JPS6328436B2 - - Google Patents

Description

[Detailed description of the invention]

At first glance, the present invention is based on the formula () (wherein X is hydrogen or hydroxy, R ₁ is hydrogen or methyl and one of R ₂ and R ₃ is methoxy and the other of R ₂ and R ₃ is hydrogen) and its Pharmaceutically acceptable acid addition salts are provided. These compounds are named as follows. 4-demethoxy-4'-O-methyl-daunorubicin (a: R ₁ = R ₃ = X = H R ₂ = OCH ₃ ) 4-demethoxy-4'-epi-4'-O-methyl-
Daunorubicin (b: R ₁ = R ₂ = X = H R ₃ = OCH ₃ ) 4-demethoxy-2,3-dimethyl-4'-O-
Methyl-daunorubicin (c: R ₁ =CH ₃ , R ₂ =OCH ₃ , R ₃ =X=H) 4-demethoxy-2,3-dimethyl-4'-epi-4'-O-methyl-daunorubicin (d : R ₁ = CH ₃ , R ₂ = X = H, R ₃ = OCH ₃ ) 4-demethoxy-4'-O-methyl-doxorubicin (e: R ₁ = R ₃ = H, R ₂ = OCH ₃ , =OH) 4-demethoxy-4'-epi-4'-O-methyl-
Doxorubicin (f: R ₁ = R ₂ = H, R ₃ = OCH ₃ , X = OH) 4-demethoxy-2,3-dimethyl-4'-O-
Methyl-doxorubicin (g: R ₁ = CH ₃ , R ₂ = OCH ₃ , R ₃ = H
=OH) 4-demethoxy-2,3-dimethyl-4'-epi-4'-O-methyl-doxorubicin (h: R ₁ = CH ₃ , R ₂ = H, R ₃ = OCH ₃ , X
=OH) In another aspect, the present invention provides a method of making an anthracycline glycoside of formula (). The process according to the invention involves converting 4-demethoxy-daunomycinone or 2,3-dimethyl-4-demethoxydaunomycinone (see US Pat. No. 4,046,878) into 2,3,6-trideoxy-3-trifluoroacetamide-4 -O-methyl-L-lyxo-hexopyranosyl chloride or 2,3,6
-trideoxy-3-trifluoroacetamide-4-O-methyl-L-arabino-hexopyranosyl chloride (see U.S. Pat. No. 4,183,919) to form formulas a to d. a: R ₁ = R ₃ = H, R ₂ = OCH ₃ b: R ₁ = R ₂ = H, R ₃ = OCH ₃ c: R ₁ = CH ₃ , R ₂ = OCH ₃ , R ₃ = H d: The protected α-glycoside with R ₁ =CH ₃ , R ₂ =H, R ₃ =OCH ₃ was obtained and the N-trifluoroacetyl protecting group was removed by mild alkaline hydrolysis to form the corresponding daunorubicin derivative a. ~d and optionally the resulting 14- by bromination and aqueous sodium formate.
It consists of converting the daunorubicin derivatives into the corresponding doxorubicin derivatives e to h by treatment with the bromo derivative. The conversion of daunorubicin derivatives a to d into the corresponding doxorubicin derivatives e to h is according to the method described in US Pat. No. 3,803,124. In yet another aspect, the present invention provides a pharmaceutical composition comprising an anthracycyl glycoside of formula ( ) or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable diluent or carrier. be. The invention is illustrated by the following examples and biological data. Example 1 4-Demethoxy-4'-O-methyl-daunorubicin (a) 4-demethoxy in 400 ml of anhydrous methylene dichloride containing 1.4 g of 1-chloro-4-O-methyl-N-trifluoroacetyl-daunosamine. - Pour a solution of 3.68 g of daunomycinone through a molecular sieve (30
Stir vigorously in the presence of 1.3 g of silver trifluoromethanesulfonate (Merck, 4A Merck) and 1.3 g of silver trifluoromethanesulfonate. After 10 minutes at room temperature, add 0.55 ml of symmetric collidine to the reaction mixture.
Neutralize with. After 40 minutes, the suspension is filtered and the organic phase is washed with 0.01N aqueous hydrochloric acid, water, saturated aqueous sodium bicarbonate solution and finally water until neutral. The residue obtained by evaporating the solvent under vacuum is dissolved in 150 ml of acetone;
Treat with 600 ml of 0.2N aqueous sodium hydroxide and dilute with 450 ml of water. After 5 hours at 0°C, the solution was
Adjust the pH to 8.5 and extract with chloroform until the chloroform extract is no longer colored.
The organic extracts are combined and acidified to pH 5 with 0.1N methanolic hydrogen chloride. Pure 4-demethoxy-
4'-O-methyl-daunorubicin (0.6 g) is precipitated. The mother liquor is purified on a column of silica gel buffered to PH7 with phosphate buffer M/15 using a chloroform/methanol/water (10:2:0.2 by volume) solvent system. The eluate containing the pure compound is diluted with water and the organic phase is separated, washed with water and evaporated to a small volume and then acidified to PH5 with 0.1N methanolic hydrogen chloride. 4 more
-Demethoxy-4'-O-methyldaunorubicin hydrochloride (0.4g) is obtained. Melting point 189-190℃ (decomposition).
Kieselgel plate (Merck F254) and chloroform/methanol/water (volume ratio 10:2:
0.2) TLC using a solvent system. Microbondapak C ₁₈ column and mobile phase of water/acetonitrile (69:31 by volume) at pH 2 adjusted with 10% orthophosphoric acid (flow rate 1.5 ml/min, retention time
HPLC using 22 min). FD-MS: m/z511 (M ⁺ ) Example 2 4-demethoxy-4′-O-methyl-doxorubicin (e) Methanol (8 ml) and dioxane (20 ml)
A solution of 0.5 g of 4-demethoxy-4'-O-methyl-daunorubicin prepared as described in Example 1 in a mixture of is treated with bromine to form the 14-bromo derivative. This 14-bromo derivative was treated with an aqueous solution of sodium formate for 18 hours at room temperature to produce 4-bromo derivatives.
Demethoxy-4′-O-methyl-doxorubicin
Obtain 0.310g. It is isolated as its hydrochloride salt. Melting point 164-165°C (decomposition). R _f 0.18 for TLC on Kieselgel plates (Merck F254) and chloroform/methanol/water (10:2:0.2 by volume) solvent system. The experimental conditions for HPLC were a column Microbondapak _C18 and pH2 adjusted with 10% orthophosphoric acid.
A water/acetonitrile (69:31 by volume) mobile phase (flow rate 1.5 ml/min, retention time 10) is used. Example 3 4-demethoxy-2,3-dimethyl-4'-O-
Methyl-daunorubicin (c) 4-demethoxy-2,3-dimethyl-daunomycinone and 1 under the conditions described in Example 1
A coupling reaction between -chloro-4-O-methyl-N-trifluoroacetyl-daunosamine gives the title compound. Example 4 4-demethoxy-2,3-dimethyl-4'-O-
Methyl-doxorubicin (g) The conversion of compound c to the title compound is carried out using the procedure described in Example 2. Example 5 4-demethoxy-4'-epi-4'-O-methyldaunorubicin (b) and 4-demethoxy-
2,3-dimethyl-4'-epi-4'-O-methyl-daunorubicin (d) 4-demethoxy-daunomycinone and 4-demethoxy-2,3-dimethyl-daunomycinone with 2,3 as described in Example 1 ,6-trideoxy-3-trifluoroacetamide-4-O-
By carrying out a coupling reaction with methyl-L-arabino-hexopyranosyl chloride, the title compound is obtained after hydrolysis of the N-protecting group. Example 6 4-demethoxy-4'-epi-4'-O-methyldoxorubicin (f) and 4-demethoxy-
2,3-Dimethyl-4'-epi-4'-O-methyl-doxorubicin (h) Compounds b and d are converted to compounds f and h, respectively, via the 14-bromo derivative under the conditions described in Example 2. Biological Activities of a and e To confirm the cytotoxicity, antitumor activity and cardiotoxicity of compounds a and e in experimental animals, we tested the original compounds daunorubicin (DNR) and doxorubicin (respectively) in several experimental systems. Compounds a and e were tested against DX).

【table】

Table 1 The data shown in Table 1 show that a is about 5 times more cytotoxic than DNR and e is about 9 times more cytotoxic than DX. Primary screening in in vivo testing
P388 ascites leukemia (10 ⁶ cells/mouse)
It was carried out in CDF-1 strain mice. The results are shown in Table 2. Both a and e were found to be more toxic and more potent than the original compound. Comparisons in maximum tolerated doses (MxTD) show that a is as effective as DNR (giving a similar increase in lifespan in mice) and e has as good an antitumor activity as DX.

【table】

[Table] Several studies have shown that C3H with gross leukemia (2 x 10 ⁶ cells/mouse) was injected intravenously.
It was carried out in mice. The data for a are shown in Table 3. When administered intravenously on day 1 after tumor inoculation, a is significantly more toxic and more potent than DNR. 1.25～
In MxTD of 1.3mg/Kg, a is 10mg/Kg
It is even more effective than DX in MxTD.
It is well known that DNR is not active unless given in very high doses (≓50 mg/Kg) when administered by the oral route. The data presented in Table 3 show that it has good antitumor activity against gross leukemia even when given orally. When administered orally on the first day only, it is active at a dosage of 1.25-1.3 mg/Kg, which is also the optimum dosage for intravenous treatment. This result suggests that the absorption of a by the gastrointestinal tract is very efficient. When administered by oral route on days 1, 2 and 3,
a was more active than when administered only on day 1, and at an optimal dosage of 0.66 mg/Kg/day, it was investigated in parallel at an optimal dosage of 1.9 mg/Kg/day. -Demethoxy-Has antitumor activity of the same magnitude as daunorubicin.
The observation that the optimal dosage of a is lower than that of 4-demethoxy-daunorubicin suggests more efficient absorption by the gastrointestinal tract compared to this compound.

[Table] Data on the antitumor activity of e compared with DX against gross leukemia are shown in Table 4. Compounds were administered 1 day after tumor cell inoculation.
DX was given intravenously and e was given intravenously and orally. When administered intravenously, 1
At mg/Kg of MxTD, e shows good anti-tumor activity similar to that observed after DX treatment. Furthermore, e is also active when administered orally at a usage dose of 1.3 mg/Kg.

【table】

[Table] Intraperitoneally (ascites form) or intravenously
CDF-1 mice were inoculated (10 ⁵ cells/mouse).
e was tested against L1210 leukemia. Treatments were performed intraperitoneally or intravenously, respectively, on the first day after tumor cell inoculation. The data shown in Table 5 shows that in the i.p.-i.p. experiment, MxTD of 0.83 mg/Kg
e in indicates that it is as active as DX at 4.4 mg/Kg of MxTD. Intravenous - In intravenous experiments, e at the accepted doses of 1 mg/Kg and 1.3 mg/Kg shows antitumor activity superior to that of DX.

Table: To evaluate antitumor activity against solid tumors, e was tested against C3H female mammalian carcinoma in comparison to DX. A third generation tumor implant was used. Treatment started 15 days after tumor implantation and 4 days after tumor implantation.
It is performed intravenously once a week. Tumor measurements are performed weekly with calipers. Tumor-free mice were treated in parallel to assess general and cardiotoxicity. These toxicities were tested in 5 mice treated with the highest doses of the two compounds and sacrificed 5 weeks after the last treatment. The results of this experiment are shown in Table 6. DX is highly effective and it inhibits tumor growth by 93-94% (compared to untreated controls) at both doses tested (6 and 7.5 mg/Kg). Toxic death (2/3) in tumor-free mice treated with DX at 7.5mg/Kg
All mice observed and examined showed histologically detectable cardiac lesions. e at a dosage of 0.4mg/Kg is slightly active and
At doses of 0.6 and 0.75 mg/Kg it significantly inhibits tumor growth. No atrial lesions are observed in tumor-free mice treated with 0.75 mg/Kg e and only 2 out of 5 mice show detectable ventricular lesions. This lesion is much less than that observed after DX treatment.

TABLE The data presented herein indicate that a and e are novel anthracycline congeners that confer very interesting biological properties. DNR
compared to about 3 when administered i.p.
twice as potent and about eight times more potent when administered intravenously. In DxTD, it is
It has antitumor activity against ascites P388 and systemic gross lymphoma, which is equivalent to that of DNR. Furthermore, it is also active when administered by the oral route, especially when the treatment is carried out for three consecutive days at a lower dosage than that of 4-demethoxy-daunorubicin. e is approximately 10 times more potent than DX in in vivo tests. The comparison in MxTD is that
DX to be equally active against ascites P388 and L1210 leukemia, systemic gross leukemia and solid mammalian carcinoma and more effective than DX against systemic (injected intravenously) L1210 leukemia shows. Furthermore, it is also active against gross leukemia when administered orally and in primary cardiotoxicity studies in C3H mice chronically treated intravenously, it causes only minimal cardiac lesions. Compound a was tested in vitro and in vivo against P388 leukemia cells resistant to doxorubicin (P388/DX). P388 leukemia cells resistant to doxorubicin (DX) (identified by Schierbel) are maintained by serial metastases in mice treated intraperitoneally with DX. For experimental purposes, BDF-1 mice are injected intraperitoneally with 10 ⁴ leukemia cells and treated intraperitoneally on day 1 after tumor inoculation. The results listed in Table 7 show that daunorubicin (DNR) is not active against this tumor, but Compound a increases the lifespan of treated mice at the optimal dosage of 0.8 mg/Kg. P388 and P388/DX leukemia cells are obtained from mouse ascites fluid and used for growth in suspension in vitro. Cytotoxicity tests are performed by exposing cells to various drug concentrations for 48 hours. At the end of this exposure time, the cells were counted by Coulter cell counter and ID ₅₀
Calculate (the amount used to give 50% production of cell numbers compared to the untreated control). The results shown in Table 8 indicate that the cytotoxicity of a to P388 leukemia cells was
about twice that of DNR and is also highly active against P388/DX leukemia cells, whereas
We show that DNR is 162-152 times less active against resistant lines than against susceptible lines.

【table】

【table】

Table: Compound e is further investigated against mammalian carcinoma in C3H mice. Mice with measurable tumors (third generation implants) were incubated once weekly for 4 weeks.
Or process with DX. Normal mice are processed in parallel for toxicity evaluation. The results shown in Table 9 confirm that e is about 10 times more potent than DX and has significant antitumor activity in this experimental system at non-toxic doses (DX is toxic).

【table】

Table: Colon 26 and Colon 38 glands where compound e was implanted subcutaneously into two solid tumors, BALB/c mice and BDF-1 mice, respectively, for good antitumor activity against mammalian carcinomas. Tests against cancer. Treatment was performed on day 1 after tumor inoculation (early) or when the tumor was already evident (advanced).
It is administered intravenously starting at the time of the infection and once a week or 3 or 4 times every 6th day. Tumor growth is assessed by caliper measurements. Tumor-free mice are treated in parallel for toxicity evaluation and observed for 90 days. The results of the three experiments are shown in Table 10. For early colon 26 adenocarcinoma, e at the maximum allowable dose of 0.9 mg/Kg/day
is the DX at the maximum allowable dose of 7.5mg/Kg/day
more active than For advanced colon 26 adenocarcinoma, e at the highest tested dose of 0.7 mg/Kg/day was nontoxic and produced greater inhibition of tumor growth than DX at the highest tolerated dose of 6 mg/Kg/day. give. For advanced colon 38 adenocarcinoma
e at the maximum tested dose of 0.9mg/Kg/day
was as active as DX at 9 mg/Kg/day in inhibiting tumor growth, but did not result in a highly increased survival time.

[Table] In conclusion, all the data described herein demonstrate that compounds a and e have superior potency over DX, activity by the oral route, activity against anthracycline-resistant tumors (a), and solidity. It confirms that it is a very interesting new anthracycline that confers activity against tumors and is not cardiotoxic (e).

Claims (1)

[Claims] 1 Formula () (wherein, X is hydrogen or hydroxy, and R ₁
is hydrogen or methyl and one of R ₂ and R ₃ is methoxy and the other of R ₂ and R ₃ is hydrogen) and pharmaceutically acceptable acid addition salts thereof. 2. The compound according to claim 1, which is 4-demethoxy-4'-O-methyl-daunorubicin. 3. The compound according to claim 1, which is 4-demethoxy-4'-O-methyl-doxorubicin. 4. Claim 1, which is 4-demethoxy-4'-epi-4'-O-methyl-daunorubicin.
Compounds described in Section. 5 Said claim 1 which is 4-demethoxy-4'-epi-4'-O-methyl-doxorubicin
Compounds described in Section. 6 4-demethoxy-2,3-dimethyl-4'-O
-Methyl-daunorubicin. 7 4-demethoxy-2,3-dimethyl-4'-O
-Methyl-doxorubicin. 8. The compound according to claim 1, which is 4-demethoxy-2,3-dimethyl-4'-epi-4'-O-methyl-daunorubicin. 9. The compound according to claim 1, which is 4-demethoxy-2,3-dimethyl-4'-epi-4'-O-methyl-doxorubicin. 10 4-demethoxy-daunomycinone or 2,3-dimethyl-4-demethoxy-daunomycinone dissolved in anhydrous methylene dichloride in the presence of silver trifluoromethane-sulfonate and molecular sieves to 2,3,6-trideoxy-3-
Trifluoroacetamide-4-O-methyl-L
-lyxo-hexopyranosyl chloride or the corresponding N-trifluoroacetyl protection by reaction with 2,3,6-trideoxy-3-trifluoroacetamide-4-O-methyl-L-arabino-hexopyranosyl chloride The α-glycoside is obtained and the N-trifluoroacetyl protecting group is removed by mild alkaline hydrolysis with 0.2N aqueous sodium hydroxide to give the corresponding daunorubicin derivative (formula: X=H), optionally. is brominated and thereby formed 14-
formula (), consisting of converting the compound into the corresponding doxorubicin derivative (formula: X=OH) by treating the bromo-intermediate with aqueous sodium formate. (wherein, X is hydrogen or hydroxy, and R ₁
is hydrogen or methyl and one of R ₂ and R ₃ is methoxy and the other of R ₂ and R ₃ is hydrogen). 11 Formula () together with an inert carrier (wherein, X is hydrogen or hydroxy, and R ₁
is hydrogen or methyl and one of R ₂ and R ₃ is methoxy and the other of R ₂ and R ₃ is hydrogen).