DK145857B - ANALOGY PROCEDURE FOR PREPARING DI-3,4,5-TRIMETHOXYBENZOIC ACID ESTATE OF PYRIDINE-2,6-DIMETHANOL - Google Patents

Description

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DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Application No. 1376/74 (51) IntCI.3 C 07 D 213/30 (22) Filing Day 13 · Mar. 1974 (24) Race day 13 · Mar. 1974 (41) Aim. available 15 · S ep. 1974 (44) Presented 21. March 1983 (86) International application no.

(86) International filing day (85) Continuation day - (62) Master application no. -

(30) Priority l4. March 1973, 412609, ES

(71) Applicant INSTINTERSA INSTITUTO INTERNACIONAL THERAPEUTICO S.A., Bar »celona, ES.

(72) Inventor José Prous Ciochs, ES.

(74) Associate Engineering Company Budde, Schou & Co.

(54) Analogous procedure for the preparation of di-3,4,5-trimethoxybenzoate = the acid ester of pyridine-2,6-dimeth = hanol.

The present invention relates to an analogous process for the preparation of the di-3,4,5-trimethoxybenzoic acid ester of pyridine-2,6-dimethanol of the formula

Hoc ° | In the OCH- ^ h3co - // ^ -cooci ^ ch2ooc- / \\ - och3 -OH, CO ^ OCH, J · 3, which process is characterized by reacting 3,4,5-trimethoxy-benzoic acid chloride with pyridine -2,6-Dimethanol according to the following reaction scheme: H3 CO0 H, CO - / - COCl + I fl - »3> = /, ΑνΑ H3CO / H0H2C ^ 2 ° 11 H, CO OCH- --► \ - In a inert organic solvent in the presence of a tertiary amine which acts as an acid acceptor for the hydrochloric acid formed during the reaction. the crude product is separated by pouring the reaction mass onto water and recrystallized from water miscible solvents.

The starting material 3,4,5-trimethoxybenzoic acid chloride is prepared by reacting the corresponding acid with an excess of thionyl chloride. The acid chloride produced can be used directly in the raw state and does not require purification by distillation.

For this reaction, 3 moles of 3,4,5-trimethoxybenzoic acid chloride are used per liter. mole of pyridine-2,6-dimethanol.

As tertiary amine, e.g. triethylamine, pyridine and tri-n-butylamine are used. Good results are obtained using pyridine, as it simultaneously acts as a solvent and binds hydrochloric acid. Pyridine is used in an amount of between 9 and 10.8 liters per liter. kg of pyridine-2,6-dimethanol.

In the process of the present invention, the reaction is preferably carried out by mixing the pyridine-2,6-dimethanol solution with the acid chloride at ambient temperature, whereupon the reaction mass is heated to ca. 80 ° C by means of a hot bath for the time needed to complete the reaction, usually between 2 and 3 hours. When the reaction is over, water is added. The reaction mass is then filtered and the crude product is collected on the filter and recrystallized. In this way, a reaction is obtained which varies between 60 and 68%.

In order to obtain a good-quality ester, it is important to rinse the crude reaction product with water to remove the colored impurities usually produced by the reaction, which are difficult to remove later by recrystallization, even using activated carbon .

Finally, the recrystallization of the crude product is carried out in various types of solvents, with methanol, ethanol and isopropanol being preferably used in this operation, where it is considered that the solubility of the product increases with the temperature from methanol to isopropanol.

The structure of the present diester has been confirmed by examination of its NMR and IR spectra.

Closely related pyridine methanol esters are known from Danish Patent Nos. 69,137 and 116,942, United States Patent Nos. 2,524,838 and DE Publication No. 1,645,918. These known pyrdine methanol esters are vasodilatorally effective.

The di-3,4,5-trimethoxybenzoic acid ester of pyridine-2,6-dimethanol, on the other hand, has been found to have an unexpected, potent hypolipidemic effect, together with a decreasing effect on increased platelet aggregation in both test animals and humans. The hypolipidemic effect is evidenced by a specific lowering of low-density lipoproteins. These therapeutic effects are demonstrated as described below.

The wide use of rabbits in experimentally induced atherosclerosis has provided the basis for comparative studies, cf. P. Constantinides, J. Booth, and G. Carlson: Production of advanced cholesterol atherosclerosis in the rabbits, Arch. Path., 70. (I960), pp. 712? GL Duff and G.C. MacMillan: The effect of alloxan diabetes on experimental cholesterol atherosclerosis in the rabbits, J. Exp. Med., 89 ^ (1949), p. 611 * D. Kritchevsky, J.L. Moynihan, J. Langan, S.A. Carpets and M.L. Sachs: Effect of D- and L-thyroxine and of D- and L-3,5,5'-triiodothyronine on development and regression of experimental atherosclerosis in rabbits * J · Atheroschler. Res., 1 (1961), pp. 211 ^ and M.R. Parwarish, Ch. Mader, W. Hill and K. Wiswedel: A Combined Procedure for Producing Arteriosclerosis in Rabbits in Short-Time Trials,

Res. Exp. Med., 160 (1973), p. 269.

Rabbits maintained on an atherogenic diet develop hyperlipidemia with elevation of β-zone lipoproteins (d <1.063 g / ml) and decrease of α-lipoproteins, cf. L. Mettler and M.R. Parvary: Morphological and Biochemical Investigations Concerning the Preventive Effect of Estrogen (Oestradiol) on a Lipid Induced 4 145857

Arteriosclerosis, Geburtsh.Frauenheilk., 2 (1972), p. 426. the surrounding soft tissue (cf. P. Constantinides et al. and D. Kritschevsky et al. above).

The hypolipidemic and antiatherogenic effect of the di-3,4,5-trimethoxybenzoic acid ester of pyridine-2,6-dimethanol (MBP) is carried out as follows: 145 male white bred New Zealand rabbits with a age of 6 months and a weight of 3212 - 480 g is divided into 3 groups. The compound is used suspended at a concentration of 75 g / liter in 30% ethanol. The control groups (1 and 2) are administered daily 1 ml of 30% ethanol. Every four weeks, blood samples are taken from ear veins for cholesterol determination. A complete analysis of lipid and lipo proteins is performed only at the end of the experiment.

After 12 weeks, the rabbits are bled and killed, and all animals are subjected to a thorough dissection. The aortas are removed from the surrounding soft tissue. Samples of the coronary arteries are immediately taken subostially at the beginning of the three main branches and histological specimen vials are prepared in the usual manner. The aortas are photographed and the affected surface area is measured planimetrically.

Similarly, the stenotic surface area of the coronary lumen is determined planimetrically using a scaled Zeiss microscope. The aortas are frozen in dry ice and acetone, and an acetone powder is prepared and weighed and suspended in 2 ml of chloroform / methanol in 2: 1 v / v.

The lipids are extracted as described by J. Folch, M.Lees and G.H. Stanley: A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1975), p. 497, and the amount of cholesterol in these extracts is determined. Total cholesterol (TCh), esterified cholesterol (ECh) and free cholesterol (Ch) are measured using the enzymatic method of P. Eøcklau, Ξ. Bernt and W. Gruber: Enzymatic Determination of Gesamb-Cholesterins im Serum, Z. Klin. Chem. Klin. Biochem., 12 (1974), p. 403. Serum lipoproteins (EP) are separated by density gradient ultracentrifugation, cf. F.T. Hatsch and R.S. Read: Practical methods for plasma lipoprotein analysis,

Adv. Lipid Res., (1968), p. 1. LP with d <1.063 g / ml is designated low-density LP (LDL) or β-zone LP after chylomicrons c 145857

D

and VLDL (very low density LP) were removed using a density gradient of d = 1.006 g / ml. LPs with d> · 1.063 g / ml are referred to as high-density LP (HDL) or α-zone LP. The purity of LP fractions is investigated by polyacrylamide disk electrophoresis of pre-stained LP fractions, cf. M.R. Parwaresch, H.J. Radzon and Ch. Ways: Diagnosis of Primary Hypolipoproteinemia in Nabelschnur blut, Nachr. Children's Saints, 125 (1977). p. 865. For quantitative assessment, mean values are compared using Student's t-test for independent populations. The distribution of values is expressed at 95% upper and lower confidence limits.

Results.

The results of the experiment are listed in Table I. The mortality rate does not exceed 10% in any of the groups. Rabbits kept on normal feed (group 1) did not show any change in lipid values during: the experiment. The atherogenic diet in group 2 of the animals leads to a massive increase in serum cholesterol and LDL cholesterol with mean values of respectively. 958 and 939 mg / dl at the end of the experimental period. These animals develop severe atherosclerosis. On average, 57% of the aortic surface is affected. The planimetric studies of the coronary arteries show an atheromatous stenosis, which, on average, occupies 37% of the vessel's lumen. The intramural cholesterol content of the aorta is in good agreement with the extent of vascular lesions.

The group maintained on the MPB shows a significantly lower rate of atherosclerosis in aorta and coronary arteries than the control group 2. Cholesterol levels in the aorta and serum cholesterol levels are consistent with the vessel changes. Animals treated with MPB show cholesterol values of 481 mg / dl.

145857 6

Table I

Inhibitory effect of MPB on experimental hypercholesterolaemia and atherosclerosis in rabbits after a 3-week experimental period.

Percent affected surface area

Group No. Serum HDL-TCh / Aortic Aortic Coronary- Death- Treat- TCh LDL-TCh Aortic-ChECh Disclosure Similarity (ml / dl) Ratio TCh Ratio Flat _ χ NO 59 22/37 7 6.7 / 0.4 0 0 0/10 (50-68) 0.59 (5-9) 16.7 2D 958 19/939 85 31/53 57 37 3/29 (868-1049) 0.02 (76-93) ) 0.6 (51-62) (32-42) 3 D / MBP 481 87/394 46 24/21 30 13 2/30 (408-533) 0.22 (40-52) 1.19 (24- 23) (10-16) NR = normal feed. D = atherogenic diet. TCh = total cholesterol.

ECh = esterified cholesterol.

The values indicated are averages. The values in parentheses are 95% upper and lower confidence limits.

The results show the distinct cholesterol-lowering effect of HPB that inhibits the development of experimentally induced atherosclerosis in rabbits.

Effect of MPB on atherogenic risk factors in humans.

1. Effect on Platelet Aggregation.

2. Effect on lipids and lipoproteins in primary type II a hyperlipoproteinemia (hyperbetalipoproteinemia).

In rabbit experiments, MBP exhibits a clear anti-atherosclerotic effect. It has been thought that the antiatherosclerotic effect is due to a possible, hypolipemic effect. This hypothesis has been investigated as described below.

24 Patients (both sexes, mean age: 48 years) with an endogenous hyperbetalipoproteinemia classified as type IIa hyper-lipoproteinemia according to a WHO proposal are given a dose of 25 mg MBP / kg body weight per day. day for 42 days.

7 145857

The lipid and lipoprotein assay as well as the platelet aggregation test are performed before (0) and 7, 14, 21, 28 and 42 days of medical treatment. The same studies are performed 1, 2 and 3 weeks after replacing the medicinal product with placebo.

Blood samples are taken after 12 hours of fasting and subjected to a modified platelet aggregation test according to Breddin, which determines the percentage of aggregated platelets.

Serum samples are subjected to gravimetric determination of total lipid (TL) and enzymatic determination of free and fed cholesterol (Ch, ECh) and of triglycerides (TG). Furthermore, phospholipids (Ph) and non-esterified fatty acids (UFA) are determined by thin layer chromatography.

After ultracentrifugation separation of chylomicrons (d · 0.93) VLDL (d ^ l, 006), LDL (d-cl, 063) and HDL (d-1.12) by a gradient fractionation, the above lipids (TL , ECh,

Ch, Ph, TG and UFA) in the separated fractions.

results:

Blodpladeaggregationsforsøg:

The compound MBP shows a clear effect on the platelet aggregation tendency, where there is increased platelet aggregation. Table II summarizes the individual values during the drug treatment period, baseline values, and values obtained after 3 weeks of placebo administration. The differences assessed using a distribution test according to Wilcoxon (modified according to Mann and Withney) are significant at p- £ 0.05.

Table II

Aggregated platelets in% of initial values.

Placebo for 0 days 7 days 14 days 21 days 28 days 3 weeks 88 36 33 59 65 92 87 30 31 36 32 86 76 30 28 31 32 79 63 28 25 25 31 68 60 28 22 20 31 68 60 23 20 20 28 66 52 22 20 18 18 58 30 18 20 18 18 23 16 18 18 15 17 18 13 12 18 11 15 18 11 10 18 11 12 15 8 145857

Of the lipid values in Table III, LDL ester cholesterol is greatly elevated prior to medical treatment. Already in the first week of treatment, this value falls on average 14%.

It is evident from the test results that the MBP compound clearly inhibits increased platelet aggregation. The results are statistically significant.

The main effect of MBP on serum lipids and lipoproteins is a lowering effect on elevated LDL-ECh values. The results are statistically significant.

The results prove that the compound has a useful anti-atherogenic effect, which is mainly due to its action against the two main factors responsible for thrombotic complications.

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The process of the invention is illustrated in the following examples.

Example 1.

15 kg (70.7 mole) of 5,4,5-trimethoxybenzoic acid and 65 liters of benzene are charged to a reactor and 27.4 liters of thionyl chloride are added to this mixture. The mass is heated to 56-70 ° C for 5 hours. The excess benzene and thionyl chloride are distilled off under vacuum. The residue is kept under vacuum at 120-123 ° C for 1 hour to give a hard crystalline solid.

A solution of 3.24 kg (23.3 mol) of pyridine-2,6-dimethanol in 35 liters of pure pyridine is added to the residue and the mass is heated to 80 ° C for 2.5 hours. The reaction mass is a brown color. The pyridine hydrochloride thus formed is cooled and crystallized. Then, the resulting reaction mass is poured into water and the resulting precipitate is filtered off and then repeatedly rinsed with water. Finally, it is dissolved in 400 liters of methanol and the resulting solution is filtered with activated carbon. From this filtrate, 50 liters of methanol are distilled off at atmospheric pressure and crystallized. 8.35 kg (15.8 moles) of the di-3,4,5-trimethoxybenzoic acid ester of pyridine-2,6-dimethanol are obtained, corresponding to a 68% yield.

The product is a white crystalline solid which melts at 119-126 ° C. After recrystallization from methanol, a product is obtained which melts at 126-127 ° C. An elemental analysis of this gives the following results: C = 6l, 24 $, H = 5.46 $, N = 2.82 $, while the calculated values for formula ^ 27Η29Ν010 are: C = 6l, 47 $, H = 5.54 $, N = $ 2.65. IR spectrum: The ester structure is confirmed by a C = O absorption band at 1720 cm @ + and strong absorption by C-O-C. The band at 760 cm 2 is added to the 3 hydrogen atoms on the pyridine ring.

NMR Spectrum: (CDCl3, TMS): 3.94T singlet -OCHy 5.52 Τ 'singlet -OCH₂ 8.00 - 7.27T multiplied aromatic protons.

Example 2.

15 kg (70.7 mol) of 3,4,5-trimethoxybenzoic acid and 65 liters of benzene are charged to a reactor and then 27.4 liters of thionyl chloride are added. The mass is heated to 65-70 ° C for 5 hours. The excess benzene and thionyl chloride are evaporated under vacuum. The residue is heated under vacuum for 1 hour at 120-123 ° C to give a hard crystalline solid.

The resulting residue is dissolved in 60 liters of benzene, to which is added 3.24 kg (23.3 moles) of pyridine-2,6-methanol and 8 liters of pyridine. The mass is heated at reflux for 5 hours. The solvents are then removed by distillation under vacuum and the residue is poured into water. The resulting precipitate is filtered off and rinsed repeatedly with water, then dissolved in 400 liters of methanol and the resulting solution is filtered with activated charcoal (charcoal).

For the filtrate, 50 liters of methanol are equilibrated at atmospheric pressure and crystallized. In this way, 8.98 kg (17 moles) of the di-3,4,5-trimethoxybenzoic acid ester of pyridine-2,6-dimethanol is obtained, corresponding to a yield of 73%. After recrystallization from methanol, a product having a melting point of 126--127 ° C is obtained. Elemental analysis gives the following results: C = 61.24 $, H = 5.46 $, N = 2.82 $ against the calculated values of formula C27H29N010: C = 61.47 $, H = 5.54 $, N = 2,65 $.