resulting in compound 3 .
- the synthesis of 2′-malylpaclitaxel ( 5 ) via coupling of 3 to the C2′-hydroxyl group of paclitaxel, leading to 4 , and removal of protective isopropylidine functionality, is presented. Finally it contains in conversion of 5 into the corresponding sodium salt 6 .
- FIG. 2 illustrates the synthesis of 2′,7-bis(malyl)paclitaxel ( 8 ), carried out similar to the synthesis of 5 .
- the coupling reaction of 3 with paclitaxel, leading to 7 was carried out at 40° C.
- FIG. 3 illustrates the synthesis of 7-malylpaclitaxel ( 11 ), via 2′-Trocpaclitaxel ( 9 ), which was coupled to 3 , leading to 10 , followed by removal of the protecting groups.
- Paclitaxel was obtained from Pharmachemie BV Haarlem. Proton magnetic resonance spectra were measured on a Bruker AC-100 or a Bruker AM-400 spectrometer. Chemical shift values are reported as ⁇ -values relative to tetramethylsilane as an internal standard; deuterochloroform was used as solvent. Mass spectra were obtained with a double focusing VG 7070E spectrometer. Elemental analyses were carried out on a Carlo Erba Instruments CHNSO EA 1108 element analyzer. Melting points were determined with a Reichert Thermopan microscope and are uncorrected.
- 1,2-O-(Propane-2,2-diyl)-malic acid ( 3 ) was obtained after treatment with malic acid ( 2 ) with acetone, in the presence of p-toluenesulfonic acid.
- 2′-malylpaclitaxel ( 5 ) was synthesized by reaction of paclitaxel ( 1 ) with 1.1 equivalent of 3 in the presence of diisopropylcarbodiimide (DIPC) and 4-dimethylaminopyridine (DMAP) at 0° C.
- DIPC diisopropylcarbodiimide
- DMAP 4-dimethylaminopyridine
- 1,2-O-(Propane-2,2-diyl)-malic acid ( 3 ) was coupled to paclitaxel ( 1 ) in the presence of DIPC and DMAP at 40° C. to yield 2′,7-bis(1,2-O-(propane-2,2-diyl)-malyl)-paclitaxel ( 7 ).
- This compound 7 was converted to 2′,7-bis(malyl)paclitaxel ( 8 ) by treatment with a mixture of HOAc/THF/H 2 O: 4/1/2.
- Compound 8 can be further converted into for example sodium salts analogously to the procedure described for compound 6 .
- Paclitaxel or paclitaxel prodrugs ( 5 , 6 , 8 , 11 .) were suspended in water or PBS-buffer (pH 7.4) until a concentration was reached of 2 mg/ml. The suspensions were sonicated for 15 minutes and centrifuged (13000 g) for 10 minutes (Nicolaou, K. C. et al. Nature 1993, 364, 464-466). The above fluid was analyzed, using HPLC. The paclitaxel (prodrug) concentration was determined using paclitaxel standards in methanol.
- HPLC Rheodyne injection valve (20 ⁇ l loop); Lichrospher 5RP18 column (200 ⁇ 3 mm, Chrompack); UV-detector (Model 759A, Applied Biosystems); eluent: CH 3 CN/MeOH/H 2 O: 5/1/4 in 10 mM NH 4 OAc (pH 5.0) (Willey, T. A. J. Chromatography 1993, 621, 231-238).
- the detection of the (pro)drugs was performed at 226 nm, where it is supposed that the extinction coefficients of paclitaxel and paclitaxel prodrugs are equal. The concentrations were determined by measuring the relative area of paclitaxel or the paclitaxel prodrugs.
- the paclitaxel prodrugs ( 5 , 6 , 8 , 11 ,) were dissolved in water, sonicated and centrifuged. 100 ⁇ l of the above fluid was mixed with 400 ⁇ l of plasma (heparin) or PBS-buffer (pH7.4), respectively, in such way that the concentration of the prodrug was about 0.5 mg/mL.
- MCF7 is estrogen receptor ER+/Progesterone receptor PgR+ and EVSA-T is ER-/PgR-.
- the compounds of this invention were dissolved to a concentration of 177147 ng/ml as follows: Paclitaxel 5% DMSO in full RPMI growth medium 5 5% DMSO in full RPMI growth medium 8 5% DMSO in full RPMI growth medium 11 5% DMSO in full RPMI growth medium
- the cells were stained for at least 15 min. with 0.4% SRB, dissolved in 1% acetic acid, and subsequently washed with 1% acetic acid to remove the unbound stain.
- the plates were air dried and the bound protein was dissolved by using 150 ⁇ l 10 mmol/l tris base.
- the absorbance was read at 540 nm using an automatic microplate reader (Titertec, Flow Laboratories LtD., Irvine, Scotland). Data were used for construction of concentration-response curves and determination of the IC 50 -value.
- OVCAR-3 The human tumor cell line OVCAR-3 was used.
- OVCAR is an ovarium carcinoma.
- paclitaxel or paclitaxel prodrugs were dissolved in DMSO to give a concentration of 5 mM. Concentrations were verified by measuring the OD at 226 nm. The antiproliferative effects of drugs and prodrugs were determined with the use of OVCAR-3 cells. Cells in supplemented tissue culture medium (DMEM, 10% fetal calf serum with 50 IU/ml penicillin and 50 microgram/ml streptomycin) were seeded in triplicate in 96-well culture plates (5000/well, 100 microliter). After 24 h, 100 microliter of culture medium containing drug or prodrug was added to give final concentrations ranging from 1 picomolar to 10 micromolar.
- tissue culture medium fetal calf serum with 50 IU/ml penicillin and 50 microgram/ml streptomycin
- IC 50 values are the (pro)drug concentration that give 50% growth inhibition when compared with control cell growth (Houba et al. Bioconj. Chem. 1996, 7, 606-611).
- IC 50 values of the compounds of this invention are given in table II.
- TABLE II IC 50 a (ng/ml) IC 50 a (nM) Compound MCF7 EVSA-T WIDR IGROV M19 MEL A498 H226 OVCAR-3 paclitaxel ⁇ 3 ⁇ 3 ⁇ 3 33 ⁇ 3 5 ⁇ 3 0.25 5 ⁇ 3 ⁇ 3 ⁇ 3 ⁇ 3 3 39 10 — 6 ⁇ 3 ⁇ 3 ⁇ 3 233 ⁇ 3 ⁇ 3 ⁇ 3 0.80 8 69 59 167 49 311 436 241 — 11 390 300 589 241 1344 1435 706 —
- Compound 5 showed similar cytotoxic activity as paclitaxel, which can probably be explained by the degradation of these compounds to the parent drug, under the conditions used to determine the activity.
- the 2′-analog 5 is stable in PBS-buffer (pH 7.4). After 24 hours, only traces of paclitaxel were detected. Whereas in human plasma only after 20 hours 50% of the analog is degraded to paclitaxel.
- Compound 6 showed a comparable against OVCAR-3 cells, when compared to paclitaxel. Of compound 6 about 50% was degraded to paclitaxel within 4 hours. Furthermore, compound 6 is sixty times more watersoluble than paclitaxel.
- the present invention discloses a method for the preparation of paclitaxel analogs of paclitaxel having a malate moiety at C2′ and/or C7-position. It is apparent that many modifications of the present invention are possible, for example the use of counterions other than sodium, which may give rise to higher solubilities. It is therefore understood that the invention may be practiced otherwise than specifically described.
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Epoxy Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to water soluble antitumor analogs of paclitaxel of formula (I) wherein R1═C(O)CH2CH(OH)COOX, R2═H, C(O)CH2CH(OH)COOX, X═H, Li, Naa or any other pharmaceutically acceptable counterion, as well as to a pharmaceutical composition comprising an antineoplastically effective amount of such analogs as an active ingredient.
Description
- The invention relates to water soluble prodrugs in which the solubulizing groups are non toxic acids which are attached to paclitaxel as an ester functionality to the C2′- and/or C7-hydroxyl position. These prodrugs are stable in aqueous solution but are readily hydrolyzed at physiological conditions to the parent drug.
-
- Although paclitaxel has demonstrated to be an unique antitumor agent, it has several disadvantages. One of the major problems is its poor solubility in water, which makes formulation difficult in relation to the intravenous administration. Due to this poor solubility, paclitaxel is formulated, using a 1:1 mixture of cremophor EL (a polyethoxylated castor oil) and ethanol (Rowinsky, E. K. et al.J. Natl. Cancer Inst. 1990, 82, 1247). This mixture is diluted with 5% dextrose in water or saline prior to lengthy infusion. Unfortunately, various hypertensive reactions have been reported in patients who were treated with paclitaxel, partly due to cremophor EL, which is responsible for histamine release, causing the effects (Rowinsky, E. K. et al. Ibid.) . Premedication, using antihistaminic drugs can deminish these side effects, but results in additional medication, cost and discomfort to the patient.
- The solubility problems with paclitaxel could be overcome by the development of a more water soluble, chemically stable, and therefore more easily formulated analog/prodrug of paclitaxel. Prodrug strategies consist of temporary modification of the physiochemical properties of a compound through chemical derivatization. Such temporary chemical modification is usually designed to alter aqueous solubility and biodistribution while the pharmacological properties of the parent drug remain intact. Prodrugs can be designed to be converted in a predictable way, in vivo, to the active drug by either an enzymatic mechanism or by hydrolysis initiated under physiological pH conditions.
- SAR studies have shown that some modifications at C7 of paclitaxel are allowed ((a) Mellado, W. et al.Biochem. Biophys. Res. Comm. 1984, 124, 329-336. (b) Kingston, D. G. I et al. New Trends in Nat. Prod. Chem. 1986, 26, 219-235. (c) Horwitz, S. B. et al. Ann. New York Acad. Sci. 1986, 466, 733-740. (d) Kingston, D. G. I. et al. J. Nat. Prod. 1990, 53, 1-12. (e) Ringel, I. et al. J. Pharmacol. Exp. Ther. 1987, 242, 692-698. (f) Chaudhary, A. G. et al. J. Org. Chem. 1993, 58, 3798-3799. (g) Chen, S. et al. J. Org. Chem. 1993, 58, 5028-5029).
- For instance, 7-acetylpaclitaxel has shown to be as active as paclitaxel in microtubule assembly assays. SAR studies have also shown that introduction of an acetyl group at C2′ resulted in the loss of the ability to promote microtubule assembly. However the cytotoxic activity of 2′-acetyl-paclitaxel is almost the same as for paclitaxel, probably due to the fact that the C2′-acetyl group is either being hydrolyzed under the conditions of the bioassay or converted intracellularly to paclitaxel or an active paclitaxel metabolite. These observations suggest that the C2′- and C7-positions of paclitaxel are suitable for (temporary) structural modifications. The C2′-position seems more suitable for reversible derivatization.
- Several research groups have reported the syntheses and biological evaluations of water soluble prodrugs of paclitaxel. These analogs have a polar substituent either at the C2′- or at the C7-hydroxyl group. In most cases the polar substituents are coupled to these hydroxyl groups via an ester, carbonate or carbamate functionality. Deutsch et al. (Deutsch, H. M. et al.J. Med. Chem. 1989, 32, 788-792) reported that some salts of 2′-succinylpaclitaxel and 2′-glutarylpaclitaxel had improved antitumor activities compared to the corresponding free acids. The triethanolamine and N-methyl-glucamine salts showed improved aqueous solubility and were more active than the sodium salts. Zhao et al. (Zhao, Z. et al. J. Nat. Prod. 1991, 54, 1607-1611) introduced sulfonate groups to improve the water solubility of paclitaxel. These sulfonate-paclitaxel analogs showed improved water solubility and had about the same (in vivo) activity compared to paclitaxel. Mathew et al. (Mathew, A. E. et al. J. Med. Chem. 1992, 35, 145-151) reported the synthesis and evaluation of some 2′- and 7-amino acid analogs of paclitaxel. The methane sulfonic salts of both 2′- and 7-amino acid esters of paclitaxel showed increased water solubility. The 2′-analogs showed activity to an extent similar to that of paclitaxel, while the others showed reduced activity. Vyas et al. (Vyas, D. M. et al. Bioorg. Med. Chem. Lett. 1993, 3, 1357-1360) synthesized and vevaluated 2′- and 7-phosphate paclitaxel analogs. These analogs showed improved water solubility. However in vitro as well as in vivo these derivatives were non-toxic compared to paclitaxel. Ueda et al. (Ueda, Y. et al. Bioorog. Med. Chem. Lett. 1993, 3, 1761-1766) synthesized 2′- and 7′-phosphonoxyphenylpropionatepaclitaxel, which both showed increased water solubility. The 2′-analog was inactive, whereas the 7-analog was as active as paclitaxel in vivo. Greenwald et al. ((a) Greenwald, R. B. et al. J. Org. Chem. 1995, 60, 331-336. (b) Greenwald, R. B. et al. J. Med. Chem. 1996, 39, 424-431) prepared some 2′- and 7-polyethyleneglycol esters of paclitaxel. These analogs were extremely water soluble. The 2′-analogs had in vitro and in vivo activities in the same extent as paclitaxel, whereas the 7-analogs showed reduced activity. Greenwald et al. claimed that by choosing the appropriate weight for the 2′-PEG moiety, a prodrug was produced that is as efficacious as paclitaxel/cremophor EL/ethanol in an in vivo model. Nicolaou et al. (Nicolaou, K. C. et al. Nature 1993, 364, 464-466) synthesized some 2′-(2-thio-aryl)ethylcarbonate analogs of paclitaxel as well as some 2′- and 2′,7-(bis)-C(O)CH2XCH2COOH (wherein X═O, S or SO2) analogs of paclitaxel, which were all more water soluble and showed increased in vitro cytotoxic activities compared to paclitaxel. Nicolaou et al. ((a) Nicolaou, K. C. et al. Angew. Chemie 1994, 106, 1672-1675. (b) Paloma, L. G. et al. Chem. Biol. 1994, 1, 107-112) also synthesized 2′- and 7-methylpyridiniumacetate analogs of paclitaxel. Both compounds showed increased water solubility. The 2′-analog was as active as paclitaxel in in vivo models, whereas the 7-analog was far less cytotoxic. Kingston et al. (Kingston et al. US patent 1995, U.S. Pat. No. 5,411,984A) prepared some 2′- and 2′,7-bis-O-aroyl analogs. These analogs showed improved water solubility. The 2′-analogs showed in vivo activities in the same extent as paclitaxel and some even better.
- Chemical stability is critical to the formulation and storage of any water soluble analog/prodrug of paclitaxel, since partial degradation to the poorly soluble parent drug is likely to lead to precipitation of paclitaxel. The enzymatic stability (in rat, human plasma or in vivo) is important in relation to the degradation of the prodrugs to paclitaxel or to an active metabolite of paclitaxel.
- From the water soluble paclitaxel prodrugs described so far the pharmacological properties of the used solubilizing functionalities, which are released once paclitaxel is liberated have not been studied. It might be possible that these solubilizing moieties or their metabolites have some undesired and/or unknown side effects. The prodrugs described in this patent release after hydrolysis a non-toxic acid.
- It is therefore an object of this invention to provide water soluble paclitaxel analogs/prodrugs, using a body innocuous solubulizing moiety, for the treatment of cancer.
- A further object of this invention is to produce water soluble analogs of paclitaxel which possess (in vitro or in vivo) antitumor activity in the same extent as paclitaxel.
- It is a further object of this invention to produce prodrugs of paclitaxel which are stable in aqueous solutions, but which upon hydrolysis at physiological (in vitro and/or in vivo) conditions exhibit the same or similar level of antitumor activity as paclitaxel.
-
- Wherein:
- R1═C(O)CH2CH(OH)COOX
- R2═H, C(O)CH2CH(OH)COOX,
- X═H, Li, Na or any other pharmaceutically acceptable counterion.
- FIG. 1 illustrates the protection of one of the carboxylic acids together with the α-hydroxyl group of malic acid (2) as an isopropylidene functionality, resulting in
compound 3. In this figure also the synthesis of 2′-malylpaclitaxel (5), via coupling of 3 to the C2′-hydroxyl group of paclitaxel, leading to 4, and removal of protective isopropylidine functionality, is presented. Finally it contains in conversion of 5 into the correspondingsodium salt 6. - FIG. 2 illustrates the synthesis of 2′,7-bis(malyl)paclitaxel (8), carried out similar to the synthesis of 5. The coupling reaction of 3 with paclitaxel, leading to 7, was carried out at 40° C.
- FIG. 3 illustrates the synthesis of 7-malylpaclitaxel (11), via 2′-Trocpaclitaxel (9), which was coupled to 3, leading to 10, followed by removal of the protecting groups.
- Unless clearly indicated by context or statement to the contrary, the terms used herein have the meanings as conventionally used in chemical arts, and definitions incorporate those used in standard texts.
- Paclitaxel was obtained from Pharmachemie BV Haarlem. Proton magnetic resonance spectra were measured on a Bruker AC-100 or a Bruker AM-400 spectrometer. Chemical shift values are reported as δ-values relative to tetramethylsilane as an internal standard; deuterochloroform was used as solvent. Mass spectra were obtained with a double focusing VG 7070E spectrometer. Elemental analyses were carried out on a Carlo Erba Instruments CHNSO EA 1108 element analyzer. Melting points were determined with a Reichert Thermopan microscope and are uncorrected. Thin layer chromatography was carried out on Merck precoated silica gel 60 F-254 plates (thickness; 0.25 mm). Spots were visualized with UV or a 6.2 a H2SO4 aqueous solution, (1L) containing ammonium molybdate (42 g) and ceric ammonium sulfate (3.6 g), followed by charring. Column chromatography was carried out using silica 60 or silica 60H (Merck). Unless otherwise stated, materials were obtained from commercial sources and used without further purification. When necessary, solvents were distilled and dried according to standard procedures. All reactions, if necessary, were carried out under argon atmosphere.
- The synthesis of prodrugs from paclitaxel in which the 2′-OH or the 7-OH group is esterified by a dicarboxylic acid needs a protection strategy for one of the carboxylic acid groups.
- With reference to FIG. 1, 1,2-O-(Propane-2,2-diyl)-malic acid (3) was obtained after treatment with malic acid (2) with acetone, in the presence of p-toluenesulfonic acid. Next, 2′-malylpaclitaxel (5) was synthesized by reaction of paclitaxel (1) with 1.1 equivalent of 3 in the presence of diisopropylcarbodiimide (DIPC) and 4-dimethylaminopyridine (DMAP) at 0° C. to afford 2′-(1,2-O-(propane-2,2-diyl)-malyl)paclitaxel (4), which reacted with a mixture of HOAc/THF/H2O: 4/1/2 at 45° C. to give 2′-malylpaclitaxel (5). This
compound 5 was subsequently eluted with a mixture of H2O/acetone: 4/1 (v/v) from DOWEX 50W×2, pretreated with 1N NaOH, yieldingsodium salt 6. -
- With reference to FIG. 3, the C2′-hydroxyl group of paclitaxel (1) was protected, using 2,2,2-trichloroethyl chloroformate (Troc-Cl) in pyridine, leading to 2′-Trocpaclitaxel (9) (Magri, N. F. et al. J. Org. Chem. 1986, 51, 797-802). Compound 9 was coupled to 1,2-O-(Propane-2,2-diyl)-malic acid (3), in the presence of DIPC and DMAP to give 2′-Troc-7- (1,2-O-(propane-2,2-diyl) -malyl)paclitaxel (10). This
compound 10 was converted to 7-malylpaclitaxel (11) by treatment with a mixture of HOAc/THF/H2O: 4/1/2, in the presence of zinc powder (Zn). - The following nonlimiting examples provide specific synthesis methods for preparing prodrugs/analogs of paclitaxel of the present invention. All technical and scientific terms used herein have the same meaning as commonly understood by persons of ordinary skill in the art. Other methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
- 2′-Malylpaclitaxel (5)
- a. 1,2-O-(Propane-2,2-diyl)-malic acid (3)
- To a solution of malic acid (2) (5.0 g, 37 mmol) and acetone (22 g, 0.37 mol) in pentane (300 ml), PTS (1.0 g, 5.8 mmol) and H2SO4 (10 drops) were added. The reaction mixture was heated to reflux temperature and then stirred for 18 hours. The formed water is removed azeotropically and trapped by molecular sieves (4 Å), using a Dean-Stark apparatus. After 18 hours, the reaction mixture was concentrated in vacuo. The residue was purified via chromatography (CHCl3/CH3CN: 1/1), yielding 3 (3.78 g, 21.7 mmol, 59%). m.p. 113° C.; 1H-NMR (100 MHz, CDCl3): δ 4.51 (1H, m, CH-malyl), 2.68 (2H, m, CH2-malyl), 1.43 (3H, s acetonide) , 1.38 (3H, s, acetonide).
- b. 2′-(1,2-O-(Propane-2,2-diyl)-malyl)-paclitaxel (4).
- A solution of paclitaxel (1) (100 mg, 0.117 mmol) and 1,2-O-(Propane-2,2-diyl)-malic acid (3) (22 mg, 0.13 mmol) in CH2Cl2 (10 ml) was stirred at 0° C. Next, DIPC (36 μl, 0.24 mmol) and DMAP (15 mg, 0.12 mmol) were added. After stirring for 3 hours at 0° C., the mixture was diluted with EtOAc (25 ml) and washed with a saturated NaHCO3 solution. The organic layer was washed with water, dried over Na2SO4 and concentrated in vacuo. The residue was purified via chromatography (EtOAc/hexane: 1/1), yielding 4 (103 mg, 0.102 mmol, 87%). m.p. 138° C.; 1H-NMR (400 MHz, CDCl3): δ 8.15 (2H, d, J=7.2 Hz, H-Ph), 7.80 (2H, d, J=7.4 Hz, H-Ph), 7.61 (1H, t, J=7.2 Hz, H-Ph), 7.45 (10H, m, H-Ph), 7.01 (1H, d, JNH-3′=9.2 Hz, NH), 6.29 (1H, s, H10), 6.26 (1H, m, H13) , 6.00 (1H, dd, J3′-NH=9.2 Hz, J3′-2′=3.0 Hz, H3′) , 5.69 (1H, d, J2-3=7.1 Hz, H2), 5.52 (1H, d, J2′-3′= 3.0 Hz, H2′), 4.97 (1H, d, J5-6=8.0, H5), 4.44 (1H, m, H7), 4.32 (1H, d, J20a-20b=8.4 Hz, H20a), 4.21 (1H, d, J20b-20a=8.4 Hz, H20b), 4.12 (1H, m, CH-malyl), 3.82 (1H, d, J3-2=7.1 Hz, H3), 2.96 (2H, m, CH2-malyl), 2.51 (1H, m, H6), 2.46 (3H, s, OCOCH3) , 2.36 (1H, m, H14a), 2.23 (3H, s, OCOCH3), 2.19 (1H, m, H14b), 2.03 (1H, m, H6), 1.93 (1H, s, H18), 1.69 (3H, s, H16), 1.57 (3H, s, acetonide), 1.51 (3H, s, acetonide), 1.25 (3H, s, H17), 1.13 (3H, s, H19); FAB-MS, 1010 [M+H]+, 1032 [M+Na]+.
- c. 2′-Malylpaclitaxel (5)
- Compound4 (100 mg, 0.099 mmol) was dissolved in a mixture of HOAc/THF/H2O (8/2/4 ml) . The mixture was stirred at 45° C. for 6 hours. Next, the organic solvents were removed by evaporation in vacuo. The residue was diluted by water (100 ml) and freeze dried, yielding 5 (91 mg, 0.093 mmol, 94%). m.p. 148-151° C. 1H-NMR (400 MHz, CDCl3): δ 8.16 (2H, d, J=7.6 Hz, H-Ph), 7;93 (2H, d, J=7.6 Hz, H-Ph), 7.61 (1H, t, J=7.3 Hz, H-Ph), 7.36 (11H, m, H-Ph and NH), 6.30 (1H, s, H10), 6.28 (1H, m, H13), 6.08 (1H, dd, J3′-NH=9.2 Hz, J3′-2′=2.8 Hz, H3′), 5.68 (1H, d, J2-3=7.3 Hz, H2), 5.51 (1H, d, J2′-3′=2.8 Hz, H2′), 4.99 (1H, d, J5-6=8.0, H5), 4.46 (1H, m, H7), 4.33 (1H, d, J20a-20b= 8.4 Hz, H20a), 4.27 (1H, m, CH-Malyl), 4.22 (1H, d, J20b-20a=8.4 Hz, H20b), 3.82 (1H, d, J3-2=7.3 Hz, H3), 3.03 (2H, m, CH2-malyl), 2.55 (1H, m, H6), 2.53 (3H, s, OCOCH3), 2.40 (1H, m, H14a), 2.23 (3H, s, OCOCH3), 2.13 (1H, m, H14b), 1.93 (1H, s, H18), 1.88 (1H, m, H6), 1.69 (3H, s, H16), 1.21 (3H, s, H17), 1.14 (3H, s, H19); FAB-MS, 992 [M+Na]+.
- Sodium salt of 2′-malylpaclitaxel (6)
- 2′-Malylpaclitaxel (5) (30 mg, 0.031 mmol) was brought on DOWEX 50W×2, which was pretreated with 1N NaOH (aq). Using a mixture of H2O and acetone (4/1, v/v) as eluent, sodium salt 6 (30 mg, 0.030 mmol, 98%) was isolated after removal of the acetone in vacuo and freeze drying. m.p. 195° C.; 1H-NMR (400 MHz, CDCl3): in accordance with the structure of
compound 5; FAB-MS, 992 [M+H]+, 1014 [M+Na]+. - 2′,7-Bis(malyl)paclitaxel (8)
- a. 2′,7-Bis(1,2-O-(propane-2,2-diyl)-malyl)-paclitaxel (7) A solution of paclitaxel (1) (50 mg, 0.0586 mmol) and 1,2-O-(Propane-2,2-diyl)-malic acid (3) (51 mg, 0.29 mmol) in CH2Cl2 (5 ml) was stirred at 0° C. Next, DIPC (100 μl, 0.064 mmol) and DMAP (7.5 mg, 0.061 mmol) were added. After 1 hour, the mixture was heated to reflux temperature and stirred for 3 days. The mixture was filtered over Hyflo. The filtrate was diluted with CH2Cl2 (30 ml) and washed with a saturated NaHCO3 solution. The organic layer was washed with water, dried over Na2SO4 and concentrated in vacuo. The residue was purified via chromatography (EtOAc/
hexane 1/1), yielding 7 (49 mg, 0.0421 mmol, 72%) . m.p. 139 ° C.; 1H-NMR (400 MHz, CDCl3) : δ 8.14 (2H, d, J=7.6 Hz, H-Ph), 7.80 (2H, d, J=7.6 Hz, H-Ph), 7.61 (1H, t, J=7.4 Hz, H-Ph), 7.39 (10H, m, H-Ph), 7.02 (1H, d, JNH-3′=9.2 Hz, NH), 6.24 (1H, s, H10), 6.23 (1H, m, H13), 5.99 (1H, dd, J3′-NH=9.2 Hz, J3′-2′=3.0 Hz, H3′), 5.69 (1H, d, J2-36.9 Hz, H2), 5.66 (1H, m, H7), 5.55 (1H, d, J2′-3′=3.0 Hz, H2′), 4.97 (1H, d, J5-6=9.4, H5), 4.84 (1H, m, CH-malyl), 4.64 (1H, m, CH-malyl), 4.33 (1H, d, J20a-20b=8.4 Hz, H20a), 4.19 (1H, d, J20b-20a=8.4 Hz, H20b), 3.94 (1H, d, J3-2=6.9 Hz, H3), 3.10 (2H, m, CH2-malyl), 2.97 (2H, m, CH2-malyl), 2.60 (1H, m, H6), 2.45 (3H, s, OCOCH3), 2.35 (1H, m, H14a), 2.25 (1H, m, H14b), 2.21 (3H, s, OCOCH3), 1.97 (1H, s, H18), 1.85 (1H, m, H6), 1.81 (3H, s, H16), 1.58 (6H, s, acetonide), 1.56 (6H, s, acetonide), 1.21 (3H, s, H17), 1.16 (3H, s, H19); FAB-MS, 1166 [M+H]+. - b. 2′,7-Bis(malyl)paclitaxel (8)
- Compound7 (40 mg, 0.0343 mmol) was dissolved in a mixture of HOAc/THF/H2O (4/1/2 ml). The mixture was stirred at 45° C. for 6 hours. Next, the organic solvents were removed by evaporation in vacuo. The residue was diluted by water (50 ml) and freeze dried, yielding 8 (33 mg, 0.0304 mmol, 89%) . m.p. 166-168° C.; 1H-NMR (400 MHz, CDCl3): δ 8.11 (2H, d, J=7.6 Hz, H-Ph), 7.84 (2H, d, J=7.5 Hz, H-Ph), 7.63 (1H, t, J=7.5 Hz, H-Ph), 7.37 (11H, m, H-Ph and NH), 6.25 (1H, s, H10), 6.16 (1H, m, H13), 5.99 (1H, dd, J3′-NH=8.8 Hz, J3′-2′=2.9 Hz, H3′), 5.67 (1H, d, J2-3=6.4 Hz, H2), 5.66 (1H, m, H7), 5.63 (1H, d, J2′-3′=2.8 Hz, H2′), 4.94 (1H, d, J5-6=7.6, H5), 4.48 (2H, m, CH-malyl) , 4.32 (1H, d, J20a-20b=7.9 Hz, H20a), 4.17 (1H, d, J20b-20a=7.9 Hz, H20b), 3.88 (1H, d, J3-2=6.4 Hz, H3), 2.97 (4H, m, CH2-malyl), 2.51 (1H, m, H6), 2.44 (3H, s, OCOCH3), 2.36 (2H, m, H14), 2.07 (3H, s, OCOCH3), 1.93 (1H, S, H18), 1.86 (1H, m, H6), 1.79 (3H, s, H16), 1.20 (3H, s, H17), 1.18 (3H, s, H19); FAB-MS, 1108 [M+Na]+.
- 7-Malylpaclitaxel (11)
- a. 2′-Trocpaclitaxel (9)
- A solution of paclitaxel (1) (80 mg, 0.0938 mmol) in CH2Cl2 (2 ml) and pyridine (0.2 ml) was stirred at −23° C. Next, 2,2,2-trichloroethyl chloroformate (13 μl, 0.095 mmol) was added. After stirring for one hour at −23° C., the mixture was diluted with CH2Cl2 (20 ml) and washed with 1N HCl (aq). The organic layer was washed with a saturated NaHCO3 solution, with water, dried over Na2SO4 and concentrated in vacuo. The residue was purified via chromatography (EtOAc/hexane: 1/1), yielding 9 (63 mg, 0.0612, 65%). m.p. 171° C. (dec); 1H-NMR (400 MHz, CDCl3): δ 8.15 (2H, d, J=7.4 Hz, H-Ph), 7.75 (2H, d, J=7.5 Hz, H-Ph), 7.61 (1H, t, J=7.4 Hz, H-Ph), 7.44 (10 H, m, H-Ph), 6.93 (1H, d, JNH-3′=9.4 Hz, NH), 6.29 (1H, s, H10), 6.25 (1H, t, J13-14=8.8 Hz, H13), 6.05 (1H, dd, J3′-NH=9.3 Hz, J3′-2′=2.8 Hz, H3′), 5.69 (1H, d, J2-3=7.3 Hz, H2), 5.54 (1H, d, J2′-3′=2.7 Hz, H2′), 4.97 (1H, dd, J=9.4 Hz, J= 1.6 Hz, H5), 4.78 (2H, d, J=8.0 Hz, CH2 (Troc)), 4.43 (1H, m, H7), 4.32 (1H, d, J20a-20b=8.4 Hz, H20a), 4.21 (1H, d, J20b-20a=8.4 Hz, H20b), 3.82 (1H, d, J3-2=7.2 Hz, H3), 2.55 (1H, m, H6), 2.48 (3H, s, OCOCH3), 2.40 (1H, m, H14a), 2.23 (3H, s, OCOCH3), 2.20 (1H, m, H14b), 1.91 (3H, s, H18), 1.88 (1H, m, H6), 1.69 (3H, s, H16), 1.24 (3H, s, H17), 1.14 (3H, s, H19); FAB-MS, 1030 [M+H]+, 1052 [M+Na]+.
- b. 2′-Troc-7-(1,2-O-(propane-2,2-diyl)-malyl)paclitaxel (10)
- A solution of9 (63 mg, 0.0612 mmol) and 3 (21 mg, 0.12 mmol) in CH2Cl2 (5 ml) was stirred at 0° C. Next, DIPC (125 μl, 0.80 mmol) and DMAP (20 mg, 0.16 mmol) were added. After 1 hour, the mixture was heated to reflux temperature and then stirred for 3 days. The mixture was filtered over Hyflo. The filtrate was diluted with CH2Cl2 (50 ml) and washed with a saturated NaHCO3 solution. The organic layer was washed with water, dried over Na2SO4 and concentrated in vacuo. The residue was purified via chromatography (EtOAc/hexane: 2/5), yielding 10 (38 mg, 0.0320, 52%). m.p. 140-145° C.; 1H-NMR (400 MHz, CDCl3) : δ 8.14 (2H, d, J= 7.3 Hz, H-Ph), 7.76 (2H, d, J=7.5 Hz, H-Ph), 7.61 (1H, t, J=7.5 Hz, H-Ph), 7.45 (1OH, m, H-Ph), 6.92 (1H, d, JNH-3′=9.5 Hz, NH), 6.26 (1H, s, H10), 6.25 (1H, m, H13), 6.04 (1H, dd, J3′-NH=9.6 Hz, J3′-2′=2.8 Hz, H3′), 5.69 (1H, d, J2-36.8 Hz, H2), 5.65 (1H, m, H7), 5.54 (1H, d, J2′-3′=2.8 Hz, H2′), 4.99 (1H, d, J5-6=8.4, H5), 4.84 (1H, m, CH-malyl), 4.78 (2H, d, J=9.4 Hz, CH2 (Troc)), 4.33 (1H, d, J20a-20b=8.4 Hz, H20a) , 4.20 (1H, d, J20b-20a=8.4 Hz, H20b), 3.95 (1H, d, J3-2=6.8 Hz, H3), 3.07 (2H, m, CH2-malyl), 2.63 (1H, m, H6), 2.47 (3H, s, OCOCH3), 2.40 (1H, m, H14a), 2.25 (1H, m, H14b), 2.16 (3H, s, OCOCH3), 1.97 (1H, s, H18), 1.87 (1H, m, H6), 1.81 (3H, s, H16), 1.60 (3H, s, acetonide), 1.57 (3H, s, acetonide), 1.24 (3H, s, H17), 1.14 (3H, s, H19); FAB-MS, 1187 [M+Na]+, 1209 [M+Na]+.
- c. 7-Malylpaclitaxel (11)
- Compound10 (28 mg, 0.0236 mmol) was dissolved in a mixture of HOAc/THF/H2O (4/1/2 ml). Zinc powder (20 mg) was added. The mixture was stirred for 3 hours at 45° C. Next, the organic solvents are evaporated in vacuo. The residue was diluted with water (50 ml) and freeze dried, yielding 11 (23 mg, 0.0237 mmol, 100%). m.p. 237-240° C.; 1H-NMR (400 MHz, CDCl3) : δ 8.13 (2H, d, J=7.3 Hz, H-Ph), 7.76 (2H, d, J=7.3 Hz, H-Ph), 7.61 (1H, t, J=7.4 Hz, H-Ph), 7.38 (1OH, m, H-Ph), 6.93 (1H, d, JNH=3′=9.2 Hz, NH), 6.25 (2H, m, H10 and H13), 5.75 (1H, dd, J3′-NH=9.2 Hz, J3′-2′=2.8 Hz, H3′), 5.69 (1H, d, J2-3=7.1 Hz, H2), 5.66 (1H, m, H7), 4.96 (1H, d, J5-6=8.4, H5), 4.83 (1H, d, J2′-3′=2.8 Hz, H2′), 4.74 (1H, m, CH-malyl), 4.28 (1H, d, J20a-20b=8.0 Hz, H20a) , 4.14 (1H, d, J20b-20a=8.0 Hz, H20b) , 3.95 (1H, d, J3-2=7.1 Hz, H3), 3.06 (2H, m, CH2-malyl), 2.60 (1H, m, H6), 2.47 (3H, s, OCOCH3) , 2.38 (1H, m, H14a), 2.25 (1H, m, H14b), 2.16 (3H, s, OCOCH3), 1.97 (1H, s, H18), 1.81 (3H, s, H16), 1.25 (3H, s, H17), 1.14 (3H, s, H19); FAB-MS, 970 [M+H]+, 992 [M+Na]+.
- Solubility and stability
- Methods
- Water Solubility: Paclitaxel or paclitaxel prodrugs (5, 6, 8, 11.) were suspended in water or PBS-buffer (pH 7.4) until a concentration was reached of 2 mg/ml. The suspensions were sonicated for 15 minutes and centrifuged (13000 g) for 10 minutes (Nicolaou, K. C. et al. Nature 1993, 364, 464-466). The above fluid was analyzed, using HPLC. The paclitaxel (prodrug) concentration was determined using paclitaxel standards in methanol.
- HPLC: Rheodyne injection valve (20 μl loop); Lichrospher 5RP18 column (200×3 mm, Chrompack); UV-detector (Model 759A, Applied Biosystems); eluent: CH3CN/MeOH/H2O: 5/1/4 in 10 mM NH4OAc (pH 5.0) (Willey, T. A. J. Chromatography 1993, 621, 231-238). The detection of the (pro)drugs was performed at 226 nm, where it is supposed that the extinction coefficients of paclitaxel and paclitaxel prodrugs are equal. The concentrations were determined by measuring the relative area of paclitaxel or the paclitaxel prodrugs.
- Stability in human plasma and PBS-buffer
- The paclitaxel prodrugs (5, 6, 8, 11,) were dissolved in water, sonicated and centrifuged. 100 μl of the above fluid was mixed with 400 μl of plasma (heparin) or PBS-buffer (pH7.4), respectively, in such way that the concentration of the prodrug was about 0.5 mg/mL. The plasma or PBS-buffer, respectively, was incubated at 37° C. and on different points in time (T=0, 0.5, 1, 4, 20, 48 hours) 50 μl was extracted with 150 μl of EtOAc. After mixing for 2 minutes (using a vortex), this mixture was centrifuged (2 minutes, 13000 g) and 100 μl EtOAc was evaporated (30 minutes, in vacuo) . The (pro)drugs were dissolved in 50 μl eluent and analyzed by HPLC (Longnecker, S. M. Cancer Treat Rep. 1987, 71, 53-59). The efficiency for the extraction of paclitaxel was about 80%.
- Solubility and stability values for compounds of the present invention are shown in table I.
TABLE I Solubility and Stability of some Water-soluble Analogs of Paclitaxel Water T½ a (hours) 2′-(R1) paclitaxel 7-(R2) paclitaxel Solubility Human No R1 = R2 = (mg/ml) pH 7.4 Plasma 1 H H 0.01 — — 5 C(O)CH2CH(OH)COOH H 0.2 >24 20 6 C(O)CH2CH(OH)COONa H 0.6 no pacl.b 4 8 C(O)CH2CH(OH)COOH C(O)CH2CH(OH)COOH 0.5 no pacl.b no pacl.b 11 H C(O)CH2CH(OH)COOH 0.003 no pacl.b no pacl.b - With the exception of 11, all prodrugs showed increased water solubility compared with paclitaxel. Best water solubility was found for the 21-
malyl prodrug 6. Allmalyl prodrugs Prodrug 6 showed also a fast hydrolysis rate in human plasma. These results show that the most promising drug for further evaluation of the drugs presented in table I is themalyl prodrug 6. - Biological Evaluation
- Material and Methods
- Determination of the IC50-values for the new synthesized prodrugs have been determined with a variety of cell lines and have been compared with the IC50-value of paclitaxel 1 (see table II)
- Compounds1, 5, 8, 11,
- The following human tumor cell lines were used:
- MCF7 Breast cancer
- EVSA-T Breast cancer
- WIDR Colon cancer
- IGROV Ovarian cancer
- M19 MEL Melanoma
- A498 Renal cancer
- HA266 non small cell lung cancer
- MCF7 is estrogen receptor ER+/Progesterone receptor PgR+ and EVSA-T is ER-/PgR-.
- Cell lines WIDR, M19 MEL, A498 and IGROV belong to the currently used anti-cancer screening panel of the National Cancer Institute, USA (Skeham et al.,J. Nat. Cancer Inst. 85: 1107-1112, 1990).
- Prior to the experiments a myocoplasma test was carried out on all cell lines and found to be negative. All cell lines, except ETSA-T, were maintained in a continuous logarithmic culture in RPMI medium with Hepes and Phenol red supplemented with 10% bovine calf serum (BCS), penicillin 111 IU/ml, streptomycin 111 μg/ml, gentamycin 46 μg/ml and insulin 10.6 μg/ml. EVSA-T was maintained in DMEM with 5% BCS and antibiotics as described. The cells were mildly trypsinized for passage and for use in experiments.
- The Experiment
- The compounds of this invention were dissolved to a concentration of 177147 ng/ml as follows:
Paclitaxel 5% DMSO in full RPMI growth medium 5 5% DMSO in full RPMI growth medium 8 5% DMSO in full RPMI growth medium 11 5% DMSO in full RPMI growth medium - On day 0, 200 μl of trypsinized tumor cells (2*103 cells/well) were plated in 96-wells flatbottom microtiter plates (Costar, no. 3799, Badhoevedorp, The Netherlands). The plates were preincubated 24 hr at 37° C,, 5% CO2 to allow the cells to adhere.
- On
day 2, 100 μl of the highest drug concentration was added to the wells of column 12 and from there diluted 3-fold tocolumn 3 by serial transfer of 100 μl using an 8 channel micropipette. The final volume ofcolumn 3 was adjusted to 200 μl with PBS.Column 2 was used for the blank. Tocolumn 1 PBS was added to diminish interfering evaporation. - On
day 7 the incubation was terminated by washing the plates twice with PBS. Subsequently the cells were fixed with 10% trichloroacetic acid in Milli Q water (Millipore, Etten Leur, The Netherlands) and placed at 4° C. for one hour. - After five washings with tap water, the cells were stained for at least 15 min. with 0.4% SRB, dissolved in 1% acetic acid, and subsequently washed with 1% acetic acid to remove the unbound stain. The plates were air dried and the bound protein was dissolved by using 150
μl 10 mmol/l tris base. The absorbance was read at 540 nm using an automatic microplate reader (Titertec, Flow Laboratories LtD., Irvine, Scotland). Data were used for construction of concentration-response curves and determination of the IC50-value. - Compounds1, 6
- The human tumor cell line OVCAR-3 was used. OVCAR is an ovarium carcinoma.
- In vitro antiproliferative effects: paclitaxel or paclitaxel prodrugs were dissolved in DMSO to give a concentration of 5 mM. Concentrations were verified by measuring the OD at 226 nm. The antiproliferative effects of drugs and prodrugs were determined with the use of OVCAR-3 cells. Cells in supplemented tissue culture medium (DMEM, 10% fetal calf serum with 50 IU/ml penicillin and 50 microgram/ml streptomycin) were seeded in triplicate in 96-well culture plates (5000/well, 100 microliter). After 24 h, 100 microliter of culture medium containing drug or prodrug was added to give final concentrations ranging from 1 picomolar to 10 micromolar.
- The cells were incubated for an additional 72 h, fixed with 10% trichoroacetic acid and stained with sulforhodamine B. The absorbance at 540 nm was read and the antiproliferative effects were expressed as IC50 values, which are the (pro)drug concentration that give 50% growth inhibition when compared with control cell growth (Houba et al. Bioconj. Chem. 1996, 7, 606-611).
- The IC50 values of the compounds of this invention are given in table II.
TABLE II IC50 a (ng/ml) IC50 a (nM) Compound MCF7 EVSA-T WIDR IGROV M19 MEL A498 H226 OVCAR-3 paclitaxel <3 <3 <3 33 <3 5 <3 0.25 5 <3 <3 <3 <3 3 39 10 — 6 <3 <3 <3 233 <3 <3 <3 0.80 8 69 59 167 49 311 436 241 — 11 390 300 589 241 1344 1435 706 — - Evaluation and Conclusions
- With exception of
compound 11, all the analogs of paclitaxel show increased water solubility relative to paclitaxel. - Compounds8 and 11 showed reduced cytotoxic activity, when compared to paclitaxel, probably due to the functional group at C7-OH. The 7-analog (11) and the 2′,7-analog (8) is very stable: in PBS-buffer (pH 7.4) as well as in human plasma, no degradation to the parent drug was observed, since no liberated paclitaxel has been detected.
-
Compound 5 showed similar cytotoxic activity as paclitaxel, which can probably be explained by the degradation of these compounds to the parent drug, under the conditions used to determine the activity. The 2′-analog 5 is stable in PBS-buffer (pH 7.4). After 24 hours, only traces of paclitaxel were detected. Whereas in human plasma only after 20 hours 50% of the analog is degraded to paclitaxel. -
Compound 6 showed a comparable against OVCAR-3 cells, when compared to paclitaxel. Ofcompound 6 about 50% was degraded to paclitaxel within 4 hours. Furthermore,compound 6 is sixty times more watersoluble than paclitaxel. - The present invention discloses a method for the preparation of paclitaxel analogs of paclitaxel having a malate moiety at C2′ and/or C7-position. It is apparent that many modifications of the present invention are possible, for example the use of counterions other than sodium, which may give rise to higher solubilities. It is therefore understood that the invention may be practiced otherwise than specifically described.
Claims (4)
2. A pharmaceutical composition comprising an antineoplastically effective amount of the analog of as an active ingredient, and pharmaceutically acceptable carrier, optionally in combination with further additives.
claim 1
3. A method for the treatment of tumours comprising administering an effective amount of the analog as defined in .
claim 1
4. A method in accordance with , wherein said tumour is a mammary carcinoma or a colon tumour.
claim 3
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/NL1998/000473 WO2000010988A1 (en) | 1998-08-21 | 1998-08-21 | Water soluble analogs and prodrugs of paclitaxel |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/NL1998/000473 Continuation WO2000010988A1 (en) | 1998-08-21 | 1998-08-21 | Water soluble analogs and prodrugs of paclitaxel |
Publications (2)
Publication Number | Publication Date |
---|---|
US20010018531A1 true US20010018531A1 (en) | 2001-08-30 |
US6344571B2 US6344571B2 (en) | 2002-02-05 |
Family
ID=19866454
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/788,344 Expired - Fee Related US6344571B2 (en) | 1998-08-21 | 2001-02-21 | Water soluble analogs and prodrugs of paclitaxel |
Country Status (11)
Country | Link |
---|---|
US (1) | US6344571B2 (en) |
EP (1) | EP1105379B1 (en) |
JP (1) | JP2002523407A (en) |
AT (1) | ATE218560T1 (en) |
AU (1) | AU8889998A (en) |
CA (1) | CA2341234A1 (en) |
DE (1) | DE69805860T2 (en) |
DK (1) | DK1105379T3 (en) |
ES (1) | ES2179526T3 (en) |
PT (1) | PT1105379E (en) |
WO (1) | WO2000010988A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2045270A2 (en) | 2002-10-18 | 2009-04-08 | FIDIA FARMACEUTICI S.p.A. | Taxanes covalently bounded to hyaluronic acid or hyaluronic acid derivatives |
CN101029034B (en) * | 2007-03-30 | 2010-05-26 | 浙江大学 | Polyenic taxol soluble derivative, its preparation and use |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6414015B1 (en) | 2000-01-28 | 2002-07-02 | Utah State University | Laulimalide microtubule stabilizing agents |
AU7007001A (en) | 2000-06-22 | 2002-01-02 | Nitromed Inc | Nitrosated and nitrosylated taxanes, compositions and methods of use |
US7239636B2 (en) | 2001-07-23 | 2007-07-03 | Broadcom Corporation | Multiple virtual channels for use in network devices |
US20050148534A1 (en) * | 2003-09-22 | 2005-07-07 | Castellino Angelo J. | Small molecule compositions and methods for increasing drug efficiency using compositions thereof |
BRPI0608173A2 (en) * | 2005-02-24 | 2010-11-09 | Elan Pharma Int Ltd | composition, use thereof, and method of producing a nanoparticulate or analogous docetaxel composition thereof |
TW200731991A (en) * | 2005-12-20 | 2007-09-01 | Sonus Pharma Inc | Lipophilic di(anticancer drug) compounds, compositions and related methods |
CN106800542A (en) * | 2016-12-31 | 2017-06-06 | 辰欣药业股份有限公司 | A kind of hydrophily paclitaxel analog compound and preparation method thereof |
CN108424405A (en) * | 2018-03-06 | 2018-08-21 | 华东师范大学 | The total prodrug and the preparation method and application thereof of Vorinostat and taxol |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5059699A (en) * | 1990-08-28 | 1991-10-22 | Virginia Tech Intellectual Properties, Inc. | Water soluble derivatives of taxol |
WO1994005282A1 (en) * | 1992-09-04 | 1994-03-17 | The Scripps Research Institute | Water soluble taxol derivatives |
-
1998
- 1998-08-21 AU AU88899/98A patent/AU8889998A/en not_active Withdrawn
- 1998-08-21 PT PT98940677T patent/PT1105379E/en unknown
- 1998-08-21 EP EP98940677A patent/EP1105379B1/en not_active Expired - Lifetime
- 1998-08-21 AT AT98940677T patent/ATE218560T1/en not_active IP Right Cessation
- 1998-08-21 DK DK98940677T patent/DK1105379T3/en active
- 1998-08-21 WO PCT/NL1998/000473 patent/WO2000010988A1/en active IP Right Grant
- 1998-08-21 CA CA002341234A patent/CA2341234A1/en not_active Abandoned
- 1998-08-21 ES ES98940677T patent/ES2179526T3/en not_active Expired - Lifetime
- 1998-08-21 JP JP2000566261A patent/JP2002523407A/en active Pending
- 1998-08-21 DE DE69805860T patent/DE69805860T2/en not_active Expired - Fee Related
-
2001
- 2001-02-21 US US09/788,344 patent/US6344571B2/en not_active Expired - Fee Related
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2045270A2 (en) | 2002-10-18 | 2009-04-08 | FIDIA FARMACEUTICI S.p.A. | Taxanes covalently bounded to hyaluronic acid or hyaluronic acid derivatives |
CN101029034B (en) * | 2007-03-30 | 2010-05-26 | 浙江大学 | Polyenic taxol soluble derivative, its preparation and use |
Also Published As
Publication number | Publication date |
---|---|
WO2000010988A8 (en) | 2000-06-15 |
DE69805860T2 (en) | 2003-01-02 |
EP1105379B1 (en) | 2002-06-05 |
JP2002523407A (en) | 2002-07-30 |
AU8889998A (en) | 2000-03-14 |
ATE218560T1 (en) | 2002-06-15 |
DK1105379T3 (en) | 2002-08-19 |
CA2341234A1 (en) | 2000-03-02 |
ES2179526T3 (en) | 2003-01-16 |
EP1105379A1 (en) | 2001-06-13 |
PT1105379E (en) | 2002-10-31 |
DE69805860D1 (en) | 2002-07-11 |
WO2000010988A1 (en) | 2000-03-02 |
US6344571B2 (en) | 2002-02-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5278324A (en) | Water soluble derivatives of taxol | |
US5352805A (en) | Water soluble derivatives of taxol | |
US5422364A (en) | Water soluble taxol derivatives | |
US5411984A (en) | Water soluble analogs and prodrugs of taxol | |
Mathew et al. | Synthesis and evaluation of some water-soluble prodrugs and derivatives of taxol with antitumor activity | |
Damen et al. | Paclitaxel esters of malic acid as prodrugs with improved water solubility | |
US5157049A (en) | Method of treating cancers sensitive to treatment with water soluble derivatives of taxol | |
US5614549A (en) | High molecular weight polymer-based prodrugs | |
AU738536B2 (en) | Taxoid reversal agents for drug-resistance in cancer chemotherapy and pharmaceutical compositions thereof | |
US4942184A (en) | Water soluble, antineoplastic derivatives of taxol | |
Du et al. | Synthesis and evaluation of water-soluble docetaxel prodrugs-docetaxel esters of malic acid | |
US5622986A (en) | 2'-and/or 7-substituted taxanes | |
NZ243548A (en) | Taxol analogues, preparation and pharmaceutical compositions | |
US6344571B2 (en) | Water soluble analogs and prodrugs of paclitaxel | |
US5750562A (en) | 10-deacetylbaccatine III and 10-deacetyl 14β-hydroxybaccatine III derivatives, a process for the preparation thereof and pharmaceutical compositions containing them | |
US8133888B2 (en) | Hydrazide containing taxane conjugates | |
CA2253912C (en) | Taxane derivatives, the preparation thereof and formulations containing them | |
SK285683B6 (en) | Semi-synthetic taxanes, pharmaceutical composition containing thereof and use thereof | |
JP4499383B2 (en) | Water-soluble prodrug | |
Kapoor et al. | Recent advances in structure modifications of Taxol (Paclitaxel) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: PHARMACHEMIE B.V., NETHERLANDS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WIEGERINCK, PETER H.G.;SPERLING, DUNCAN;BRAAMER, LESLY;AND OTHERS;REEL/FRAME:011749/0393 Effective date: 20010108 |
|
REMI | Maintenance fee reminder mailed | ||
LAPS | Lapse for failure to pay maintenance fees | ||
STCH | Information on status: patent discontinuation |
Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362 |
|
FP | Lapsed due to failure to pay maintenance fee |
Effective date: 20060205 |