WO2008083634A1

WO2008083634A1 - 1-(2-deoxy-alpha-d-erythro-pentofuranosyl)-5-azacytosine for use as drug

Info

Publication number: WO2008083634A1
Application number: PCT/CZ2007/000019
Authority: WO
Inventors: Antonin Holy; Miroslav Otmar; Alois Piskala; Ivan Votruba; Ales Kovarik; Miloslava Fojtova; Eva Bartova
Original assignee: Ustav Organicke Chemie A Biochemie Av Cr, V.V.I.
Priority date: 2007-01-09
Filing date: 2007-03-27
Publication date: 2008-07-17
Also published as: CZ200718A3

Abstract

The invention relates to 1-(2-deoxy-alpha-D-erythro-pentofuranosyl)-5-azacytosine of formula (I) for use as a hypomethylating drug applicable for epigenetic therapy of cancer and other epigenetically determined diseases and further includes the use of the compound of formula (I) for manufacturing of a pharmaceutical composition for epigenetic therapy of cancer and other epigenetically determined diseases.

Description

l-(2-Deoxy-alpha-D-eryt/zro-pentofuranosyl)-5-azacytosine for use as drug

Technical Field

The invention relates to 1 -(2-deoxy-alpha-D-er>'t/zro-pentofuranosyl)-5-azacytosine (alpha anomer of 5-aza-dCyd) for use as hypomethylating agent applicable for epigenetic therapy of cancer and other epigenetically determined diseases.

Background Art

The most considerable compound of the group of deoxynucleosides of 5-azacytosine is 5 -aza-2'-deoxycytidine (Dacogen Decitabine, l-(2-deoxy-beta-D-erμt/zro- pentofuranosyl)-5-azacytosine), which is a potent chemotherapeutic effective against a broad variety of malignant tumor cells (Momparler, R. L. Semin. Hematol. 2005, 42, (Suppl. 2), S9-16). In 2006 this nucleoside was approved in the U.S.A. for the treatment of all types of myelodisplastic syndromes (Hennessy, B. T.; Garcia- Manero, G.; Kantarjian, H. M.; Giles, F. J. Expert. Opin. Investig. Drugs 2003, 12, 1985-1993).

The efficacy of 5-aza-2'-deoxycytidine is based on its ability to decrease the level of DNA methylation by a specific and potent inhibition of DNA methyltransferase, which represents the basic principle of the epigenetic therapy of cancer. 5-Aza-2'- deoxycytidine (5-aza-dCyd) exhibits the most potent hypomethylating activity from the compounds studied so far. DNA hypomethylation leads to reactivation of the genes which are essential for the normal cell function, and which were during tumorogenesis silenced because of aberrant methylation. Thus, the malignant cell population is reverted to a more normal stage, which is the principle of the epigenetic therapy of cancer diseases (Yoo, C. B.; Jones, P. A.: Nature Rev. 2006, 5, 37-50). Screening of new drugs targeting the specific enzymes involved in epigenetic regulation of gene expression represents an efficient and valuable approach to the chemotherapy as well as the chemoprophylaxis of cancer. It is known that l-(2-deoxy-alpha-D-erj;t/2ro-ρentofuranosyl)-5-azacytosine, alpha anomer of 5-aza-2'-deoxycytidine (compound of formula I), exhibits a potent antileukemic activity, though in higher concentrations than 5-aza-dCyd (5-aza-2'~ deoxycytidine), but at a considerably lower toxicity and higher stability, solution than 5-aza-2'-deoxycytidine (Vesely, J.; Piskala, A. Cancer Res. 1984, 44, 5165— 5168).

It is supposed that the biological activity of l-(2-deoxy-alpha-D-eryt/zrσ- pentofuranosyl)-5-azacytosine (alpha anomer of 5-aza-dCyd, compound of formula I) is caused by its successive anomerization to the highly efficient 5-aza-2'- deoxycytidine in aqueous solutions. This conversion consists in a reversible hydrolytic cleavage of the 5-azacytidine ring to form iV-(formylamidino)-Af -(beta-D- 2'-deoxy-ribofuranosyl)-urea with a subsequent epimerization on the C-I' carbon and recyclization to 5-aza-2'-deoxycytidine.

Recently we found that the alpha anomer of 5-aza-dCyd of formula I is able to induce DNA hypomethylation, and that the efficacy of this process is comparable with that of 5-aza-2'-deoxycytidine without incorporating into DNA and without being intracellularly deaminated by cytidine deaminase (Tab. 1). With regard to the low toxicity, higher stability in aqueous solution, and easier manufacturing compared to 5-aza-2'-deoxycytidine, the alpha anomer of 5-aza-dCyd of formula I can be applied for epigenetic therapy of cancer (Fig. 2).

Disclosure of the Invention

Object of the present invention is l-(2-deoxy-alpha-D-er>tf/zro-pentofuranosyi)-5- azacytosine (alpha anomer of 5-aza-dCyd) of the formula I

NH₂

HO

HO-^{1 U} (I) for use as drag inducing DNA hypomethylation applicable for epigenetic therapy of cancer and other epigenetically determined diseases.

Another aspect of the present invention is the use of the compound of formula I for the manufacturing of a drug for epigenetic therapy of cancer and other epigenetically determined diseases.

A further aspect of the invention is a pharmaceutical composition containing the compound of formula I as the active component.

Another aspect of the present invention is a pharmaceutical composition for epigenetic therapy of cancer and other epigenetically determined diseases comprising the compound of formula I.

The present invention relies on the findings of the ability of the alpha anomer of 5- aza-dCyd of formula I to induce DNA hypomethylation, whose efficacy is comparable with that of the clinically used 5-aza-2'-deoxycytidine, which enables its use as a hypomethylating drug in epigenetic therapy of cancer and other epigenetically determined diseases.

Comparison of the hypomethylating efficacy of the alpha anomer of 5-aza-dCyd of formula I and 5-aza-dCyd (5-aza-2'-deoxycytidine) in CCRF-CEM cells is shown in Tab. 1.

CCRF-CEM cells were incubated in the presence of alpha anomer of 5-aza-dCyd (compound of formula I) and 5-aza-dCyd (5-aza-2'-deoxycytidine) for 72 h, counted and harvested for DNA analysis. The level of methylation is expressed as the ratio of methylated/hypomethylated fraction, which was monitored in two repetitive sequences of DNA: centromeric satellite 2 and in genes for ribosomal rRNA (18S rDNA). Table 1. DNA methylation levels in CCRF-CEM cells treated with alpha anomer of 5-aza-dCyd of formula I and 5-aza-dCyd (5-aza-2'-deoxycytidine)

Concentration Relative methylation (%)

(μM) (locus)

Satellite 1 I 18S rDNA

Control - 100 100

Alpha anomer of 5-aza-dCyd 0.4 28 77

Alpha anomer of 5-aza-dCyd 2.0 1 31

5-Aza-dCyd (5-Aza-2^!- 0.1 20 64

deoxycytidine)

5-Aza-dCyd (5-Aza-2⁵- 0.5 0.7 18 deoxycytidine)

The following examples illustrate the details of this invention, without thereby limiting it in any manner. As described in the examples below, the compound of formula I induces DNA hypomethylation, whose efficacy is comparable with that of the clinically used 5-aza-2'-deoxycytidine.

Description of Drawings

Fig. 1 shows a methylation analysis of genomic DNA isolated from control cells and cells incubated with compound of formula I („ Alpha") and 5-aza-2'-deoxycytidine (,,Beta"), respectively. After cleavage with methylation-sensitive restriction enzymes, genomic DNA was hybridized with 18S rDNA and satellite 2 probes. Methylation of cytosine in 18 rDNA and satellite 2 was monitored in restriction sites for £ϊtBI and /φαII. Fig. 2 shows inhibition of CCRF-CEM cells growth by the treatment with alpha anomer of 5-aza-dCyd (compound of formula I) (3) and 5-aza-2'-deoxycytidine (2), respectively; IC_5O = 2.5 μM for alpha anomer of 5-aza-dCyd (compound of formula I) and 0.6 μM for 5-aza-dCyd (5-aza-2'-deoxycytidine). Line (1) represents growth of the control cells.

Fig. 3 is l-(2-deoxy-alpha-D-er>'tAro-pentoruranosyl)-5-azacytosine (alpha anomer of 5-aza-dCyd) of formula (I).

Examples

Materials and methods

Cells. Human small lung carcinoma cells A549 (ATCC:CCL 185) were plated at a density of 2 x 10⁵/ml in D-MEM (PAN Germany) supplemented with 10% fetal calf serum, and these cells were treated the next day with methylation inhibitors and Trichostatin A (TSA 75 nM; Sigma). Cells were harvested after the 72 h (methylation inhibitors) or 48 h (TSA) of treatment. The CCRF-CEM T lymphoblastoid cells (human acute lymphoblastic leukemia, ATCC: CCL 119) were cultivated in RPMI 1640 medium supplemented with 10% calf fetal serum and treated by the same way as above.

Chemicals. The alpha anomer of 5-aza-dCyd of formula I and 5-aza-dCyd (5-aza-2'- deoxycytidine) were synthesized as described in Piskala and Sorm (Piskala, A.; Sorm F. Anomeric 4-amino-l-(2-deoxy-D-erythro-pentafuranosyl)-5-triazin-2(lH)-ones (2'-deoxyazacytidine) and its α-D-anomer. In: Townsend, L. B.; Tipson, R. S; editors. Nucleic Acid Chemistry. John Wiley & Sons, Inc., New York, 1978.). The purity of both compounds was established by elemental analysis, 500 MHz ¹H NMR spectra, and HPLC analysis using a commercially packed octadecylsilane column (Spherisorb ODS, 5 μm), which was eluted at a flow rate of 1 ml/min with 10^"2 M phosphate buffer (pH 7.0). The column eluate was monitored at 244 nm.

Southern blot hybridization. Total genomic DNA was isolated from approximately 5 x 10⁷ cells using standard phenol-chloroform method (Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning A laboratory manual. Cold Spring Harbor Laboratory Press, New York, 1988.). The cells were lysed in a buffer containing 1% SDS and Protease-K at 55 ⁰C overnight. After the lysis the DNA was extracted by phenol- chloroform-isoamylalcohol (25:24:1) and ethanol precipitated. Purified genomic DNAs (1-3 μg) were digested with an excess of the enzymes (5 U μg^'1 DNA) and subjected to electrophoresis on a 1.0 % agarose gel. After electrophoresis, the gels were alkali-blotted onto Hybond-XL membranes (Amersham Biosciences, Little Chalfont, UK). Southern hybridization was carried out in 0.25 M Na-phosphate buffer, pH 7.0, supplemented with 7% sodium dodecyl sulfate (SDS) at 65 ° C for 16 h followed by washing with 2x SSC (Ix SSC - 150 mM NaCl, 15 niM Na₃-citrate, pH 7.0), 0.1% SDS (twice 5 min), 0.2x SSC, and 0.1% SDS (twice 15 min). After washing under high stringency conditions, the hybridization bands were visualized with a Phosphorlmager (STORM; Molecular Dynamics, Sunnyvale, CA) and the data were processed by ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Western blotting. Cells were washed with PBS and lysed in sodium dodecyl sulphate (SDS) lysis buffer (50 mM Tris-HCl, pH 7.5; 1% SDS; 10% glycerol) and subjected to SDS PAGE. After being electrotransferred onto a polyvinylidene difluoride membrane (Immobilon-P, Sigma, Czech Republic), proteins were immuno-detected using appropriate primary and secondary antibodies, and visualized by ECL+Plus reagent (Amersham Pharmacia Biotech, Czech Republic) according to the manufacturer's instructions. To monitor apoptosis goat polyclonal antibody against the carboxy terminus of mouse lamin B (sc-6217, Santa Cruz) which cross-reacts with the human homolog, was used. Western blots were performed according Pachernik et al. (Pachernik, J.; Hampl, A.; Soucek, K.; Kovarikova, M.; Andrysik, Z.; Hofmanova, J.; Kozubik, A. Arch. Dermatol. Res. 2002; 293, 626-633.) and experimental data were normalized to the selected band of total nuclear proteins.

DNA probes. 18S rDNA probe was generated by PCR amplification using the following primers designed according to the sequence in database (Ml 0098): 18S for: 5'-CTGGATTTGCTGGTGATGAT-S'; 18S rev: 5' -TGGCCTC AGTTCCGAA- AACC-3'. Human satellite 2 probe was generated by amplification of genomic DNA using primers derived from conserved region of the repeat (X06199): SAT2 for: 5'- TTCGAGTCCATTCGATGAT-S'; SAT2 rev: 5'-ATGGAATCAACATCAAATGG- 3'. The PCR products were gel purified, sequenced and used as probes.

Example 1

Profiles of methylation in cells before and after the treatment with hypomethylating alpha anomer of 5-aza-dCyd of formula I in comparison with 5-aza-dCyd (5-aza-2'- deoxycytidine).

In course of the study of DNA hypomethylation the CCRF-CEM (human acute lymphoblastic leukaemia; ATCC:CCL 119) and A549 cells (human small lung carcinoma cells; ATCC:CCL 185) were incubated in two different concentrations of compound of formula I and 5-aza-dCyd (5-aza-2'-deoxycytidine). Genomic DNA was extracted from the cells after 72 hours of incubation with the drug and analyzed with methylation-sensitive restriction enzymes using Southern blot hybridization (Fig. 1). The DNAs were digested with BstBl and Hpall enzymes and hybridized against the satellite 2 and 18S rDNA probes. BstBl is sensitive to methylation of CG dinucleotide in TTCGAA recognition sequence; Hpall is sensitive to CG methylation in CCGG sites. In control (non-treated) CCRF-CEM cells the satellite 2 and 18S rDNA probes hybridized to high molecular weight (relict) DNA, which reflects high level of 5-mC in both monitored sequences. In A549 cells, the DNA was digested to a ladder of low molecular weight BstBl fragments hybridizing with the satellite 2 probe. Similarly, most 18S hybridization fragments resulting from Hpall digestion of 18S rRNA genes migrated in the front of gel in A549 cells. These patterns are consistent with very low or negligible methylation in A549 in repetitive DNA sequences.

Treatments of cells with alpha anomer of 5-aza-dCyd of formula I or 5-aza-dCyd (5- aza-2'-deoxycytidine) clearly enhanced digestion of CCRF-CEM DNA with methylation-sensitive restriction enzymes. In hypomethylated DNAs the satellite 2 probe hybridized to a ladder of fragments indicating loss of methylation at BstBl sites. Similarly, the 18S probe hybridization revealed stronger Hpall bands in the low molecular weight fraction compared to non-treated controls. The extent of digestion was higher in DNA samples isolated from cells exposed to higher concentrations of the drugs indicating the dosage-dependent hypomethylation effect. In contrast to CCRF-CEM cells, the drugs did not alter the hybridization patterns in A549 cells confirming negligible CG methylation in this line. The signals were quantified and expressed as mC/C ratio (TAB. 1). The fractions used for radioactivity scanning (Phosphorlmager) are indicated on right margin.

Example 2

Cytostatic effect of alpha anomer of 5-aza-dCyd of formula I in comparison with 5- aza-dCyd (5-aza-2'-deoxycytidine)

For the monitoring of the cytostatic effect of both drugs, the cells were counted (Celltac MEK 5208 NIHON KOHDEN haematological analyzer) after the treatment (Fig. 2). The IC₅₀ value (defined as a concentration of drug causing 50% inhibition of cell proliferation) was 2.4 μM for alpha anomer of 5-aza-dCyd of formula I and 0.6 μM for 5-aza-dCyd (5-aza-2'-deoxycytidine), respectively indicating lower antiproliferative activity of the compound of formula I in CCRF-CEM cells.

Industrial Applicability

l-(2-deoxy-alpha-D-er>'t/zro-pentofuranosyl)-5-azacytosine of formula I is useful for the preparation of a pharmaceutical composition for epigenetic therapy of cancer and other epigenetically determined diseases.

Claims

1. l-^-Deoxy-dpha-D-eryt/zro-pentofuranosyty-S-azacytosine of formula I

for use as a drug inducing DNA hypomethylation.

2. l-(2-Deoxy-alpha-D-eryt/zro-pentofuranosyl)-5-azacytosine of formula I

for use as a drug inducing DNA hypomethylation for epigenetic therapy of cancer and other epigenetically determined diseases.

3. Use of l-(2-deoxy-alpha-D-eryt/zro-pentofuranosyl)-5-azacytosine of formula I for manufacturing of a pharmaceutical composition for epigenetic therapy of cancer and other epigenetically determined diseases.

4. A pharmaceutical composition for epigenetic therapy of cancer and other epigenetically determined diseases comprising the compound of formula I.