DK160828B - METHOD FOR PREPARING A REACTION PRODUCT OF 2-METHYL-2,5-DIMETHOXY-2,5-DIHYDRO-FURAN AND L-ASCORBIC ACID - Google Patents

Description

DK 160828 B

The present invention relates to a process for the preparation of a novel reaction product of 2-methyl-2,5-dimethoxy-2,5-dihydrofuran and L-ascorbic acid of formula 5

H.. H HH

εΗΛΑρ0 -—

Λ O I

hoch

10 H% H CH 2 OH

and with Rf values of 0.65 and 0.3 on silica gel thin-layer chromatography plates using a solvent mixture of chloroform and methanol in the ratio of 9: 1, and this method is characterized by reacting 2-methyl -2,5 - dimethoxy-2,5-dihydrofuran of formula H3COCH3 H3CO0> OO;> Vh with L-ascorbic acid of formula

HO OH

25χΠOχΠCHOH-CH₂OH 1 an aqueous medium in a nitrogen atmosphere.

Since the reaction product produced is inherently difficult to handle, it is first converted with succinic anhydride into a crystalline complex which is then administered by the therapeutic treatment described below.

In use, this complex is decomposed, whereby the active substance, i.e. the subject reaction product is released.

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DK 160828 B

It has been found that the present reaction product exhibits cytostatic, hypotensive and analgesic properties and gives good results in animal cancer treatment.

The reaction product is obtained e.g. 93 g of 2-methyl-5 -2,5-dimethoxy-2,5-dihydrofuran (prepared according to the procedure described by N. Clauson-Kaas and F. Lindberg,

Acta Chem. Scand., 1, 619 (1947)), with vigorous mechanical stirring, an aqueous solution of 75 g of L-ascorbic acid is added under a nitrogen atmosphere. The furan compound is used in a 50% excess. Over 15-20 minutes, the solution becomes homogeneous. The reaction is followed by a high-pressure liquid chromatograph equipped with a UV indicator. The initial peak of Rf = 1.6 for L-ascorbic acid at 254 nm in 20% aqueous methanol disappears, and a new peak of Rf = Rf = 5.9-6.0 appears under 64 atmospheric pressure. At the same time, it is observed that the solution no longer consumes iodine, which shows that the ascorbic acid is completely consumed.

The aqueous solution is left to stand overnight at 20 ° C and then freeze-dried to give a slightly yellowish foam. This shows well-defined IR, 13 C-NMR and 1 H-NMR spectra. The product exhibits Rf values of 0.65 and 0.3 on silica gel thin-layer chromatography plates using a solvent mixture of chloroform and methanol in the ratio 9il.

25 64.0 g (0.25 mole) of the crude product (mp 55 ° C) and 25.0 g (0.25 mole) succinic anhydride are added to a 1 liter round bottom flask equipped with a reflux condenser and a nitrogen intake. After addition of 200 ml of HPLC grade ethyl acetate, the mixture is heated at reflux for 4 hours.

The homogeneous solution is cooled to room temperature and then added to an ice-water bath. A white precipitate forms, which is separated by suction filtration and washed with a few milliliters of cold ethyl acetate. 29.9 g of the crude product is obtained (yield 46.6%). The filtrate is concentrated to half the volume and cooled in an ice-water bath. The second yield of the precipitate 3

DK 160828 B

separated by filtration to give 13.5 g of solid containing some unreacted succinic anhydride.

The product is recrystallized from a solvent mixture of ethyl acetate and chloroform at a ratio of 20:80, and a total yield of 22.5 g (35.2%) of pure product is obtained in the form of long white needles, m.p. 134 to 134.5 ° C.

Analysis calculated for C26H28 ° 17: C = 50.99%, H = 4.61%, O = 44.41% Found: C = 50.67%, H = 4.52%, O = 44.58% .

The formula for the reaction product prepared by the process of the invention is confirmed by X-ray crystallography as follows: fl 15 ° = Ck> h ,, 0 "75 o} ~ \" °, ° c "3 This reaction product is identified by NMR and IR spectra as shown in the drawing, in which Fig. 1 shows the NMR spectrum of the reaction product, and Fig. 2 shows the iR spectrum of the product.

The peaks of the NMR spectrum (60 MHz) confirm the presence of hydroxyl groups and of H atoms as present in the above formulas. The NMR spectrum shows the presence of a mixture of the indicated compounds, dominating in the formula of the reaction product on the left the compound.

The 30 IR spectrum shows the presence of several hydroxyl acids and two different carbonyl groups.

Study results.

Experiment 1.

A single oral dose of the reaction product is administered to CD-I mice and CD rats. 5 g per kg is dissolved in 0.85% NaCl-4

DK 160828 B

solution containing 0.0238 M of a bicarbonate buffer, pH 6.8, and administered continuously orally. A constant volume dose of 10 ml per ml is used. kg of reaction product dissolved in buffer and buffer alone. The mice and rats observed-5 are taken 14 days after administration. After 14 days, the animals are killed and autopsied to obtain an approximate detection of the toxicity, but no evidence of toxicity is found.

Experiment 2.

10 Early early latent tumor stage (TDS) DBA / 2 mice are used 10 days after a viable L 5178Y tumor cell immunity test to measure the effect of the reaction product on cytotoxic lymphocyte (CTL) activity. Two doses of the compound, 10 and 100 µg per dose, respectively. and 15 buffers alone are added in 1Q βΐ volumes to MLTC microculture dishes containing adult splenic lymphocytes from the DBA / -2 mice, nerve end organ (E) and L 5178Y target cells (T) in various E / T ratios . The MLTC reaction is carried out in the presence or absence of irradiated L 5178Y stimulator cells.

Table I-A.

Effect of the reaction product on cytotoxic lymphocyte activity with TDS spleen lymphocytes from DBA / 2 mice, measured by MTLC assay using effector cells co-cultured with stimulator cells.

% Light E / T Ratio 30 -

Reaction product, wife. 50: 1 25: 1 12.5: 1, 6.25: 1 100 Mg / ml 71.6 79.8 75.7 56.3 35 10 / Ltg / ml 75.7 72.0 67.1 60 , 5 µg / ml 63.9 61.9 45.0 35.5 5

DK 160828 B

Table I-B.

Testing as in Table I-A, but using effector cells without stimulator cells.

5% Light E / T ratio

Reaction product, wife. 50: 1 25: 1 12.5: 1 6.25: 1 10 -: - 100 µg / ml 21.8 16.6 11.1 6.1 10 µg / r & l 12.8 8.1 6.9 0 , 7 0 µg / ml -7.5 -3.0 -3.5 -1.3 15

The reaction product investigated causes a significant improvement in cytotoxic lymphocyte activity, expressed as% lysis at several different concentrations, regardless of the E / T ratios used and whether or not unstimulated cells are present.

Experiment 3.

The effect of the reaction product on the incidence of L 5178Y latent tumor stage (TDS) is investigated. 40 DBA / 2 mice, 25 of which have artificially induced a latent tumor state, are treated with the reaction product. Two days after interperitoneal administration of 50,000 viable L5178Y tumor cells, 20 of the mice on 7 consecutive days are treated interperitoneally with the reaction product (100 mg per kg) and the remaining 30 mice are treated with buffer alone. Twenty-five days after administration of the last dose of the reaction product, a peritoneal partial wash is performed and the peritoneal exudate flow «is erased to determine the number of tumor cells (quantitative testing). The survival time is noted 90 days after the treatment with viable L 5178Y tumor cells. Mortality rates are calculated for each group.

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Table II — A.

Distribution range for tumor cells in TDS-DBA / 2 mice, as measured by tumor cell quantity measurement.

5 Control group Treated group%%

Number of Tumor Cells Total Frequency Total Frequency Total 0 1 6 4 22 10. 1000 6 35 6 33 1000-100,000 8 47 7 39 100,000 2 12 16 15 Table II-B.

Mortality statistics for TDS-DBA / 2 mice after 50 days.

Compound, Number% 20 concentration survivor survivor 100 mg / kg 10 55.6 0 mg / kg 3 17/6 25

In animals treated with the compound (the reaction product), a significant reduction in the number of tumors relative to the control animals can be observed. The treated group shows more mice with fewer tumor cells in the peritoneal 30 washes. Also, the chances of survival for the treated mice are significantly greater than for the untreated control animals.

Experiment 4.

Mice of the BDF-β strain infected with L-1210-leu-35 germ cells are treated for 7 days with 25, 50, 100, 200 and 400 mg / kg of the reaction product as well as with buffer alone.

Thereafter, work is done according to a regulation prepared by the National Cancer Institute to investigate new anti-cancer agents and the T / C ratios are calculated.

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Table III.

Effect of the reaction product in the treatment of L-1210 leukemia, measured by T / C ratio 5 in mice of the BDF ^ strain.

Compound, concentration Average

(mg / kg) survival time (days) T / C

10 - 400 25 1.25 200 30 1.50 100 30 1.50 50 21 1.05 15 25 20 1.00 0 20

Proposal 5 «20 Mice of the BDFi strain are infected with L-1210 leukemia cells and treated for 7 days with 100 mg of the reaction product per ml. with 20 mg of the known anti-cancer agent 5-fluoro-uracil (5-FU) per kg and with buffer alone. The National Cancer Institute's method for investigating new 25 anti-cancer agents is being developed and T / C ratios calculated.

Table IV.

Efficacy of the reaction product and of 5-FU in the treatment of L-1210 leukemia, measured by the 30 T / C ratios of BDFi strain mice.

Concentration Average Drug (mg / kg) survival time T / C

Reaction product 100 28 1.40 5-FU 20 30 1.50

Buffer - 20

DK 160828 B

p

A dose of 100 mg / kg of the reaction product gives a T / C value of lf40 compared to 1.50 for 5-FU, ie. a positive result. This underlines the positive results of Experiment 4 and shows the anti-cancer effect of the reaction-5 product compared to the action of a known and accepted agent, namely 5-FU.

10 15 20 25 30 35

Claims (1)

DK 160828 B Patent claims. Process for the preparation of a reaction product of 2-methyl-2,5-dimethoxy-2,5-dihydrofuran and L-ascorbic acid of the formula HH HH v / oho Jrij! 10 o ^ ΟΗ HOCH h CH 2 OH and with Rf values of 0.65 and 0.3 on silica gel thin-layer chromatography plates using a solvent mixture of chloroform and methanol in a ratio of 9: 1, characterized by reacting 2-methyl-2,5-dimethoxy-methanol. 2,5-dihydro-furan of formula H3c ..... OCH3 HgCO * \ 0 ^ '* -H with L-ascorbic acid of formula HO OH OT ^ · 0' ^^ CHOH-CH ^ H in an aqueous medium in a nitrogen atmosphere.