DK152677B - METHOD OF ANALOGUE FOR THE PREPARATION OF 4'-O-TETRA-HYDROPYRANYLADRIAMYCINE OR A POISONOUS ACID ADDITION SALT - Google Patents

Description

DK 152677 B

The present invention relates to an analogous process for the preparation of 4'-O-tetrahydropyranyladriamycin of formula M

AND, 'O OH

J 0 (I) 5 or an nontoxic acid addition salt thereof, 41-O-Tetrahydro-pyranyladriamycin is a novel compound having antibiotic action.

The process of the invention is characterized in that adriamycin of formula I is reacted. | H »

1 C-CHOOH

vvvv

AND 0 OH I

10. O (II)

H0 K

or an acid addition salt thereof with 3,4-dihydro-2H-pyran in an inert organic solvent and in the presence of an acidic catalyst eliminates the tetrahydropyranyl group at the C-14 position by partial hydrolysis or alcoholysis 2

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of the thus-obtained tetrahydropyranyl compound of formula COO>

CCH., O OH I

3 0 (I) d? and, if desired, converting the formed compound of formula (I) into a non-toxic acid addition salt thereof or, if necessary, converting a formed acid addition salt of the compound of formula (I) into the free compound or to another, non-toxic acid addition salt thereof.

A number of anthracycling glycosides are described in the literature. Among these, adriamycin (U.S. Patent Nos. 3,590,028 and 3,803,124), which is the known compound most closely related to the compound of Formula I, which is recovered from the culture fluid from the cultivation of certain streptomyces species, has a broad antitumor spectrum against various experimental 15 tumors and it is used clinically as a strong chemotherapeutic agent. Despite the usefulness of adriamycin as a clinical antitumor agent, it is known to have serious side effects such as alopecia, leukopenia and cardiotoxicity.

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It has been found that 4'-O-tetrahydropyranyladriamycin has a stronger anticancer activity and lower toxicity than adriamycin.

The term "non-toxic acid addition salts" encompasses acid addition salts of the compound of formula I with all the organic and inorganic acids commonly used to prepare substantially non-toxic salts of drugs containing amine functions. Such salts may be formed, for example, from acids such as sulfuric acid, phosphoric acid, hydrochloric acid, hydrogen bromide, nitric acid, phosphoric acid, acetic acid, propionic acid, maleic acid, voleic acid, palmitic acid, citric acid, succinic acid, tartaric acid, glutamic acid, pantothenic acid, pantothenic acid, pantothenic acid. , methanesulfonic acid and naphthalenesulfonic acid.

The compounds of formula I may be in the form of the individual diastereomers (herein arbitrarily designated as isomer a) and isomer b) which have different configuration at the C-2 position of the tetrahydropyranyloxy group or as mixtures of such isomers. The present invention encompasses the preparation of both the individual diastereomers and the diastereomer mixtures.

In the drawing, FIG. 1 shows the infrared absorption spectrum (KBr) of 4'-O-tetrahydropyranyladriamycin (isomer a); 2 shows the infrared absorption spectrum (KBr) of 4'-0-25 tetrahydropyranyladriamycin (isomer b); and FIG. 6-7 proton NMR spectra (100 MHz, CDCl 3) of the compounds in the same order as the above-mentioned infrared absorption spectra; The 3-4 proton NMR spectra (100 MHz, CDCl3) of 4 ', 14-di- (O-tetrahydropyranyl) -adriamycin, isomer a), respectively.

30 isomer b), FIG. 5 proton NMR spectra (100 MHz, CDCl 3) of 14-O-tetrahydropyranyldriamycin.

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Adriamycin used as starting material may be the free base or an acid addition salt. Since the acid addition salts of the tetrahydropyranyl ether reaction products can be converted by the known methods to the corresponding free bases or to 5 non-toxic acid addition salts, it is not necessary for a starting material to be non-toxic.

Any non-reactive organic solvent can be used for the tetrahydropyranylation reaction. Suitable solvents are, for example, benzene, toluene, xylene, dimethylformamide, acetonitrile and tetrahydrofuran. The reaction solvent may be a single solvent or a mixture of solvents. A preferred solvent is anhydrous dimethylformamide.

The acidic catalyst can be any organic acid (e.g.

Formic acid or trifluoroacetic acid) or an inorganic acid (e.g. hydrochloric or phosphoric acid). A preferred class of acidic catalysts comprises the organic sulfonic acids.

Particularly preferred catalysts are aromatic sulfonic acids such as p-toluenesulfonic acid and benzenesulfonic acid. A particularly preferred catalyst is p-toluenesulfonic acid.

The reaction temperature is not critical. Good results in the ethereal reaction have been obtained at room temperature, although higher and lower temperatures can also be used ...

The reaction time varies according to the selected reaction conditions, e.g. temperature, catalyst and solvent. Selection of the optimal reaction time can be done by routine experimentation using the thin layer test described below. Generally, reaction times of approx. 20 to approx. 50 hours proved to yield favorable results. 1

While reaction products and the reaction yield as mentioned above depend on factors such as the concentration of the starting materials, the ratio of the reactants, etc., the main products are 14-O-tetrahydropyranyladriamycin (14-O-PA) with Rf = 5

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0.12 and two components which are diastereomers of 4 ', 14-di (O-tetrahydropyranyl) adriamycline, i.e. 4 ', 14-di (O-tetrahydro-pyranyl) -adriamycin (isomer a), abbreviated as 41,14-di-O-PAa at Pf 0.55 and 41,14-di (O-tetrahydropyranyl) adriamycin (isomer) 5 b), abbreviated as 4 ', 14-di-O-PAb at Rf 0.73. The compound 4 ', 14-di (O-tetrahydropyranyl) adriamycin may also be called 41,14-bis (O-tetrahydropyranyl) adriamycin. These products can be detected in the reaction mixture by thin layer chromatography on silica gel (Merck Co. "6OF254") using a chloroform-methanol-acetic acid mixture (80: 20: 4 v / v) as the eluent.

By utilizing the difference in reactivity between the primary C-14 hydroxyl group and the secondary C-4'-hydroxyl group, the tetrahydropyranyl group on C-14 is removed in 4 ', 14-di-O-PAa and 15 4', 14-di-0 -PAb (or an acid addition salt thereof) selectively by hydrolysis or alcoholysis to give the corresponding diastereomers of 4'-O-PAa and 4'-O-PAb in good yield. The conversion of the tetrahydropyranyloxy group to a hydroxyl group can e.g. is carried out by hydrolysis with acidified water 20 (e.g., an aqueous inorganic acid) or by alcoholysis with an alcohol or phenol (e.g., a C 1-6 alkanol). A suitable process comprises treating with dilute acetic acid or p-toluenesulfonic acid-methanol solution at room temperature for from ca. 30 minutes to 5 hours. Side reactions 25 can be diminished by carrying out the hydrolysis or alcoholysis reaction in the dark.

The products of formula I can be isolated from the reaction mixture by conventional methods. Thus, the products of the tetrahydropyranylation reaction can be recovered by neutralizing the reaction mixture with a basic substance (e.g., an alkali metal carbonate or bicarbonate), extracting the neutralized reaction mixture with a water-immiscible organic solvent (e.g., ethyl acetate, chloroform, methylene chloride or methyl isobutyl ketone), extraction of the organic extract with a dilute aqueous acid solution (organic or inorganic), neutralization of the aqueous acidic layer with a basic substance, 6

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extraction of the neutralized aqueous layer with a water-immiscible organic solvent and concentration of the organic extract to dryness. The dark red dried powder thus obtained can be purified by silica gel column chromatography or, in the case of a smaller amount, by preparative thin layer chromatography. The products of the hydrolysis or alcoholysis reaction can be recovered from the reaction mixture by neutralization with a basic substance, extraction of it, neutralized reaction mixture with a water-immiscible organic solvent and concentration of the organic extract to dryness.

Products obtained in the form of a mixture of diastereomers (a and b isomers) can be separated as described above by silica gel thin layer chromatography in the individual a and b isomers substantially in pure form.

Products obtained by the above reactions can be recovered in the form of the free base or a non-toxic acid addition salt. The free bases can easily be converted into non-toxic acid addition salts whose therapeutic activity is substantially equivalent to that of the corresponding free bases. The salts are formed, isolated, purified and formulated by the methods generally used for salt formation of antibiotics, e.g. anthracyclinglycosidanti-antibiotics. Thus, the free base can be reacted with a non-toxic acid in a suitable solvent and the salt is recovered by lyophilization 25 or by precipitation with a precipitant, ie. a solvent in which the desired salt is only slightly soluble. Products in the form of an acid addition salt can be converted to the corresponding free base by neutralization with a basic substance. Finally, toxic acid addition salts can be converted to non-toxic acid addition salts by neutralization and treatment with a non-toxic acid as described above.

Physico-chemical properties

The present compounds exist in solid form as an amorphous or crystalline red powder. As free bases, they are soluble in ethyl acetate, chloroform and ethanol and

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heavily soluble in i.a. water, n-hexane and petroleum ether. Ethanolic solutions and acidic solutions of the compounds are red, give positive ninhydrin reaction and do not reduce Fehling's liquid. FIG. 1-2 and 5-7 and Table 1 shows elemental analysis, melting point (decomposition), specific rotation (C = 0.2 i.CHCl 3), absorption spectrum for UV light and visible light (methanol), infrared absorption spectrum (KBr tablet) and NMR spectrum (100 MHz, CDCl ^).

Table 1 10 Compound_4 1 -O-PAa_4 1 -Q-PAb_

Analytical data________ (1) Elementary C: 59.65 (59.52) C: 59.71 (59.52) Analysis H: 6.33 (6.10) H: 6.24 (6.10) () calculated % * N: 2.21 (2.17) N: 2.05 (2.17) (2) Molecular weight 627.70 627.70 (3) Melting point 172 ~ 177 188-192 (° C) (decomp. (decomp.) (4) Specific 0, 7C rotation [a] (2 + 150 ° [a] _ + 150 C = 0.2 CHCl 3 (5) Rf value ** 0.32 0.49 20 ( 6) Absorption 220s (350), 234 (515) 220s (365), 234 (480) spectrum for 252.5 (360), 290 (120) 252 (350), 290 (110) UV light and 480 ( 150), 497 (160) 480s (135), 498 (140) visible light (nm) 532 (100), 577 (30) 531.5 (100), 580 (45) (E “m) __ * Calculated scan Monohydrate ** Silica Gel TLC: CHCl 3 OH = 10: 1 (v / v), 26 ° C (Merck Co. 60F254)

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With respect to the structure of compounds 4 ', 14-di-O-PAa, 4', 14-di-O-PAb, 14-O-PA, 4'-O-PAa and 4'-O-PAb, the number of tetrahydropyranyl groups bound to the compounds / analyzed to be either one or two from the signal intensity of the methine proton in the O 2 and methylene protons in the 03/04/05 and Οβ positions of the tetrahydropyranyl group. The binding position of the tetrahydropyranyl group can be analyzed by chemical displacement of the 04 'proton in the daunosamine portion toward a lower field (compared to that of dauno-mycin) due to formation of a glycosidic bond of 04'.

The difference in configuration between 4, -0-PAa and 4'-O-PAb and 4 ', 14-di-O-PAa and 4', 14-di-O-PAb is presumed to be a difference in absolute configuration R and S of O 2 in the tetrahydropyranyl group, with chemical shift and coupling constants (J value) of the O 2 and O 3 protons being different from each other. However, the absolute configuration of the a and b isomers is still unknown. Table 2 shows the chemical shift (from Figures 3-7) of the O 2 proton in the tetrahydropyranyl group 20 and of the O 4 'proton in the daunosamine moiety.

Table 2

Proton "4'-THP1 · 14-THP1 _ (ppm) _ (ppm) _

Compound 4 '-O-PAa 4/36 · - -. 4'-O-PAb 4.72 4 ', 14-di-O-PAa 4.38 4.70 41.14-di-O-PAb 4.72 4.72 14-O-PA - 4, 71 THP: Chemical displacement in the C-2-methine group in a substituted tetrahydropyranyl group 9

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From the above, the structure of the compounds prepared by the process of the present invention was determined to be as indicated above.

Antibiotic Activity The compounds of formula I have been shown to have antimicrobial activity against many different pathogenic microorganisms. The minimum inhibitory concentrations (determined by the broth dilution method) of the compounds prepared by the method of the present invention are shown in Table 3.

Table 3 Minimum Inhibitory Concentration (MIK / µg / ml)

Tested compound

Test-organism_41 -O-PAa_41 -0-PAb

Staph, aureus 6.25 6.25

FDA 209P

15. Staph, aureus 6, 25 6.25.

Smith

Bacillus subtilis 3.12 3.12 NRRLB-558

Bacillus cereus 6.25 6.25 ATCC 10702

Bacillus megaterium 3.12 3.12

APF

Sarcina lutea 0.78 0.78 PCI 1001 20 Micrococcus flavus 3.12 3.12 FDA 16

Corynebacterium bovis 3.12 3.12 1810

Pseudomonas aeruginosa> 100> 100 A3

Escherichia coli> 100> 100

NIHJ

Mycobacterium smegmatis 100 100 ATCC 607 25 Candida albicans> 100> 50

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As shown in Table 3, the compounds of the invention are useful as antibiotic agents, especially against gram-positive bacteria.

Antitumor activity

Compounds of formula I show pronounced antitumor activity with low toxicity in standard tests.

A. The compounds of the invention were found to have a strong inhibitory effect on the growth and nucleic acid synthesis of L1210 leukemia cells in culture.

Eg. For example, L122 cells (5x10 5 cells / ml) are included in RPMI-1640-10 medium (Roswell Park Memorial Institute 1640) containing 20% calf serum and grown at 37 ° C in the presence of 0.1 and 0.5 µg / ml. ml of the subject compounds in a CC1 incubator. The number of cells is counted periodically and the growth inhibition (%) of control was determined as shown in Table 4.

Table 4 Growth inhibitory effect of tetrahydropyranyl derivatives on L1210 cells in culture.

Concentration_Inhibition (%) _

Compound 0.1 / 0.5 µg / ml 4'-0-PAa 65.9 81.1 4'-0-PAb 78.1 92.9

Adriamycin -70.7 84.2

The effect of the subject compounds on nucleic acid synthesis was investigated as follows: 5

1x10 celler cells / ml of L210 cells were suspended in RPMI medium 25 containing 10% calf serum and first grown at 37 ° C.

for 1 to 2 hours in a CC1 incubator, after which the subject compounds were added to the medium at various concentrations.

After 15 minutes of incubation, CC-uridine (0.05 µCi / 14 11) was added

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ml) or C-thymidine (0.05 µCi / ml) and incubated at 37 ° C for 60 minutes. 10% trichloroacetic acid (TCA) was added to the incubation medium to stop the reaction and precipitate the acid-insoluble materials and then the precipitate was washed three times with 5 to 10% 1s TCA dissolved in formic acid. Radioactivity was measured and expressed as 50% inhibition concentration for incorporation.

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Table 5 50% inhibition concentration of C-thymidine 14 and C-uridine incorporation into L122 cells in culture.

50 inhibition concentration yg / ml

Compound_Uridine_Thymidine 4'-O-PAa 0.20 0.37 4'-O-PAb 0.24 0.50

Adriamycin 0.50 2.1 15 B. When tested against various experimental tumors in animals, the compounds of this invention exhibit a marked antitumor activity with reduced toxicity to adriamycin and daunomycin. Accordingly, the compounds are therapeutically useful for inhibiting the growth of mammalian tumors.

For example, BDF-1 mice were inoculated intraperitoneally with r 1x10 cells / mice of L1210 leukemia cells. After 24 hours of inoculation, the subject compounds were administered intraperitoneally to the mice once daily 25 for ten consecutive days, and the mice were kept under observation for 45 days. The antitumor activity was shown at the prolongation of life (T / C,%) relative to the lifetime of control mice injecting physiological saline. The results are shown in Table 6.

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Table 6 Anti-tumor activity of tetrahydropropyranil derivatives (T / C,%). Dose (mg / kg / day)

Compound_5_2.5 1.25 0.6 0.3 0/15 5 4'-0-PAa - 173 180 187 120 127 41-0-PAb> 375> 360> 373 293 160 113

Adriamycin toxic 231 218 230 165 128 dead

From the results of toxic deaths and weight loss in mice in this experiment, it has been shown that the toxicity of the subject compounds is 1/3 to 1/2 lower than that of adriamycin.

C. The pronounced antitumor effects seen by A and B above were confirmed by the stability of the compounds of the invention against inactivation with hepatic NADPH cytochrome P450 reductase. Specifically, NADPH cytochrome P450 reductase isolated from rat liver homogenate and purified was incubated with the subject compounds at 25 ° C for 25 minutes in nitrogen gas phase and the resulting incubation product, 7-deoxyagiycone, was determined as shown in Table 7.

Table 7 Stability of tetrahydropyranyl derivatives against rat NADPH cytochrome P450 reductase.

Product (nmol / tube)

Compound (7-deoxyaglycon) 4'-O-PAa 10.9 4'-O-PAb 15.8 Adriamycin 47.2

Composition of the reaction mixture:

NADPH 0.2 mM

Tris-HCl (pH 8.0) 0.1 M

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Substrate 0.1 mM

Enzyme 4.6 µg / ml (Tris-HCl = tris- (hydroxymethyl) -aminomethane).

Therapeutic Use 5 As mentioned above, the compounds 4'-0-PAa and 4'-0-PAb and their non-toxic acid addition salts are novel antibiotics useful in both human and veterinary medicine and also exhibit inhibitory action against malignant mammalian tumors, including both solid and ascitic types.

The present compounds may be formulated as a pharmaceutical composition containing a therapeutically effective anti-microbial or tumor-inhibiting amount of 4'-O-PAa or 4'-O-PAb, or a mixture thereof or a non-toxic acid addition salt thereof. in combination with a pharmaceutical carrier or diluent. Such preparations may be prepared in any pharmaceutical form suitable for parenteral administration.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. They may also be prepared in the form of sterile solid preparations which may be dissolved in sterile water, physiological saline or other sterile injectable medium immediately prior to use.

The preferred dose size depends on the nature of the compound used, the formulation of the preparation and the mode of application, the situs and host, and the nature of the disease. In general, the compounds are injected intraperitoneally, intravenously, subcutaneously or locally in mammals other than humans and intravenously or locally in humans. Many factors that modify the effect of the drug must be taken into consideration by those skilled in the art, e.g. age, body weight, sex, diet, time of administration, route of administration, rate of excretion, patient's condition, drug combination.

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14 reactions, reaction sensitivities and severity of the disease. Administration may be performed continuously or periodically within the maximum tolerated dose. Optimal application rates for a given set of conditions can be determined by those skilled in the art using conventional dosing determination tests according to the above guidelines.

For use as antimicrobial agents, the compounds are generally administered such that the concentration of the active ingredient is greater than the minimum inhibitory concentration of the affected organism.

The method according to the invention is further illustrated in the following example.

Example A. To a solution of 130 mg of adriamycin hydrochloride in 10 ml of anhydrous dimethylformamide is added 2 ml of 3,4-dihydro-2H-pyran and a catalytic amount of p-toluenesulfonic acid. After standing for 48 hours at room temperature in the dark, the reaction mixture was added to 20 ml of 0.1 N aqueous sodium bicarbonate solution and extracted with 5 x 20 ml of ethyl acetate. After extracting the ethyl acetate layer with 4 x 40 ml of 1% acetic acid, the acidic aqueous layer was neutralized with sodium hydrogen carbonate and extracted with 10 x 20 ml of chloroform. The chloroform layer was dried over anhydrous sodium sulfate and concentrated to dryness. The resulting 160 mg of solid was eluted and purified by preparative thin layer chromatography on silica gel using a chloroform-methanol (10: 1) (v / v) mixture. The bands of Rf 0.73 and 0.55 were scraped off the thin-layer plate, eluted with the chloroform-methanol mixture (10: 1) (v / v) and concentrated to dryness. 1

16 mg of a red solid of 4 ', 14-di-O-PAa and 14 mg of a red solid of 4', 14-di-O-PAb were obtained from Rf 0.55 and 0 ', 73 respectively. fractions.

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4 ', 14-di-O-PAa and 41,14-di-O-PAb are diasteromers of 4', 14-bis- (O-tetrahydropyranyl) adriamycin.

B. 12.4 mg of 4 *, 14-di-O-PAa was dissolved in 1.5 ml of 10% acetic acid and left for 4.5 hours at room temperature in the dark. The reaction mixture was added to 10 ml of water, neutralized with sodium bicarbonate powder and extracted with 2 x 15 ml of chloroform.

The chloroform layer was dried over anhydrous sodium sulfate and concentrated to dryness. The resulting 11 mg of residue 10 was purified by thin layer chromatography on silica gel as described above using a chloroform-methanol mixture (10: 1) (v / v). The main band at Rf 0.32 was scraped off and eluted with the chloroform-methanol mixture (10: 1) (v / v).

The eluate was concentrated to dryness. The residue was dissolved in methylene chloride, t-butanol was added under cooling to freezing and dried under reduced pressure. 7 mg of a red solid of 4'-O-PAa was obtained and its physicochemical properties are shown in Table 1.

C. 16 mg of 41,14-di-O-PAb was dissolved in 5 ml of 0.005 N p-toluene-sulfonic acid methanol solution and left for 1 hour at room temperature in the dark. The reaction mixture was neutralized with 10 ml of 0.01 N aqueous sodium hydrogen carbonate solution and extracted with 4 x 10 ml of chloroform. The chloroform layer was dried over anhydrous sodium sulfate and treated as shown in (B). 7.2 mg of a red solid of 4'-O-PAb was obtained which shows Rf 0.49 on a silica gel thin sheet under the conditions described above. Its physicochemical properties are shown in Table 1.

4'-O-PAa and 4'-O-PAb are diastereomers of 4'-O-tetrahydro-pyranyl-adriamycin.

D. The HCl acid addition salts are prepared by dissolving it

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16 free base of 41-O-PAa and 4'-O-PAb in ethyl acetate, respectively, and add approx. 1 equivalent HCl. Upon lyophilization, 41-O-PAa-HCl, respectively, is obtained with m.p. 140-142 ° C (dec.) And 4'-O-PAb-HCl, m.p. 147-152 ° C (dec.).

Claims (2)

0 OH f OCH-O OH 30 (I) 5 or a non-toxic acid addition salt thereof, known g-net by reacting adriamycin of the formula 0 OH tt OCH_ O OH I ,. (II) H ° ™ 2 or an acid addition salt thereof with 3,4-dihydro-2H-pyran in an inert organic solvent and in the presence of an acidic catalyst eliminates the tetrahydropyranyl group at the C-14 position at partial hydrolysis or alcoholysis of the thus-obtained di-tetrahydropyranyl compound of the formula DK 152677 B li f «/ ° -V yw ^ WN / C-CH2 OV \ JT JT ^ Y Y-OH x-x ) Æ \ | 09 / if desired, the formed compound of formula (I) converts to a non-toxic acid addition salt thereof or, if necessary, converts a formed acid addition salt of the compound of formula (I) to the free compound or to another non-toxic acid addition salt.