CH615198A5 - Process for the preparation of daunomycin - Google Patents

Description

The present invention relates to a new process for the chemical synthesis of daunomycin and its yS anomer, the 4'-epi-daunomycin mixture of a and β anomer, and each individual anomer.

Daunomycin and daunomycinone are described and claimed in GB-PS 35 1033 383.

The full name of 4'-epi-daunomycin-a-anomer is 7-0- (3'-amino-2 ', 3', 6'-trideoxy-a-L-arabinohexopyran-osyl) -daunomycinone.

The full name of 4'-epi-daunomycin - /? - anomer 40 is 7-0- (3'-amino-2 ', 3', 6'-trideoxy - /? - L-arabinohexopyrano-syl) -daunomycinone.

The process according to the invention for the production of daunomycin and of 4'-epi-daunomycin and the corresponding / β anomers is characterized in that permanent 45-nomycinone with 1-chloro-2,3,6-trideoxy-3-trifluoroacetamido-4- trifluoroacetoxy-aL-lyxohexopyranose or l-chloro-2,3,6-tri-deoxy-3-trifluoroacetamido-4-trifluoroacetoxy-aL-arabinohexo-pyranose in an organic solvent in the presence of a mercury salt as catalyst and a hydrogen chloride acceptor and then the Trifluoroacetyl protective groups in the glycoside compounds formed are removed and the a and / J anomers are separated.

The method according to the invention can be reproduced as follows:

Daunomycinone of the formula

NH

CO

I.

CF

to form the corresponding glycosides from which the trifluoroacetyl protective groups are removed and the a- and β-anomers are separated, so that the pharmacologically active glycosides of the following formulas are obtained

Q OH COCHs

CHsO O OH

50

55

ÖH

(IV)

(Daunomycin),

NHa

Q OH COCHs

ÖH

(I)

CHsO O OH \

OH

is condensed with l-chloro-2,3,6-trideoxy-3-trifluoroacet

60

65

CHsO O

COCHs

ÖH

(V),

Daunomycin (IV) 1/2 Anomer

3rd

615198

O OH

COCHs

OH

(VI) and

4'-epi-daunomycin

COCHs

OH

CHsOO OH |

O

NHs I

O

HO

10th

15

20th

(VII)

/ S anomer of (VI)

25th

30th

The N-trifluoroacetyl protecting groups in the compounds II and III can be removed satisfactorily by mild alkaline treatment.

The process according to the invention is preferably applied to the starting products l-chloro-2,3,6-trideoxy-3- 35 trifluoroacetamido-4-trifluoroacetoxy-L-lyxohexopyranose and l-chloro-2,3,6-trideoxy-3-trifluoroacetamido-4 -trifluoroacetoxy-L-arabinohexopyranose, which are obtained by reacting 2,3,6-trideoxy-3-amino-L-lyxo-hexopyranose or 2,3,6-trideoxy-3-amino-L-arabinohexopyranose at 0 ° C with 40 trifluoroacetic anhydride to form the new tri-tri-fluoroacyl derivatives, which after treatment with anhydrous hydrogen chloride gas at 0 ° C l-chloro-2,3,6-trideoxy-3-tri-fluoroacetamido-4-trifluoroacetoxy-L-lyxoh * exopyranose and 1-chloro-2,3,6-trideoxy-3-trifluoroacetamido-4-trifluoroacetoxy-45 L-arabinohexopyranose.

The 1-chlorhexoses are stable solid compounds that can be stored under anhydrous conditions for several days.

As for suitable reaction conditions for synthesis 50 of the glycoside bond, the well-known Koenigs-Knorr reaction (J. Conchie, GA Lewy and CA Marsh, Advances in Carbohydrate Chemistry, 1957, 12, 157) enables a range of different conditions, the modifications of the solvent, the Include temperature, the catalyst, and 55 the hydrogen chloride acceptor. The modifications are important because an optimum of individual conditions is usually necessary in order to achieve an acceptable reaction rate.

The application of standard conditions to the 1-Ha 60 log derivatives of 1-deoxy sugar leads to the corresponding glycals (Zorbach et al., See above).

The following examples are intended to explain the present invention in more detail, without, however, being restricted thereto.

example 1

1 g of hydrochloride of daunosamine (II) is in water65

free diethyl ether and treated at 0 ° C with 8 ml of trifluoroacetic anhydride. After standing for 2 hours at 0 ° C. and 1 hour at room temperature, the solvent is removed under reduced pressure and the residue is crystallized from dichloromethane, 1.1 g of tri-trifluoroacetyldaunosamine being obtained, mp 132 to 134 ° C., mass spectrum m / e 391 (M-44), 322 (M-113). 0.5 g of this product are treated in anhydrous diethyl ether at 0 ° Celsius with anhydrous hydrogen chloride gas. After standing overnight at 5 ° C., the solvent is removed in vacuo, l-chloro-N, 0-di-trifluoroacetyldaunos-amine being obtained as an oily product (1-chloro-2,3,6-trideoxy- 3-trifluoroacetamido-4-trifluoroacetoxy-L-lyxo-hexopyranose).

NMR (CDCls): 1.22 ô (doublet, J = 6.5 Hz, 3H, CHS),

2.05 to 2.70 ó (multiplet, 2H, C (2) H2), 4.46 ô (doublet quartet, J = 6.5 Hz and J <1 Hz, 1H, C (5) H),

4.60 to 5.10 Ò (multiplet, 1H, C (3) H), 5.37 ò (C, Wh = 6.0 Hz, 1H, C (4) H), 6.29 <5 (multiplet , Wh ö 6.5 Hz, 1H, C (l) H) and

6.37 <5 (broad singlet, IH, NH).

300 mg (0.75 mmol) of finely powdered daunomycinone (I) are dissolved in 75 ml of anhydrous chloroform and treated with 600 mg of mercury oxide, 150 ml of mercury bromide and a molecular sieve (3Â, Merck).

The suspension is stirred for 1 hour, after which 600 mg of l-chloro-N, 0-trifluoroacetyldaunòsamin are added. The mixture is stirred at room temperature for 64 hours and then filtered. The solution is evaporated in vacuo. The residue is taken up in 200 ml of methanol and refluxed for 15 minutes. The residue that remains after removal of the solvent is chromatographed on a silica column using a mixture of chloroform: benzene: methanol 100: 20: 3 (by volume) as the eluent.

In addition to unconverted daunomycinone, 220 mg of N-trifluoroacetyldaunomycin, mp. 169 to 171 ° C (after recrystallization from tetrahydrofuran and hexane) and 20 mg of N-trifluoroacetyldaunomycin (^ isomer), mp. 138 to 140 ° C, [a] j ^ ° + 440 ° (c = 0.1 in chloroform) obtained.

0.20 g of N-trifluoroacetyldaunomycin are dissolved in 20 ml of 0.1N aqueous sodium hydroxide. The solution obtained, after standing at room temperature for 30 minutes, was treated with 0.1N aqueous hydrogen chloride to bring the pH to 8.6 and extracted repeatedly with chloroform.

The extracts are dried over anhydrous sodium sulfate, concentrated to a small volume and acidified with 0.1N methanolic hydrogen chloride to a pH of 4.5 in order to allow the crystallization of daunomycin hydrochloride. This is identical in every respect to the product obtained by fermentation (see F. Arcamone et al., Gazzetta Chimica Italiana, 1970, 100, 949).

The yield is practically quantitative.

Example 2

1 g of 2,3,6-trideoxy-3-trifluoroacetamido-L-arabinohexose is suspended in 20 ml of anhydrous diethyl ether and treated at 0 ° C. with trifluoroacetic anhydride. The mixture is further treated as described in Example 1, a quantitative yield of 2,3,6-trideoxy-N, 0-di-trifluoroacetyl-L-arabinohexopyranosyl chloride being obtained. NMR (CDCls): 1.30 ô (doublet, J = 6.0 Hz, 3H, CHS),

2.25 to 2.80 Ò (multiplet, 2H, C (2) H2), 4.20 to 4.65 <5 (multiplet, 1H, C (5) H),

615198

4th

4.65 to 5.15 ô (multiplet, 2H, C (3) H and C (4) H),

6.25 Ô (multiplet, Wh = 6.0 Hz, 1H, C (l) H) and

6.45 ô (broad single, IH, NH).

A solution of 0.5 g daunomycinone in anhydrous chloroform is mixed with 1 g mercury oxide, 0.25 g mercury bromide and 10 g molecular sieve (3Â, Merck) and 0, 5. g 2,3,6-trideoxy-N-O-di-trifluoroacetyI-L-arabinohexopyranosyl chloride treated. The mixture is stirred for 24 hours, the solids are filtered off and it is evaporated in vacuo. The residue is taken up in methanol, held in reflux for 15 minutes, evaporated to dryness and chromatographed on a silica column using a mixture of chloroform: benzene: methanol 10: 20: 3 as eluent. The main product that is obtained is a mixture in the ratio of 70:30 a- and / 3-7-0- (N-trifluoroacetyl-4'-epi-daunosamin-yl) -daunomycinone (yield after crystallization from chloroform 0, 3 g). After treatment with 0.1N sodium hydroxide, as described above, this material is converted quantitatively into a mixture of the corresponding a- and / 3-glycosides in the form of free bases. The product is separated by silica gel chromatography using a solvent system consisting of chloroform: methanol: water 135: 20: 2 (based on the volume) as the eluent into the α and β anomers. There are 0.16 g. "- Anoxner (4'-epi-Dauno-mycin (VI)) 'M 23 ° + 32o ° (c = 0.045 in methanol), mp. 199

to 201 ° C, and 0.06 g / j anomer (VII), mp. 182 to 184 ° C obtained.

Connect

dose

Mycotic. index

Colony counts dung

Ottg / ml)

2 hours 4 hours

8 hours

2 hours 8 hours 24 hours

0.025

226 *

100

117

113 88 48

5 VI

0.05

189 *

103

122

115 56 23

0.1

79

140

0

77 23 3

IDso

0.16 0.056 0.027

0.25

137

103

85

108 86 70

ltf VII ■

0.5

95

67

88

101 37 18

1

52

40

0

98 17 6

IDS0

> 1 0.47 0.33

. Dauno-

15 myein

IDso

0.098 0.036 0.021

: metaphasic block

20 "

Table 2

Effect of compounds (VI) and (VII) on foci formation and cell proliferation in cultured mouse fibroblasts infected with Moloney sarcoma virus 25 "(3-day treatment).

Biological activity of the compounds (VI) (4'-epi-daunomycin) and (VII) <4'-epi-daunomycin, / J-anomer)

The compounds (VI) and (VII) show excellent biological properties as strong inhibitors of cell mitosis and proliferative activity in culture cells in vitro. They also show a significant effectiveness on time transformations induced by oncogenic viruses. They have anti-tumor activity in mice, such as an increase in mean survival in non-toxic doses in animals that have a number of experimental tumors; shown (Tables 1 to 4).

The in vitro cardiotoxic activity of compound (VI) is very low (lower than that of daunomycin), as is evident from the experiment below.

The method consists of cultivating individual heart cells that have been isolated from the heart of newborn mice by tripsinization. After 3 to 4 days, the cultures that showed clustering cells could be studied from both the frequency and rhythm points of view (Necco A., Dasdia T. IRCS, 2: 1293, 1974).

Connect

dose

Foci education

Cell proliferation

B / A

dung

Cug / ml)

(° / o

ID50

(%

IDso

Con

(/.tg/ml)

Con

(«G / ml)

30th

troll)

(A)

troll)

(B).

0.0062

51

75

VI

0.025

0

0.006

23

0.013

2.1

.0.1

0

14

35

0.4.

0

1

0.0062

.48

87

VII

0.025

44

0.01

80

0.064

6.4

0.1,

5

28

40

0.4

.0

14

Dauno

mycin

0.006

0.0086

1.4

Table 3

Effect of the compounds (VI) and (VII) on the mean survival time and on the number of survivors in female mice (Swiss CDI) to whom intraperitoneal sarcoma 180 abdominal addiction (1 X 10® cells / mouse) had been introduced. The compounds were administered on the day following tumor transplantation.

60

Table 1

Effect of compounds (VI) and (VII) on the mycotic index and proliferative activity of cultured HeLa cells at different exposure periods.

The results are expressed as a percentage of untreated controls.

65

Connect

Medium dose

Number of dung

(mg / kg)

Survival time

Survivors

(0/0 controls)

on the 60th day

0.22

111.1

1/10

VI

1.1

120.8

1/10

• 5'7

174.5

0/10

0.26

96.6

0/10

VII

1.3

114.8

1/10

6.7

118.6

1/10

6/10 animals died as a result of drug toxicity

Table 4

Effect of compounds (VI) and (VII) on transplantable gross leukemia. Mean survival of CsH female mice intravenously vaccinated with a suspension of leukemic lymph nodes and spleen (2.5 X 106 cells / mouse). The compounds were administered once a day for 5 days beginning on the day following the tumor's transplant.

615198

Compound daily mean

dose

Survival time

(mg / kg)

(% Controls)

1.5

115

2.25

143

VI

3rd

162

3.75

122

4.5

120

1.5

106

VII

2.25

102

3rd

121

M

Claims (2)

615198 2nd PATENT CLAIMS 1. A process for the preparation of daunomycin and 4'-epi-daunomycin and the corresponding / Î-anomers, characterized in that daunomycinone with 1-chloro 2,3,6-trideoxy-3-trifluoroacetamido-4-trifluoroacetoxy- aL- 5 lyxohexopyranose or l-chloro-2,3,6-trideoxy-3-trifluoroacet-amido-4-trifluoroacetoxy-aL-arabinohexopyranose in an organic solvent in the presence of a mercury salt as catalyst and a hydrogen chloride acceptor and then the trifluoroacetyl protective groups in the 10 glycoside compounds formed are removed and the a and β anomers are separated. 2. Application of the method according to claim 1 to starting products l-chloro-2,3,6-trideoxy-3-trifluoroacetamido-4-trifluoroacetoxy-L-lyxohexopyranose and l-chloro-2,3,6-tri-deoxy-3- trifluoroacetamido-4-trifluoroacetoxy-L-arabinohexopyranose, which are obtained by reacting 2,3,6-trideoxy-3-amino-L-lyxo-hexopyranose or 2,3,6-trideoxy-3-amino-L- Arabineohexopyranose at 0 ° C with trifluoroacetic acid anhydride to form the new tri-trifluoroacetyl derivatives, which after treatment with anhydrous hydrogen chloride gas at 0 ° C l-chloro-2,3,6-trideoxy-3-trifluoroacet-amido-4-trifluoroacetoxy -L-lyxohexopyranose and 1-chloro-2,3,6-trideoxy-3-trifluoroacetamido-4-trifluoroacetoxy-L-arabi-nohexopyranose. 25th amido-4-trifluoro-acetoxy-a-L-lyxohexopyranose of the formula II (II) CO IjîH CFs CO I CFs 15 or with l-chloro-2,3,6-trideoxy-3-trifluoroacetamido-4-trifluoro-acetoxy-a-L-arabinohexopyranose of the formula III 20th (III) 30th