FI71300C - FOERFARANDE FOER FRAMSTAELLNING AV SALTER OCH ESTRAR AV SYRAN SOM SVARAR MOT MONACOLIN-K VILKA HAR ANTIHYPERKOLESTEREMISK EFFEKT - Google Patents

Description

TD1 ANNOUNCEMENT

WgSgl tBJ (11) UTLÄGGNIN GSSKRIFT 7130 0 C («) 1 ::: j: c 7/51 \ Kv Ik Vint Cl 4 C ° 7 C 59 / 1,6» 69/30 (51) Kv.lk. /1nt.CI. uc 07 0 309 / 3o ^ c 12 P 7 / Q6 FINLAND — FINLAND (21) Patent application - Patentansökning 802357 (22) Application date - Ansökningsdag 2 5.0 7.8 0 (23) Starting date - Gilcighetsdag 25.07.80 (41) Publication - Blivit offentlig 28 Oj 8l

National Board of Patents and Registration ..... ... ,,,,,,, •, (AA) Date of launch and hearing publication—. Q.

Patent application and registration certificate issued in accordance with the Public Procurement Act "3 · 09. Oo (86) International application - Int. Ansökan (32) (33) (31) Privilege requested - Begärd priority 27.07.79 26.09.79 Japan -Japan (JP) 95814/79, 123461/79 (71) Sankyo Company Limited, 1-6, 3-chome, Nihonbashi Honcho, Chuo-ku, Tokyo, Japan-Japan (JP) (72) Yoshio Tsujita, Tokyo, Kazuhiko Tanzawa, Tokyo, Masao Kuroda, Tokyo, Seigo Iwado, Tokyo, Kiyoshi Hamano, Tokyo, Akira Terahara, Tokyo, Sadao Sato, Tokyo, Japan-Japan (JP) (74) Forssin 6 Salomaa Oy (5 * 0 Method against hypercholesteremia For the preparation of salts and esters of an acid corresponding to monacolin-K - For the preparation of salts and esters corresponding to monacolin-K, monocolin-K, a brittle antihypercholesterolemic effect

The present invention relates to a process for the preparation of salts and esters of an acid corresponding to Mona-Kolin-K against hypercholesterolemia.

Our patent application GB 8005748 discloses the compound Monacoline K and its preparation using microorganisms of the genus Monascus, in particular Monascus ruber strain 1005 (FERM 4822). This application discloses the valuable and unexpected efficacy of Monacoline K as an anti-hypercholesterolemic agent. Subsequently, our patent application GB 8007240 discloses the preparation of Monacoline K by culturing many other microorganisms of the genus Monascus. We have now found that salts and esters of the free acid of which the lactone Monacolin K is have similar antihypercholesterol activity to Monacoline K, but to the same or greater extent. For convenience, these compounds are hereinafter referred to as "salts or esters of monacoline K" and this term is to be understood as meaning salts and esters of the acid of which the lactone Monacoline K is present.

2 71300

The salts and esters of Monacol K provided by the present invention have the formula: HO.

x ^ ^ COO (R) n k / ° H o li

OF

wherein: R is sodium, calcium, a lower alkyl group or a benzyl group; and n is the inverse of the valence of the group or atom represented by R.

Salts of monacolin K can be easily converted to Monacollin K or its corresponding acid by acid treatment. The Monacollin K or acid thus obtained returns to the salt in the presence of an alkali, e.g. alkali metal hydroxide or carbonate.

This step can be performed quantitatively and reproducibly. We have found that changes between Monacollini K or its equivalent acid and metal salt are in close association with the pH of the medium. The critical pH is about 5.0. Provided that metal ions are available, Monakollini K is always in salt form at pH above 5.0. On the other hand, when the pH is below 5.0, Monacollin K itself, the corresponding acid or a mixture of the two is present in varying proportions.

The esters of Monacollin K provided by the present invention are compounds of the above formula wherein R represents a substituted or unsubstituted lower alkyl group. When R represents an unsubstituted alkyl group of 3,713,000, preferably having up to 8 carbon atoms, examples of such alkyl groups include methyl, ethyl, propyl, isopropyl, butyl and hexyl groups.

Of the esters, methyl, ethyl, butyl and benzyl esters are particularly preferred.

The salts and esters of Monacollin K of the present invention can be readily prepared by salt formation or esterification. From Monakolllni K itself or from a reactive derivative of Monakolllni K. Examples of suitable reactive derivatives are the free acid corresponding to Monacollin K and, in the preparation of esters, the salts of Monacollin K (e.g. the salt groups described above, in particular sodium, potassium, calcium, magnesium, aluminum, iron, zinc, copper , nickel or cobalt salt).

The salts of Monacollin K can be prepared by a simple reaction of Monacollin K or a corresponding free acid with an oxide, hydroxide, carbonate or bicarbonate of a selected metal, preferably a hydroxide or carbonate. It must be ensured that the reaction is carried out at a pH above 5.0 and preferably above 7.0 in order to guarantee complete conversion of Monacollin K or acid to the desired salt. The Monacoline K used in this reaction is preferably prepared, as described in our aforementioned patent application, by culturing a fungus belonging to the genus Monascus and isolating Monacoline K from the culture medium. Monacoline K may be isolated from the culture medium and purified prior to salt formation, or more preferably, salt formation is accomplished during isolation and purification of Monacoline K from the culture medium so that Monacoline K is isolated as its desired metal salt.

Whichever method is chosen, the metal salt can be isolated from the reaction medium or culture medium by methods well known in the art, including those described below in connection with isolating metal salts of Monacoline K from cultures of fungi producing salts of Monacoline K.

Esters of monacoline K can be prepared by a simple ester bin of Monacoline K or a reactive derivative thereof. The reaction can be carried out by reacting an alcohol of formula RÖH (or a reactive derivative thereof) wherein R represents a lower alkyl group with Monacoline K or a corresponding free acid, preferably in the presence of a water-binding agent, e.g. an acid halide (such as acetyl chloride). Alternatively, esters of Monacoline K can be prepared by reacting a salt of Monacoline K with a halide of formula RX (wherein R represents a lower alkyl group and X represents a halogen atom, preferably an iodine atom).

The process according to the invention is thus characterized in that the salts and the ester of the acid corresponding to Monacoline-K have the formula according to page 2, in which R is sodium, calcium, a lower alkyl group or a benzyl group; and n is the inverse of the valence of the atom or group represented by R is prepared by forming a salt or ester of the free acid corresponding to Monacoline-K or a reactive derivative thereof by reaction of one of the latter with a compound of formula ROH, wherein R is as defined above; or with an oxide, carbonate or bicarbonate of R, wherein R is sodium or calcium; or by reacting a reactive derivative of Monacollin-K with a compound of formula RX wherein R is a lower alkyl or benzyl group and X is a halogen atom.

The physiological efficacy of the salts and esters of Monacoline K can be studied and quantified by the following experimental methods, which can also be used in modification to monitor the production of salts of Monacoline K during the fermentation process of the present invention.

1. Inhibition of cholesterol biosynthesis

Like monacoline K, its salts and esters specifically inhibit 3-hydroxy-3-methyl-glutaryl-CoA reductase, a rate-controlling enzyme in cholesterol biosynthesis. The following Table 1 gives the concentrations (ng / ml) of the compounds of the invention which inhibit 3-hydroxy-3-methyl-glutaryl (HMG) -CoA reductase by 50% / measured by the method described in Analytical Biochemistry, 31, 383 (1969). ) and the concentrations (ng / ml) of the compounds of the invention which inhibit cholesterol biosynthesis by 50% as measured by the method described in Journal of Biological Chmistry, 247, 4914 (1972). Similar results have also been given for the known compound ML-236B (which compound is known to have a similar potency and is produced by culturing microorganisms of the genus Penicllllum, as disclosed in our patent publication GB 1 453 425). In addition, the concentration of Monacoline K itself, which inhibits cholesterol biosynthesis by 50%, has been given. It is observed that the required concentration of ML-236B for a 50% inhibition of cholesterol biosynthesis is 10.0 ng / ml, while the corresponding concentration for monacolin K esters is of the order of 1 ng / ml (i.e. about 10 times better) and the corresponding the concentration of the sodium salt of Monacoline K is about 0.14 ng / ml (i.e., about 70-fold better). Salts and esters of Monacoline K are comparable in potency to Monacoline K itself or better.

Table 1 ^ 50% concentration required for inhibition (Ng / ml)

Compound HMG-CoA Cholesterol Reductase Biosynthesis Methyl Ester 70.0 1.2 Ethyl Ester 12.0 1.3 Butyl Ester 15.0 2.0 Benzyl Ester 11.0 1.1 Sodium Salt 2.0 0.14 Calcium Salt 12.0 1.8

Monacoline K - 2.0 ML-236B 10.0 10.2 2. Decrease in blood cholesterol levels The animals used in this experiment were rats of the Wistar Imamichi strain, each weighing about 300 g. The experiments were performed on rat rhymes, each comprising 5 animals. Each animal was injected intravenously with 400 mg / kg of Triton WR-1339 (trade name for a substance known to increase blood cholesterol) and at the same time orally administered one of the compounds listed in Table 6 71300 2 in the amount indicated in the table. After 20 hours of oral drug administration, rats were sacrificed by blood counting, blood and liver were collected, and their cholesterol levels were determined by conventional means. The results are shown in Table 2, which shows a decrease in blood and liver cholesterol levels compared to a control group of rats given Triton WR-1339 alone.

For comparison, similar results have been reported for Monacoline K and ML-236B, but these compounds were administered at substantially higher doses than the compounds of the invention to provide a comparable reduction in cholesterol levels.

Table 2

Dose Decrease in cholesterol (%)

Compound mg / kg in blood liver methyl ester 5 23.8 18.4 ethyl ester 5 23.3 18.0 butyl ester 5 19.5 15.7 benzyl ester 5 24.4 18.5 sodium salt 2 27.5 18.9 calcium salt 5 21, 4 17.1

Monacoline K 10 22.4 16.7 ML-236B 40 24.6 20.9

The reduction in blood or liver cholesterol was calculated from the formula: 1 - (C - B) --x 100

A - B

where: A * concentration in Triton WR-1339 treatment alone in the group; B = concentration in the untreated control group; C = concentration in the experimental group.

1 71300 3. Acute toxicity

The compounds studied were the methyl, ethyl, butyl and benzyl esters of Monacoline K, as well as the sodium and calcium salts. It was found that all of these compounds had a 50% lethal dose (LD, _q) of at least 2000 mg / kg orally and at least 500 mg / kg intraperitoneally. The compounds thus have very low toxicity.

These results indicate that the compounds of the invention inhibit cholesterol biosynthesis and thereby lower blood cholesterol levels. They are thus valuable drugs in the treatment of hyperlipemia and atherosclerosis.

The compounds of the invention may be administered orally or parenterally in the form of a capsule, tablet, injectable preparation or any other known composition, although it is usually preferred to administer them orally. The dosage will vary depending on the age and weight of the patient and the severity of the condition, but in general the daily dose for the onset is preferably 0.1 to 100 mg, more preferably 0.1 to 10 mg, using salts of Monacoline K and 0.5 to 100 mg, more preferably 0, 5-10 mg, using esters of Monacoline K.

The preparation of the compounds of the invention is further illustrated by reference to the following examples.

Example 1

Sodium salt of monacoline K 300 liters of culture medium, pH 5.5 before sterilization, containing 5% by weight glucose, 0,5% by weight of malt soaking water, 2% by weight of peptone (quality Kyokuto, available from Kyokuto Seiyaku KK, Japan) and 0.5 wt% ammonium chloride were charged to a 600 liter fermentor and seeded with the microorganism Monascus ruber SANK 18174 (Ferm. 4957). The organism was cultured for 116 hours at 26 ° C with an aeration power of 300 liters per minute and stirring at 190 rpm. The pH of the culture solution containing the organism was then adjusted to 3.4 by adding 6-N hydrochloric acid, and then the solution was extracted with 800 liters of methanol.

8 71300

6 kg of Hyflo Super Cel was added and the extract was filtered using a filter press to give 1100 liters of methanol extract. This extract was washed with 200 liters of saturated aqueous sodium chloride solution and then with 180 liters of ethylcyclohexane. The resulting solution was extracted with 600 lirts of ethylene dichloride. To the ethylene dichloride extract was added 50 g of trifluoroacetic acid and allowed to react at 80 ° C for 30 minutes. The reaction mixture was then washed successively with 200 liters of a 2% by weight aqueous solution of sodium bicarbonate and 200 liters of a 10% by weight aqueous solution of sodium chloride. The mixture was then concentrated by evaporation under reduced pressure to give 135 g of an oily substance.

This oily substance was dissolved in 400 ml of methanol. 20 ml of the resulting methanol solution (containing 6.8 g of oil) was subjected to preparative flash liquid chromatography using Waters Co. Limited System 500 equipped with a Prepac C ^ g column (counter-phase column). An 85:15 methanol / water mixture (v / v) was used as developer. The development was performed at a flow rate of 200 ml / min (development time about 10 minutes), while monitoring the differential refractometer connected to the body, and the fraction which gave the main peak on the differential refractometer was separated. This procedure was repeated and the resulting main peak fractions were collected and concentrated to give 10.2 g of an oily substance. This oily substance was dissolved in 30 ml of methanol and 6 ml of a methanol solution (containing about 2 g of oil) was again treated with the same preparative flash liquid chromatography and developed with an 80:20 methanol / water mixture (v / v) at a flow rate of 200 ml / min. The fraction giving the main peak was separated. This procedure was repeated and the fractions corresponding to the main peak were collected and concentrated. The residue was treated with aqueous methanol to give 1170 mg of crude crystals, which were recrystallized several times from ethanol to give 864 mg of crystals.

To these crystals, 19.4 ml of 0.1 N aqueous sodium hydroxide solution was added, and the mixture was stirred at 50-60 ° C for 3 hours. The insoluble matter was removed by filtration, and the filtrate was lyophilized to give 900 mg of the sodium salt of Monacoline K, which had the following properties: 1. Color and shape: white powder 9,71300 2. Elemental analysis: calculated: C, 64.84%; H, 8.38 Z; Na, 5.17 Z Found: C, 64.78%; H, 8.55 Z; Na, 5.21 Z.

3. Molecular weight: 444 [mass spectrometry (F.D. mass) J.

4. Gross formula: C24H37 ° 6Na 5. UV absorption spectrum:

According to the attached Figure 1, λ max 232 nm (log * 4.30); 239 nm (log = »4.36); 248 nm (log * 4.19).

6. IR absorption spectrum (in KBr tablet):

According to the attached Figure 2.

7. NMR spectrum (DO)

According to the attached Figure 3.

8. Flash liquid chromatography:

Dwell time: 8.5 minutes;

Column, Microbondapac C18; 60% v / v methanol / water + 0.1% PIC-A (product of Waters Co. Limited), flow rate, 1.5 ml / min According to the attached Figure 4.

9. Solubility:

Soluble in water; insoluble in organic solvents.

10. Specific rotation: = + 266 ° (c = 0.33, in water).

10 71 300

Example 2

Sodium salt of monacoline K 300 liters of culture medium, pH 7.4 before sterilization, containing 1.5% by weight of soluble starch, 1.5% by weight of glycerol, 2.0% by weight of fishmeal and 0.2% by weight of % calcium carbonate, was charged to a 600 liter fermentor and seeded with the microorganism Monascus ruber SANK 17075 (CBS 832.70). The organism was cultured for 120 hours at 26 ° C with an aeration power of 300 liters per minute and stirring at 190 rpm. The culture broth was then filtered on a filter press to give 35 kg (wet weight) of the organism.

To this was added 100 liters of water, and the pH of the mixture was adjusted to 12 by adding sodium hydroxide with stirring. The mixture was allowed to stand at room temperature for 1 hour, after which 2 kg of Hyflo Super Cel was added and the mixture was filtered on a filter press. The pH of the filtrate was adjusted to 10 by the addition of hydrochloric acid, and the resulting mixture was then adsorbed onto a column containing 5 liters of HP-20 resin washed with 15 liters of water and 15 liters of a 10% v / v aqueous solution of methanol. The column was then eluted with 10% v / v aqueous methanol. The eluate was concentrated to about 10 liters and then its pH was adjusted to 2 by adding hydrochloric acid, and the mixture was extracted with ethyl acetate.

The extract was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and then concentrated by evaporation to dryness to give 50 g of an oily substance containing Monacoline K acid. To this oily substance was added methanol to a total volume of 100 ml. 20 ml of the solution thus obtained was subjected to preparative flash liquid chromatography using a counter-column (as described in Example 1) eluting with a 20% v / v aqueous solution of methanol (containing 2% acetic acid) at a flow rate of 200 ml / min. The fraction which was separated on the antoln main peak by differential refractometer was separated (7-10 minutes). The remaining 80 ml of the methanol solution was then treated by the same method. The resulting fractions corresponding to the main peak were collected, concentrated and extracted with ethyl acetate. The extract was evaporated to dryness after the addition of heptane to give 1.2 g of an oily substance.

This oily substance was dissolved in 20 ml of methanol and subjected to flash liquid chromatography as described above to give 150 mg of Monacoline K acid. To this were added 2 ml of methanol and 100 ml of water, and the resulting solution of n 71300 was adjusted to pH 8.0 by adding 1-N aqueous sodium hydroxide solution to give a clear aqueous solution. This solution was passed through a column containing 10 ml of HP-20 resin and then the column was washed with 10 ml of water and eluted with methanol containing 20% by volume of water. The eluate was lyophilized to give 130 mg of the sodium salt of Monacoline K as a white powder. The properties of the product were identical to those reported for the product of Example 1.

Example 3

Calcium salt of monacoline K.

The culture, extraction, concentration, preparative flash liquid chromatography and recrystallization from ethanol described in Example 1 were repeated. 500 mg of the crystals thus obtained were then dissolved in 50 ml of methylene chloride, and the resulting solution was filtered through a Millipore filter. To the filtrate was added 20 ml of saturated aqueous calcium hydroxide solution, and then the mixture was stirred vigorously at room temperature. When the pH of the solution dropped below 8, a 10 ml portion of saturated aqueous calcium hydroxide solution was added and stirring was continued. When the pH no longer dropped (a total of 50 ml of aqueous calcium hydroxide solution was dissolved), distilled water was added and the whole solution was transferred to a separatory funnel. The organic phase was collected and the solvent was removed by distillation. The residue was treated with heptane and then sonicated to give a powder. The powder was rotated by filtration and dried to give 450 mg of the calcium salt of Monacolin-1-K as a white powder.

This calcium salt had the following properties: 1. Molecular weight: 882 (mass spectrometry).

2. Gross formula: (C2 & 7 ° t> 2 C ’3. Melting point: 155-165 ° C (decomposes).

i2 71 3 0 0 4. Specific rotation: = + 209 ° (c = 1.48, in chloroform).

5. IR absorption spectrum (KBr):

According to the attached Figure 5.

6. NMR spectrum (CD 1 OD):

According to the attached Figure 6.

Example 4

Monocolyl K methyl ester

The process steps described in Example 1: culture, extraction, concentration, preparative flash liquid chromatography, and recrystallization from ethanol were repeated to give 864 mg of Monacolil K. 200 mg of this Monacolil K was dissolved in 20 ml of methanol (previously dehydrated on a 3A molecular sieve). After a few drops of acetyl chloride were added, the solution was stirred at room temperature for 3 hours. The solvent was removed by distillation, and the residue was extracted with 50 ml of ethyl acetate. The extract was washed with 2% by weight aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution, and then dried over anhydrous sodium sulfate, after which the solvent was removed by distillation. The residue was adsorbed onto a column containing 10 g of silica gel (Wakogel C-100) previously treated with benzene. The fractions eluted with a 6:94 mixture of ethyl acetate and benzene (v / v) were collected and the solvent was removed by distillation to give 65 mg of Monacolyl K methyl ester as a colorless oil.

This product had the following properties: 1. Molecular weight: 436.6 (mass spectrometrically).

2. Gross formula: C25H40 ° 6 13 71300 3. Specific rotation: λ ς C? * 1 jj = + 208 ° (c - 1.05, in methanol).

4. IR absorption spectrum:

According to the attached Figure 7.

5. NMR spectrum:

According to the attached Figure 8.

Example 5

Methyl ester of monacoline K

The sodium salt of monacoline K was prepared as described in Example 2.

100 mg of this sodium salt was then dissolved in 2 ml of dimethyl sulfoxide, and then 50 methyl iodide was added to the solution. The solution was heated to reflux with stirring at 40-50 ° C for 5 hours in a refluxed reactor. The reaction mixture was then diluted with 5 ml of water and extracted with 10 ml of methylene chloride. The solvent was removed by distillation from the extract, and the residue was adsorbed onto a column containing 10 g of silica gel (Wakogel C-100) previously treated with benzene. The fractions eluting with a 4:96 (v / v) mixture of ethyl acetate and benzene were collected and the solvent was removed by distillation to give 35 mg of Monacoline K methyl ester as an oil.

Example 6

Ethyl ester of monacoline K 100 mg of monacoline K (prepared as in Example 1) was dissolved in 15 ml of ethanol previously dehydrated with a 3A molecular sieve, and a few drops of acetyl chloride were added to the solution. The mixture was then stirred at room temperature for 3 hours, after which the solvent was removed by distillation, and the residue was extracted with 50 ml of ethyl acetate. The extract was washed with 2% by weight aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution, and then dried over anhydrous sodium sulfate. The solvent was removed by distillation on a dehydrated column containing 10 g of silica gel (Wakogel C-100) previously treated with benzene. The fractions eluting with a 6:94 (v / v) mixture of ethyl acetate and benzene were collected and the solvent was removed by distillation to give 30 mg of ethyl ester of Monacoline K as a colorless oil.

The properties of this compound were as follows: 1. Molecular weight: 450.6 (mass spectrometry).

2. Gross formula: C26H42 ° 6 3. IR absorption spectrum (CHCl3):

According to the attached Figure 9.

4. NMR spectrum (CDCl 3):

According to the attached Figure 10.

Example 7

Butyl ester of monacoline K 200 mg of monacoline K (prepared as in Example 1) was dissolved in 20 ml of butanol previously dehydrated with a 3A molecular sieve.

After a few drops of acetyl chloride were added to the solution, the resulting mixture was stirred at room temperature for 2 hours. The solvent was then removed by distillation, and the residue was extracted with 50 ml of ethyl acetate. The extract was washed with 2% aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution. The solution was then dried over anhydrous sodium sulfate, and the solvent was removed by distillation. The residue was adsorbed onto a column containing 10 g of silica gel (Wakogel C-100) previously treated with benzene. The fractions eluting with a 4:96 (v / v) mixture of ethyl acetate and benzene were collected and the solvent was removed by distillation to give 80 mg of Monacoline K butyl ester as a colorless oil.

15 71 30 0 This compound had the following properties: 1. Molecular weight: 478.7 (mass spectrometry).

2. Gross formula: C28H46 ° 6 3. IR absorption spectrum (CHCl3).

According to the attached Figure 11.

4. NMR spectrum (CDCl 3):

According to the attached Figure 12.

Example 8

Benzyl ester of monacoline K 200 mg of monacoline K (prepared as in Example 1) was dissolved in 200 ml of benzyl alcohol previously dehydrated on a 3A Mole alkyl screen. After a few drops of acetyl chloride were added to the solution, the resulting mixture was stirred at room temperature for 3 hours. The solvent was then removed by distillation, and the residue was extracted with 50 ml of ethyl acetate. The extract was washed with 2% aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution. The solution was then dried over anhydrous sodium sulfate, and the solvent was removed by distillation. The residue was adsorbed onto a column containing 10 g of silica gel (Wakogel C-100) previously treated with benzene. The fractions eluting with a 4:96 (v / v) mixture of ethyl acetate and benzene were collected and the solvent was distilled off to give 70 mg of benzyl ester of Monacoline K as a colorless oil.

This compound had the following properties: 1. Molecular weight: 512.6 (mass septrometrically).

16 71 300 2. Gross formula: C31H44 ° 6 3. IR absorption spectrum (CHCl3):

According to the attached Figure 13.

4. NMR spectrum (CDCl 3):

According to the attached Figure 14.

Claims (1)

A process for the preparation of salts and esters of an acid corresponding to Monacoline-K against hypercholesteremia, characterized in that the above compounds of the formula: kaνγ / ^ οοο (Ρ) η o wherein: R is sodium, calcium, lower an alkyl group or a benzyl group; and n is the inverse of the valence of the atom or group represented by R, prepared by forming a salt or ester of the free acid corresponding to Monacoline-K or a reactive derivative thereof by reaction of one of the latter with a compound of formula RHH, wherein R is as defined above; or with an oxide, carbonate or bicarbonate of R, wherein R is sodium or calcium; or by reacting a reactive derivative of Monacoline-K with a compound of formula RX wherein R is a lower alkyl or benzyl group and X is a halogen atom.