Oxazolinodoxorubicin – a Promising New Anthracycline

Materials and Methods

Antibiotics. Doxorubicin hydrochloride (the substrate for the synthesis of compound 1) with a purity of 98.8% according to HPLC was synthesized by Synbias Pharma Ltd (Donieck, Ukraine). Dimethyl acetal of N-formylmorpholine was obtained according to Abdulla's and Brinkpreyer's method (7). All solvents for the synthesis were p.a. grade and those used in HPLC had a purity of 99.8%.

Physical measurement. HPLC analyses (purity and stability evaluations) were performed using a Waters liquid chromatographic system with a DAD detector (Waters USA). A Chromolith Performance RP-18e column (100-4.6 mm) (E.Merck Darmstadt, Germany) was used at 1.0 ml/min constant flow rate. The mobile phase, consisting of a laurylosulfate buffer and acetonitrile (1:1, v/v) was filtered under vacuum, mixed, and degassed with helium before use. ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectra were recorded on a Bruker DRX500 Advance spectrophotometer in d₆DMSO, at room temperature. The chemical shifts are given in Δppm relative to TMS and the coupling constants (J) in Hz, respectively. Elemental analyses were performed at the Analytical Laboratory of the Department of Chemistry of the Warsaw University of Technology, Poland.

Synthesis of oxazolinodoxorubicin. A solution of 0.365 ml (0.225 mmol) dimethyl acetal of N-formylmorpholine and 8 ml of methanol was added to a solution of 0.870 g of doxorubicin hydrochloride in 190 ml of methanol, and the mixture was stirred for 3 h. The precipitation of crystals began after 1.5 h of stirring. The mixture was then stirred for a further 5 h at a temperature of 5-7°C. The obtained crystals were filtered off, washed with methanol and then with diethyl ether, and dried; 295.1 mg of oxazolinodoxorubicin was obtained (35.5% theoretical yield). For the isolation of the formamidinodoxorubicin, 10 ml of diethyl ether was added to the filtrate which was condensed to a volume of ca. 14 ml. The obtained mixture was kept for 7 h at a temperature of 5-7°C. The crystalline precipitate was then filtered off, washed with methanol and diethyl ether, and dried; 536.2 mg of formamidinodoxorubicin were obtained (55.8% of theoretical yield). The purity of both products according to HPLC method was 98.2% and 98.5%, respectively.

Determination of stability of oxazolinodoxorubicin and formation of N-formyldoxorubicin. Oxazolinodoxorubicin was dissolved in methanol and in the following mixtures: DMSO/water 1:1 v/v, methanol/water 1:1 v/v, and methanol/water 1:2 v/v. The obtained 0.06% mixtures were kept at 37°C and at room temperature for 48 h. Concentrations of oxazolinodoxorubicin, N-formyldoxorubicin and doxorubicin were determined every 12 h by HPLC. The structure of oxazolinodoxorubicin and N-formyldoxorubicin were confirmed by elemental analysis and ¹HNMR as well ¹³CNMR spectra (Table I).

Elemental analysis of oxazolinodoxorubicin: for C₂₈H₂₇NO₁₁: calc.: C 60.74%, H 4.92%, N 2.53%; found: C 60.84%, H 5.07%, N 2.44%. Elemental analysis of N-formyldoxorubicin: for C₂₈H₂₉NO₁₂: calc.: C 58.83%, H 5.12%, N 2.45%; found: C 58.72%, H 5.19%, N 2.34%.

Cell lines. The cell lines used in these studies were obtained from the following collections: American Type Culture Collection (ATCC; Rockville, Maryland, USA), German Cell Line Collection (DKFZ; Tumorbank, Heidelberg, Germany), supplied to us by Professor J. Konopa (Gdansk University of Technology, Poland) and European Collection of Animal Cell Cultures (ECACC; Salisbury, UK).

The following cancer cell lines were used: MCF-7 breast cancer (ATCC); PC-3 prostate cancer (ATCC); colon cancer: SW-707, LoVo, and LoVo/DX (DKFZ); uterine sarcoma: MES-SA and MES-SA/DX5 (ATCC) and leukemia: HL-60, multidrug-resistant variant HL-60/Vinc (DKFZ) and variant HL-60/MX2 resistant to mitoxantrone due to the lack β isoform of topoisomerase II and expression of a truncated the α isoform (ATCC); RPMI 8226 human myeloma and ACHN human renal adenocarcinoma (ECACC).

Cellular uptake of agents was measured in HL-60 (sensitive to doxorubicin) and HL-60/MX2 cells (resistant to topoisomerase II traping agents). These cell lines were chosen for uptake studies to answer the question whether oxazolinodoxorubicin is able to overcome doxorubicine resistance in cancer cells as a result of enhanced uptake or as a result of a different mechanism of cytotoxicity which is not related to topoisomerase inhibition.

The cell lines were cultured in vitro or in vivo at the Cell Culture Collection of the Institute of Immunology and Experimental Therapy, Wroclaw, Poland and at the Institute of Biotechnology and Antibiotics, Warsaw, Poland. All cell lines were maintained under standard conditions as previously described in detail (4).

Antiproliferative assay in vitro. The cells were plated in 96-well plates at a density of 10⁴ cells per well and cultured at 37°C in a humid atmosphere, saturated with 5% CO₂, for 24 h before application of the tested compounds. The cells were then exposed to different concentrations of the tested compounds for 72 h. Stock solutions of oxazolinodoxorubicin and doxorubicin (1.0 mg/ml) were prepared ex tempore for each test by dissolving them in DMSO and in water for injection, respectively. The solutions were then diluted in culture medium to obtain final concentrations ranging from 0.1 to 10000 ng/ml. The antiproliferative effect in vitro was determined by SRB (sulforhodamine B colorimetric assay) or the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay) (for leukemia cells) method (8, 9). The optical densities of the samples were measured on a Multiskan RC photometer (Labsystem, Helsinki, Finland) at 540 nm (SRB) or 570 nm (MTT). The results were calculated as 50% inhibitory concentration (IC₅₀), namely the dose of the tested compound inhibiting proliferation of the cancer cells by 50% as compared to the untreated control cells. Each concentration of the compounds was tested three times in every experiment. Each experiment was repeated 3-5 times.

Resistance indices. The resistance index (RI) was calculated, using the obtained IC₅₀ values, as the ratio of the IC₅₀ for the resistant cell line to that for the sensitive cell line. According to Harker et al. (10), the cells can be assigned to one of three categories depending on the RI value: for RI values of 0-2, the cells are drug sensitive, for values of 3-10, the cells are moderately drug resistant, and for values greater than 10, the cells are markedly drug resistant.

Anthracycline uptake and efflux. The fluorimetric procedure of Riganti et al. (11) was applied to examine the transport of oxazolinodoxorubicin and doxorubicin through cell membranes. HL-60 and HL-60/MX2 cells were concentrated to 10⁷ cells/ml and incubated in growth medium with 10 mM of the compounds at 37°C. Aliquots of cell suspension (0.2 ml) were taken at 15, 30, 60, 90, 120 and 150 min from the start of incubation and mixed with 5 ml of ice-cold phosphate-buffered saline (PBS) and centrifuged at 1500 ×g for 5 min. They were then washed twice with ice-cold PBS. The final cell pellets were resuspended in 2 ml of a 1:1 mixture of ethanol/0.3 M HCl and the fluorescence intensity of these suspensions was measured at 20°C by a Perkin-Elmer LS 55 spectrofluorometer (Massachusetts, USA). Optimal excitation and emission wavelengths were 485 nm and 590 nm, respectively. Calibration curves in the 0.01-0.5 μM concentration range were prepared to estimate the concentrations of compounds. As the fluorescence was slightly quenched in the presence of cells, each point on the calibration curves was measured in the presence of 2×10⁶ cells suspended in a mixture of ethanol/HCl. The relative uptake is the ratio of the drug uptake measured for resistant cells to that of the sensitive cell line multiplied by 100%.

Animals. For toxicity evaluation, female and male mice (BALB/c xDBA/2) (CD2F1), (weight 19-22 g), supplied from the Wroclaw Medical University, were maintained under standard laboratory conditions. For evaluation of the anticancer activity towards the HL-60/Vinc cell line, female NOD/SCID mice (weight 19-22 g), supplied from the University Children's Hospital, Cracow, Poland, were maintained in specific pathogen-free conditions. All animal experiments were performed according to the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketing and Education, as issued by the New York Academy of Sciences Adhoc Committee on Animal Research and approved by the First Local Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland.

Preliminary toxicity evaluation. Oxazolinodoxorubicin was dissolved ex tempore in physiological solution. Mice (three animals per dose) were intraperitoneally (i.p.) administered with the test solution in a single dose of 2, 5, 10 or 50 mg/kg. The animals were observed for three weeks. The body weight was measured every day.

Evaluation of in vivo anticancer activity. On day 0 of the experiment, HL-60/Vinc leukemia cells (6×10⁶, in 0.2 ml of 1:1 saline and Matrigel) were injected subcutaneously (s.c.) into NOD/SCID mice. The mice were then randomly divided into groups of seven mice each, to receive different treatment agents. The test solutions were prepared ex tempore in DMSO, and were then diluted with 0.9% sodium chloride solution (final DMSO concentration=10%). The solutions were injected i.p. at a volume of 0.01 mL/g of body weight on the 10th and 17th day after tumor transplantation. The control mice received a 10% DMSO solution at a volume of 0.01 mL/g of body weight according to the same schedule. The animals were observed for a period of 91 days.

Evaluation of the therapeutic effect. The tumor volume was calculated using the formula (a²×b)/2, where a=shortest tumor diameter in mm and b=longest tumor diameter in mm. The tumor diameter was measured by caliper and monitored throughout the study (three times a week).

Statistical evaluation. The statistical analysis was performed using STATISTICA version 7.1 (StatSoft Inc., USA). The ANOVA assumptions were checked using PP-plots, Shapiro-Wilk's test and Levene's test. In the case of violation of ANOVA assumptions, a non-parametric stratified permutation Kruskal-Wallis test was used with subsequent pairwise comparisons and Bonferroni correction. The survival data in experimental groups in vivo were compared using Cox's F-test with Bonferroni correction for multiple comparisons (p adjusted=p counted × N, where N=number of pairwise comparisons). p-Values less than 0.05 were considered significant. The significance of the differences in compound uptake was analyzed by the Student's t-test.