PL212279B1 - New derivatives of epirubicines and their medical application and pharmaceutically admitted form of drug - Google Patents

Description

The subject of the invention are new 7-O- (3'-amino-2 ', 3', 6'-trideoxy-α, eL-arabinohexo-pyranosyl) -adriamycinone (epirubicin, 4'-epidoxorubicin) derivatives of formula 1, in which at most two of the four R1, R2, R3 and R4 substituents are the same or different and represent a hydrogen atom, and at least two of the four substituents are an alkyl group, an alkene or alkyne group with 1 to 5 carbon atoms, an alkylcarbonyl group of an alkyl chain of 1 to 3 carbon atoms, especially acetyl, where if R1 and R4 are both hydrogen, then R2 and R3 together with the nitrogen atom to which they are attached represent a dimethylformamidyl group. The invention also relates to a new medical use of these derivatives for the preparation of drugs that actively inhibit hepatitis C virus (HCV) replication, and to a pharmaceutically acceptable form of the drug.

Hepatitis C virus (HCV) is of particular importance: it is a highly pathogenic virus belonging to the Flaviviridae family (genus hepacivirus), very widespread in the world (according to WHO data, c.a. 300 million infected people). Chronic active hepatitis occurs / develops in approx. 85% of highly infected carriers and leads to cirrhosis of the liver and to hepatocellular carcinoma (Hagedorn and Rice, ed. The Hepatitis C Viruses, Springer, and Heidelberg 2000).

Surprisingly, the applicant has found that epirubicin hydrochloride, the known anti-cancer drug, is an extremely potent agent against the hepatitis C virus (HCV). Epirubicin hydrochloride is toxic to mammalian haemopoietic cells and cardiac tissue cells, but causes less haematological and cardiac toxicity than adequate doses of another widely used anti-cancer drug, doxorubicin (adriamycin). In addition, the likelihood of congestion in a human heart and a total sk ₂ mulowanej dose not exceeding 150 mg per m ^{2 of} human body surface area by the Kaplan-Meier plot (Launchbury and Habooubit, Cancer Treatment Reviews 1993, 19, 197 - 228) is negligible. On July 7, 2005, the applicant filed a patent application with the Polish and European Patent Office (PL375008 and EP05460019.2, respectively).

It has now surprisingly been found that epirubicin derivatives of formula 1 as described below exhibit potent hepatitis C virus (HCV) replication inhibitory activity with significantly lower cytotoxicity than epirubicin to Huh7 and PBMC cells, better in vitro therapeutic index ( TI) (Table 1) and lower in vivo toxicity in mice.

Accordingly, the new epirubicin derivatives can be advantageously used for the treatment of HCV infections. The present invention also relates to pharmaceutically acceptable dosage forms containing said derivatives of formula 1.

The new 7-O- (3'-amino-2 ', 3', 6'-trideoxy-a, eL-arabinohexopyranosyl) -adriamycinone (epirubicin) derivatives are represented by the formula 1, where at most two of the four R1, R2 substituents , R3 and R4 are the same or different and represent a hydrogen atom, and at least two of the four substituents are an alkyl group, an alkene or alkyne group with 1 to 5 carbon atoms, an alkylcarbonyl group with an alkyl chain of 1 to 3 carbon atoms, especially an acetyl group, wherein if R 1 and R 4 are both hydrogen, then R 2 and R 3 together with the nitrogen atom to which they are attached represent a dimethylformamidyl group.

Medical use of the novel epirubicin derivatives represented by the formula 1 for the preparation of drugs actively inhibiting the replication of the hepatitis C virus (HCV).

Medical use of the new epirubicin derivatives according to the invention for the production of drugs that actively inhibit the replication of the hepatitis C virus (HCV) and provide low toxicity to Huh7 and PBMC cells, and a therapeutic index (TI) higher than epirubicin alone and lower than epirubicin toxicity in vivo.

Alternatively, the new epirubicin derivatives can be used to treat HCV infections.

The pharmaceutically acceptable form of the drug against HCV infections according to the invention, containing known carriers and additives, is characterized in that it comprises the active ingredient epirubicin derivatives represented by the formula 1, wherein R1, R2, R3 and R4 are as defined above.

The studies presented below demonstrate for the first time that the new epirubicin derivatives represented by the formula 1 exert a strong inhibitory effect on HCV replication at nanomolar concentrations and can be used as effective drugs against HCV infections.

PL 212 279 B1

The following are non-limiting embodiments of the invention.

P r z k ł a d I

₃ N-Acetyl-4'-epidoksyrubicyna (1 BNE01)

4'-epidoxyrubicin hydrochloride (58 mg, 0.1 mmol) was dissolved in acetone (6.8 mL) and 18 µl of diisopropylethylamine was added. The solution was cooled to 0 ° C in an ice bath and 10 µL (0.11 mmol) of acetic anhydride was added with constant stirring. The reaction mixture was allowed to warm to room temperature and stirred for an additional 2 h. The solution was concentrated to dryness in vacuo, the residue was dissolved in ca 13 mL of CHCl3, and the solution was washed three times with 0.1 M phosphate buffer pH 7.0 and twice with water. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness in vacuo. The residue was chromatographed on a silica gel column (CHCl3 to CHCl3: MeOH 1: 1). Compound of formula 1 was obtained, (R1: H, R2: CH3CO, R3: H, R4: H) Temp. top 180 ° C. MS (ES +) 586.14; NMR (CDCl3) δ 1.36 (d, 3, 5'-CH3), 2.01 (s, 3, 3'-N-COCH3), 2.06 (m, 2, 2'-H2), 2.19 (d, 1, 8A -H), 2.41 (d, 1,8B-H), 3.02 - 3.12 (m, 3, 3'-H and 4'-H / 4'-OH), 3.28 (d, 1, 10A-H), 3.32 (d, 1, 10B-H), 3.78 - 3.81 (m, 1.5'-H), 4.09 (s, 3, 4-OCH3), 4.80 (dd, 3, 14-H2 / 14-OH) , 5.30 (br s, 1,9-OH), 5.43 (d, 1,1'-H), 5.51 (d, 1,7-H), 7.41 (d, 1,3-H), 7.80 (t , 1,2-H), 8.05 (d, 1,1-H), 13.26 (s, 1,1-OH), 14.02 (s, 1,6-OH).

P r z x l a d II

N ₃ ', O ₄ ', O ₁₄ -triacetyl-4'-epidoxyrubicin (2, BNE02)

4'-epidoxyrubicin hydrochloride (100 mg, 0.17 mmol) was dissolved in 10 mL of dry chloroform and 20 mg of dimethylaminopyridine was added. The solution was cooled to 0 ° C in an ice bath and 400 µL (4.24 mmol) of acetic anhydride was added with constant stirring. The mixture was allowed to warm to room temperature and further stirred. The progress of the reaction was followed by TLC (CHCl3: MeOH 9: 1). The resulting solution was washed 3 times with 0.1 M phosphate buffer pH 7.0 and 2 times with water. The organic layer was dried over anhydrous sodium sulfate and evaporated in vacuo. The residue was chromatographed on a silica gel column (CHCl3 to CHCl3: MeOH 1: 1). Compound of formula 1 was obtained, (R1: CH3CO, R2: CH3CO, R3: H, R4: CH3CO) Temp. top 178 ° C. MS (ES +) 670.25. NMR (DMSO-d6) δ 1.09 (d, 3, 5'-CH3), 1.69 (s, 3, 14-O-COCH3), 1.97 (s, 3, 4'-O-COCH3), 2.08 - 2.12 ( m, 5, 3'-N-COCH3, 8H2), 2.94 / 3.08 (d / d, 1/1, 2'-H2), 4.05 (s, 3, 4-OCH3), 4.10-4.13 (m, 1 , 5'-H), 4.43 (t, 1, 1'-H), 4.96-4.97 (m, 1, 7-H), 5.22 (d, 2, 14-H2), 5.79 (s, 1, 9 -OH), 7.66 (t, 1,2-H), 7.72 (d, 1,3-H), 7.92 (d, 1,1-H), 13.28 (s, 1,1-OH), 14.01 ( s, 1,6-OH).

P r z x l a d III

3'-N, N-dimethyl-4'-epidoxyrubicin (3, BNE06)

1.3 mL of acetonitrile was added with stirring to a solution of 4'-epidoxyrubicin hydrochloride (100 mg, 0.17 mmol) in 630 µL of H ₂ O and the mixture was heated to 30 ° C. 130 µL (1.31 mmol) of 37% aqueous formaldehyde solution was added and the solution was stirred for 20 minutes at 23-25T. Then 23 mg (0.37 mmol) of NaCNBH3 in 1.3 mL of acetonitrile was added dropwise over 15 minutes and stirred at 24 ° C for 20 minutes. The reaction mixture was diluted with water (4 mL) and extracted 2 times with 4 mL of chloroform. The chloroform extracts were combined, dried over anhydrous sodium sulfate and evaporated in vacuo. The residue was chromatographed on preparative gel plates (Merck No 105717) PLC plates using CHCl3: MeOH: H2O 40: 10: 1. Compound of formula 1 was obtained, (R1: H, R2: CH3, R3: CH3, R4: H) Temp. top 220 ° C. MS (ES +) 572.28, (ES-) 570.22. NMR (DMSO-d6) δ 1.20 (d, 3.5'-CH3), 1.56 (dt, 1,2'-H), 1.72 (dd, 1,2'-H), 2.17 (br s, 8, 3'-N (CH3) 3, 8-H2) 2.91 - 3.08 (m, 4, 10-H, 4'-OH, 4'-H, 3'-H), 3.80 - 3.85 (m, 1 , 5'-H), 3.99 (s, 3, 4-OCH3), 4.55 (br s, 2, 14-H2), 4.85 (br s, 1, 14-OH), 4.97 (t, 1, 1 ' -H), 5.32 (d, 1,7-H), 5.45 (s, 1,9-OH), 7.62 (d, 1,3-H), 7.89 (t, 1,2-H), 7.94 ( d, 1,1-H), 13.24 (br s, 1,1-OH), 14.30 (br s, 1,6-OH).

P r x l a d IV

3'-N- (W '', N '' - dimethylformamidinyl) -4'-epidoxyrubicin (4, BNE06)

To a solution of 4'-epidoxyrubicin hydrochloride (150 mg, 0.26 mmol) in 7.5 mL of dry methanol was added dropwise under argon N, N-dimethylformamidodimethyl acetal (182 µL, 1.30 mmol). The reaction mixture was stirred for 4 h and then the solvent was evaporated in vacuo. The residue was chromatographed on a silica gel column (CHCl3 to CHCl3: MeOH 9: 1). Compound of formula 1 was obtained, (R1: H, R2: = CH-N (CH3) 2, R4: H) Temp. top 206 ° C. MS (ES +) 599.13, (ES-) 597.24; NMR (DMSO-d6) δ 1.24 (d, 3, 5'-CH3), 1.99 (d, 2, 8H2), 2.19 (m, 2, 2'-H2), 3.01 - 3.11 (mim, 11, 10- H2, 4'-OH, 4'-H, 3'-H, N '' - Me2), 3.93 (m, 1, 5-H), 4.00 (s, 3, 44

PL 212 279 B1

OCH3), 4.57 (d, 2, 14-H2), 4.87 (t, 1, 14-OH), 4.99 (t, 1, 1-H), 5.29 (t, 1, 7-H), 5.48 (s , 1,9-OH), 7.68 (t, 1,2-H), 7.94 (d, 2, 1-H and 3-H), 8.08 (s, 1,3'-NC ”-H), 13.26 (s, 1,1-OH), 14.04 (s, 1,6-OH).

Investigation of antiviral activity and cytotoxicity

The potential antiviral (anti-HCV) activity of the epirubicin derivatives represented by Formula 1 was tested in vitro in cell culture using a system containing HCV subgenomic replicon stably transfected into Huh7 cells (BM 4.5 clone) (Ju-Tao Gao et al., J Virol. 2001) .

Huh7 cells containing the HCV replicon were grown in the culture medium at various concentrations of the epirubicin derivative. The media and drug were changed each day for 5 days. After 5 days of drug treatment, cells were lysed and completely RNA extracted. The isolated RNA was then tested for the presence of HCV replication by Northern Blot analysis using an HCV sample bearing the HCV NS5B region. Moreover, hybridization with a labeled β-actin sample allowed the normalization of these results, i.e. the attribution of the drug effect on the intracellular gene expression (housekeeping).

Cell lines were treated daily with the appropriate epirubicin derivative in the concentration range of 2 - 900 nM for 5 days and RNA was isolated from the treated cells at the end of the treatment. Total RNA was examined with a specific HCV sample using Northern blot analysis for HCV RNA determination. The resulting chromatogram was then densitometric using "Phosphor Imager" and viral replication was quantified.

The results of the analysis carried out depending on the treatment time showed that epirubicin derivatives in concentrations of 0.018 - 0.9 μ inhib effectively inhibit HCV replication in vitro in the subgenomic replicon system (Table 1).

Table 1 also shows that the 50% concentration (IC50) of epirubicin derivatives with anti-HCV activity measured in the HCV replicon system is low and lies in the range of 0.018 - 0.9 μΜ, while their concentrations causing 50% inhibition (CC50) of Huh7 cells are 0.06-0.9 μΜ, preferably N3 ', O4', O14-triacetylepirubicin (BNEO2) with a therapeutic index of 10.

The inhibition of HCV replication by epirubicin (BNE00), N3'-acetylepirubicin (BNE01) and N3 ', O4', O14-triacetylepirubicin (BNE02) (Figure 1, Table 2) was determined as follows. Lymphocytes were cultured in medium enriched with known concentrations of BNE00, BNE01 and BNE02. On the fifth and fourteenth days of culture, cells were harvested and, after extraction, HCV RNA was qualitatively assayed using Real Time Polymerase Chain Reaction (RT PCR) measurement. Quantification of HCV RNA was also performed using RT PCR (Figure 2). RNA was isolated using the "Total RNA extraction kit" (A&A Biotechnology, Gdynia, Poland, www.abiot.com). HCV RNA amplification was performed as follows: RT-PCR was performed in one step for 60 minutes at 42 ° C and for 15 minutes at 75 ° C. For the first PCR, which included 30 cycles of amplification (10s at 65 ° C and 20s at 72 ° C), 3 µL of cDNA was used. 2 μL of the product from the first process was used for the second amplification (35 cycles, each cycle 10s at 94 ° C, 10s at 65 ° C and 20s at 72 ° C and then extended for 7 min at 72 ° C. 10 pmoles were used in the first process. NTR 1 LOW primers (GGTGCACGGTCTACGAGACCT) and NTR 1 UP (CGACACTCCACCATAGAT) primers and NTR 2 LOW (CACTCGCAAGCACCCTAT CAGGCAGT) and NTR 2 UP (CCACCATAGATCACTCCCCTGT) products were analyzed using the gel of the PCCTGT. FMC) ”by staining with ethidium bromide.

The cytotoxicity (CC50) of epirubicin derivatives in PBMCs was very low, 39-41 μΜ (Fig. 3). Acute toxicity in mice (LD50) was determined according to OECD Guidelines for the Testing of Chemicals (Guideline No. 401). (OECD Guidelines for Testing of Chemicals, Guideline No 401). " Compound 2 (BNEO2) preferably has a very low in vivo acute toxicity (LD50 = 1300 mg / kg mouse body weight), while epirubicin hydrochloride (BNE00) has a value of 37 mg / kg.

In this study it was demonstrated for the first time that epirubicin derivatives exert an inhibitory effect on HCV replication at nanomolar concentrations and can be used as effective drugs against HCV infections with negligible toxicity in vitro and in vivo.

PL 212 279 B1

T a b e l a 1

Anti-HCV activity in the HCV ¹ replicon system and cytotoxicity of epirubicin and its derivatives against Huh-7 and PBMC cells.

Relationship PBMC CC50 cytotoxicity (μΜ) Cytotoxicity to Huh7 CC50 cells (μΜ) ² Anti-HCV IC50 activity (μΜ) ³ Therapeutic index CC50 / IC50 Epirubicin Hydrochloride (BNE00) 1.7 0.003 0.002 1.5 _1.N-3- acetylepirubicin (BNE01) 40.9 0.9 > 0.9 > 1 2. N3 ', O4', O14-triacetylepiribin (BNE02) 39 5 0.5 10.0 3. 3'-W, W-Dimethyl-4'-epidoxorubicin (BNE06) - 0.06 0.018 3.0 4. 3'-N- (N '', N "-Dimethylform- dinyl) -4-epidoxorubicin (BNE04) - > 0.05 > 0.05 > 1

¹ HCV subgenomic replicon stably transfected into Huh7 cells (clone BM4.5) (Ju-Tao Gao et al., J. Virol. 2001,) ² cytotoxicity (CC50) - 50% cytotoxic concentration ³ antiviral activity (IC50) - required concentration to inhibit virus replication by 50%

Table 2 presents data on the cytotoxicity (CC50 and CC90), anti-HCV activity (IC50 and IC90) and the therapeutic index (TI50 and TI90) of epirubicin and its derivatives in lymphocyte culture.

T a b e l a 2

Relationship CC50 CC90 IC50 IC90 Tl50 Tl90 BNE00 1.8 2.4 0.32 0.46 5.6 5.2 BNE01 33.00 91.0 0.36 0.47 92.0 194.0 BNE02 49.00 120.0 > 0.10 0.50 > 490 240.0

Patent claims

Claims (4)

Patent claims 1. New epirubicin derivatives, i.e. 7-O- (3'-amino-2 ', 3', 6'-trideoxy-a, eL-arabinohexo-pyranosyl) -adriamycinone represented by the formula 1, in which at most two of the four R1 substituents , R2, R3 and R4 are the same or different and represent a hydrogen atom, and at least two of the four substituents are an alkyl group, an alkene or alkyne group with 1 to 5 carbon atoms, an alkylcarbonyl group with an alkyl chain of 1 to 3 atoms carbon, especially acetyl, wherein if R 1 and R 4 are both hydrogen, then R 2 and R 3 together with the nitrogen atom to which they are attached represent a dimethylformamidyl group. 2. Medical use of the novel epirubicin derivatives as defined in claim 1 for the preparation of medicaments actively inhibiting the replication of the hepatitis C virus (HCV). 3. The use of derivatives according to claim 1 2, for the production of drugs actively inhibiting the replication of hepatitis C virus (HCV), providing low toxicity to Huh7 and PBMC cells, higher therapeutic index (TI) than epirubicin alone and lower toxicity than epirubicin in vivo. 4. A pharmaceutically acceptable form of the drug against HCV infections, containing known carriers and additives, characterized in that the active substance is epirubicin derivatives represented by the formula 1, in which at most two of the four substituents R1, R2, R3 and R4 are the same or different and represent a hydrogen atom, and at least two of the four substituents represent an alkyl group, an alkene or alkyne group with 1 to 5 carbon atoms, an alkylcarbonyl group with an alkyl chain of 1 to 3 carbon atoms, especially acetyl, whereby if R1 and R4 represent hydrogen simultaneously, then R2 and R3 together with the nitrogen atom to which they are attached represent a dimethylformamidyl group.