NO762464L - - Google Patents

Description

Smoking material, as well as flavoring agent for this

The invention relates to substances which are useful as flavourings, more particularly as smoke flavorings for tobacco, reconstituted tobacco and tobacco substitutes, by which is meant materials which, although not of tobacco origin, are smokeable in the same way as cigarettes, cigars, pipes, etc.

Proposals have been made regarding the replacement of tobacco with substitutes for it because tobacco smoke is known to be productive in diseases of the bronchi and lungs. The known substitutes, despite improved health characteristics compared to tobacco, are of limited value due to their smoke aroma which does not resemble tobacco. In order to give a pronounced degree of tobacco aroma, it has hitherto been necessary to mix in large proportions of tobacco and/or extracts of tobacco, whereby the achievable health benefits are reduced.

The invention provides a substance comprising a hydroxy- or glycosyloxy-mono- or di-unsaturated spirovetione.

Spirovetivone has the following formula and the numbering used in the following when naming its derivatives as shown.

The above formula and all other formulas used in the following are conventional, in that each angle or unattached valence line represents a carbon atom with all four of its valences occupied, either in the manner indicated or otherwise by attached hydrogen atoms.

More particularly, the invention provides a substance comprising a compound of the formula

where in formula I one of the radicals and R2 is hydrogen and the other represents either a hydroxyl group or a glycosyloxy group -OX where X is the radical of a mono- or disaccharide, and the dashed lines represent possible positions for a double bond, and in formula II represents R| either a hydroxyl group or the above-mentioned glycosyloxy group -OX.

The substances according to the invention are useful as aroma substances, especially as smoke aroma substances for tobacco, tobacco substitutes and mixtures thereof.

The compounds of formula I which contain a glycosyloxy group -OX are the preferred compounds.

The applicant has identified glucosides of formulas I and II (where X represents a glucosyl group) as constituents of tobacco, where they are present in exceedingly small quantities.

Strongly concentrated preparations, containing for

eg 25 wt% or more of the glucbsides, can be obtained from tobacco. One method of obtaining such preparations comprises extracting tobacco with a low-boiling ketone solvent, for example acetone, successive removal from

the extracts of the parts soluble in aliphatic hydrocarbon, for example hexane, and then a low-boiling chlorinated hydrocarbon, for example chloroform, and obtaining a water-soluble fraction from the residue, and subjecting the aqueous solution thereof to treatment with ion-exchange resins to remove basic and acidic fractions, so that a neutral residue is obtained, after which the residue is extracted with a lower alkanol, -especially butanol, and the solution is subjected to gel permeability and liquid chromatography for

redissolve it in fractions rich in individual glucosides... The glucosides according to the invention are concentrated in the last eluted fraction from the gel permeability separation, and individual, pure glucosides can be isolated by liquid chromatography if desired. Other fractions from gel permeability chromatography contain glucosides of various natures, some of which are described in the applicant's contemporaneous British patent no.

(Applications 32286/75, 9989/76). The separation technique used by the applicant is more specifically described in the examples.

At each step in the process, the tobacco aroma that . is produced by burning the fraction, more pronounced.

The final glucosides are, when they are. in essentially pure form, colourless, solid substances. They have been attributed to chemical structures by a• v<13>C NMR (nuclear magnetic resonance) spectroscopy and by enzymatic hydrolysis to the corresponding aglycones, containing -OH groups instead of the glucosyloxy groups, and these have been isolated as color-

loose oils.

13

C NMR spectroscopy and PMR (proton magnetic resonance) spectroscopy have also been used to determine the structure of the aglycones.

13'

In the C NMR spectra of the substances according to the invention, the spirocarbon atom (number 5) produces a characteristic resonance at 50.6 - 1.0 ppm relative to tetramethylsilane

(TMS)..

As a further feature that can define the substances 13

x according to the invention, the C NMR spectra of the aforementioned substances therefore exhibit a characteristic resonance at 50.6 1.0 ppm in relation to tetramethylsilane.

Glycosides according to the invention are obtainable synthetically using as starting materials a mono- or disaccharide (especially glucose) and a hydroxy-mono- or di-unsaturated spirovetione. Thus, the mono- or disaccharide can be O-acylated and converted into a functional derivative which is reactive towards hydroxyl groups, then reacted with the hydroxy-spirovetiion compound and the O-acyl groups in the product hydrolysed. In particular, glucose can be converted to tetraacetoxyglucosyl bromide which can be condensed with the hydroxy-spirovetivon compound and the acetyl groups. can be hydrolyzed to produce the spiro-vetivone glucoside.

Preferred glycosides are derived from hexoses or pentoses, glucosides being particularly preferred.

Specific examples of the preferred glucosides are the compounds of the following formulas t

where G represents an Ø-D-glucosyl group.

Such compounds can exist in many stereobisomeric forms, all of which are within the scope of the invention.

The aglycon compounds according to the invention can be synthesized by the following processes: 1. A first method for producing an aroma-giving substance of the formula:

comprises treating the enolate of a spirin compound of the formula

with a trimethylhalosilane to produce a trimethylsilyl-

ether, reaction of the product with an organic percarboxylic acid and hydrolysis of the product to remove the trimethylsilyl group therefrom.

One diastereoisomer with the above spiran form1 is known as solavetivon. This compound has already been isolated from infected potato tubers. A mixture of four diastereoisomers is obtainable synthetically from Orange Oil condensate by a method described in the examples.

By carrying out the above procedure, solavetivon or the synthetic mixture of Orange Oil condensate can be enolized, for example by treatment with butyl lithium in the presence of a base (for example diisopropylamine). Trimethylchlorosilane, neutralized with a base, for example triethylamine, is added,

for formation of the trimethylsilylenol ether. A solution of this ether in inert solvent is added at low temperature (eg -10°C) to a stirred suspension of the percarboxylic acid (eg m-chloroperbenzoic acid, giving 3-trimethylsiloxy-solavetivon, which can then be hydrolysed with dilute acid,

for example hydrochloric acid, and the product can be purified in a conventional manner.

2. Another method for producing an aroma-giving substance of the formula

comprises treating the enolate of a compound of formula

with a complex formed by a molybdenum peroxide, an organic tertiary amine and hexamethylphosphor amide.

As with the former method, solavetivon, or the synthetic mixture of diastereoisomers obtained from Orange Oil condensate can be enolized, for example with butyl lithium. The complex used to carry out the oxidation is known in the art, described in the Journal of the American Chemical Society, 1974, pp. 5944-5945. To carry out the oxidation, the complex can be added in powder form to the enolized solavetivon at a very low temperature (for example -70°c). When the reaction is complete, the oily product can be extracted from the mixture and

cleaned in a conventional manner.

3. A method for producing an aroma substance of the formula

includes treatment of a compound of the formula

with osmium tetroxide to form an osmate ester and cleavage

of the product in a known manner for cleavage of osmate 1,2-esters to thereby form a corresponding 1,2-diol;.

The process can be carried out by contacting solavetivon or the synthetic mixture of diastereoisomers obtainable from Orange Oil condensate with osmium tetroxide in inert

environment, for example diethyl ether, at approx. 0°C. The osmate ester can

is decomposed, for example by treatment with an aqueous solution of sodium sulphite, and the product can be isolated and purified in a conventional manner.

An aglycone of the formula

can be made by isomerization of the compound or alternatively by dehydration of the compound

The substance according to the invention, especially the glycosides, gives a distinct tobacco aroma to the smoke from tobacco substitutes which would otherwise be completely devoid of such aroma. Only small proportions of the pure glycosides, for example 1% by weight, are necessary to produce such an effect. This is indeed remarkable, as the glycosides themselves are not volatile. The aglycones from which the glycosides are obtainable, by comparison, impart only a small (albeit defined) amount of tobacco smoke flavor to tobacco substitutes. The aglycones are principally useful as intermediates from which the glycosides can be prepared.

Although the synthetic glycosides constitute a special feature, aroma-setting substances in the form of concentrates isolated from tobacco are also included within the scope of the invention, as such concentrates contain, for example, at least 25% of the previously indicated glucosides.

If desired, the substances according to the invention can also be used to improve or strengthen the tobacco aroma, especially for reconstituted tobacco or mixtures of tobacco and tobacco substitutes.

According to a further feature of the invention, smoking materials are therefore provided which comprise tobacco (including reconstituted tobacco), tobacco substitute or a mixture of tobacco with a tobacco substitute, and to which is added a small proportion by weight, for example 1%, of a substance as defined in the preceding (especially a glycoside).

Tobacco substitutes can, for example, be based on materials of carbohydrate origin, for example such materials as are described in British patents 1,055,473 and 1,143,500 as well as in US patent 3,106,209, namely cellulose, oxidized cellulose or salad leaves. Cellulose ethers can also be used. Modified carbohydrates are preferred, as the term "modified" means chemically modified and implies that the original carbohydrate has undergone a change of a chemical nature.

The tobacco substitute can advantageously be a heat-degraded carbohydrate, made for example by exposing carbohydrate

(especially cellulose) for a catalyzed degradation process at a temperature above 100°C (for example 100-250°C as indicated in

applicant's British patent 1,113,979, or above 250°C as stated in applicant's British patent 1,415,893) until the weight of degraded carbohydrate is less than 90% of the weight of the original carbohydrate. A similar substance is obtainable as described in the applicant's British patent specification 1,289,354 by acid- or base-catalyzed condensation of a compound of the formula

where R 1 and R 2 , which may be the same or different, each represent a hydrogen atom or an alkyl, hydroxyalkyl or formyl group or a precursor of such a compound

(IV).

The substances according to the invention can be mixed into smoking materials in any suitable way. Other known ingredients in smoking materials can be included in order to provide desired physical properties and burning characteristics. For example, the smoking materials may contain glow-regulating catalysts, materials that improve the ash coherence and colour, nicotine, other aroma substances, drugs, humectants or film-forming agents.

In particular, the smoking materials according to the invention may contain protein, in accordance with the applicant's British patents 1,312,483 and 1,312,786.

The smoking materials according to the invention are usually produced in sheet form, containing a film-forming agent, for example a natural gum or pectin or a cellulose ether, especially carboxymethyl cellulose or a salt thereof. For the production of such sheets, the ingredients of the smoking material, preferably together with the tobacco or tobacco substitute in finely divided form, can be mixed with a sufficient quantity of water to produce a slurry which is then cast on a surface and dried. Sheet material produced from the slurry can be unraveled so that a material is obtained in a form suitable for smoking. ,;

Partly preferred smoking materials according to the invention are those which are based on tobacco substitutes and more particularly on thermally decomposed carbohydrates as previously defined. Such smoking materials according to the invention provide smoke that contains relatively small proportions of harmful ingredients, and as they themselves have a distinct tobacco aroma, it belongs to the invention

that they can be mixed with tobacco in relatively large proportions to provide mixtures which are acceptable to habitual smokers of tobacco cigarettes.

In what follows, the invention shall be illustrated, but not limited, by examples in which all parts and percentages indicate weight.

Example 1

Isolation and characterization of spirovetivine glycosides from FCV tobacco

An acetone extract from 100,000 parts of smoke-cured Virginia (PCV) tobacco was fractionated by successively removing the parts soluble in hexane and then chloroform. The residue was extracted with water.

The water-soluble fraction (4,000 parts) was treated with ion-exchange resins to remove: (a) bases (using "Amberlite" IRC-50(H)/ethanol)

(b) acids (using "Amberlite" IRA-45(0H)/water)

and the neutral residue was contacted with n-butanol. The butanol-soluble fraction (200 parts) was subjected to the gel permeability technique (using "Sephadex" LH 20/water) and resolved into four major bands. The fraction which was last eluted (20 parts) ,. was rich in the spirovetivon glycosides along with other glycosides. Preparative pressure liquid chromatography (phase - silicon dioxide; solvent system - dichloroethane (85), acetonitrile (8.5), ethanol (8.5), water (0.6)) gave resolution of this fraction,

and four spirovetivone glycosides C^tega®^ Gl (1 part), G2 (2 parts), G3 (2 parts) and G4 (2 parts) were isolated. The glycosides were obtained as colorless, freeze-dried solids.

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• C NMR spectroscope! and enzymatic hydrolysis were important techniques in the characterization of these glycosides. The glycosides G2, G3 and G4 were easily hydrolysed by Ø-glucosidase, whereby a 1/3-glucosidic bond was established in these materials. The corresponding aglycones that were released, A2, A3bg A4, were

characterized in a conventional way. Glycoside Gl released

not readily an aglycone under these conditions. Aglycone A2 was isolated as a colorless oil with the following characteristics: MS: M<+>234 (C15H22O2); m/e 232, 217, 205, 176, 161, 148,

133, 121, 109, 108, 91, 79, 68, 53, 41.

IR (CC14): 3480, 3080, 1680, 1645, 890 cm"<1>

UV (EtOH) : ^240 nm (£= 13,800)

PMR (CDCl 3 ): 1.22 (3H, d, J = 7Hz); 1.76 (3H, s); 2.03 (3H, d,

J = 1.5Hz); 3.82 (1H, d, J » 1-2 .5Hz)J 4*74 (2H, extended); 5.83 (lH, d, J = 1.5Hz) é ppm

13

C NMR data (shown in Table 1) and PMR lanthanide exchange/cleavage experiments completed the characterization studies, identifying aglycone A2 as 3-hydroxy-spirovetiva-1, (10), 11-diene-2-one, where the secondary methyl group in the ring and the hydroxy function are trans and diequatorial.

Aglycone A3 was isolated as a colorless oil with the following characteristics: MS: M<+>234 (C15H22O2); m/e 219, 216, 206, 191, 174, 159,

145, 137, 121, 108, 91, 79, 67, 55, 41.

IR (CC14): 3620, 3450, 3080, 1676, 890 cm"<1>

UV (EtOH): A 241 nm (£ = 13,000)

PMR (CDCl 3 ): 0.99 (3H, d, J = 7Hz); 1.95 (3H, d, J^v 1Hz)j

4.14 (2H, s); 4.95 (1H, p); 5.07 (1H, s);

5.76 (1H extended s) ppm

13

C NMR data (shown in Table 1) and PMR lanthanide exchange/cleavage experiments completed the characterization studies. Aglycone Å3 was thus identified as 13-hydroxy-spirovetiva-1 (10), 11-dien-2-one.

Aglycone A4 was isolated as a colorless oil with the following characteristics: MS: M<+>252 (<C>15<H>24<0>3); m/e 234, 222, 221, 203, 175, 161,

137 (100%), 135, 133, 121, 109, 107, 95, 93, 91, 81, 79, 75, 69, 57, 43 (100%), 41.

IR (CC14): 3620, 3450, 1670 cm"<1>

UV (EtOH): A max. 240 nm (6 11,800)

PMR (CDC13): 0.98 (3H, d, J= 7Hz)?1.25 (3H, s)* 1.97 (3H, d,

J^1Hz)j 3.50 (2H, q); 5.75 (1H, extended s) é ppm 13

C NMR data are shown in Table 1 and confirmed the designation of aglycone A4 as 11, 12-dihydroxy-spirovetiva-1(10)-en-2-one.

Glycoside identifications

The corresponding glycosides G2, G3 and G4 were consequently constitutionally identified as the fl-D-qlucoside of 3-hydroxy-spirovetiva-1 (10), ll-dien-2-one, / 3-D-qlucoside of 13-hydroxy-spirovetiva - 1 (10), 11-dien-2-one and fl-D-glucoside of 11, 12-dihydroxyspirovetiva-1,10-en-2-one. Satisfactory spectral data were obtained for these glucosides, and ■ 13C NMR data for

the chemical exchange for these glucosides and also the unhydrolyzable glucoside Gl is shown in table 1/

The spectral data, especially<13>C NMR, of glycoside G1 were very similar to the data for glycoside G2. Small differences observed (underlined in Table 1) suggested some slight variation in structure centered around the C-3 atom. Accordingly, glycoside G1 was constitutionally identified as the C-3 epimer of glucoside G2, in that the ring methyl group and the glucosidic bond in this case have a cis-relationship.

Example 2

Aroma- assessment of glycoside Gl - spirovetiva- 1 ( 10), llrdien- 3-01- 2- on- j3- D-. glucoside (epimer 1)

5.18 parts of a material prepared by yarmenic decomposition of cellulose by heating at 250° in the presence of 5% ammonium sulfamate until a weight loss of more than 10% had occurred was mixed with 60 parts of water and ground in a disintegrator.

1.2 parts glycerol and 0.4 parts ammonium sulfate in 20 parts yann were added. A dry mixture consisting of 3.3 parts calcium carbonate, 1.0 part bentonite was added, followed by 3.0 parts sodium carboxymethylcellulose, 5.72 parts magnesite and 0.2 part spirovetiva-1 (10) , ll-dieh-3 -ol-2-one-/3-D-glucoside (epimer 1). The resulting slurry was stirred for at least one hour and then cast onto a dryer which produced a film with a dry basis weight of 48-52 g/m<2>.

The film was unraveled and the fibers processed into cigarettes and these cigarettes were assessed with regard to aroma. A tobacco flavor was determined by smoking the test cigarettes. Instead of the /3-D-glucoside used in this example, the corresponding galactoside can be used.

Example 3

Aroma assessment of glycoside G2 - spirovetiva- 1 ( 10), ll- diene- 3- ol-2- one-/ 3- D- glucoside ( epimer 2)

5.18 parts of a material prepared by thermal decomposition of cellulose by heating at 250°C in the presence of 5% ammonium sulfate until a weight loss of more than 10% had occurred was mixed with 60 parts of water and ground in a disintegrator .. 1.2 parts of glycerol and 6.4 parts of ammonium sulfate in 20 parts of water were added. A dry mixture consisting of 3.3 parts calcium carbonate, 1.0 part bentonite was added, followed by 3.0 parts sodium carboxymethyl cellulose, 5.72 parts magnesite and 0.2 part spirovetiva-1 (io) , ll-diene-3 -ol-2-one-/3-D-glucoside (epimer 2). The resulting slurry was stirred for at least one hour and then cast onto a drier which produced a film with a dry basis weight of 48-52 g/m 2 .

The film was unraveled and the fibers processed into cigarettes and these cigarettes were assessed with regard to aroma. A tobacco aroma was detected when smoking the test cigarettes.

Likewise, a tobacco flavor can be recognized when smoking cigarettes made from a mixture containing only sodium carboxymethyl cellulose, inorganic filler and spirovetiva-1 (10), , 11-dien-3-ol-2-one-/3-D-glucoside (epimer 2) .

Example 4

Example 3 was repeated using 0.2 part of the aglycone of epimer 2 instead of its glucoside. A less pronounced but recognizable tobacco aroma was observed when smoking the cigarettes.

Example 5

Aroma assessment of glycoside G3 - spirovetiva- 1 ( 10), ll- diene- 13- ol-2- one-/ ?- D^- glucoside

,5,18 parts of a material produced by thermal decomposition of cellulose by heating at 250°C in the presence of 5% ammonium sulphate until a weight loss of more than 10% had occurred was mixed with 60 parts of water and ground in one disintegrator. 1.2 parts of glycerol and 0.4 parts of ammonium sulfate in 20 parts of water were added. A dry one. mixture which consisted of 3.3 parts calcium carbonate,. 1.0 part bentonite was added, followed by 3.0 parts sodium carboxymethyl cellulose, 5.72 parts magnesite and 0.2 part spirovetiva-1 (10) , 11-dien-3-ol-2-one-/3-D- glucoside. The resulting slurry

was stirred for at least one hour and then cast on a dryer which produced one film with a dry basis weight of o 48-52 g/m

The film was unraveled and the fibers processed into cigarettes and these cigarettes were assessed with regard to aroma. A tobacco aroma was detected when smoking the test cigarettes.

Example 6

Example 5 was repeated using 0.2 part of spirovetiva-1 (10), 11-dien-13-ol-2-one instead of its glucoside. A less pronounced but recognizable tobacco aroma was observed when smoking the cigarettes.

Example 7

Aroma assessment of glycoside G4 - 12-( g- D- glucosyloxy)- spirovetiva-1-( 10)-en-ll-ol-2-one ........

5.18 parts of a material produced by the thermal decomposition of cellulose by heating at 250°C in the presence of 5% ammonium sulphate until a weight loss of more than 10% had taken place

hit, was mixed with 60 parts water and ground in a disintegrator. 1.2 parts of glycerol and 0.4 parts of ammonium sulfate in 20 parts of water were added. A dry mixture consisting of 3.3 parts calcium carbonate, 1.0 part bentonite was added, followed by 3.0 parts sodium carboxymethylcellulose, 5.72 parts magnesite and 0.2 part 12-(Ø-D-glucosyl-oxyJ-spirovetiva -1-(10)-en-ll-ol-2-one The resulting slurry was stirred for at least one hour and then cast onto a drier which produced a film with a dry basis weight of 48-52 g/m 2 .

The film was unraveled and the fibers processed into cigarettes and these cigarettes were assessed with regard to aroma. A tobacco aroma was determined by smoking; of the test cigarettes.

Example 8

Example 7 was repeated using 0.2 part

of spirovetiva-1-(10)-en-11,12-diol-2-one instead of its glucoside. A less pronounced but recognizable tobacco aroma was observed when smoking the cigarettes.

Example 9

Isolation and synthesis of spirovetiva-1(10),ll-dien-2-one (solavetivon)

(a) Isolation of solavetivon from infected potato tubers

The isolation of this compound from infected potato tubers is described by D.T. Coxon et al. (Tetrahedron Letters,

34, 2921 (1974)). In the work described here, using Coxon's methods, the inoculation of 250,000 parts of potatoes with the fungus Phytophthora infestans, and subsequent processing, gave 17 parts of optically pure solavetivon. The material was identically identical in all respects to that described in

13

the literature. A C NMR comparison is shown in Table 2 below.

v

(b) Synthesis of spirovetiva-1(10),ll-dien-2-one from Orange Oil condensate

The Isolation of (+)Valences from Orange Oil Condensate

and its conversion to (+)Nootkatone is described by G.L.K. Hunter and W.B. Brogdeh (Journal of Food Science 30, 876 (1965)). The conversion of (+)Nootkatone to (+)anhydro-β-rotunol has also been described (D.S. Caine and C.Chu Tetrahedron Letters, 703 (1974)).

These methods were followed to prepare (+)anhydro-β-rotunol,

with spectral properties identical to those described in the literature.

Lithium/ ammonia reduction of arihydro- ff- rotunol

Approximately 0.01 part lithium was added to 60 parts liquid ammonia which was stirred and cooled to -70° and protected with nitrogen. After 10 minutes of stirring, the blue color that initially developed began to fade. Another 0.016 part of lithium was added, followed immediately by the addition of a solution of 0.218 part of (+)anhydro-O-rotunol,

0.074 part t-butanol and 10 parts diethyl ether at such a rate that the blue coloration was not destroyed. 1 part of ammonium chloride was added to the reaction mixture after stirring for 30 minutes at

-70°, and the mixture was warmed to room temperature to remove ammonia. Water was added, and the resulting mixt

was ether extracted. The organic phase was subjected to the usual treatments of washing, drying and evaporation and yielded 0.219 part of an evaporation residue. which contained spirovetiva-1(10), 11-dien-2-pn as the major product, together with a trace of anhydro-/3-rotunol.

The purified product obtained by column chromatography proved, by means of C NMR, to be a diastereoisomeric mixture of spirovetivones comprising all four possible

diastereoisomers in the relative amounts of Isis3:3. As shown by the data in Table 2, one of the minor isomers had identical spectral characteristics to naturally occurring solavetivon,

which was confirmed by spectral "spiking" experiments.

Example 10

3R- and 3S- hydroxy- spirovetiva- l( 10), ll- diene- 2-ones (a) From solavetivon via the trimethylsilylenol ether (see Rubottom et al. Tetrahedron Letters 1974, pp. 4319-4322).

A solution of lithium diisopropylamide was prepared by adding under nitrogen .4.3 parts of a 1.5n solution of butyllithium in n-hexane to a stirred solution of 0.61 parts of diisopropylamine in 10 parts of dry THF at -30°. After 10 minutes of stirring at -30°, a solution of 1.09 parts of solavetivon in 20 parts of THF was added, and the resulting reaction mixture was stirred for an additional 45 minutes at -30° to -50°.

Separately, 0.922 part of trimethylchlorosilane was added by syringe through a serum jacket to a solution of 0.25 part of triethylamine in 20 parts of dry THF. The precipitated amine hydrochloride was consolidated by centrifugation, and the supernatant solution was added to the above reaction solution at -30°. The reaction mixture was allowed to warm to room temperature within 1 hour. Hexane was added to the reaction product, and the mixture was washed with ice-cold bicarbonate solution, followed by water, and the organic phase was dried over sodium sulfate. Removal of solvent after filtration gave 1.43 parts of an oily material characterized as the trimethylsilylenol ether of solavetivon.

A solution of the crude TMS enol ether in 20 parts n-hexane was added to a stirred suspension of 0.91 part m-chloroperbenzoic acid in 40 parts n-hexane cooled to -10°. After stirring for 1 hour at -10°, the reaction mixture was filtered and evaporated. The crude product, 1.6 parts, was found to contain 3-trimethylsiloxy-solavetivon as the main component.

Without purification, the crude 3-trimethylsiloxy solavétivone was taken up in 40 parts of diethyl ether and stirred vigorously with 20 parts of 1.5N HCl solution. After stirring for 10 hours, the mixture was separated and the acidic layer extracted with diethyl ether. The combined ether extracts were washed with sodium bicarbonate solution, followed by water, dried over sodium sulfate and filtered. Removal of solvent gave 1.08 parts of an oily residue, which after purification by column chromatography over neutral alumina (eluent 1s 15s85, ethyl acetate cyclohexane) gave 0.35 part (30% total) of 3-hydroxy-spirovetiva-1(10) ,11-dien-2-one isolated as a colorless oil, with the following spectral characteristics: MS: M<+>234 (C15H22O2): m/e 219, 216, 205, 176, 161, 148,

133, 109, 108, 91, 79, 68, 67, 41, 39.

IR (CC14): 3490, 3080, 1680, 1645, 1610, 885 cm"<1>

UV (EtOH): A240 nm ( 6 = 10,200)

PMR (CDClg): The spectrum indicated that the sample was a mixture of C-3 epimers, of which In was identical to aglycone A2, isolated after hydrolysis from tobacco fractions. Chemical shift PMR data (&ppm from TMS) and their designations are shown in Table 3.

The H spectrum also indicated that the ratio between the epimers SA1:SA2 13 was of the order of 1:2. C NMR studies confirmed this relationship, and the corresponding chemical shift data are shown in Table 4 below. No attempt was made to separate the epimers in this step. (b) From solavetivon by molybdenum peroxide complex reaction (see Vedijs Journal bf the American Chemical Society 1974, p. 5944).

A solution of lithium diisopropylamide was prepared by adding 4.3 parts of a 1.5N solution of butyllithium in n-hexane to 0.61 part of diisopropylamine in 10 parts of dry tetrahydrofuran at -40° under nitrogen stirring. After 10 minutes of stirring at -40°, a solution of 1.09 parts of solavetivon in 20 parts of dry tetrahydrofuran was added and the solution stirred at -40° for 30 minutes. After further cooling to -70°, 2.82 parts of powdered molybdenum peroxide were added (the complex MoOg.Py.HMPAs abbreviated to MoOPH was used) and the mixture stirred for 1 hour at

-70°. The mixture was allowed to warm to room temperature, water was added and the mixture was extracted with ether. They combined

extracts, were washed with 5% sodium carbonate, 5% HCl solution and water, dried and the solvent removed after filtration.

The oily residue, 0.76 parts, was chromatographed over neutral alumina (eluent 1: 15:85 ethyl acetate:cyclohexane) to give 0.22 parts of 3-hydroxy-solavetivon (20% yield).

This material exhibited the spectral characteristics

for the product obtained by route (a). However, indi-

13

kerte H and C data that the ratio of the epimers SAl:SA2

in this product was 1:3.

Example 11

3R- and 3S-(j3-D-glucosyloxy)-spirovetiva-1 (10), 11-dien-2-ones

(Synthetic Gl and G2 - designated SGl and SG2)

To a stirred solution of 0.47 part of synthetic 3-hydroxy-spirovetiva-l(lO),ll-dien-2-ones (SAl and SA2) in 50 parts of dry 1,2-dichloroethane was added 1.8 parts of freshly prepared powdered sjSSlv carbonate. The system was flushed with nitrogen and heated to reflux. The volume of the reaction mixture was reduced to half by distillation, and an iodine crystal was added. 2.25 parts of 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide in 300 parts of 1,2-dichloroethane were added dropwise to the stirred solution over the course of 3 hours, and during this time the solution the agent distills over at approximately one speed. A further 200 parts of 1,2-dichloroethane were added dropwise with simultaneous removal by distillation. The reaction mixture was filtered and the solvent removed to give 2.9 parts of a syrupy residue.

This residue was dissolved in 350 parts methanol and treated with a solution of 2.9 parts potassium bicarbonate in 120 parts water. The reaction mixture was flushed with nitrogen and then allowed to stand at room temperature for 5 days. The solution was concentrated to remove methanol, and the aqueous phase was extracted sequentially with (I) ether, (II) chloroform, (III) chloroform:methanol (9:1) and (IV) chloroformmethanol (2:1). High-speed liquid chromatography detected the desired glucosides in fractions (II) and (III), which after freeze-drying gave 0.13 parts of amorphous material (yield in total

about. 16%). Preparative pressure liquid chromatography (phase-silica-dioxide: solvent system - dichloroethane (85), acetonitrile (8.5), ethanol (8.5), water (0.6)) gave resolution of this fraction bg glucosides, SGl, 0, 04 part and SG2, 0.05 part, were obtained as colorless, freeze-dried solids.

The retention times (namely TMS/GC and HSLC) of SG1 and SG2 were in good agreement with the values for the naturally occurring glucosides, G1 and G2, respectively. Satisfactory • spectral data were also obtained, and the C NMR chemical shift data are shown in Table 4.

Example 12

11R- and 11S- 12- dihydroxy- Bpyrovetiva- 1( 10),- en- 2-ones

0.5 part osmium tetroxide in 25 parts diethyl ether was added to a solution of 0.335 part solavetivon in 25 parts diethyl ether. The solution was kept at 0° for 2 days. The solvent was removed and 40 parts ethanol added. The osmate ester was cleaved by heating on a water bath with a solution of sodium sulphite (8 parts in 80 parts water). The precipitate was removed by filtration and extracted with hot ethanol. The combined filtrates were extracted with chloroform, and the organic phase was washed and dried in the usual manner. Removal of solvent gave an oily residue, 0.32 part, which was chromatographed over neutral alumina (eluent 1: chloroform:hexane 1:1) to give 11,12-dihydroxy-spirovetiva-1(10),-en-2 -on, 0.26 part

(67% yield), as a colorless, viscous oil. The product showed the following spectral characteristics: MS: M<+>2<52>(<C>1<5>H24°3)* m/e 221'161'137'107'91'

84, 79, 75, 44, 43 (100%) 41.

IR ("neat"): 3430, 1655, 1610, 1045 cm"<1>

UV (EtOH): Xmax. 243 nm (E = 14,000)

PMR (CDCl3): 0.96 (3H, d, J = 7Hz), 1.17 and 1.20 (both 3H, S,

which indicated an approximately equal mixture of pyrites): 1.95 (3H, d, J = lHz ) : 3.45 (2H, q);

5.75 (lH, extended s) h ppm

<13>C NMR (CDCl3 ) : 119.6, 167.3 and 167.5, 125.5 and. 125.6, 73.7, 69.4,

50.2 and 49.9, 46.1, 42.9, 38.7 and 38.5, 36.3 and 37.0, 33.9 and 34.1, 28.3 and 27.4, 21.9 and 21.7, 21.1, 15.9 h ppm

Examination of relative peak intensities indicated that the product was an approximately 1:1 mixture of the C-11 epimers, namely 11-R- and 11-S-12-dihydroxy-spirovetiva--1(10) ,-en-2- oners.

Example 13

11R- and llS- hydroxy- 12( ff- D- glucosyloxy)- spirovetiva- 1( 10),- en-2-ones ( SG4 and epimerl

To a stirred solution of 0.25 parts of synthetic 11,12-dihydroxy-spirovetiva-1(10),-en-2-ones, in 25 parts of dry 1,2-dichloroethene was added 0.9 parts of freshly prepared, powdered silver -carbonate. The system was flushed with nitrogen and heated to reflux. The volume of the reaction mixture was reduced to half by distillation, and an iodine crystal was added. 1.13 parts of 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide in 150 parts of 1,2-dichloroethane was added dropwise to the stirred solution over 3 hours, during which time the solvent distill over at an approximately constant rate. A further 100 parts of 1,2-dichloroethane were added dropwise with simultaneous removal by distillation. The reaction mixture was filtered and the solvent removed to give 1.3 parts of a syrupy residue.

This residue was dissolved in 175 parts methanol and treated with a solution of 1.3 parts potassium bicarbonate in 60 parts water. The reaction mixture was flushed with nitrogen and then allowed to stand at room temperature for 5 days. The solution was concentrated to remove methanol, and the aqueous phase was extracted sequentially with (I) ether, (II) chloroform, (III) chloroform-methanol (9:1) and (IV) chloroform-methanol (2:1). The desired glucosides were detected in fractions (II) and (III) by high-speed liquid chromatography. These fractions were combined and freeze-dried and then gave 0.8 parts of 11-hydroxy-12-(Ø-D-glucosyloxy)spirovetiva-1(10),-en-2-ones (total yield approx. 19%).<13 >C NMR data for this material is shown below and confirmed the designations and indicated the presence of both C-II epilayers in approximately equal amounts.

<13>C NMR (Dg - DMSO): 197.8, 167.3, 124.6, 103.5, 76.8, 76.8,

74.0 and 73.1, 73.6, 71.6 and 71.3, 70.1, 61.2, 49.5, 45.3, 42.7, 33.3, 26.8, 23, 8 and 23.1, 20.5 and 15.6 h ppm.

Carbon atoms 4 and 6 were masked by solvent resonances.

Claims (30)

1. Substance in concentrated form, characterized in that it essentially consists of a hydroxy- or glycosyloxy-mono- or di-unsaturated spirovetione. 2. Substance as specified in claim 1, characterized in that it essentially consists of a compound of the formula where one of the radicals R^ and Rj is hydrogen and the other represents either a hydroxyl group. or a glycosyloxy group -OX where X is the radical of a mono- or disaccharide and the dashed lines represent possible positions for a double bond. 3.. Substance as stated in claim 1, characterized by the fact that it essentially consists of a compound of the formula where R^ represents either a hydroxyl group or a glycosyloxy group -OX where X is the radical of a mono- or disaccharide. 4. Substance as stated in claim 1, characterized in that the glycosyloxy compound originates from a hexose. 5. Substance as stated in claim 4, characterized in that the glycosyloxy compound is a glucoside. 6. Substance as stated in claim 5, characterized in that the glycosyloxy compound is a/3-D-glucoside. 7. Substance as stated in claim 1, characterized in that it comprises a glycosyloxy compound of the formula where G represents a /3-D-glucosyl group. 8. Substance as stated in claim 1, characterized in that it comprises a glycosyloxy compound of the formula where G represents a /3-D-glucosyl group. 9. Substance as stated in claim 1, characterized in that it comprises a glycosyloxy compound of the formula where G represents a/ 3-D-glucosyl group. 10. Substance as stated in claim 1, characterized in that it comprises a hydroxy compound of the formula 11. Substance as stated in claim 1, characterized in that it comprises a hydroxy compound of the formula 12. Substance as specified in claim 1, characterized in that it comprises a hydroxy compound of the formula 13. Substance as specified in claim 1, characterized in that it is present in practically pure form. 14. Substance as stated in claim 1, characterized in that it has a <13> C NMR spectrum which exhibits a characteristic resonance at 50.6 - 0.1 ppm in relation to tetramethylsilane. 15. Smoking material comprising tobacco, reconstituted tobacco or a tobacco substitute or a mixture thereof and which has added from 0.01 to 2% by weight of a substance as specified in claim 1. 16. Smoking material as specified in claim 15, characterized in that they contain from 0.1 to 1% by weight of the substance. 17.. Smoking material as stated in claim 15, characterized in that it comprises a tobacco substitute where the principal smoke-producing fuel is a modified carbohydrate. 18. Smoking material as stated in claim 17, characterized in that the modified carbohydrate is a cellulose ether. 19. Smoking material as stated in claim 18, characterized in that the cellulose ether is sodium carboxymethyl cellulose. 20. Smoking material as stated in claim 17, characterized in that the modified carbohydrate is a heat- broken down carbohydrate. 21. Smoking material as stated in claim 20, characterized in that the heat-decomposed carbohydrate is heat-decomposed cellulose. 22. Smoke material as stated in claim 15, characterized in that it further comprises protein, the ratio between protein and smoke-producing fuel being in the range 1:1 to 1:60. 23. Process for the production of a glycosyloxy-mono- or -di-unsaturated spirovetione, characterized by condensing an O-acyl glycosyl halide with a hydroxy-mono- or -di-unsaturated spirovetione and then hydrolyzing the O-acyl groups in the product to hydroxy groups. 24. Method as stated in claim 23, characterized in that the O-acylglycosyl halide is tetraacetoxy-glucosyl bromide. 25. Method as stated in claim 23, characterized in that the hydroxy-spirovetivone has the formula where one of the radicals R. and R2 represents hydrogen and the other represents OH and the dashed lines represent possible positions for a double bond. 26. Method as stated in claim 23, characterized in that the hydroxy-spirovetivone has the formula 27. Method as stated in claim 23, characterized in that the spirovetivone has the formula and is made by treating the enolate of an enf compound of the formula with a trimethylhalosilane to produce a trimethylsilyl ether, reacting the product with an organic percarboxylic acid and hydrolysis of the product, to remove the trimethylsilyl group therefrom. 28. Method as stated in claim 27, characterized in that the trimethylhalosilane is trimethylchlorosilane and that the organic percarboxylic acid is m-chloroperbenzoic acid. 29. Method as stated in claim 23, characterized in that the spirovetivone has formula and is made by treating the enolate of a compound of the formula with a complex formed by a molybdenum peroxide, an organic tertiary amine and a hexamethylphosphoramide. 30. Method as stated in claim 23, characterized in that the spirovetivone has formula and is made by treating a compound of the formula with osmium tetroxide to form an osmate ester and cleavage of the product in a known manner for cleavage of osmate 1,2-esters, thereby forming the corresponding 1,2-diol.