Derivatives of camptothecin, a method of producing them and their use

Abstract

The subject of the present invention are water-soluble derivatives of camptothecin, their synthesis and use. These compounds exhibit preferable biological properties for use in anti-neoplasm therapy.

Claims

1. A compound defined by the general formula (I) ##STR00005## where R.sup.1 denotes (N-piperidinyl)methyl, (N-piperazinyl)methyl, (N(N-methylpiperazinyl)methyl, (N-morpholinyl)methyl, (N-pyrrolidinyl)methyl, (N-azetidinyl)methyl, (N-aziridinyl)methyl, (N-methylamino)methyl; R.sup.2 denotes ethyl; R.sup.3 denotes H, (N-piperidinyl)methyl, (N-piperazinyl)methyl, (N(N-methylpiperazinyl)methyl, (N-morpholinyl)methyl, (N-pyrrolidinyl)methyl, (N-azetidinyl)methyl, (N-aziridinyl)methyl, (N-methylamino)methyl.

2. A method of obtaining the compound defined in claim 1, comprising reacting 7-ethyl-10-hydroxycamptothecin with an amine and formaldehyde in an environment of acetonitrile and acetic acid at boiling temperature, wherein the amine is piperidine, piperazine, N-methylpiperazine, morpholine, pyrrolidine, azetidine, aziridine, methylamine, or diethylamine.

3. A compound selected from a group consisting of: 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin, 7-ethyl-11-(N-morpholinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-morpholinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N-piperidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-11-(N-piperidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-piperidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N-piperazinyl)methyl-10-hydroxycamptothecin, 7-ethyl-11-(N-piperazinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-piperazinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N(N-methylpiperazinyl)methyl-10-hydroxycamptothecin, 7-ethyl-11-(N(N-methylpiperazinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N(N-methylpiperazinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N-pyrrolidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-11-(N-pyrrolidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-pyrrolidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N-azetidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-11-(N-azetidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-azetidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N-aziridinyl)methyl-10-hydroxycamptothecin, 7-ethyl-11-(N-aziridinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-aziridinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin, 7-ethyl-11-(N-methylamino)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-methylamino)methyl-10-hydroxycamptothecin, 7-ethyl-11-(N,N-diethylamino)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N,N-diethylamino)methyl-10-hydroxycamptothecin.

4. An intermediate compound (-QM) o-methylenequinone defined by the formula (II): ##STR00006## where R.sup.1 denotes (N-piperidinyl)methyl, (N-piperazinyl)methyl, (N(N-methylpiperazinyl)methyl, (N-morpholinyl)methyl, (N-pyrrolidinyl)methyl, (N-azetidinyl)methyl, (N-aziridinyl)methyl, (N-methylamino)methyl, (N,N-diethylamino)methyl; R.sup.2 denotes ethyl; or R.sup.1 denotes H; R.sup.2 denotes ethyl.

5. An intermediate compound (-QM) o-methylenequinone defined by the formula (III): ##STR00007## where R.sup.2 denotes ethyl; R.sup.3 denotes H, (N-piperidinyl)methyl, (N-piperazinyl)methyl, (N(N-methylpiperazinyl)methyl, (N-morpholinyl)methyl, (N-pyrrolidinyl)methyl, (N-azetidinyl)methyl, (N-aziridinyl)methyl, (N-methylamino)methyl, (N,N-diethylamino)methyl; or R.sup.2 denotes ethyl; R.sup.3 denotes (N-piperidinyl)methyl, (N-piperazinyl)methyl, (N(N-methylpiperazinyl)methyl, (N-morpholinyl)methyl, (N-pyrrolidinyl)methyl, (N-azetidinyl)methyl, (N-aziridinyl)methyl, (N-methylamino)methyl, (N,N-diethylamino)methyl.

6. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutical carrier.

7. A pharmaceutical composition comprising the compound of claim 3 and a pharmaceutical carrier.

8. A method of treating breast cancer, leukemia or colon cancer in a subject comprising administering to the subject the compound of claim 1.

9. A method of treating breast cancer, leukemia or colon cancer in a subject comprising administering to the subject the compound of claim 3.

10. The compound according to claim 3, characterised in that it is a compound selected from a group consisting of: 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-morpholinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N-piperidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-piperidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N-piperazinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-piperazinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N(N-methylpiperazinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N(N-methylpiperazinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N-pyrrolidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-pyrrolidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N-azetidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-azetidinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N-aziridinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-aziridinyl)methyl-10-hydroxycamptothecin, 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin, 7-ethyl-9,11-bis(N-methylamino)methyl-10-hydroxycamptothecin.

11. An intermediate compound (-QM) o-methylenequinone defined by the formula (II): ##STR00008## where R.sup.1 denotes (N-piperidinyl)methyl, (N-piperazinyl)methyl, (N(N-methylpiperazinyl)methyl, (N-morpholinyl)methyl, (N-pyrrolidinyl)methyl, (N-azetidinyl)methyl, (N-aziridinyl)methyl, (N-methylamino)methyl; R.sup.2 denotes ethyl; or R.sup.1 denotes H; R.sup.2 denotes ethyl.

Description

(1) The subject of the present invention in its example embodiments is shown in the figures in which:

(2) FIG. 1 represents PFGSE NMR analysis results for derivatives of DNA/TPT prior to UV exposure. The denoted lines indicate diffusion coefficients, Di, for DNA (with a non-covalently bound derivative of TPT), ca. 1.710.sup.10 m.sup.2/s, and unbound derivative of TPT in a rapid exchange with DNA, 2.310.sup.10 m.sup.2/s;

(3) FIG. 2 represents a PFGSE NMR experiment using derivatives of DNA/TPT following UV exposure. The denoted lines indicate diffusion coefficients, Di, ca. 1.410.sup.10 m.sup.2/s for the DNA-TPT bioconjugate;

(4) FIG. 3 represents a MALDI MS diagram for alkylation products of d(GCGATCGC).sub.2 using the salt [Me.sub.3N.sup.+CF.sub.3SO.sub.3.sup.] TPT following UV exposure; the double-stranded (ds) region of the alkylated conjugate is shown alkylated. Solid arrows indicate unalkylated dsDNA, whereas dashed arrows indicate the monoalkyl conjugate of TPT;

(5) FIG. 4 represents biological analysis results using 7-ethyl-9-dimethylaminomethyl-10-hydroxycamptothecin with three evaluated cell lines: L-1210-murine leukaemia, MCF-7human breast cancer cells and Caco-2 (human colon cancer cells);

(6) FIG. 5 represents biological analysis results using 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin with three evaluated cell lines: L-1210-murine leukaemia, MCF-7human breast cancer cells and Caco-2 (human colon cancer cells);

(7) FIG. 6 represents the ESI MS spectrum of the compound defined in claim 1 with a visible o-methylenequinone signal, m/z 405.1371.

(8) Due to the present invention, it was possible to obtain a covalent, spontaneous or UV-induced, DNA alkylation reaction by derivatives of camptothecin, as was shown in the following example embodiments using DNA oligomers.

(9) The results obtained are very significant in terms of the use of the compounds according to the present invention in medicine, in particular in anti-cancer therapy.

(10) In the embodiments of the present invention we give example compounds and prove the formation of conjugates using PFGSE NMR, which facilitates the easy differentiation of molecular complexes from covalent hybrid conjugates through a measurement of their diffusion coefficients. Moreover, the present invention was tested in biological systems using cancer lines, yielding a preferable IC.sub.50 parameter value at a 20 M concentration.

(11) In light of the above, the present invention may constitute a breakthrough in chemotherapy due to the uncommonly preferable properties of the claimed compounds, thus delivering an effective tool for combating neoplastic diseases.

(12) The present invention is disclosed using the following example embodiments, without limiting in any way the scope nor methods in which it may be embodied.

EXAMPLE I

Production of 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin

(13) SN38 (7-ethyl-10-hydroxycamptothecin) (3.3 mg; 810.sup.3 mmol) was suspended in 2.4 ml CH.sub.3CN, and then supplemented with acetic acid (103 L), 37% aqueous CH.sub.2O (8 L; 0.0986 mmol), 8.5 l morpholine (0.098 mmol). The resulting mixture was mixed at a temperature of 80 C. After 10 h the solvent was evaporated off under a vacuum (at 40 C.) and rinsed with diethyl ether (31 mL). The resulting residue was lyophilized using 2 ml H.sub.2O and dissolved in deuterated DMSO. On the basis of .sup.1H NMR spectra, we detected the presence of two substances: SN38 and 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin (efficiency: 33%). LR-MS (ESI): m/z 393 [M+H].sup.+, 492 [M+H].sup.+. The deuteriated DMSO was evaporated off under a vacuum. The residue was extracted using 0.5% HCl (31 ml) in order to extract water-soluble derivatives of morpholinylcamptothecin. The insoluble 7-ethyl-10-hydroxycamptothecin was removed through filtration. Next, this was purified using HPLC on an RP-C18 LPH column (150 mm10 mm) using the following liquid phase system: 20% CH.sub.3CN/80% aqueous 0.1% HCOOH at a flow rate of 3 ml/min. The course of the chromatography was monitored using UV detection at a wavelength of 260 nm. Fractions were collected (retention time: 8.5 min) and were lyophilized yielding a pure product (>98%).

(14) Spectral data: 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin

(15) LR-MS (ESI): m/z 492 [M+H].sup.+

(16) .sup.1H NMR (D.sub.2O, TSPA) 500 MHz: 1.01 (t, J=7.3 Hz, 3H), 1.45 (t, J=7.3 Hz, 3H), 2.00 (m, 2H), 3.13 (m, 1H), 3.22 (m, 1H), 3.32 (m, 4H), 3.97 (m, 4H), 4.87 (d, J=15.0 Hz, 1H), 4.88 (d, J=15.0 Hz, 1H), 4.93 (d, J=18.7 Hz, 1H), 4.96 (d, J=18.7 Hz, 1H), 5.41 (J=16.14 Hz, 1H), 5.55 (J=16.14 Hz, 1H), 7.25 (d, J=9.17 Hz, 1H), 7.26 (s, 1H), 7.59 (d, J=9.17 Hz, 1H).

(17) .sup.13C NMR (D.sub.2O, TSPA); HSQC 6.1, 13.4, 24.6, 30.5, 42.9, 49.8, 55.0. 63.4, 65.8, 97.8, 121.5, 130.7; HMBC 73.2, 117.5, 146.0. 146.9, 150.5, 174.30

(18) UV nm: 378, 366, 329, 268, 224, 202.

(19) IR (KBr) cm.sup.1:1038, 1056, 1108, 1162, 1261, 1303, 1383, 1423, 1463, 1513, 1593, 1656, 1744, 2853, 2922, 2958, 3401

EXAMPLE II

Production of 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin

(20) SN38 (2.9 mg, 710.sup.3 mmol) was suspended in 2.1 ml CH.sub.3CN, and then supplemented with acetic acid (190 L), 37% aqueous CH.sub.2O (6.8 L, 0.084 mmol), and morpholine (7.3 L, 0.084 mmol). The resulting mixture was mixed at a temperature of 80 C. After 8 h was evaporated off under a vacuum soluble (w 40 C.) and rinsed with diethyl ether (31 ml). The resulting residue was lyophilized using 2 ml H.sub.2O and dissolved in deuterated DMSO. On the basis of .sup.1H NMR spectra, we noted the presence of two substances: SN38 and 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin (efficiency: 25%). LR-MS (ESI): m/z 393 [M+H].sup.+, 492 [M+H].sup.+. The deuterated DMSO was evaporated off under a vacuum. The residue was extracted using 0.5% HCl (31 ml) in order to extract water-soluble derivatives of camptothecin. The insoluble camptothecin was removed through filtration. The purification of the residue was performed using HPLC in the following manner: the residue was dissolved in the liquid phase (20% CH.sub.3CN, 80% 0.1% aqueous HCOOH) and loaded onto an RP-C18 LPH column 150 mm10 mm, and then eluted at a rate of 3 ml/nm. The course of the chromatography was monitored using UV detection at 260 nm. Fractions were collected (retention time: 8.5 min) and were lyophilized yielding a pure product (>95%).

(21) Spectral data: 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin as in example I

EXAMPLE III

Production of 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin

(22) SN38 (2.27 mg, 5.5410.sup.3 mmol) was suspended in 1.65 ml CH.sub.3CN, and then supplemented with acetic acid (142 l), 37% aqueous CH.sub.2O (10.8 l; 0.133 mmol), morpholine (11.6 l 0.133 mmol). The resulting mixture was mixed at a temperature of 80 C. After 6 h was evaporated off under a vacuum soluble (w 40 C.) and rinsed with diethyl ether (31 ml). The resulting residue was lyophilized using 2 ml H.sub.2O and dissolved in deuterated DMSO. On the basis of .sup.1H NMR spectra, we noted the presence of two substances: SN38 and 7-ethyl-9-N-morpholinylmethyl-10-hydroxycamptothecin (efficiency: 37%). LR-MS (ESI): m/z 393 [M+H].sup.+, 492 [M+H].sup.+. The deuterated DMSO was evaporated off under a vacuum. Residue was extracted using 0.5% HCl (31 ml) in order to extract water-soluble derivatives of camptothecin. The insoluble camptothecin was removed through filtration. The purification of the residue was performed using HPLC in the following manner: the residue was dissolved in the liquid phase (20% CH.sub.3CN, 80% 0.1% aqueous HCOOH) and loaded onto a RP-C18 LPH column 150 mm10 mm, and then eluted at a rate of 3 ml/min. The course of the chromatography was monitored using UV detection at 260 nm. Fractions were collected (retention time: 8.5 min) and were lyophilized yielding a pure product (>96%).

(23) Spectral data: 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin as in example I.

EXAMPLE IV

(24) Alkylation of TPT.sup.23

(25) Methyl trifluoromethanesulphonate (0.1 ml; 0.9 mmol) was dissolved in CH.sub.2Cl.sub.2 (3 ml), and then supplemented with a small quantity of K.sub.2CO.sub.3. The mixture was mixed for 30 min. in order to neutralize trifluoromethanesulphonic acid (CF.sub.3SO.sub.3H). The solution was filtered free of K.sub.2CO.sub.3, added to dry free TPT (1.8 mg; 4.410.sup.3 mmol) and was mixed for 24 h at room temperature. The mixture was evaporated off until dry under a vacuum at room temperature and dissolved in deuterated DMSO-d.sub.6. On the basis of .sup.1H NMR spectra, we detected the presence of five products: N-methylated TPT-([Me.sub.3N.sup.+CF.sub.3SO.sub.3.sup.], TPT salt); N,O-dimethylated TPT, protonated TPT-[TPTH.sup.+CF.sub.3SO.sub.3.sup.], and 23-CH.sub.2OH alcohol. N-methylated TPT ([Me.sub.3N.sup.+CF.sub.3SO.sub.3.sup.] salt TPT) constituted up to 50% of the mixture. We performed an LR ESI MS of the reaction mixture; LR-MS (ESI): m/z: 149 [CF.sub.3SO.sub.3].sup. 422 [M+H].sup.+, 436 [M].sup.+, 450 [M].sup.+.

(26) The evaporated to dryness the reaction mixture was rinsed with water (51 ml). The precipitate was filtered off and the aqueous layer was lyophilized. Next, this was purified using HPLC on an RP-C18 LPH column (150 mm10 mm) using the following liquid phases: 10% CH.sub.3CN/90% aqueous 0.1% HCOOH for the first 15 min, whereafter the system was changed for 20% CH.sub.3CN/80% aqueous 0.1% HCOOH with a steady flow rate of 3 ml/min. The course of the chromatography was monitored using UV detection at a wavelength of 260 nm. Fractions were collected (retention time: 25 min) and were lyophilized yielding 2 products.

(27) LR-MS (ESI): m/z 422 [M+H].sup.+-[TPTH.sup.+CF.sub.3SO.sub.3]-non-reactive in UV; 436 [M].sup.+[Me.sub.3N.sup.+CF.sub.3SO.sub.3.sup.] reactive UV.

(28) Experimental Details of the HPLC

(29) Chromatography in a reversed phase system, C-18, was performed using an analytical column (Bionacom Velocity C18 LPH, 4.6250 mm, 5 microns) and a semi-preparative column (Bionacom Velocity C18 LPH, 10 mm150 mm, 5 microns) in a Shimadzu LC-8A HPLC apparatus equipped with a Shimadzu SPD-6A UV spectrophotometric detector.

(30) ESI MS Experimental Data

(31) Solvent: 0.1% HCOOH, ACN 1:1; ion source: ESI; injected volume 1 L; m/z range: 150-1500; ion retention time in the ion trap: 10 ms; detector voltage: 1.7 kV; interface voltage: 4.5 and 4.5 kV; CDL temperature: 200 C; Heat Block temperature: 200 C; carrier gas: 1.5 L/min; flow rate: 0.1 ml/min; wavelength: Ch1: 254 nm, Ch2: 220 nm; Apparatus: Shimadzu LCMS-IT-TOF.

EXAMPLE V

(32) NMR Experiments Using Diffusion

(33) We performed a PFGSE analysis using a DNA/TPT derivative prior to UV exposure (FIG. 1) and after UV exposure (FIG. 2). Next, we separated the unbound TPT derivative and analyzed it chromatographically using HPLC.

(34) Based on the resulting spectrum, we detected a clear difference between the molecular complex and bioconjugate. The experimental results constitute evidence of the existence of a covalent bond between the chemically modified CPT unit and a DNA oligomer.

EXAMPLE VI

(35) Sample Preparation and MALDI-TOF-MS Analysis

(36) A dry sample prepared according to the above procedure, containing up to 50% TPT salt [Me.sub.3N.sup. CF.sub.3SO.sub.3.sup.], was added in excess (2 eq.) to an aqueous solution containing 1 mmol of the oligonucleotide d(GCGATCGC).sub.2 without buffer, in quartz NMR tubes. The solution was exposed using a UV lamp with a wavelength of 365 nm, for 20 hrs. The formed precipitate was filtered off, and the solution was evaporated off until dry under reduced pressure. In order to perform MALDI, the solid was dissolved in deionised water. The mass spectrum for negative MALDI ions was recorded using a Voyager-Elite apparatus (PerSeptive Biosystems Inc., Framingham, Mass., USA), equipped with a nitrogen laser (337 nm) in linear mode with an accelerating voltage of 20 kV and delayed ion extraction.

(37) A mixture of 50 mg/ml of a 3-hydroxypicolinic acid solution in 50% acetonitrile and 50 mg/ml of a diammonium hydrocitrate solution in deionised in water (8:1, vol/vol) was used as a template. One L of the oligonucleotide solution in deionised water at a concentration of 0.01 OD/L was mixed on a MALDI plate with one L of template and left to air dry. The mass spectrum was obtained from at least 100 laser impulses and transformed using Data Explorer Ver. 4 software (Applied Biosystems, Foster City, Calif.). MALDI-TOF analysis made it possible to obtain an m/z mass with an accuracy higher than 0.1% (i.e. 1 mass unit for [M1].sup. of 1000).

(38) MALDI MS analysis results for products of the d(GCGATCGC).sub.2 alkylation using a TPT salt [Me.sub.3N.sup.+CF.sub.3SO.sub.3.sup.] following UV exposure show the existence of a double-stranded region in the alkylated conjugate of DNA, as shown in FIG. 3.

EXAMPLE VII

(39) Biological Analysis of Cells

(40) L1210 Murine Leukaemia Cell Line

(41) The culture of L1210 murine leukaemia cells was conducted in RPMI-1640 medium, supplemented with 10% thermally inactivated foetal bovine serum, penicillin (100 UI/ml), streptomycin (100 g/ml), amphotericin (250 ng/ml), L-glutamine (2 mM) and 1% non-essential aminoacids. The cells were cultured at 37 C., in an atmosphere of 5% CO.sub.2 and 95% air. The medium was exchanged every 48 hours. For all experiments, the cells were inoculated at density of 110.sup.5 cells/ml.

(42) MTT Assay

(43) This test is based on a measurement of the amount of formazan formed by the ability of mitochondrial dehydrogenase (part of the respiratory chain) to reduce the water-soluble 3-(4,5-dimethylthiazolo-2-ylo]-2,5-diphenyltetrazole (MTT) bromide. Damaged or dead cells exhibit a low or no dehydrogenase activity. This test may be used to determine cell viability. The solution of 7-ethyl-9-dimethylaminomethyl-10-hydroxycamptothecin or 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin was prepared in DMSO. In order to determine the cytotoxicity of the evaluated compounds, L1210 cells were incubated for 24 hours with a solution of 7-ethyl-9-dimethylaminomethyl-10-hydroxycamptothecin or 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin at concentrations of 1 M, 5 M, 10 M, 20 M, 30 M, 40 M and 50 M. After centrifugation and rinsing with PBS buffer, the cells were incubated for 3 hours in MTT solution, and the crystals formed were dissolved using isopropanol. Formazan absorbance was measured on a Power Wavex microplate spectrophotometer (Biotek Instruments) at a wavelength of 570 nm.

(44) Statistical Analysis

(45) To evaluate the statistical significance of changes in the level of cytotoxicity we used analysis of variance (ANOVA) with Dunnett's post-hoc test at a significance level of p<0.05. Calculations were performed using the Statistica Ver. 9.0 package (StatSoft, Inc. USA).

(46) MCF-7 Human Breast Cancer Line

(47) The culture of the MCF-7 human breast cancer cell line was conducted in Eagle's Minimum Essential Medium (MEM), supplemented with 10% thermally inactivated foetal bovine serum, penicillin (100 UI/ml), streptomycin (100 g/ml), amphotericin (250 ng/ml), L-glutamine (2 mM) and 1% non-essential aminoacids. The cells were cultured at 37 C., in an atmosphere of 5% CO.sub.2 and 95% air. The medium was exchanged every 48 hours, and after 80% confluence was attained, the cells were passaged with a 0.25% trypsin solution. In all experiments, cells were inoculated at a density of 510.sup.4 cells/ml.

(48) Test MTT

(49) This test is based on a measurement of the amount of formazan formed by the ability of mitochondrial dehydrogenase (part of the respiratory chain) to reduce the water-soluble 3-(4,5-dimethylthiazolo-2-ylo]-2,5-diphenyltetrazole (MTT) bromide. Damaged or dead cells exhibit a low or no dehydrogenase activity. This test may be used to determine cell viability. The solution of 7-ethyl-9-dimethylaminomethyl-10-hydroxycamptothecin or 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin was prepared in DMSO. In order to determine the cytotoxicity of the evaluated compounds, cells MCF-7 were incubated for 24 hours with a solution of 7-ethyl-9-dimethylaminomethyl-10-hydroxycamptothecin or 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin at concentrations of 1 M, 5 M, 10 M, 20 M, 30 M, 40 M and 50 M. After rinsing with PBS buffer, cells were incubated for 3 hours in MTT solution, and the crystals formed were dissolved using isopropanol. Formazan absorbance was measured w Power Wavex microplate spectrophotometer (Biotek Instruments) at a wavelength of 570 nm.

(50) Statistical Analysis

(51) To evaluate the statistical significance of changes in the level of cytotoxicity we used analysis of variance (ANOVA) with Dunnett's post-hoc test at a significance level of p<0.05. Calculations were performed using the Statistica Ver. 9.0 package (StatSoft, Inc. USA).

(52) Caco-2 Human Colon Cancer Line

(53) The culture of Caco-2 human colon cancer cells was conducted in Eagle's Minimum Essential Medium Eagle (MEM), supplemented with 20% thermally inactivated foetal bovine serum, penicillin (100 UI/ml), streptomycin (100 g/ml), amphotericin (250 ng/ml), L-glutamine (2 mM) and 1% non-essential amine. The cells were cultured at 37 C., in an atmosphere of 5% CO.sub.2 and 95% air. The medium was exchanged every 48 hours, and after 80% confluence was attained, the cells were passaged with a 0.25% trypsin solution. In all experiments, cells were inoculated at a density of 6.510.sup.4 cells/ml.

(54) Test MTT

(55) This test is based on a measurement of the amount of formazan formed by the ability of mitochondrial dehydrogenase (part of the respiratory chain) to reduce the water-soluble 3-(4,5-dimethylthiazolo-2-ylo]-2,5-diphenyltetrazole (MTT) bromide. Damaged or dead cells exhibit a low or no dehydrogenase activity. This test may be used to determine cell viability. The solution 7-ethyl-9-dimethylaminomethyl-10-hydroxycamptothecin or 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin was prepared in DMSO. In order to determine the cytotoxicity of the evaluated compounds, cells Caco-2 were incubated for 24 hours with a solution of 7-ethyl-9-dimethylaminomethyl-10-hydroxycamptothecin or 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin at concentrations of 1 M, 5 M, 10 M, 20 M, 30 M, 40 M and 50 M. After rinsing with PBS buffer cells were incubated for 3 hours in MTT solution and the crystals formed were dissolved using isopropanol. Formazan absorbance was measured in Power Wavex microplate spectrophotometer (Biotek Instruments) at a wavelength of 570 nm.

(56) Statistical Analysis

(57) To evaluate the statistical significance of changes in the level of cytotoxicity we used analysis of variance (ANOVA) with Dunnett's post-hoc test at a significance level of p<0.05. Calculations were performed using the Statistica Ver. 9.0 package (StatSoft, Inc. USA).

(58) The biological analysis results using 7-ethyl-9-dimethylaminomethyl-10-hydroxycamptothecin in the three evaluated cell lines: L-1210-murine leukaemia cells, MCF-7-human breast cancer cells and Caco-2 human colon cancer cells are shown in FIG. 4. The biological analysis results using 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin in the three evaluated cell lines: L-1210-murine leukaemia cells, MCF-7-human breast cancer cells and Caco-2 human colon cancer cells are shown in FIG. 5.

EXAMPLE VIII

(59) Title: Materials and Experimental Methods Used

(60) Experimental Details

(61) DNA oligomers were purchased from Integrated DNA Technologies, Inc. and purified as described..sup.12 The oligonucleotide d(GCGATCGC).sub.2 from Integrated DNA Technologies, Inc. was purified using ion exchange chromatography, using a HiTrap-Q column (Pharmacia Biotech) using gradient elution with ammonium bicarbonate (0.1 M-0.8 M) and desalted on a column packed with Sephadex G-10. Purity>95+

(62) Topotecan hydrochloride was purchased from Alexis Biochemicals and used directly without additional purification. Purity>98+

(63) In order to obtain the free TPT base, topotecan hydrochloride (2 mg, 4.410.sup.3 mmol) was dissolved in H.sub.2O (0.5 mL) and pH was set to 7 using aqueous NaHCO.sub.3. Free TPT was extracted using CH.sub.2Cl.sub.2 and dried under a vacuum.

(64) Sample Preparation

(65) .sup.1H NMR spectra were obtained using a VARIAN VNMRS 500 MHz spectrometer using a Nalorac ID probe equipped with a gradient unit generating a gradient of 60 Gs/cm along the Z axis, at a temperature of 25.0 C. Chemical shifts were made in reference to TSPA as an internal standard dissolved in D.sub.2O.

(66) The parameters of the .sup.1H NMR analysis were: 25.3 C.; sw=8000 Hz (spectrum range); nt=16, or 64, or 256; (number of scans), 32 K memory points; at=2 s (acquisition time), d1=6 s (relaxation delay), satdly=2 s (presaturation delay), 1b=0.3 Hz (line broadening).

(67) Analysis parameters .sup.13C NMR; 25.0 C.;

(68) The .sup.1H/.sup.13C-HSQC. .sup.1H-.sup.13C HSQC spectra (heteronuclear single quantum correlation) were obtained using a spectrum range of 5000 Hz, 2048 points in the .sup.1H domain and 8000 Hz, 8002 increments in the .sup.13C domain; 128 scans per increment t.sub.1, with a relaxation delay of 1 s and .sup.1J(C,H)=135 Hz. The data were linearly extrapolated to 1600 points and filled with zeros to 4096 points in F.sub.1 prior to a Fourier transformation.

(69) .sup.1H/.sup.13C-HMBC spectra (heteronuclear multiple bonds correlation) HMBC spectra with a gradient coherence of .sup.1H-.sup.13C were obtained, using an acquisition time of 0.2 s, .sup.1H-90 impulse width of 7.8 s, .sup.13C-90 impulse width of 11.5 s, spectrum range of 5000 Hz, 2048 given points in the .sup.1H dimension and 25000 Hz, 1024 increments in the .sup.13C dimension, and a relaxation delay of 1.2 s. Data were obtained in the form of absolute values, using 64 scans per t.sub.1 increment. The experiment was optimized for .sup.nJ(C,H)=8 Hz, and for .sup.1J(C,H)=140 Hz with a low-pass filter. The data were linearly extrapolated to 2048 points and filled with zeros to 4096 points in F.sub.1 prior to a Fourier transformation.

(70) (PFGSE) (Pulsed Field Gradient Spin Echo) (Diffusion Measurement Experiments)

(71) DOSY NMR is a method with a high potential for analysing mixtures of chemical compounds in solution, used to measure the translation coefficient of diffusion D.sub.t. Larger molecules diffuse much slower than smaller ones. Each spectral line in the .sup.1H-1D NMR spectrum of a given chemical individual is characterised in terms of the same D.sub.t coefficient, differing for each molecule with a different molecular mass.

(72) The experiments were performed using the following conditions: 16 spectra were collected using the PFGDSTE sequence.sup.18 (Double Stimulated Echo with convection compensation) with an initial saturation of the residual water signal. The gradient coil value was calibrated using the known water diffusion coefficient (D.sub.t=19.0210.sup.10 m.sup.2/s).sup.19. The gradient strength was increased as a square function in the range from 2 to 50 G/cm. The diffusion time () and duration of the magnetic field gradient () were 150 ms and 1.0 ms, respectively. The remaining parameters were: sweep width of 8000 Hz, 32K memory points, 64 scans and a data acquisition time of 2.7 s and a relaxation delay of 2 s and 16 dummy scans. Data are transformed using a Varian package for DOSY.sup.17.

LITERATURE

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