CS251755B2 - Method of verbenone and myrtenale production from mixtures of oxidized terpenes - Google Patents

Abstract

When a mixture resulting from the oxidation of alpha - and ss-pinene is subjected to an oxidising treatment, it is possible to isolate verbenone, myrtenal and pinocarveol from the reaction product by fractional distillation. These compounds have therapeutic properties in bronchopulmonary diseases.

Description

The invention relates to a process for the production of verbenone and myrtenal from mixtures of oxidized terpenes useful for the treatment of bronchopenum-related diseases.

The preparation of verbenone / a myrtenal from alpha-foam by autocatalysted oxidation in the presence of salts of greyhound, cobalt and others was repeatedly demanded in a yield of 15% to 20%. One of the reasons for these low reaction yields is the formation of by-products of the radical reaction (radical reactions), such as verbenols and myrtenals, and these alcohols cannot be further oxidized to ketones in specific environmental reactions.

Swiss Patent No. 542,163 of the same Applicant discloses a process for the preparation of terpene fractions which are useful for the treatment of asthmatic diseases, the method being based on the oxidation of mixtures containing predominantly alpha-foam. Prepared according to the above Swiss patent are acquired mainly two fractions of terpenes which decSiljjí ranging te ^p Lot from ⁴⁰ to ⁶⁰ ° C and from ⁶⁵ to ¹⁰³ ° C.

It has now been found, and it is an object of the present invention, that such oxidized mixtures can be subjected to further processing, which in the case of verbene and myytenal production is further oxidation by chromium trioxide in sulfuric acid. ‘

According to the present invention, the process for the preparation of the amine and myytenal from oxidized teypene mixtures is treated with a mixture obtained by oxidation of alpha and beta-pinene with a mixture of chromium trioxide and sulfuric acid in an aqueous medium, followed by separation of Clrbtnyl compounds. at the temperatures and ICs selected in the range in which these compounds decompose. The process is preferably carried out such that after separation of carbonyl compounds is performed by fractional deetilace for separating myytenalu (which deettluje at 90 and ⁹⁵ ° C and at ^2, 6, ⁷ mbar) and усгЬсм ^ (which can give away ^ ESI ^ U is ^p s ^p te Lot ^with Ш and ^{from 113} ° C at the same pressure).

It has been found that in the case of verbene and olyteial during the oxidation of the teypene mixture according to the Swiss Patent Specification, chromium trioxide in sulfuric acid oxidation leads to the complete disappearance of the alcohols and the formation of?

Isolation of the thus obtained ketone compounds after the oxidation with chromium trioxide in sulfuric acid is carried out by means of the sulfide complex and traction distillation.

The opening of the oxirane ring in the case of pentacrylate (preferably is carried out with isopropyl isopropyl acetate), then the carbonyl compounds are removed and fractional distillation is performed to isolate piitClothiol.

Regarding therapeutic poιljití compounds obtained by the process of this invention is therapeutic posjití fractions of terpenes referred in Swiss Pat. No. 542,163 already ^Ziami, namely ^ o ^ three fyakce, ^kt Era de ^ i ^ U is between 65 and ^{f 103} ° C (hereinafter referred ^to as fraction 2 ').

And the analeptic effects of fraction 2 are already known, and the therapeutic assessment of this fraction depends on such effects.

In addition to the scientific uncertainty regarding the precise identification of the effective principles that are responsible for the therapeutic efficacy of pharmaceutical fractions based on such fraction 2, the industrial disadvantage is the obvious disadvantage of processing the smmsi compounds of a composition that even in a certain extent. In addition, it should be noted that the fraction 2 formulation has a general indication as a healing agent with few side effects, without any то ^ЬЬ! Advantageously, one of these effects over the other or improve general efficiency.

It has now been found that said terpene compounds have, in addition to the already modest effects, such as a healing and anaaeptic effect, also a marked deficiency, annealing, antioxidant and antiplatelet effect.

Demonstration of the pharmacological efficacy of verbenone and myytenal is based on the following examples:

1.

2.

3.

4.

5.

6.

Effect

Effect

Effect

Effect

Annibaakeeial effect.

toxicity bronchial muscles inflammatory process hemolysis platelet aggregation na na na na

In particular, all of these tests compared the efficacy of the various compounds with the efficacy of fraction 2 above and the efficacy of the terpenic alcohol therapeutic preparations, sobrerol.

The individual compounds of the present invention will now be evaluated:

A. Verbenon

1. Acute toxicity

Table 1 lists the LD, .g (and 95% spot life expectancy) values set by Licitfeedtovtu and

By the WcxOxon method (J. Pharmacco. Exp. Ther. 26, 99, 1949), administration of verbenone intrapeeitoneally (i.p.) and orally (p.o.) to a single group of 10 mice and 6 rabbits.

Compound

ZvVře

Sex

Method of administration

LD50 in mg / kg body.

^ ο ^ ο ^ Ι

GLeason ^ and classification

Verbenon

IN

mouse male female and. P- 361 (337 to 385) low 325 (308 to 343) mouse male p. O. 1 410 (1 165 up to 1 706) moderate female 1 530 (1 124 up to 1,912) rabbit male and. p. 310 (250 to 384) low female 232 (197 to 274) rabbit male P · O. 790 (608 to 1 027) low female 655 (570 to 573)

On the basis of these data, verbenone appears to be a well tolerated substance because its toxicity by Gleason and animal species and route of administration is moderate or low,.

2. Bringing efficiency

Verbenon exhibited distinct in vitro and in vivo brtochothilatory activity, which differs statistically significantly from the other drug compounds present in fraction 2 of Swiss Patent Specification No. 542,163 from the comparative terpenes, i.e., sobrerol.

(a) in vitro

On the trachea urine, isolated by the method of Conntannin (J. Phaam. PharmacoO.,

17, 384, 1965), causes verbenon to relax the smooth muscles of the stomach at the end of the

125 to 2000 µg per milliliter at a rate that is tignifiaantly higher than that of fractions # 3 # 2 and also higher than that of sobrerol (Table 2). In addition, in the concentration range of 10 to 5 x 10 3 m, he inhibits contraction induced by hisaamine on isolated marrow trachea (hittamine 10 ^{- 5} M, see Table 3).

Table 2

Percent relaxation (mean + standard deviation of 4 preparations) of isolated guinea pig witch induced by concentrations of 125-500 and 2000 µg per ml.

Compound Preparations count 125 µg / ml 500 yg / mi 2000 / Hg / ml Verbenon. 4 standard diameter 29.25 61.25 71.37 deviation 2.39 2.39 1.15 Fraction 2 diameter · 13,5 '” 33.75 52.25 4 standard deviation 1.70 1.49 2.95 Soorerol diameter 1.87 5.62 20.75 4 standard deviation .1,19 2.13 1.49 T a b at 1 k and 3 Percentage of histamine spasms caused by smooth relaxation muscle worm verbenonem (mean + standard deviation of 4 prepaates). - 3 , _- 3 Concentration 10 ³ M 5.10 ³ M

29.2 96.8

5.1 16.6 average standard deviation

(b) in vivo

Verbenone given to intaveenous anesthetized dogs at doses ranging from 0.6 µM per kg of body weight to 4.8 milligams per kg of body weight induces a marked reduction in lung resistance as determined by Diamond (Arch. Int. ΡΙ ^^ ο ^ ο ^. 168, 239, 1967) (see Table 4). In addition, perfumed verbenone intaveenous to rabbits at a single dose of 0.25 milliliters per minute of body mass per minute, inhibits hisaamine experimentally induced bronchial spasm (100 micrograms of histamine to a ^^ gaBm body mass) higher than that statistically significant. fraction 2 as well as sorbrerol (Table 5).

Table 4

Verbenon: Percentage reduction in lung resistance after intravenous administration to dogs (mean + standard deviation in 4 animals;).

Dosage mg / kg

physical body 0.6 1,2 2.4 4.8 Diameter 7,175 10.85 22,225 30.6 Standard deviation 6,786 7.7 18,636 13,507 Table 5 Percentage inhibition of hisraoineo-induced bronchial spasms after iotaavloosis peafusion (mean in 4 animals + standard deviationa • . * Perfusion time Compound 15 Dec 30 60 minutes Diameter 36.25 46.27 73.32 Verbenon Standard deviation 1.65 9.00 7.17 Diameter 16.30 24.20 38,92 Fraction 2 Standard deviation 3.62 4.39 3.67 Diameter 2.15 1.15 3.97 Sobrerol Standard deviation 4.30 6.39 8.73 3. Protective efficacy Verbenon given iotraplriOonllloa in a dose 30 оИ ^ гол ^ to С-.Ъ ^ сгг <^ о body weight! white rats of the COBS strain (Chhales River) exacerbates the caea- genin (Winter, C.A. et al., Proc. Soc. Exp. Biol. Mee., 111, 544 (1962), i

is statistically significant in rats and adrenal removed animals to a degree significantly better than fraction 2 and sobrerol (Table 6).

After intrapertoneonal administration to rats at doses of 36 and 120 milligam per kioogaam of body weight, verbenone exhibits significant antiexsuda activity in rats. experiential pleuritis induced by terpentnee (Nuuley, J.V. with J. Paah. sp., 91, 575, 1966) to a degree that can be compared to rcelylotali acid (Table 7).

Table 6

Anti-inflammatory activity in rats with carrageenin-induced edema

Compound

Intraperitoneal doses mg / kg body weight% inhibition of plantar edema one hour after administration (mean + standard deviation in 6 animals)

Verbenon

Fraction 2

Sobrerol

4. Hemolytic activity in vitro

Verbenon protects in vitro rat red blood cells from surfactant-induced hemolysis (Tween 80) at an effective concentration of 50% (ЕС 50) of 639.5 / дд per kg body weight (95% confidence limits; 518.0 to 760.11).

5. In vitro anti-aggregation activity

Verbenon at concentrations from 160 to 1280 yug / ml inhibits thrombocyte aggregation in vitro, according to the method of Born and Cross (J. Physiol., London, 168, 178, 1963), to a degree higher than that of fractions 2 and sobrerol. (Table 8).

Table7

Efficacy of verbenone and terpentine-induced plauritis in rats

Intraperitoneal exudate + stand. inhibition of significance ml deviation of% P

control (solvent) 1.97 0.24 - - Aspirin 100 mg / kg 1.30 0.13 34 0.01 Verbenone (36 mg / kg) 1,23 0.24 27.6 0.01 Verbenon (120 mg / kg 0.97 0.22 50.1 0.01

al

Percentage of platelet aggregation inhibition (average of repeat tests)

Final concentration of µg / ml% inhibition

Kottrbla 0 verbe ^ n 1 280 100 ALIGN! verbe ^ n 640 44 verbe ^ n 320 6 verbe ^ n 160 0 0 sobrerol 4 000 32 sobrerol 2 000 24 sobrerol 1 500 12 sobrerol 1 000 4 Console 0 Fraction 2 1 280 70 Fraction 2 1 016 18 Fraction 2 806 14 Fraction 2 640 0

Antibacterial efficacy

Verbenon exhibits a low degree of antibiotic activity against gram-positive and gram-negative microorganisms with a MIC (minimum concentration concentration) of 800 µg / ml against Staphylococcus aureus and Escheeichia ® (Table 9) and is as effective as fraction 2 and more effective than sobrerol.

and

Minimum гпСгЬ ^ п! concentration (MIC) in micrograms per milliliter

Microorganism compound

Staphylococcus aureus

Eschhrichia

Verbenon

Fraction 2

Sobberol

800

800 oo

800

800

O®

On the whole, the compound has been shown to have distinct efficacy which differs in a statistically significant manner from both the active ingredients in fraction 2 of Swiss Patent No. 542,163 and the trrort serving as a standard (sofobe). _

Because of these important properties, verbenone acts as an active ingredient of the pharmaceutical formulation that is. particularly useful in the treatment of bronceopneumoniais of diseases which are associated with damage to the airways by inflammation and agents causing infection. The dose recommended for the therapeutic application ranges from 10 to 100 mg per day.

Myrtenal

1. Acute toxicity

LD ₅₀ determined by the method of Lichtfield and Wilcoxon (J. PharmacoO. Exp. Thee., 96,

99, 1949) administration of myrtencl to intensive care groups of 10 mice was 170 mg nc kg body weight.

2. Bronchodilation efficiency

Myrtenal showed good bronchial efficacy both in vitro and in vivo.

(a) in vitro

On an isolated passageway as measured by (J. Pharm. J., 17, 384, 1965), the oyytenal showed relaxation of smooth musculoskeletal muscles at concentrations of 125 to 200 µg / ol at a rate that is statistically higher than fraction 2 and sobrerol (Table 10).

Table- 10

Percentage of relaxation (mean + standard determined by means of endings of 125 to 500 and 2 deviation of 4 preparations of isolated drkdušnioe guinea pigs 000 yig / ml Compound Preparations count 125 µg / ml 500 µg / ol 2,000 yig / ml Myytenal 4 diameter 7.5 47.5 62.5 standard deviation 5.0 15.0 20.7 Fraction 2 4 diameter 13.5 33.75 52.25 standard deviation 1.70 1.49 2.95 Sooreeol 4 standard diameter deviation 1.87 5.62 20.75

(b) in vivo

Myrtenal given by ave is added per minute and inhibits the expecation of e. mine per kirogcao body weight (intravenously) to a degree that is statistically higher than that of sobrerol and slightly lower than that of fraction 2 (Table 11).

perfusion of rabbits at a dose of 2.5 ml per kg of bodyweight hmoonnosi spasm bronchus induced by hisComineo (100 micrograms histaTable 11

Percent inhibition of hisComine-induced bronchial spasms by iitcaveiosis perfusion to rabbits (mean in 4 animals + standard deviation).

Perfusion time Compound minutes

Myytenal mean standard deviation

25.4 20.4 28.8

10.4 16.2 13.5

Table 11 - continued

Perfusion time 15 30 60 minutes

Compound

Fraction 2 standard diameter 16.30 24.20 38,92 deviation 3.62 4.39 3.67 Sooberol standard diameter 2.15 1.15 3.97 deviation 4.30 6.39 8.73

3. Anti-inflammatory activity

Myrtenal, when administered intraperiooneally to white rats of the COBS strain (Chaales River) at doses of 30 milligrams per kiogram of body amotinti, inhibits the experimental paw edema induced by carrageenin injection (CA Winter et al. Proc. Soc. Exp. Biol. Med. 111 544, 1962) degree with a greater intensity than fraction 2 as well as sobrerol (Table 12).

Table 12

Prooizant efficacy in carrageenan edema in rats

Compound

Dosage mg / kg body weight iotraolritonlálrlt% inhibition of plaque edema 4 hours after administration (mean of 6 animals + standard deviation

Myrtenal 3030

Fraction 2 3021

Sooberol 3011

4. Anothiothytic efficacy in vitbo

Myrtenal at 80 to 500 / dd / ml concentrations protects rat erythrocytes from surfactant-induced haemolysis (Tween 80) with an effective concentration of 50% 157.09 / µg / ml (95% = 112.3 to 189.9).

5. Anti-aggregation efficiency

Myrtenal at end-concentrations of 160 to 1280 µg / ml ioaibulated in vitro platelet aggregation induced by adenine diphosphoric acid, evaluated according to the method of Born and Cross (J. Physiol., London, 168, 179, 1963), at a higher rate than Fraction 2. and sobrerol.

Table 13

Platelet aggregation:% ioaibicl (average of repeat tests)

Final concentration ug / ml % ioaibicl Konnrola 0 Myrtenal 1 280 ' 75 Myrtenal 840 20 May

Table 13 - continued

Final concentration / µg / ml% inhibition

Myrtenal 320 12 Myrtenal 160 0 K ^ n ^ trol ^ a 0 SoOberol · 4 000 32 Sobberol 2 000 24 SoOberol 1 500 12 SoObebOl 1 000 4 Kooinbola 0 Fraction 2 1 280 70 Fraction 2 1 016 ' 18 Fraction 2 806 14 Fraction 2 640 0

6. Antibackerial efficacy

Myrtenal shows remarkable anti-bacterial activity against Gram-positive strains, less intense prod Gram-negative strains while minimizing endocrine inhibition (MIC) 200 µg / ml prod Staphylococcus aureus and 800 µg / ml prod EscCheichia cooi and is more effective than fraction 2 and sobrerol (Table 14).

Table 14

Minimum inhibitory concentration (MIC) in ml <r ^ (^ cJgramrgram ^ ί.Иl.l.l: r

Microor ga nsm ^ us Staphyloenceut Escheeichia compound aureus c ^ O: i

Myytenal 200 800 Fraction 2 800 800 Suderol C- oa

This compound, i.e. myytmal, showed in conclusion that it has significant antibacterial activity, which is statistically significantly different from both the smmsi terpenes of fraction 2 according to the cited Swiss patent specification and the other medicinal standard substances mentioned. Myytenal has a low antisense activity, exhibits antihrmotic activity, and finally has broncho-type activity and anti-aggregation activity.

In light of these particular facts, myytenal is indicated as an active ingredient of drugs that are particularly useful in the treatment of bronchopneumoneal diseases when the složkaе ^ ее И složka složka component is present, or when abattoirs are required to be treated with antiЬiodt. For such indications, a daily dose of 10 to 100 mg myytenal is recommended.

The pharmaceutical compositions of the compounds of the present invention may be present in the form of oral supplements with normal or delayed action, for example, as soft capsules or injection ampoules, suppositories, sprays in the form of various solutions, maize and balsams with conventional bases, fillers, etc., as commonly used in pharmaceutical practice, either as an active ingredient or in conjunction with drugs indicated for the aforementioned diseases, i.e., andbiotics and baccicic agents, active substances, corbizone preparations and anaphores.

The following example is intended to illustrate the preparation of the compounds of the invention without, however, limiting the invention.

Example

(a) Oxidation of alcohol

100 g of a terpene mixture of fraction 2 according to Swiss Patent No. 542 163, which contains up to 30% by weight of compounds having a carboxyl residue (predominantly verbenone and myrtenal), while 50 to 60% by weight contains compounds with an alcoholic residue (mainly verbenol and myrtenol) Dissolve in 2000 ml dry acetone.

Separately, an oxidation solution is prepared by carefully mixing 37.41 g of chromium trioxide with 32.2 ml of concentrated sulfuric acid in an amount of water sufficient to give a final volume of exactly 140 ml.

The chromic acid is then slowly added dropwise by stirring to the acetone solution while cooling in an ice bath so that the temperature is kept below 30 ° C. After the addition was complete, filtration was performed on Celite (trademark) and the filtrate was freed from acetone by evaporation under reduced pressure at 40 ° C. The residue is then diluted with water, neutralized under cooling with 10% sodium hydroxide and extracted exhaustively with chloroform. The combined organic extracts were dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure at 40 ° C.

In this way, about 100 g of a crude product, consisting of 50 to 60% by weight, of compounds with a carbonyl residue (predominantly verbenone and myrtenal) are obtained.

b) Separation of carbonyl compounds from the raw material obtained by oxidation

A suitable reaction vessel is charged with 100 g of the crude mixture obtained in step a) of this method, which is dissolved in 500 ml of ether and in a solution prepared from 120 g of sodium sulfite and 72 g of sodium bicarbonate in 2000 ml of water. The mixture was shaken vigorously for about 20 hours at room temperature and then transferred to a separatory funnel and the organic phase removed. The aqueous phase is washed twice with 500 ml of ether each time and the aqueous sulphite solution is separated and subjected to steam distillation. Working up the distillate gives 50-55 g of a mixture consisting of pure verbenone and myrtenal.

c) Obtaining pure verbenone and myrtenal g of the product obtained in the preceding step is subjected to fractional distillation under vacuum at 20 psig using a distillation column packed with nickel shavings. Distillation is performed slowly (5-8 mL / h) maintaining a reflux ratio of 40: 1 throughout the operation. The fraction distilling at 90 ° C to 95 ° C consists of myrtenal (19 g) and has the following physicochemical properties:

Пд ^С · - 1.50 d ₂₀ = 0.98

Infrared analysis: bands at

684 cm 1 gamma C = O (conjugated)

623 cm ¹ gamma C = C (conjugated)

471 cm 1 gamma C-H

423 to 1,387 to 1,372

Ultraviolet analysis λ _max (EtOH) = 246 nm (£ about 8,400)

The semicarbazone melts at 210-215 ° C.

The fraction which distills later^při¹¹⁰ to¹¹³ Noc: 2 ° C^por lacquer^2.67 kPa, consists of verbe nonu (28.5 g) and has the following physicochemical properties:

D

1.49

0.97

Infrared bands at 1

670 cm ' ¹ ГC = O

FRC 615 cm ⁴ = C ^{(k g} onju rate) (conjugated)

650 to 1435 to 1370

Ultraviolet spectrum A _max (EtOH) = 250 nm (€ = 7,300)

Semikatoazon ^b from the ^T 188 and ^from 190 ° C.

Claims (2)

SUBJECT OF THE INVENTION Process for the production of verbenone and myytenal from a mixture of oxidized terpenes, characterized in that the mixture obtained by oxidation of alpha and beta-pinene is treated with a mixture of chromium trioxide and sulfuric acid in an aqueous medium, followed by separation of carbonyl compounds and isolation By distillation at temperatures and those selected in the range in which these compounds distill. 2. Method according to claim 1, characterized in that after separation of carbonyl compounds ^and di- ^p rov jirakCní ^d eesilace for separate ^and the read access ^ m ^ which četoluje ^p s 90 to 95 ° C for ⁷ Daku 2.6 kPa, and verbenone that deetoluje ^P s ^{of 113} and ¹¹⁰ ° C under a pressure of ^2.67 to ^P and. Severography, n. P., MOST Price 2,40 · Kčs