SYNTHESIS AND PURIFICATION OF CANNABIGEROL AND ITS APPLICATION

Abstract

The present invention relates to a method for producing cannabigerol and purifying it from a reaction mixture. The present invention also relates to the cosmetic use of cannabigerol for the inhibition of tyrosinase activity and/or the reduction of melanin production in the skin, in particular for reducing pigmentation of the skin, preferably for the improvement of the appearance of the skin in case of hyperpigmentation, lentigo or vitiligo. Furthermore, the present invention relates to cannabigerol for use in a therapeutic method for the inhibition of tyrosinase activity and/or the reduction of melanin production in the skin, preferably for use in a therapeutic method for the treatment and/or prevention of malign skin disorders, in particular skin cancer.

Claims

1. A method for producing cannabigerol comprising the: (i) reacting olivetol with one or a mixture of two or more allylic compound(s) having a leaving group in the presence of an acidic or lewis acidic catalyst, and (ii) purifying the product by distillation or liquid-liquid extraction, wherein the allylic compound having a leaving group or at least one of the allylic compounds having a leaving group is a compound of formula (I) and/or (II) ##STR00033## wherein X represents the leaving group.

2. The method according to claim 1, wherein the olivetol is reacted with the one or the mixture of two or more allylic compound(s) having a leaving group in a molar ratio of from 1:1 to 1:2, (olivetol:allylic compound(s)).

3. The method according to claim 1, wherein the allylic compound of formula (I) and/or (II) is geraniol and/or linalool.

4. The method according claim 1, wherein (i) comprises: (ia) reacting olivetol with the compound of formula (I) and/or (II) in a molar ratio of from 1:0.5 to 1:1.2 (olivetol:compound of formula (I) and/or (II)) to form a mixture, and (ib) reacting the mixture of (ia) with a steric allylic compound having a leaving group in a molar ratio of 0.2:1 to 1:1 (steric allylic compound:olivetol), wherein the allylic residue of the steric allylic compound having a leaving group comprises at least 5 carbon atoms provided that if the allylic compound comprises 10 or less carbon atoms it includes a ring or a bridged ring system.

5. The method according to claim 4, wherein the olivetol is reacted with a sum of the compound of formula (I) and/or (II) and the steric allylic compound having a leaving group in a molar ratio of from 1:1 to 1:2 (olivetol:(compound of formula (I) and/or (II)+steric allylic compound)).

6. The method according to claim 4, wherein the allylic compound of formula (I) and/or (II) is geraniol and/or linalool and/or the steric allylic compound having a leaving group is menthadienol.

7. The method according to claim 1, wherein the reaction of (i) is conducted as a batch or continuous flow reaction process.

8. The method according to claim 1, wherein the acidic or lewis acidic catalyst is chosen from p-TsOH, formic acid, acetic acid, butyric acid, oxalic acid, phosphoric acid, sulfuric acid, benzoic acid, AlCl.sub.3, ZnCl.sub.2, FeCl.sub.3, AlBr.sub.3, (CF.sub.3SO.sub.3).sub.2Zn, BF.sub.3*Et.sub.2O, BF.sub.3*2CH.sub.3COOH, BF.sub.3*CH.sub.3OH, BF.sub.3*THF, CH.sub.3SO.sub.3H, and aluminium isopropoxide.

9. The method according to claim 1, wherein the method further comprises: (iii) crystallizing the product of (ii).

10. A method for inhibiting tyrosinase activity and/or reducing melanin production in skin comprising applying cannabigerol or salt thereof to the skin.

11. A cosmetic composition comprising cannabigerol or a salt thereof.

12. A method for treating or preventing malign skin disorders comprising applying the cosmetic composition of claim 11 to the skin.

13. The method of claim 12, wherein the malign skin disorder is a skin cancer.

14. The method of claim 10, wherein the method reduces pigmentation of the skin.

15. The method of claim 2, wherein the molar ratio is from 1:1.2 to 1:1.9 (olivetol:allylic compound(s)).

16. The method of claim 4, wherein the molar ratio of (1a) is from 1:0.9 to 1:1.1 (olivetol:compound of formula (I) and/or (II)) and the molar ratio of (ib) is from 0.3:1 to 0.5:1 (steric allylic compound:olivetol).

17. The method of claim 5, wherein the molar ratio is from 1:1.2 to 1:1.9 (olivetol (compound of formula (I) and/or (II)+steric allylic compound)).

18. The method according to claim 5, wherein the allylic compound of formula (I) and/or (II) is geraniol and/or linalool and/or the steric allylic compound having a leaving group is menthadienol.

19. The method of claim 7, wherein the reaction of (i) is conduced as a continuous flow reaction process.

Description

SHORT DESCRIPTION OF THE FIGURES

[0087] FIG. 1: Schematic overview of the extraction process

[0088] FIG. 2: Biological effects of cannabigerol (CBG) on tyrosinase activity in B16 melanocytes.

[0089] FIG. 3: Biological effects of cannabigerol (CBG) on melanin production in B16 melanocytes.

EXAMPLE 1: PRODUCTION OF OLIVETOL

[0090] Olivetol methyl ester (6) with the IUPAC name of methyl 2,4-dihydroxy-6-pentyl-benzoate is the starting material for all synthetic approaches.

##STR00022##

[0091] Olivetol (2) is obtained by saponification with subsequent decarboxylation from olivetol methyl ester (6). The saponification is done under basic conditions with potassium hydroxide in water. Solvation problems of the staring material in the beginning disappear after warming up to 50-60° C. The reaction is refluxed for 3 h with a constant separation of the generated methanol by distillation. Polymerisation can be prevented by a short heating period and high concentration. The obtained raw product can be purified by distillation to give a white/yellow solid with an overall yield of 94% and a purity of 99.4%. (Scheme 4)

##STR00023##

[0092] A suspension of olivetol methyl ester (6) (200 g, 839 mmol, 1.00 eq.), KOH (226 g, 4.03 mol, 4.80 eq.) in 600 ml of water was stirred at 400 rpm (exothermic). After solvation at 50-60° C. the reaction solution turned yellow/red. The resulting solution was stirred at 95-100° C. for 2.5 h and monitored by HPLC. After complete saponification the reaction was cooled to 20° C. and 400 mL water and 400 mL ethyl acetate were added. A pH of 4-5 was adjusted with conc. HCl (300 g in 200 mL water). The extensive foam production due to the CO.sub.2 development was reduced by the ethyl acetate. After extraction with ethyl acetate, the combined organic phase was washed with brine, dried over sodium sulfate and concentrated in vacuo to give the crude product. The residue was purified by distillation (1.7 mbar 150° C.) to give olivetol (2) (slight yellow solid, 142 g, 94% yield).

[0093] R.sub.f: 0.67 (1:1 ethyl acetate/cyclohexane) Anisaldehyde

[0094] .sup.1H NMR (400 MHz, Chloroform-d) δ=6.23 (d, J=2.1 Hz, 2H), 6.15 (t, J=2.2 Hz, 1H), 5.05 (brs, 2H, OH), 2.38 (dd, J=8.9, 6.7 Hz, 2H), 1.48 (p, J=7.2 Hz, 2H), 1.34-1.15 (m, 4H), 0.84 (t, J=6.9 Hz, 3H) ppm.

[0095] .sup.13C NMR (101 MHz, Chloroform-d) δ=156.2, 146.4, 108.2, 100.2, 35.8, 31.5, 30.7, 22.5, 14.0 ppm.

EXAMPLE 2: INCREASING THE REGIO SELECTIVITY BY BLOCKING ONE POSITION BY AN ESTER

[0096] In a strategic attempt to minimize the competitive alkylation at the regio position, olivetol methyl ester (6) was used as a starting material in the Friedel-Crafts-Alkylation. The ester function blocks one unwanted reaction center. This leads to a smaller amount of the regio isomer (16) and is resulting in less regioisomer (5) after saponification and decarboxylation. The complete suppression of the regio isomer could not be achieved as well as other accumulating byproducts made a separation more challenging. Complete conversion of the starting material could not be achieved. Extra addition of catalyst as well as allylic alcohol (3) brought negligible progress. (Scheme 5)

[0097] The first reaction step of the Friedel-Crafts-Alkylation is done with boron trifluoride etherate and geraniol (3) in dichloromethane. Due to the impurities several separation techniques were used. At first the crude product was purified by distillation from the low boiling remains of geraniol (3) as well of the high boiling polyalkylated byproducts. Two flash silica gel column chromatographies and one crystallization enabled to obtain the pure cannabigerol methyl ester (7) in a pure form with an overall yield of 6%.

##STR00024##

[0098] The saponification and decarboxylation of the cannabigerol methyl ester (7) was established under basic conditions with potassium hydroxide in methanol/water.

[0099] Alkylation:

[0100] To a solution of olivetol methyl ester (6) (5.00 g, 21.0 mmol, 1.00 eq.) and geraniol (3) (10.0 g, 64.8 mmol, 3.08 eq.) in 100 mL dichloromethane was added boron trifluoride etherate (2.98 g, 3 mL, 21.0 mmol, 1.00 eq.) at 22° C. over 1.5 h under vigorous stirring. Simultaneously to the addition of the catalyst, three portions of geraniol (3×2.00 g, 19.5 mmol, 1.08 eq.) were added (distributed over that time). The reaction was quenched by adding aq. NaOH (1 M, 200 mL) and vigorously stirred for 30 min. The organic phase was separated and the aqueous phase extracted with ethyl acetate (3×100 mL). The combined organic layer was washed with brine, dried over sodium sulfate and concentrated in vacuo to give the crude product. The crude mixture was purified by distillation (Kugelrohr distillation 0.4 mbar, 200-280° C.). The product fraction (7) was further purified by two flash column chromatography's (gradient: cyclohexane->3:7 cyclohexane: ethyl acetate) to obtain a yellow oil (487 mg, 1.30 mmol, 6% yield).

[0101] .sup.1H NMR (400 MHz, Chloroform-d) δ=12.02 (s, 1H), 6.23 (s, 1H), 5.92 (s, 1H), 5.31-5.23 (m, 1H), 5.08-5.01 (m, 1H), 3.91 (s, 3H), 3.43 (d, J=7.1 Hz, 2H), 2.83-2.77 (m, 2H), 2.14-2.01 (m, 4H), 1.82-1.78 (m, 3H), 1.69-1.65 (m, 3H), 1.59 (s, 3H), 1.56-1.47 (m, 2H), 1.37-1.29 (m, 4H), 0.93-0.87 (m, 3H) ppm.

[0102] .sup.13C NMR (101 MHz, Chloroform-d) δ=172.5, 162.5, 159.5, 145.8, 138.9, 132.0, 123.8, 121.5, 111.5, 110.8, 104.5, 51.8, 39.7, 36.8, 32.1, 31.6, 26.4, 25.7, 22.5, 22.1, 17.7, 16.2, 14.1 ppm.

[0103] Saponification/Decarboxylation:

[0104] KOH (540 mg, 9.16 mmol, 8.00 eq.) was dissolved in water (5 ml). Cannabigerolic ester (7) (450 mg, 1.20 mmol, 1.00 eq.) was dissolved in methanol (0.5 mL) and added to the solution. The resulting red/yellow solution was stirred at 95-100° C. for 3 h and monitored by HPLC. The added and newly developing methanol was distilled off during the process. After complete saponification the reaction was cooled to 25° C. and additional water (50 mL) and ethyl acetate (50 mL) were added. A pH of 3 was adjusted with conc. hydrochloric acid. After extraction with ethyl acetate, the combined organic phase was washed with brine, dried over sodium sulfate and concentrated in vacuo to give the crude product. The residue was purified by silica column chromatography to give cannabigerol (1) (191 mg, 0.60 mmol, 50% yield) as a slightly yellow/white solid.

[0105] R.sub.f: 0.52 (1:1 cyclohexane/ethyl acetate) KMnO.sub.4

[0106] .sup.1H NMR (600 MHz, Chloroform-d) 5=6.24 (s, 2H), 5.27 (th, J=7.1, 1.3 Hz, 1H), 5.07 (s, 2H), 5.05 (thept, J=7.0, 1.4 Hz, 1H), 3.39 (d, J=7.1 Hz, 2H), 2.48-2.41 (m, 2H), 2.13-2.08 (m, 2H), 2.08-2.03 (m, 2H), 1.81 (q, J=1.0 Hz, 3H), 1.67 (q, J=1.2 Hz, 3H), 1.59 (d, J=1.3 Hz, 3H), 1.58-1.53 (m, 2H), 1.36-1.25 (m, 4H), 0.88 (t, J=7.0 Hz, 3H) ppm.

[0107] .sup.13C NMR (151 MHz, Chloroform-d)=δ 154.8, 142.8, 138.9, 132.1, 123.8, 121.7, 110.6, 108.4, 39.7, 35.5, 31.5, 30.8, 26.3, 25.7, 22.6, 22.3, 17.7, 16.2, 14.0 ppm.

EXAMPLE 3: INCREASING THE REGIO SELECTIVITY IN BLOCKING ALL UNWANTED POSITIONS BY HALOGENATION

[0108] In this strategic attempt to block the wrong regio position in favor of a selective alkylation, halogens can be used. Bromine is preferred due to its unproblematic substitution. In terms of the reactivity, bromine has a reactivity advantage over SO.sub.3H, NO.sub.2, COOH, COOMe (see example 2). Bromine is a substituted first order and is only slightly deactivating the aromatic ring and therefore the alkylation. The reversibility of the bromination in the presence of acceptor groups enables its usage as a protecting group (Effenberger: Angew. Chem., 2002, 114). (Scheme 6)

##STR00025##

[0109] Bromination of Olivetol (2) (WO2017/011210 A1)

[0110] The selective double bromination of olivetol (2) with bromine in dichloromethane proceeds selectively at cold temperatures. A limitation of cooling below −30° C. is the solubility of the olivetol (2). After crystallization a pure dibrominated olivetol (8) could be obtained in a yield of 73%. (Scheme 7)

##STR00026##

[0111] To a solution of olivetol (2) (20.0 g, 111 mmol, 1.00 eq.) in 600 mL dichloromethane was added bromine (35.5 g, 11.4 mL, 222 mmol, 2.00 eq.) at −28 to −30° C. for 30 min. After additional 15 min of stirring the reaction was quenched by addition of sodium bicarbonate (200 mL) and the mixture was allowed to warm up to rt. Brine (200 mL) was added and the phases were separated. The aqueous phase was extracted with dichloromethane (2×100 mL). The combined organic phase was dried over sodium sulfate and concentrated in vacuo to give the raw product. The solid raw product was dissolved by heating in heptane (150 mL) and crystallized at −15° C. The obtained white crystalline solid product (8) (27.4 g, 81.0 mmol, 73%) was directly used in the next reaction step.

[0112] Rf: 0.53 (1:1 ethyl acetate/cyclohexane) KMnO.sub.4

[0113] .sup.1H NMR (400 MHz, Chloroform-d)=δ 6.65 (s, 1H), 5.66 (s, 2H), 2.96-2.89 (m, 2H), 1.54 (dddd, J=8.5, 5.5, 2.1, 1.1 Hz, 2H), 1.47-1.33 (m, 4H), 0.96-0.90 (m, 3H) ppm.

[0114] .sup.13C NMR (101 MHz, CDCl3) δ=152.6, 141.4, 104.1, 100.7, 37.6, 31.8, 27.7, 22.4, 14.0 ppm.

[0115] Friedel-Crafts-Alkylation of Brominated Olivetol (2):

[0116] The alkylation was done with the brominated olivetol (8) and geraniol (3). Three successful procedures are shown in Scheme 8. They differ in the choice of the catalyst and the reactor type and therefore the method of operation (batch and continuous reactions).

##STR00027##

[0117] Due to the slight deactivation of the aromatic ring by the two bromines the reaction under acidic catalysis with p-TsOH proceeded slowly (example A). The conversion of the starting material in this case took a week and gave the wanted product (9) after chromatography in a yield of 44%. The use of a strong lewis acid as boron trifluoride etherate enables a rapid transformation in 30 min of the starting material towards the product (example B). The downside to this approach was the higher amount of byproducts. After chromatography the pure product (9) could be obtained in 22% yield. The fast transformation with a strong lewis acid was repeated in a continuous reaction environment, which led to an improvement (example C) in yield.

[0118] A:

[0119] To a solution of brominated olivetol (8) (2.00 g, 5.92 mmol, 1.00 eq.), geraniol (3) (1.64 g, 10.7 mmol, 1.80 eq.) in 200 mL dichloromethane was added at rt p-TsOH (51 mg, 0.29 mmol, 0.05 eq.). The solution was stirred for 7 days and quenched by addition of brine (150 mL). The layers were separated and the organic layer was washed with brine. The organic layer was dried over sodium sulfate and concentrated in vacuo to give the crude product. After purification by silica flash column chromatography (gradient: cyclohexane/ethyl acetate) the pure product (1.24 g, 2.61 mmol, 44%) could be obtained as slightly yellow solid.

[0120] B:

[0121] To a solution of brominated olivetol (8) (10.0 g, 29.5 mmol, 1.00 eq.), geraniol (3) (13.7 g, 88.7 mmol, 3.00 eq.) in 200 mL dichloromethane at 15° C. was added boron trifluoride etherate (2.27 g, 2.0 mL, 16.0 mmol, 0.54 eq.) over 25 min. The solution was stirred for 10 min and then quenched by the addition of NaOH (100 ml, 1 M). The layers were separated and the organic layer was washed with brine. The organic layer was dried over sodium sulfate and concentrated in vacuo to give the crude product. After purification by silica flash column chromatography (gradient: cyclohexane/ethyl acetate) the pure product (3.15 g, 6.64 mmol, 22%) could be obtained as slightly yellow solid.

[0122] C:

[0123] A continuous reactor with a volume of 18 mL was fed with starting material solution A and catalyst solution B. The starting material solution A contained brominated olivetol (8) (5.00 g, 14.7 mmol, 1.00 eq.) and geraniol (3) (2.97 g, 19.2 mmol, 3.34 eq.) in 40 ml dichloromethane. The catalyst solution B contained boron trifluoride etherate (1.26 mg, 8.87 mmol, 0.60 eq.) in 40 mL dichloromethane.

[0124] Starting material solution A was pumped with 24 mL/min and catalyst solution B was pumped with 12 ml/min while the reaction was stirred at 1300 rpm. The reaction mixture left the reactor with a flow of 36 ml/min through a 1.5 m long PTFE-hose (20 mL volume) into a aq. sat. sodium bicarbonate solution.

[0125] The quenched mixture was stirred for 15 min. Cyclohexane (150 mL) was added and the phases were separated. The aqueous phase was extracted twice with cyclohexane. The combined organic phase was dried over sodium sulfate and concentrated in vacuo. The crude product was purified by silica flash column chromatography and gave the product (9) (2.24 g, 4.72 mmol, 32%) as a yellow solid.

[0126] R.sub.f: 0.64 (1:10 ethyl acetate/cyclohexane) KMnO.sub.4

[0127] .sup.1H NMR (400 MHz, Chloroform-d) δ=5.78 (s, 2H), 5.23 (tq, J=7.1, 1.3 Hz, 1H), 5.05 (tdt, J=5.8, 2.9, 1.4 Hz, 1H), 3.49-3.44 (m, 2H), 2.93-2.86 (m, 2H), 2.12-2.03 (m, 2H), 2.03-1.96 (m, 2H), 1.79 (d, J=1.3 Hz, 3H), 1.65 (t, J=1.4 Hz, 3H), 1.57 (d, J=1.3 Hz, 3H), 1.56-1.48 (m, 2H), 1.39 (ttd, J=7.2, 4.0, 3.1, 1.6 Hz, 4H), 0.96-0.88 (m, 3H) ppm.

[0128] .sup.13C NMR (101 MHz, CDCl3) δ=150.3, 138.3, 137.1, 131.5, 124.1, 121.0, 113.5, 104.3, 39.7, 37.5, 31.8, 27.9, 26.6, 25.7, 24.4, 22.4, 17.7, 16.2, 14.0 ppm.

[0129] Dehalogenation

[0130] A dehalogenation strategy was used to obtain the product cannabigerol (1) from the halogenated species (9) by heating the intermediate (9) in the presents of sodium sulfite, ascorbic acid and a base. (Scheme 9)

##STR00028##

[0131] To a solution of brominated CBG (9) (750 mg, 1.58 mmol, 1.00 eq.) in methanol (10 mL) was added a solution of sodium sulfite (1.99 g, 15.8 mmol, 10.0 eq.) and ascorbic acid (50 mg, 0.32 mmol, 0.20 eq.) in water (10 mL). Triethylamine (0.80 g, 1.1 mL, 7.90 mmol, 5.00 eq.) was added to the brown milky mixture which turned purple. The mixture was refluxed for 24 h and quenched by adding HCL (1M) at rt, followed by ethyl acetate (100 mL) and brine (100 ml). The phases were separated and the aqueous phase was extracted with ethyl acetate (3×30 mL). The combined organic layers were dried over sodium sulfate and concentrated in vacuo to give the crude product. The crude product (1) was purified by silica flash column chromatography (1:20 ethyl acetate/cyclohexane) to give a white solid product (261 mg, 0.82 mmol, 52%).

[0132] R.sub.f: 0.52 (1:1 cyclohexane/ethyl acetate) KMnO.sub.4

[0133] .sup.1H NMR (600 MHz, Chloroform-d) δ=6.24 (s, 2H), 5.27 (th, J=7.1, 1.3 Hz, 1H), 5.07 (s, 2H), 5.05 (thept, J=7.0, 1.4 Hz, 1H), 3.39 (d, J=7.1 Hz, 2H), 2.48-2.41 (m, 2H), 2.13-2.08 (m, 2H), 2.08-2.03 (m, 2H), 1.81 (q, J=1.0 Hz, 3H), 1.67 (q, J=1.2 Hz, 3H), 1.59 (d, J=1.3 Hz, 3H), 1.58-1.53 (m, 2H), 1.36-1.25 (m, 4H), 0.88 (t, J=7.0 Hz, 3H) ppm.

[0134] .sup.13C NMR (151 MHz, Chloroform-d)=δ 154.8, 142.8, 138.9, 132.1, 123.8, 121.7, 110.6, 108.4, 39.7, 35.5, 31.5, 30.8, 26.3, 25.7, 22.6, 22.3, 17.7, 16.2, 14.0 ppm.

EXAMPLE 4: DIRECT SYNTHESIS OF CANNABIGEROL (1)

[0135] In a direct synthesis of cannabigerol (1) via a Friedel-Crafts-Alkylation, olivetol (2) is alkylated with geraniol (3) or linalool (4) under acidic or lewis acid catalysis. The resulting product mixture can contain the product CBG (1), regio isomer (5), polyalkylation byproducts, remaining starting material (2) and minor impurities. It is a complex mixture which requires more than one separation technique, when chromatography should be excluded (due to costs).

[0136] Main Strategy Using Menthadienol (13) for Derivatization of the Byproduct

[0137] Olivetol (2) alkylated with equimolar amounts of geraniol (3) under catalysis of p-TsOH in toluene gives CBG (1) and the regio isomer (5) as the main compound. The product and byproduct are formed in a 1:1 ratio. Remaining olivetol (2) can be separated by extraction.

[0138] This CBG-(1)-regioisomer (5)-mixture is the starting point of this strategy. The regio isomer (5) and CBG (1) are similar in their physical properties (e.g. boiling point, polarity), which prevents the use of distillation and extraction techniques.

[0139] However, the chemical properties can be differentiated through chemical reaction like alkylations. The regioisomer (5) bears the geranyl and pentyl residue on the same side and leaves an open space for a second alkylation. This is used to derivatize the regioisomer into a polyalkylation product. This polyalkylation was done with menthadienol (13) under p-TsOH catalysis in toluene. Menthadienol (13) is a sterically hindered allylic alcohol, which leads to an excellent selective alkylation of the regioisomer (5) and hardly any alkylation of the CBG (1). This reaction step provides CBG (1) and byproducts which can be separated by a normal distillation step. A further crystallization step gives the pure product in overall yield of 13%.

##STR00029##

[0140] To a solution of olivetol (2) (20.0 g, 111 mmol, 1.00 eq.) and geraniol (3) (17.1 g, 111 mmol, 1.00 eq.) in 200 mL toluene was added p-TSOH (1.91 g, 11.1 mmol, 0.10 eq.) at 20° C. The solution was stirred at 400 rpm for 30 min. The reaction was quenched by adding aq. sat. sodium bicarbonate (100 mL) and brine (100 mL). The organic phase was separated and the aqueous phase extracted with toluene (2×100 mL). The combined organic layer was dried over sodium sulfate and concentrated in vacuo to give the crude product. The crude mixture was separated from the remaining starting material by extraction. Therefore, the raw product was dissolved in methanol (300 mL), brine (100 mL), water (40 mL) and was extracted with cyclohexane (5×50 mL). The combined cyclohexane layers were dried over sodium sulfate. The obtained product mixture was directly subjected to the next step reaction without further purification.

[0141] To the solution of product mix (31.9 g, 34% CBG (1), 24% regioisomer (5)) and menthadienole (13) (3.83 g, 25.2 mmol) in 200 mL toluene was added p-TSOH (1.73 g, 10.1 mmol) at 15° C. The solution was stirred at 500 rpm for 15 min. The reaction was quenched by adding aq. sat. sodium bicarbonate (100 mL) and brine (100 mL). The organic phase was separated and the aqueous phase extracted with toluene (2×100 mL). The combined organic layer was subjected to sodium hydroxide solution (100 mL, 1M) and stirred vigorously for 15 min. The phases were separated and the organic phase was dried over sodium sulfate and concentrated in vacuo to give the crude product. The crude product was distillated (0.4 mbar, 190-200° C.), the product fraction (13.3 g) was dissolved in heptane (15 ml) and crystallized at −20° C. The mother liquid was partly condensed and subjected to two additional crystallizations. The combined product (1) was a white crystalline powder (4.49 g, 14.2 mmol, 13%) with a purity >97%.

[0142] R.sub.f: 0.52 (1:1 cyclohexane/ethyl acetate) KMnO.sub.4

[0143] .sup.1H NMR (600 MHz, Chloroform-d) δ=6.24 (s, 2H), 5.27 (th, J=7.1, 1.3 Hz, 1H), 5.07 (s, 2H), 5.05 (thept, J=7.0, 1.4 Hz, 1H), 3.39 (d, J=7.1 Hz, 2H), 2.48-2.41 (m, 2H), 2.13-2.08 (m, 2H), 2.08-2.03 (m, 2H), 1.81 (q, J=1.0 Hz, 3H), 1.67 (q, J=1.2 Hz, 3H), 1.59 (d, J=1.3 Hz, 3H), 1.58-1.53 (m, 2H), 1.36-1.25 (m, 4H), 0.88 (t, J=7.0 Hz, 3H) ppm.

[0144] .sup.13C NMR (151 MHz, Chloroform-d)=δ 154.8, 142.8, 138.9, 132.1, 123.8, 121.7, 110.6, 108.4, 39.7, 35.5, 31.5, 30.8, 26.3, 25.7, 22.6, 22.3, 17.7, 16.2, 14.0 ppm.

[0145] Use of Geraniol for the Derivatization of the Byproduct

[0146] Geraniol (3) is added in excess. Olivetol (2) reacts with geraniol to the product CBG (1) and the regio isomer (5) in a 1:1 ratio. The product (1) and regioisomer (5) have an increased inductive effect due to the newly formed bond. The product and regioisomer (5) gain in reactivity towards a second alkylation while the regioisomer is more sterically accessible. This leads to a major polyalkylation of the unwanted regioisomer and just a slight alkylation of the CBG (1). The disadvantage of the polyalkylation can therefore be turned to an advantage.

##STR00030##

[0147] To a solution of olivetol (2) (20.0 g, 111 mmol, 1.00 eq.) and geraniol (30.8 g, 200 mmol, 1.80 eq.) in 200 mL toluene was added p-TSOH (0.86 g, 5.00 mmol, 0.05 eq.) at 40° C. The solution was stirred at 600 rpm for 30 min. The reaction was quenched by adding aq. sat. sodium bicarbonate (100 mL) and brine (100 mL). The organic phase was separated and the aqueous phase extracted with toluene (2×100 mL). The combined organic layer was dried over sodium sulfate and concentrated in vacuo to give the crude product (45.4 g). The raw product was dissolved in methanol (180 mL), water (20 mL) and was extracted with heptane (4×50 mL). The heptane phase was discarded. Brine (50 mL) was added to the aqueous phase (containing product), which was subsequently extracted with cyclohexane (5×50 mL). The cyclohexane phase was combined, dried over sodium sulfate and concentrated in vacuo. The residue (10 g) was further purified by distillation (0.8 mbar, 200-210° C.) and crystallization (seeding) in heptan (11 mL) to give a white crystalline product (2.84 g, 8.97 mmol, 8%).

[0148] R.sub.f: 0.52 (1:1 cyclohexane/ethyl acetate) KMnO.sub.4

[0149] .sup.1H NMR (600 MHz, Chloroform-d) δ=6.24 (s, 2H), 5.27 (th, J=7.1, 1.3 Hz, 1H), 5.07 (s, 2H), 5.05 (thept, J=7.0, 1.4 Hz, 1H), 3.39 (d, J=7.1 Hz, 2H), 2.48-2.41 (m, 2H), 2.13-2.08 (m, 2H), 2.08-2.03 (m, 2H), 1.81 (q, J=1.0 Hz, 3H), 1.67 (q, J=1.2 Hz, 3H), 1.59 (d, J=1.3 Hz, 3H), 1.58-1.53 (m, 2H), 1.36-1.25 (m, 4H), 0.88 (t, J=7.0 Hz, 3H) ppm.

[0150] .sup.13C NMR (151 MHz, Chloroform-d)=δ 154.8, 142.8, 138.9, 132.1, 123.8, 121.7, 110.6, 108.4, 39.7, 35.5, 31.5, 30.8, 26.3, 25.7, 22.6, 22.3, 17.7, 16.2, 14.0 ppm.

[0151] Linalool as an Alternative Allylic Alcohol

[0152] An alternative allylic alcohol to geraniol (3) is linalool (4), due to the diverse tolerance of the allylic alcohol for the alkylation. The use of Linalool results in the same product and similar byproduct mixture.

##STR00031##

[0153] To a solution of olivetol (2) (20.0 g, 111 mmol, 1.00 eq.) and Linalool (4) (30.8 g, 200 mmol, 1.80 eq.) in 200 mL toluene was added p-TsOH (1.91 g, 11.1 mmol, 0.10 eq.) at 40° C. The solution was stirred at 600 rpm for 1 h. The reaction was quenched by adding aq. sat. sodium bicarbonate (100 mL) and brine (100 mL). The organic phase was separated and the aqueous phase extracted with toluene (2×100 mL). The combined organic layer was dried over sodium sulfate and concentrated in vacuo to give the crude product (44.7 g). The raw product was dissolved in methanol (180 mL), water (20 mL) and was extracted with heptane (4×50 mL). The heptane phase was discarded. Brine (50 ml) was added to the aqueous phase (containing product), which was extracted with cyclohexane (5×50 mL). The cyclohexane phase was combined, dried over sodium sulfate and concentrated in vacuo. The residue (11 g) was further purified by distillation (0.5 mbar, 200-210° C.) and crystallization (seeding) in heptane (6 mL) to give a white crystalline product (1) (1.23 g, 3.88 mmol, 3%).

[0154] R.sub.f: 0.52 (1:1 cyclohexane/ethyl acetate) KMnO.sub.4

[0155] .sup.1H NMR (600 MHz, Chloroform-d) δ=6.24 (s, 2H), 5.27 (th, J=7.1, 1.3 Hz, 1H), 5.07 (s, 2H), 5.05 (thept, J=7.0, 1.4 Hz, 1H), 3.39 (d, J=7.1 Hz, 2H), 2.48-2.41 (m, 2H), 2.13-2.08 (m, 2H), 2.08-2.03 (m, 2H), 1.81 (q, J=1.0 Hz, 3H), 1.67 (q, J=1.2 Hz, 3H), 1.59 (d, J=1.3 Hz, 3H), 1.58-1.53 (m, 2H), 1.36-1.25 (m, 4H), 0.88 (t, J=7.0 Hz, 3H) ppm.

[0156] .sup.13C NMR (151 MHz, Chloroform-d)=δ 154.8, 142.8, 138.9, 132.1, 123.8, 121.7, 110.6, 108.4, 39.7, 35.5, 31.5, 30.8, 26.3, 25.7, 22.6, 22.3, 17.7, 16.2, 14.0 ppm.

EXAMPLE 5: BORON TRIFLUORIDE ETHERATE AS CATALYST IN BATCH AND CONTINUOUS PROCESSES

[0157] Olivetol (2) was alkylated with geraniol (3) under catalysis of boron trifluoride etherate. This was done in a normal batch reaction and in a continuous flow reactor.

##STR00032##

[0158] Batch Reaction

[0159] To a solution of olivetol (200 mg, 1.11 mmol, 1.00 eq.), geraniol (223 mg, 1.44 mmol, 1.30 eq.) in 20 mL dichloromethane at 20° C. was added boron trifluoride etherate (126 mg, 110 μL, 0.88 mmol, 0.80 eq.). The solution was stirred for 3 min and quenched by the addition of sodium bicarbonate (10 ml). The layers were separated and the organic layer was washed with brine. The organic layer was dried over sodium sulfate and concentrated in vacuo to give the crude product. After purification by silica flash column chromatography (gradient: cyclohexane/ethyl acetate) the product (6 mg, 0.02 mmol, 3%) could be obtained as a white solid.

[0160] Continuous Flow Reaction

[0161] A continuous flow reactor with a volume of 18 mL was fed starting material solution A, consisting of olivetol (2) (5.00 g, 14.7 mmol, 1.00 eq.) and geraniol (3) (5.56 g, 36.1 mmol, 1.30 eq.) in 40 ml dichloromethane. Catalyst solution B consisted of boron trifluoride etherate (4.72 mg, 33.2 mmol, 1.20 eq.) in 40 mL dichloromethane.

[0162] Starting material solution A was pumped with 24 mL/min and catalyst solution B was pumped with 12 ml/min while the reaction was stirred at 1300 rpm. The reaction mixture left the reactor with a flow of 36 ml/min through a 1.5 m long PTFE-hose (20 mL volume) into an aq. sat. sodium bicarbonate solution.

[0163] The quenched mixture was stirred for 15 min, cyclohexane (150 mL) was added and the phases were separated. The aqueous phase was extracted twice with cyclohexane. The combined organic phase was dried over sodium sulfate and concentrated in vacuo. The crude product was purified by silica flash column chromatography and gave the product (95 mg, 0.30 mmol, 2%) as a white solid.

EXAMPLE 6: SOLVING THE PURIFICATION ISSUE BY TECHNICAL SOLUTION AS SMB OR CPC

[0164] As a technical solution of purifying a mixture of CBG (1) and its byproducts non-standard purification methods (SMB, CPC) were evaluated.

[0165] SMB

[0166] The simulated moving bed (SMB) chromatography was done by using a SMB apparatus by Knauer. The concept is to use eight smaller solid adsorbents columns instead of a large one and rotate these columns in the opposite direction of the flow of the mobile phase. A solvent stream and a feed stream (containing CBG (1) and byproducts) is constantly applied. When running at a steady state, the various stages of separation are carried out simultaneously by different columns. The extract and raffinate outlet deliver under optimized conditions the product (1) in the raffinate fractions and the regioisomer (5) in the extract fractions (http://sembabio.com/simulated-moving-bed-chromatography/). Advantages of this continuous separation process are higher productivity and purity compared with a lower solvent consumption compared to standard preparative HPLC or silica flash column chromatography.

[0167] The eluent was prepared by adding 5 kg dest. water to 15.8 kg methanol.

[0168] The feed solution was prepared by dissolving 19.70 g of the CBG mix (45% CBG) in 780 mL eluent.

[0169] The pump flow of the feed, eluent and two internal pumps for zone 4 and 2 were adjusted as in the Table (1). After an equilibration time of 8 h, the raffinate and extract were collected.

TABLE-US-00001 TABLE (1) SMB conditions. Rotation time 10.81 rpm zone 4 pump 3.65 mL/min zone 2 pump 4.7 mL/min Feed pump 0.2 mL/min Eluent pump 4.6 mL/min Raffinate 1.3 mL/min Extract 3.5 mL/min

[0170] The product containing raffinate fractions were extracted with cyclohexane, dried over sodium sulfate and concentrated in vacuo. After crystallization the white solid product CBG (1) could be obtained

[0171] CPC

[0172] Centrifugal Partition Chromatography is a separation technique of the field of liquid-liquid chromatography. The separation principle is based on the different distribution of the product CBG (1) and the impurities between two immiscible liquid phases which are mixed together to form a two-phase system, and are then separated multiple times (http://www.kromaton.com/en/the-cpc/technologies).

[0173] The mixing and separation was done in a 100 mL centrifugal rotor by Gilson. The stationary phase was retained in the rotor through centrifugal force. The CPC is constantly fed with the mobile phase which is containing the extracted solutes and is collected in fractions. It is therefore necessary to find a solvent mixture (stationary and mobile phase) that has an adequate distribution of compounds (which should be separated) as well the ability to partition fast enough. An advantages of liquid-liquid techniques are as high speed, high loading and high resolution (Garrard: Phytochem Rev., 2014; 13(2), 547-572).

[0174] A usable solvent mixture is heptane:ethanol:water in a ratio of 5:4:1.5 (volume) and a rotation speed of 2500 rpm. Other test systems like heptane:ethyl acetate:methanol:water (1:1:1:1) were not successful in separation of CBG (1) and the regioisomer (5).

[0175] The solvent mixture was prepared by mixing heptane (300 mL), ethanol (240 mL), water (90 mL). The phases were left to separate in a separation funnel, from which the filling of the CPC took place. The stationary phase (upper heptane phase) was filled with a flow of 5 mL/min at a rotation of 500 rpm. (descending mode) The mobile phase (lower ethanol/water phase) was filled with a flow of 5 mL/min at a rotation of 2500 rpm.

[0176] For starting the separation 500 mg of the product/byproduct mix was dissolved in 5 mL of the stationary phase and 5 mL of the mobile phase. After injection, the collection of the fractions took place with a flow of 2 mL/min which enabled an enrichment of CBG >70%. The following crystallization gave the pure product CBG (1).

EXAMPLE 7: BIOLOGICAL TESTING

[0177] Introduction

[0178] The monooxygenase tyrosinase is a key enzyme in the melanogenesis, the production of melanin. Melanin is responsible for skin and hair pigmentation and protects the skin against radiation. However, abnormal melanin production can lead to various skin disorders like hyperpigmentation, lentigo, vitiligo and skin cancer. Additionally, inhibition of melanin production can have some cosmetic benefits due to skin whitening/lightening.

[0179] A prominent tyrosinase—and subsequently of melanin production—inhibitor is kojic acid, which is used in the following tests as a positive control.

[0180] Experimental Procedure

[0181] Cell Culture. The cell line B16 (mouse melanocytes, obtained from ATCC and part of SimDerma platform) was maintained in supplemented DMEM medium containing 10% FBS and 1% antibiotics penicillin/streptomycin (DMEM complete medium) at 37° C. in a humidified atmosphere of 5% CO.sub.2.

[0182] Measurements of Tyrosinase Activity: To measure the tyrosinase activity of the cells, B16 cells were treated for 3 days with several doses of the compounds (cannabigerol (1) or kojic acid), then lysed by incubation at 4° C. for 30 min in lysis buffer (20 mM sodium phosphate, pH 6.8, 1% Triton X-100, 1 mM PMSF, 1 mM EDTA) containing protease inhibitors cocktail. The lysates were centrifuged at 15.000 g for 10 min to obtain the supernatant as the source of tyrosinase. The reaction mixture contained 20 mM phosphate buffer, pH 6.8, 1.25 mM L-dopa (Sigma-Aldrich). After incubation at 37° C. for 30 min, dopachrome formation was monitored by measuring absorbance at a wavelength of 475 nm in a microplate reader (TriStar LB 941 Berthold Technologies, GmbH & Co. KG). Kojic acid was used as a positive control.

[0183] Determination of Melanin Content and Cytotoxicity in B16 cells: To analyse the effect of cannabigerol (1) on melanogenesis, B16 melanoma cells were used as a cellular assay system to evaluate the depigmenting activity in cell cultures. B16 cells (4A5) were treated for 3 days with several doses of the compounds (cannabigerol (1) or kojic acid). The cells were washed with phosphate buffer saline (PBS) at the end of the treatment and dissolved in 1 N NaOH containing 10% DMSO for 1 h at 60° C. The absorbance at 405/490 nm was measured (TriStar LB 941 Berthold Technologies, GmbH & Co. KG) and the melanin content quantified with a reference standard of synthetic melanin (Sigma-Aldrich, St Louis, Mo., USA). Cytotoxicity was measured in parallel by the MTT assay to discard that melanin inhibition is not masked by cytotoxicity. Kojic acid was used as a positive control.

[0184] Results

[0185] Tyrosinase Activity

[0186] Cannabigerol (1) inhibited tyrosinase activity in the doses of 0.5 μg/ml and higher. With the dose of 1 μg/ml cannabigerol (1) the inhibition of tyrosinase was as potent as the effect of 2 mM of kojic acid. Cannabigerol (1) exhibited no cytotoxic effects in any of the tested doses. FIG. 2 shows the biological effects of cannabigerol on tyrosinase activity in B16 melanocytes.

[0187] Melanin Production

[0188] As shown in FIG. 2 melanin production was dose dependently prevented by cannabigerol. The dosage of 0.5 μg/ml exhibited similar inhibitory effects as 2 mM of kojic acid, which showed approx. 20% reduction of melanin production, 1 μg/ml cannabigerol (1) was even more potent with about 30% inhibition of melanin production. Cannabigerol (1) exhibited no cytotoxic effects in any of the tested doses. FIG. 3 shows the biological effects of cannabigerol (1) on melanin production in B16 melanocytes.