IL21756A - Isovaleric acid ester sedative - Google Patents

Description

This invention relates to an isovaleric acid ester sedative and a process for its isolation.

Commercial preparations from the valerian root are not standardized «*r a definite active material; although they are used therapeutically as general sedatives with a very broad spectrum of action, their effect as a sedative on the central nervous system is disputed.

Non-aqueous sedatively active extracts from the roots and rhizomes of valerian contain, in addition to a number of hydrocarbons, ketones, carbonic acids and alcohols of the terpene series a complicated mixture of esters. If such a mixture is saponified in the usual way, then isovaleric acid is predominantly detectable as the acid component, whilst the corresponding alcohols are resinified. /©riginal investigations have shown that stimulating and sedatively acting substances are present in valerian.

The object of the present invention is the production of a previously unknown ester having a centrally sedative action according to a technically usable and reproducable procedure .

It has now been found that , although the isovaleric acid ester of valerian is decomposed on the commercial aluminium oxides used for chromatography, the ester mixture may be separated chromatographically if an aluminium oxide is used as the adsorption agent after partial inactivation by treatment with a carboiwrc acid According to the invention, therefore, a process for the isolation of an isovaleric acid ester sedative ^ comprises the chromatographic separation, from a nonaqueous valerian extract, of the isovaleric acid ester sedative on aluminium oxide which has been partially inactivated by treatment in a non-aqueous medium with a carboiKt-β acid having from 2 to 7 carbon atoms in the molecule, and elution therefrom with a non-aqueous solvent .

The treatment of the aluminium oxide is carried out in a lipophilic solvent with additives if required and the isovaleric acid ester with a sedative action on the central nervous system is eluted preferably with a solvent of the first range of the eluotropic series or with a corresponding isoeluotropic mixture.

The invention includes an isovaleric acid ester sedative when obtained by the process and the new isovaleric acid ester of the empirical formula ^22^30^8 °^ molecular weight 422. k6 , an ultra violet spectrum with λ max. = 256 my ( ε = 15010) and λ ma 2 = 20h mp ( ε = 3800) in methanol and IR- and NMR- spectra according to Figs. 1 and 2 respectively of the accompanying drawings.

For the partial inactivation neutral or basic aluminiur oxide (standardization according to Brockmann, described in Per. dtsch. chem. Ges. ¾(19 l) S.73 - 78) is treated prior to its use with a carboiwrc acid having from 2 to C atoms, if necessary with the addition of dioxane or acetone or an ester, such as for example amyl acetate pr glycerine monoacetate or mixtures thereof, in water-free, lipophilic solvents. In this partial inactivation of he aluminium oxide it is important always to operate in the water-free medium. If for example a commercial aluminium oxide is treated with an excess solution of 5 parts by volume glacial acetic acid and 100 parts by volume n-heptane or with a solution of 50 parts by volume glycerine monoacetate and 1 to 5 parts by volume glacial acetic acid and 200 parts by volume acetone, then a reaction takes place between the aluminium oxide and the added solution with the evolution of heat. Concurrently the acid components are washed out with the saae solvent as is used for the elution of the ester. The sensitive substances contained in the valerian may be chromatographed on a thus inactivated aluminium oxide without decomposition.

The elution of the ester having a centrally sedative action is best attained with n-heptane, hexane , cyclo-r hexane or other solvents of the first range of eluotropic series or with a corresponding isoeluotropic mixture. The following procedure for the isolation of the hitherto unknown ester having a centrally sedative action is recoumended .

A non-aqueous valerian extract is supplied to a column of partially inactivated aluminium oxide and is eluted with a non-aqueous solvent , preferably with a solvent of the first range of the eluotropic series 01 with a corresponding isoeluotropic mixture. The first fractions are rejected, and the fractions which contain the isovaleric acid ester wnicn i-s characterised by the properties set forth below, are worked up in the usual way.

The sedative action of the valerian drug is mainly ascribable to this hitherto unknown isovaleric acid ester having a centrally sedative action.

According to the combustion analysis and the cryo-scopic molecular weight determination in benzene .it has the empirical molecular formula of C„JI„n0o. A colour- 22 O a less highly viscous oil is produced, of aromatic odour and with a somewhat soapy, bitter and burning taste, having the following properties: 28 a = + I67 (in methanol; /= 1 . 035 1 = 1 dm ) D n = 1 33 D UV-Spectrum: λ = 256 m μ ( ε = 15010 ) in methanol = 20h my ( ε = 3800 ) in methanol X max^ The following reaction can serve for the identification of the ester: If a petrol solution of the ester is shaken up with a solution of acetic acid and hydrochloric acid, the ester is instantaneously dissolved giving rise to an intensive blue colour in the acid phase. When carrying out this reaction carefully it can be observed that it passes through a yellow intermediate stage.

The invention will be illustrated by the following non-limiting examples:- Example 1 . Ο g./aluminium oxide activity stage III according to Brocknann were suspended under rotation in a solution consisting of 600 ml acetic acid in an Erlenmeyer flask. The mixture was left tp stand for 24 hours at 20°C and was then passed into a column 700 mm high and of 30 mm diameter. ¾>hen this was washed through with n-heptane until the wash liquor indicated directly pfi = ¾.5 on a moistened jj - paper. g of a methanolic extract of the roots and ri iioraes of Indian valerian enriched in isovaleric acid esters, in accordance with the process of our oapond-^iTg ^patent tippl 1 mti nn PRQp fl'l were admitted to the column dissolved in a little n-heptane. The elution was effected with n-heptane.

The dropping speed amounted to 40 drops/min. 100 fractions were collected each of 50 ml. The control of the individual fractions was effected by thin layer chromatography.

The substances from the fractions 0 to 2 did not show any sedative action in animal experiments. The fractions 25 - 60 contained 1.4925g = 2 .85$ of a chromatographically unitary substance having a very strong sedative action in animal experiments. It had the following characteristics: Empirical formula: C^^R^O^ Molecular weight calculated: 422.46; calculated : 62.54? H calculated: 7.15; 0 calculated: 30.31 Molecular weight found: found: 7.32; 0 found: 29.92 (Cryoscopically in benzene) 28 0 = + 178 (in benzene; 1.089; 1 = 1 dm) a o £ D = + I67 (in methanol; ¾ = 1.035; 1 = 1 dm) n = 1.4-933 UV-spectrum: λ ma 1 = 256 my ( ε = 15010 ) (in methanol) λ max2 = 20k m u ( ε = 3800 ) (in methanol) IR- and NMR-spectrum of the esters are annexed in the accompanying drawings (Fig. 1 lR-spectrum; Fig. 2 NMR-spectrum) .

Example 2. 300 g 1aluminium oxide (activity stage I according Brockraann) was inactivated with 15 ml glacial acetic acid in 300 mlfk -heptane similarly to Example 1 and .paea-gd into a column of 30 mm diameter and 700 mm height; 2500 ml of a mixture of 85 parts by volume n-heptane and 15 parts by volume of ethyl methyl ketone were used for washing out the excess of acetic acid.^' ji ff « *t *t<° "^'1 2 . 1 g. of a light yellow coloured, oily isovaleric acid ester mixture produced by the process of the British ie I^ Uk atfin q l -8 nnH rnn -^^yn^ k were dissolved in a little n-heptane ethyl methyl ketone ( 85 vol! n-heptane: 15 vol J ethyl methyl ketone) and introduced 'into the column. The elution was effected with n-heptane/ethyl methyl ketone, the dropping speed amounted to 30 drops/min. , the temperature 20°C . After a preliminary run of about 500 ml 30 fractions of 20 ml each were collected. The ester having a sedative action was present mainly in the fractions 3- 5 · These were combined, de-acidified with sodium bicarbonate solution, washed with water, dried over magnesium sulphate and concentrated under vacuum at , Example 1 .

Example 3 . 250 gaaluminium oxide (activity stage I according to roocckknnffaan: n) were inactivated with a mixture consisting of 200 mlt jacetone, 50 ml«glycerine monoacetate and 1 ml glacial acjetic acid similarly to Example 1 . After standing for Zh hours at room temperature the suspension was introduced into a column of 30 mm diameter and 330 mm height, the supernatant solvent decanted and the aluminium oxide was rewashed with 500 ml ^ eptane. g of a neutral, brown coloured petrol ether extract from radix Valerianae of Indian originj^were introduced into the column and left to infiltrate. The elution was effected with n-heptane ; the dropping speed amounted to kO drops per minute, the temperature 22°C. 50 fractions each of 25 ml were collected. The combined fractions from 2k to 0 produced after de-acidifying with ^ bicarbonate solution, washing with water, drying over sodium sulphate and concentrating under vacuum, I. 767 g. = 35 . 3<¾]pure product with the empirical formula C ^-Q^ *8 ' Opftical rotation, refractive index, molecular weight, UV- , IR- and N?lR-spectra were identical -*< the corresponding values from Example 1 .

Example k 300 g. ftl 0„ were admitted diameter and 1200 mm. height whjrjrs-t avoiding tho forma- .

After uniform deposition of the aluminium oxide and j3£JA*f- the positive heat effect occuring due to the re¬ the ivater phase showed a p of to 5· E I5O g. of an ester mixture, which was extracted from Indian valerian root in accordance with the process of the British patent ^pp1 -i rati pn fih , was dissolved in 150 m hexane and added to the column layer. After penetration of the solution the elution was commenced by means of hexane. The control of the collected eluate fractions was effected by thin layer chromatography. The pure fractions containing the active substance were combined, washed free from acid with water and I'n sodium carbonate solution, decolorized with animal charcoal, and dried over magnesium sulphate. g. = 28.80^pure ester was obtained from the concentrated pure fractions as a colourless, highly viscous oil. Optical rotation, refractive index, molecular weight, UV- , IR- and NMR-spectra were the same as the cor esponding valies from Example 1. Example 3OOO ivity stage I according to Brock- mann were partially inactivated under the same conditions as described in Example k. However, in this case the aluminium oxide was rewashed with petrol (boiling range: 50 to 75 C) instead of with hexane, until the /accruing wash eluate remained constant when shaken 3 »1ίτΐΓ a similar volume of water and the water phase tion the elution'was commenced by means of petrol. The control of the collected eluate .fractions was effected by thin layer chromatography. All pure fractions containing active material were combined, washed free from acid with water and I70 sodium bicarbonate solution, decolorized with animal charcoal and dried over magnesium sulphate. colourless oil from the concentrated pure fractions.

Optical rotation, refractive index, molecular weight, UV- , IR- and NMR-spectra were identical i-e the corresponding values from Example 1.

Example 6 500 g^Al9Oq of the activity stage # I ajccordxng tp ¾¾ck- mann were added slowly^into a glass rube of 30 nm diameter addition of n-heptane was continued until the^accruing wash eluate remained constant -wolumowige on shaking up with a similar volume of water and the water phase showed a pin of to 5. 5g of the ester mixture used in Example h was then introduced to the column layer dissolved in 5 m of a mixture of 9 vol After penetration of t »he solution the eluti1on was commenced by means of n-heptane. The control of the collected eluate fractions was effected as -under Example 5· All pure fractions containing active material were then combined, washed free from acid with water, and with 1 sodium bicarbonate solution, decolorized with animal charcoal and dried over magnesium sulphate.

I. 6 g. = 29.2/ pure ester whose optical rotation, refractive index ,1molecular weight, UV- , IR- and NMR-spectra were identical to the corresponding values from Example 1, were obtained from the concentrated pure fractions.

Example 7 I5OO of the activity stage I according to Brock- mann was slowly suspended whilst being stirred in a of the positive heat effect the suspension was rinsed shaking up with an equal volume of water and the aqueous phase showed a p„ of k to 5· 22.3 g. of an isovaleric was then introduced to the column layer dissolved in a mixture of 1 mlifheiane and er pene tration of the solution the elution by means of hexane was commenced. The control of the collected eluate fractions was effected as -tmdai Example 5 · All the pure fractions containing active material were then combined, washed free from acid with water and with 1$ sodium carbonate solution, decolorized with animal charcoal and dried over magnesium sulphate. 13 « 95 g« = ester were obtained from the concentrated pure fractions. Optical rotation, refractive index, molecular weight, UV- , IR- and NMR-spectra were identical ^fc-o* the corresponding values from Example 1 .

Example 8 500 .^AlgO^ of the activity stage I according to Brock- mann were slowly suspended whilst stirring in a mixture of OQ mlrjacetone , etate and 20 mlffm-butyric acid. itive heat effect the suspension was rinsed into a column of 30 mm diameter and 1000 mm height , the solvent mixture decanted and/ washed with hexane . The washing of the column with hexane was continued until/ the accruing remained constant volumewise on shaking volume of water and the aqueous phase showed a p-. of to 5 . 6 . 3 g- of an isovaleric acid ester mixture extracted from Indian valerian root by the process of the British ftatent rr pl i nn ti nn ?ft3ifi/fi,l were then introduced into the column dissolved in 20 ral hexane. After penetration of the solution the elution with hexane was commenced. The control of the collected eluate fractions was effected as ^-u xiej--- Example 5 · All pure fractions containing active material were then combined, washed free from acid with water and 1$ sodium hydroxide, shaken up with phosphate buffer (Soerrensen) of p^ . 9 and after decolorization with animal charcoal dried over nagnesium sulphate. 2.0 g. = 31· 7 pure ester were obtained from the concentrated pure fractions. Optical rotation, refractive index, molecular weight, UV- , IR- and NMR-spectra were identical <&6 the corresponding values from Example 1.

Example 9 300 ^AlgO^ of the activity state I according to Brock- mann were slowly suspended whilst stirring in a mixture of 240 ml propionic acid. After decay of the positive heat effect ^c^ji the suspension was rinsed into a column of 30 mm diameter and 500 mm height, the solvent mixture was decanted t$ &~ washed m* with hexane . The washing of the column was continued until the^ accruing wash eluate remained constant volumewise on shaking with an equal volume of water, and the aqueous phase showed a p^-value of 4 to 5· 3 g. of an isovaleric acid ester mixture extracted from Indian valerian root by the process of British patent -a-pplic at iuu 2.839/6"^ were then dissolved in 10 ml hexane and introduced into the column.

After penetration of the solution the elution with hexane is commenced. The control of the collected eluate fractions is effected as Jiad«r Example 5· All/pure fractions containing active material are then combined, washed free from acid once he- lft sodium hydroxide solution and twice with water and once with potassium hydrogen phosphate solution (according to Soerrensen: p . )) decolorized with animal charcoal and dried over nagnesium sulphate.

O.9099 g = 30.33$Jpure ester were obtained from the concentrated pure fractions. Optical rotation, refractive index, molecular weight, UV- , IR- and NiiR-spectra are identical ,¼f the corresponding values from Example 1.

Claims (1)

1. HAVING iro particularly described and ascertained the nature of our said invention and in what manner the same is to be performed, we declare that what we claim is s What we claim is:- A process for the isolation of an isovaleric acid ester sedative^ comprising the chromatographic separation, from a non-aqueous valerian extract , of the isovaleric acid ester sedative on aluminium oxide which has been partially inactivated by treatment in a non-aqueous medium with a carboi**-e acid having from 2 to 7 carbon atoms in the molecule, and elution therefrom with a non-aqueous solvent. A process as claimed in claim 1 in which the partially inactive aluminium oxide is obtained by treating neutral or basic aluminium oxide in a lipophilic solvent. A process as claimed in claim 2 in which dioxane, acetone or an ester is added in the treatment of the aluminium oxide . A process as claimed in claim 2 in which amyl acetate, glycerine monoacetate or mixtures thereof is/are added in the treatment of the aluminium oxide. A piocess as claimed in any of the preceding claims in which the isovaleric acid ester sedative is eluted from the partially inactivated aluminium oxide x^ith a solvent of the first range of the eluotropic series or a corresponding isoeluotropic mixture. A process is n-heptane, qr petrol with a boiling range of from 50° to 75°C. A s going examples. An isovaleric acid ester sedative when prepared by the process claimed in any of the preceding claims. Isovaleric acid ester of the empirical formula C22H30^8 ' molecular weight 422.46, a UV-spectrura with λ inaxj = 256 m μ ( ε = 15010 ) and λ ma 2 = 204 my ( e = 3800) in methanol and IR- and NMR-spectra according to Figs. 1 and 2 respectively of the accompanying drawings. DATED THIS 22nd day of July, 196 . COHEN ZEDE & SPISBAGH P.O.BOX 1169» TEL AVIV Attorneys for Applicants.