Synthesis of (+)-cannabinoids and their therapeutic effects

Abstract

The present invention relates to a method of producing a compound of formula (I) or a salt of a compound of formula (I). The invention also relates to a compound of formula (I) or a salt of a compound of formula (I) for use in a therapeutic method to achieve one or more therapeutic effects as well as for use in the treatment and/or prevention of certain diseases. Furthermore, the invention provides a pharmaceutical composition comprising one or more compound(s) of formula (I) or salt(s) of compound(s) of formula (I).

Claims

1. A method for producing a compound of formula (I), or a salt thereof, ##STR00014## wherein X=H or —COOY, Y=a saturated or unsaturated, branched or unbranched alkyl group, an aryl group, or a heteroaryl group, having 1 to 12 carbon atoms, respectively, and optionally substituted with one or more amino group(s), hydroxyl group(s) and/or halogen(s), and n=2 or 4, the method comprising: i) reacting 4S-menthadienol in a halogen free solvent with a compound of formula (II) in a continuous flow reaction process, ##STR00015## wherein n=2 or 4, to obtain a compound of formula (III) ##STR00016## wherein n=2 or 4; ii) optionally, transesterification of the compound of formula (III); and iii) optionally, decarboxylation of the compound of formula (III) and/or the transesterified product of formula (III) with an acid.

2. The method according to claim 1, wherein the compound of formula (I) is selected from the group consisting of compounds (1) to (5) and/or salts thereof: ##STR00017##

3. The method according to claim 1, wherein pure 1S,4S-menthadienol or pure 1R,4S-menthadienol or a mixture thereof is used in step i).

4. The method according to claim 1, wherein in step i) a solution of a Lewis acid catalyst is provided and brought into contact with a solution of a compound of formula (II) and 4S-menthadienol.

5. The method according to claim 4, wherein the Lewis acid catalyst is boron trifluoride diethyl etherate.

6. The method according to claim 1, wherein in step ii) a transesterification with ethylene glycol and/or 1,2-pentanediol is conducted.

7. The method claim 1, wherein the halogen-free solvent is toluene.

Description

SHORT DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the CB.sub.1 (left) and CB.sub.2 (right) binding of (+)-CDB as evaluated in example 4.

(2) FIG. 2 shows the CB.sub.1 (left) and CB.sub.2 (right) binding of (+)-CBDV as evaluated in example 4.

(3) FIG. 3 shows the CB.sub.1 (left) and CB.sub.2 (right) binding of (+)-CBD-ME as evaluated in example 4.

(4) FIG. 4 shows the CB.sub.1 (left) and CB.sub.2 (right) binding of (+)-CBD-GE as evaluated in example 4.

(5) FIG. 5 shows the CB.sub.1 (left) and CB.sub.2 (right) binding of (+)-CBD-HPE as evaluated in example 4.

(6) FIG. 6 shows the effect of (+)-CBD on CB.sub.1 functional activity (agonistic activity) as evaluated in example 5.

(7) FIG. 7 shows the effect of (+)-CBDV on CB.sub.1 functional activity (agonistic activity) as evaluated in example 5.

(8) FIG. 8 shows the effect of (+)-CBD-ME on CB.sub.1 functional activity (agonistic activity) as evaluated in example 5.

(9) FIG. 9 shows the effect of (+)-CBD-GE on CB.sub.1 functional activity (agonistic activity) as evaluated in example 5. (+)-CBD-GE shows cytotoxicity at 25 μM.

(10) FIG. 10 shows the effect of (+)-CBD-HPE on CB.sub.1 functional activity (agonistic activity) as evaluated in example 5.

(11) FIG. 11 shows the effect of (+)-CBD on CB.sub.1 functional activity (antagonistic activity) as evaluated in example 5.

(12) FIG. 12 shows the effect of (+)-CBDV on CB.sub.1 functional activity (antagonistic activity) as evaluated in example 5.

(13) FIG. 13 shows the effect of (+)-CBD-ME on CB.sub.1 functional activity (antagonistic activity) as evaluated in example 5.

(14) FIG. 14 shows the effect of (+)-CBD-GE on CB.sub.1 functional activity (antagonistic activity) as evaluated in example 5. CBD-GE shows cytotoxicity at 25 μM.

(15) FIG. 15 shows the effect of (+)-CBD-HPE on CB.sub.1 functional activity (antagonistic activity) as evaluated in example 5.

(16) FIG. 16 shows the effect of (+)-CBD on CB.sub.2 functional activity (agonistic activity) as evaluated in example 5.

(17) FIG. 17 shows the effect of (+)-CBDV on CB.sub.2 functional activity (agonistic activity) as evaluated in example 5.

(18) FIG. 18 shows the effect of (+)-CBD-ME on CB.sub.2 functional activity (agonistic activity) as evaluated in example 5.

(19) FIG. 19 shows the effect of (+)-CBD-GE on CB.sub.2 functional activity (agonistic activity) as evaluated in example 5.

(20) FIG. 20 shows the effect of (+)-CBD-HPE on CB.sub.2 functional activity (agonistic activity) as evaluated in example 5.

(21) FIG. 21 shows the effect of (+)-CBD on CB.sub.2 functional activity (antagonistic activity) as evaluated in example 5.

(22) FIG. 22 shows the effect of (+)-CBDV on CB.sub.2 functional activity (antagonistic activity) as evaluated in example 5.

(23) FIG. 23 shows the effect of (+)-CBD-ME on CB.sub.2 functional activity (antagonistic activity) as evaluated in example 5.

(24) FIG. 24 shows the effect of (+)-CBD-GE on CB.sub.2 functional activity (antagonistic activity) as evaluated in example 5.

(25) FIG. 25 shows the effect of (+)-CBD-HPE on CB.sub.2 functional activity (antagonistic activity) as evaluated in example 5.

(26) FIG. 26 shows the effects of (+)-CBD on cell viability in human monocytes as evaluated in example 6.

(27) FIG. 27 shows the effects of (+)-CBDV on cell viability in human monocytes as evaluated in example 6.

(28) FIG. 28 shows the effects of (+)-CBD-ME on cell viability in human monocytes as evaluated in example 6.

(29) FIG. 29 shows the effects of (+)-CBD-GE on cell viability in human monocytes as evaluated in example 6.

(30) FIG. 30 shows the effects of (+)-CBD-HPE on cell viability in human monocytes as evaluated in example 6.

(31) FIG. 31 shows the effects of (+)-CBD on inflammatory parameters in LPS-treated human monocytes as evaluated in example 6. The columns for each concentration indicate from left to right: IL1 beta, TNF alpha, IL6, IL8, MMP9, PGE2 and Isoprostan.

(32) FIG. 32 shows the effects of (+)-CBDV on inflammatory parameters in LPS-treated human monocytes as evaluated in example 6. The columns for each concentration indicate from left to right: IL1 beta, TNF alpha, IL6, IL8, MMP9, PGE2 and Isoprostan.

(33) FIG. 33 shows the effects of (+)-CBD-ME on inflammatory parameters in LPS-treated human monocytes as evaluated in example 6. The columns for each concentration indicate from left to right: IL1 beta, TNF alpha, IL6, IL8, MMP9, PGE2 and Isoprostan.

(34) FIG. 34 shows the effects of (+)-CBD-GE on inflammatory parameters in LPS-treated human monocytes as evaluated in example 6. The columns for each concentration indicate from left to right: IL1 beta, TNF alpha, IL6, IL8, MMP9, PGE2 and Isoprostan.

(35) FIG. 35 shows the effects of (+)-CBD-HPE on inflammatory parameters in LPS-treated human monocytes as evaluated in example 6. The columns for each concentration indicate from left to right: IL1 beta, TNF alpha, IL6, IL8, MMP9, PGE2 and Isoprostan.

(36) FIG. 36 shows the effects of (+)-CBD on inflammatory parameters in IL-1-treated human dermal fibroblasts as evaluated in example 6. The columns for each concentration indicate from left to right: IL6, IL8 and PGE2.

(37) FIG. 37 shows the effects of (+)-CBDV on inflammatory parameters in IL-1-treated human dermal fibroblasts as evaluated in example 6. The columns for each concentration indicate from left to right: IL6, IL8 and PGE2.

(38) FIG. 38 shows the effects of (+)-CBD-ME on inflammatory parameters in IL-1-treated human dermal fibroblasts as evaluated in example 6. The columns for each concentration indicate from left to right: IL6, IL8 and PGE2.

(39) FIG. 39 shows the effects of (+)-CBD-GE on inflammatory parameters in IL-1-treated human dermal fibroblasts as evaluated in example 6. The columns for each concentration indicate from left to right: IL6, IL8 and PGE2.

(40) FIG. 40 shows the effects of (+)-CBD-HPE on inflammatory parameters in IL-1-treated human dermal fibroblasts as evaluated in example 6. The columns for each concentration indicate from left to right: IL6, IL8 and PGE2.

(41) FIG. 41 shows the effects of (+)-CBD on MMP1, MMP9 and TIMP1 in PolyIC-treated human keratinocytes (HaCat) as evaluated in example 6. The columns for each concentration indicate from left to right: MMP9, TIMP1 and MMP1.

(42) FIG. 42 shows the effects of (+)-CBDV on MMP1, MMP9 and TIMP1 in PolyIC-treated human keratinocytes (HaCat) as evaluated in example 6. The columns for each concentration indicate from left to right: MMP9, TIMP1 and MMP1.

(43) FIG. 43 shows the effects of (+)-CBD-ME on MMP1, MMP9 and TIMP1 in PolyIC-treated human keratinocytes (HaCat) as evaluated in example 6. The columns for each concentration indicate from left to right: MMP9, TIMP1 and MMP1.

(44) FIG. 44 shows the effects of (+)-CBD-GE on MMP1, MMP9 and TIMP1 in PolyIC-treated human keratinocytes (HaCat) as evaluated in example 6. The columns for each concentration indicate from left to right: MMP9, TIMP1 and MMP1.

(45) FIG. 45 shows the effects of (+)-CBD-HPE on MMP1, MMP9 and TIMP1 in PolyIC-treated human keratinocytes (HaCat) as evaluated in example 6. The columns for each concentration indicate from left to right: MMP9, TIMP1 and MMP1.

(46) The invention will be explained in more detail on the basis of the examples below.

EXAMPLE 1

Synthesis of (+)-CBD (3)

(47) ##STR00009##

(48) 71.4 g (300 mMol) olivetol methylester and 50 g (330 mMol) 1R,4S-menthadienol were dissolved together with toluene to reach a combined volume of 400 ml.fwdarw.Solution A. 21.3 g (150 mMol) BF.sub.3 etherate was dissolved with toluene to reach a volume of 300 ml.fwdarw.Solution B. Both reaction solutions were then put through two separate pump systems and the continuous flow reactor (rotation: 1200 U/min, solution A: 24 ml/min, solution B: 12 ml/min). Solution B started before and ended after solution A to guarantee that catalyst is always present in the reaction chamber. The reaction mixture was continuously collected in a 2 liter lab reactor (30° C. mantel temperature, 300 rpm) filled with 700 ml saturated NaHCO.sub.3 solution. The aqueous solution was discarded; the organic solution was washed at 45 degrees 4 times with 250 mL of 1% NaOH solution. After washing, the organic solution was evaporated to dryness to give 94.58 g of raw (+)-CBD methylester (purity=78%, yield 68%). The raw compound can be used further without purification.

(49) Exemplary Purification of (+)-CBD ME (1):

(50) The crude product was purified by flash chromatography (eluent system cyclohexane/ethyl acetate=40/1 v/v). GC purity: 99.1%. Chiral GC analysis: enantiomeric excess 99% (for enantiomeric pure starting material). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 11.98 (s, 1H), 6.50 (s, 1H), 6.21 (s, 1H), 5.55 (s, 1H), 4.52 (p, J=2.4, 1.4 Hz, 1H), 4.41-4.36 (m, 1H), 4.16-4.06 (m, 1H), 3.90 (s, 3H), 2.89-2.79 (m, 1H), 2.78-2.69 (m, 1H), 2.44-2.33 (m, 1H), 2.29-2.15 (m, 1H), 2.09 (dq, J=17.9, 4.0, 2.5 Hz, 1H), 1.84-1.76 (m, 2H), 1.80-1.77 (m, 3H), 1.72-1.68 (m, 3H), 1.57-1.46 (m, 2H), 1.38-1.28 (m, 4H), 0.89 (t, J=6.8 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 172.62, 163.11, 159.91, 147.23, 145.94, 140.19, 124.06, 114.38, 111.49, 111.23, 103.91, 51.73, 46.66, 36.83, 35.40, 32.10, 31.14, 30.24, 27.84, 26.92, 23.71, 22.55, 18.83, 14.10.

(51) ##STR00010##

(52) 49.2 g (103 mMol) (+)-CBD-ME (1) was dissolved at 60 degrees in 250 mL ethylene glycol and poured in a 1 L lab reactor. 5.7 g potassium hydroxide was added and the reaction mixture was started to heat under stirring to 120 degrees and a vacuum of 500 mbar. Accumulated volatile side products were distilled off. After 2 hours the reaction temperature was increased to 150 degrees and the temperature was kept for additional 3 hours. The reaction mixture was cooled to 80 degrees, following addition of 400 mL water and 130 mL n-heptane. The temperature was further decreased to 40 degrees, following the slow addition of 1.2 mL of sulfuric acid (50%) until a pH of approx. 6. The layers were separated, the organic layer was washed once with 250 mL of water and once with 250 ml of sodium hydroxide solution (0.05%). The organic layer was dried over Na.sub.2SO.sub.4 and then evaporated to dryness. Yield: 30.3 g, GC purity: 53%.

(53) Exemplary Isolation and Purification of (+)-CBD GE (2):

(54) A sample of the reaction mixture was taken after 2 hours at 120 degrees, quenched with n-heptane and water and neutralized with sulfuric acid (10% w/w). The layers were separated and the organic layer was evaporated to dryness. The crude (+)-CBD-GE (2) was purified by flash chromatography (eluent system n-heptane/ethyl acetate=4/1 v/v). GC purity: 97.8%. Chiral GC analysis: enantiomeric excess 99% (for enantiomeric pure starting material). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 11.88 (s, 1H), 6.53 (s, 1H), 6.23 (s, 1H), 5.55 (s, 1H), 4.54-4.50 (m, 1H), 4.50-4.44 (m, 2H), 4.40-4.36 (m, 1H), 4.14-4.06 (m, 1H), 3.98-3.92 (m, 2H), 2.88 (ddd, J=13.1, 8.8, 6.7 Hz, 1H), 2.78 (ddd, J=13.1, 8.7, 6.8 Hz, 1H), 2.39 (q, J=8.1 Hz, 1H), 2.29-2.16 (m, 1H), 2.10 (dq, J=17.9, 3.6 Hz, 1H), 1.84-1.76 (m, 2H), 1.80-1.77 (m, 3H), 1.71 (s, 3H), 1.60-1.49 (m, 2H), 1.36-1.29 (m, 4H), 0.89 (t, J=6.8 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 172.39, 163.27, 160.12, 147.20, 145.86, 140.31, 123.97, 114.52, 111.68, 111.28, 103.71, 66.73, 61.19, 46.65, 46.01, 36.98, 35.39, 32.06, 31.48, 30.24, 27.83, 23.72, 22.68, 18.82, 14.09.

(55) Purification of (+)-CBD (3):

(56) The crude (+)-CBD was purified in this instance by flash chromatography (eluent system cyclohexane/ethyl acetate=20/1 v/v). Flash chromatography can be substituted with thin layer distillation, following crystallisation from n-heptane. GC purity: 99,8%. Chiral GC analysis: enantiomeric excess 99% (for enantiomeric pure starting material and for starting materials with up to 5% 4R-menthadienol enantiomer). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 6.35-6.09 (m, 2H), 5.97 (s, 1H), 5.57 (dt, J=2.8, 1.6 Hz, 1H), 4.66 (p, J=1.6 Hz, 2H), 4.56 (d, J=2.0 Hz, 1H), 3.85 (ddp, J=10.7, 4.5, 2.3 Hz, 1H), 2.48-2.41 (m, 2H), 2.38 (ddd, J=10.6, 3.7 Hz, 1H), 2.30-2.17 (m, 1H), 2.09 (ddt, J=17.9, 5.1, 2.4 Hz, 1H), 1.88-1.81 (m, 1H), 1.79 (dt, J=2.6, 1.2 Hz, 3H), 1.78-1.72 (m, 1H), 1.65 (t, J=1.1 Hz, 3H), 1.62-1.50 (m, 2H), 1.37-1.22 (m, 4H), 0.88 (t, J=7.0 Hz, 3H). 13C NMR (101 MHz, CDCl.sub.3) δ 156.07, 153.90, 149.41, 143.06, 140.07, 124.12, 113.76, 110.84, 109.84, 108.00, 7.35, 77.03, 76.71, 46.15, 37.28, 35.48, 31.50, 30.64, 30.41, 28.41, 23.69, 22.55, 20.54, 14.05.

EXAMPLE 2

Synthesis of (+)-CBD HPE (4)

(57) ##STR00011##

(58) 10 g (24 mMol) (+)-CBD-ME (1) was dissolved at 60 degrees in 250 mL 1,2-pentandiol and poured in a 1 L lab reactor. 1.1 g potassium hydroxide was added and the reaction mixture was started to heat under stirring to 120 degrees and a vacuum of 500 mbar. Accumulated volatile side products were distilled off. After 2 hours the reaction mixture was cooled to 80 degrees, following addition of 400 mL water and 130 mL n-heptane. The temperature was further decreased to room temperature and neutralized with sulfuric acid (10% w/w). The layers were separated, the organic layer was washed once with 250 mL of water, dried over Na.sub.2SO.sub.4 and evaporated to dryness. The crude product (+)-CBD-HPE (4) was purified by flash chromatography (eluent system cyclohexane/ethyl acetate=10/1 v/v). GC purity: 98%. Chiral GC analysis: enantiomeric excess 99% (for enantiomeric pure starting material). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.61 (s, 1H), 9.89 (s, 1H), 6.20 (s,1H), 5.11-5.05 (m, 1H), 4.91-4.82 (m, 1H), 4.46 (d, J=2.7 Hz, 1H), 4.42 (dd, J=2.8, 1.5 Hz, 1H), 4.24-4.11 (m, 2H), 3.95-3.86 (m, 1H), 3.81-3.71 (m, 1H), 3.03 (td, J=11.4, 10.9, 3.0 Hz, 1H), 2.74 (s, 2H), 2.22-2.05 (m, 1H), 1.94 (dd, J=16.7, 4.1 Hz, 1H), 1.76-1.63 (m, 2H), 1.61 (t, J=1.8 Hz, 3H), 1.58 (s, 3H), 1.53-1.34 (m, 6H), 1.33-1.26 (m, 4H), 0.89 (t, J=7.0 Hz, 3H), 0.86 (t, J=6.7 Hz, 3H). .sup.13C NMR (101 MHz, DMSO-d.sub.6) δ 171.22, 162.07, 160.33, 148.61, 144.07, 130.72, 125.60, 114.82, 110.15, 109.84, 103.50, 68.95, 67.27, 43.32, 35.71, 35.61, 35.44, 31.36, 30.91, 30.13, 29.12, 23.13, 22.00, 18.88, 18.12, 13.86, 13.

EXAMPLE 3

Synthesis of (+)-CBDV (5)

(59) ##STR00012##

(60) 91 g (430 mMol) divarin methylester and 66 g (430 mMol) 4S-menthadienol were dissolved together with toluene to reach a combined volume of 610 ml.fwdarw.Solution A. 20 g (140 mMol) BF.sub.3 etherate was dissolved with toluene to reach a volume of 177 ml.fwdarw.Solution B. Both reaction solutions were then put through two separate pump systems and the continuous flow reactor (rotation: 1200 U/min, solution A: 93 ml/min, solution B: 29 ml/min). The reaction mixture was continuously collected in a 2 liter lab reactor (30° C. mantel temperature, 300 rpm) filled with 750 ml saturated NaHCO.sub.3 solution. The aqueous solution was discarded; the organic solution was washed at 40 degrees 7 times with 250 mL of 1% NaOH solution. After washing, the organic solution was evaporated to dryness to give 117 g of raw (+)-CBDV-ME (purity=81%, yield 70%). The raw compound was used further without purification.

(61) ##STR00013##

(62) 117 g (275 mMol) (+)-CBDV-ME was dissolved at 60 degrees in 150 mL ethylene glycol and poured in a 1 L lab reactor. 30.8 g (550 mMol) potassium hydroxide was dissolved in 100 ml ethylene glycol and added to the stirring solution. The reaction mixture was started to heat under stirring to 120 degrees and a vacuum of 500 mbar. Accumulated volatile side products were distilled off. After 2 hours the reaction temperature was increased to 150 degrees and the temperature was kept for additional 3 hours. The reaction mixture was cooled to 80 degrees, following addition of 550 mL water and 200 mL n-heptane. The temperature was further decreased to 40 degrees, following the slow addition of 45 g of sulfuric acid (50%) until a pH of approx. 6. The layers were separated, the aqueous layer was extracted once with 200 ml MTBE. Both organic layers were combined and evaporated to dryness to give 99 g of crude product. To the crude was added 23 g of Synalox oil and the resulting mixture was distilled over a thin layer distillation apparatus. The resulting distillate (62 g, according to GC analysis 83.4% product) was then crystallized from n-heptane. The resulting white crystals were recrystallized once more from n-heptane to give pure product. Yield: 27 g. GC purity: 99.6%. Chiral GC analysis: enantiomeric excess 99% (for enantiomeric pure starting material and for starting materials with up to 5% 4R-menthadienol enantiomer). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 6.37-6.08 (m, 2H), 5.98 (s, 1H), 5.57 (dt, J=2.8, 1.6 Hz, 1H), 4.71 (s, 1H), 4.66 (p, J=1.6 Hz, 1H), 4.55 (d, J=2.1 Hz, 1H), 3.85 (ddq, J=10.5, 4.5, 2.4 Hz, 1H), 2.46-2.38 (m, 2H), 2.45-2.34 (m, 1H), 2.30-2.17 (m, 1H), 2.09 (ddt, J=17.9, 5.1, 2.5 Hz, 1H), 1.87-1.81 (m, 1H), 1.79 (dd, J=2.8, 1.5 Hz, 3H), 1.77-1.72 (m, 1H), 1.65 (t, J=1.2 Hz, 3H), 1.64-1.52 (m, 2H), 0.90 (t, J=7.3 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 156.08, 153.82, 149.37, 142.78, 140.07, 124.13, 113.82, 110.85, 109.82, 108.11, 77.35, 77.04, 76.72, 46.17, 37.58, 37.25, 30.41, 28.41, 24.03, 23.69, 20.51, 13.81.

EXAMPLE 4

In Vitro Binding to CB.SUB.1 .and CB.SUB.2 .Receptors

(63) Compounds with an expected profile as CB.sub.1 and/or CB.sub.2 receptor ligands were evaluated by competition studies that allow determining the affinity of these compounds (Ki values) for both receptors against a classical cannabinoid ligand. The competition studies were conducted with membranes transfected with either CB.sub.1 or CB.sub.2 receptors. For stock solutions the compounds were dissolved at concentrations of 50 and 100 mM and stored at −20° C.

(64) Experimental Procedure

(65) Membranes from human CB.sub.1 or CB.sub.2 receptor-transfected cells (RBHCB1M400UA and RBXCB2M400UA, respectively) were supplied by Perkin-Elmer Life and Analytical Sciences (Boston, Mass.). The values of B.sub.max and K.sub.d for the CB.sub.1 or CB.sub.2 receptor membranes are variable. The used batch showed the following B.sub.max and K.sub.d values, 1.9 pmol/mg membrane protein and 0.16 nM, respectively, for CB.sub.1 receptor membranes, and 5.2 pmol/mg membrane protein and 0.18 nM, respectively, for CB.sub.2 receptor membranes. The protein concentration for the CB.sub.1 receptor membranes was 8.0 mg/ml, whereas for the CB.sub.2 receptor membranes 4.0 mg/ml. The commercial membranes were diluted (1:20) with the binding buffer (50 mM TrisCl, 5 mM MgCl.sub.2.H.sub.2O, 2.5 mM EDTA, 0.5 mg/mL BSA and pH=7.4 for CB.sub.1 binding buffer; 50 mM TrisCl, 5 mM MgCl.sub.2.H.sub.2O, 2.5 mM EGTA, 1 mg/mL BSA and pH=7.5 for CB.sub.2 binding buffer). The radioligand was [.sup.3H]-CP55940 (144 Ci/mmol; PerkinElmer) used at a concentration of 0.10 nM with a final volume of 200 μl for CB.sub.1 binding and at a concentration of 0.15 nM with a final volume of 600 μl for CB.sub.2 binding. 96-well plates and the tubes necessary for the experiment were siliconized with Sigmacote (Sigma). Membranes were resuspended in the corresponding buffer and were incubated with the radioligand and each compound for 90 min at 30° C. Non-specific binding was determined with 10 μM WIN55212-2 and 100% binding of the radioligand to the membrane was determined by its incubation with membrane and without any compound. Filtration was performed by a Harvester®filtermate (Perkin-Elmer) with Filtermat A GF/C filters pretreated with polyethylenimine 0.05%. After filtering, the filter was washed nine times with binding buffer, dried and a melt-on scintillation sheet (Meltilex™ A, Perkin Elmer) was melted onto it. Then, radioactivity was quantified by a liquid scintillation spectrophotometer (Wallac MicroBeta Trilux, Perkin-Elmer).

(66) Results

(67) Radioligand displacement assays were used to evaluate the affinity of the new compounds using membranes from cells (HEK293EBNA) transfected with the CB.sub.1 or the CB.sub.2 receptors and [.sup.3H]-CP55940 as radioligand. The evaluation of the compounds was conducted in two phases. The first phase consisted in a simple screening with a unique and high concentration of each compound (40 μM). The data was collected from at least three experiments performed in triplicate. Only those compounds that are able to displace more than 50% the binding of [.sup.3H]-CP55940 (0.10 nM for CB.sub.1 and 0.15 nM for CB.sub.2) were selected for the second phase. This consisted of a competition study with [.sup.3H]CP55940 (0.10 nM for CB.sub.1 and 0.15 nM for CB.sub.2) and different concentrations of the selected compounds (10.sup.−4-10.sup.−11M). The data was analyzed, by using GraphPad Prism® version 5.01 (GraphPad Software Inc., San Diego, Calif., USA), for the calculation of Ki values expressed as mean±SEM of at least three experiments performed in triplicate for each point. The calculated Ki values are indicated in table 2. The binding profiles are shown in FIGS. 1-5.

(68) TABLE-US-00002 TABLE 2 CB.sub.1 and CB.sub.2 binding activities of the compounds Compound CB.sub.1-Ki (nM) CB.sub.2-Ki (nM) CB.sub.1/CB.sub.2 selectivity (+)-CBD 982 40.5 24.3 (+)-CBDV 294 33.1 8.9 (+)-CBD-ME 345 28.0 12.3 (+)-CBD-GE 359 12.9 27.8 (+)-CBD-HPE 3.1 0.8 3.9

EXAMPLE 5

Functional Assays on Transfected Cells

(69) After it was established that the compounds expressed binding affinity towards the cannabinoid receptors, their function on these receptors (agonism, antagonism) was analyzed.

(70) Experimental Procedure

(71) HEK 293T-CB.sub.1 and HEK 293T-CB.sub.2 cells (stably transfected with CB.sub.1 and CB.sub.2 cDNAs) (10.sup.5/ml) were incubated in 24-well plates and transiently transfected with 0.5 μg/ml of the plasmid CRE-Luc that contains six consensus cAMP responsive elements (CRE) linked to firefly luciferase. Transient transfections were performed with Rotifect (Carl Roth GmbH, Karlsruhe, Germany) according to the manufacturer's instructions and harvested 24 h after transfection.

(72) For CB.sub.1 agonistic activity the transfected cells were stimulated either with increasing concentrations of the test compounds or with WIN 55,212-2 (positive control for CB.sub.1), during 6 h and then luciferase activity was measured in the cell lysates (1-2). Forskolin is an adenylate cyclase activator that is used at 10 μM as a positive control of cAMP signaling pathway activated by a CB.sub.1 receptor-independent mechanism.

(73) For CB.sub.1 antagonistic activity the CB.sub.1 cells were pre-incubated with the test compounds during 15 min and then stimulated with WIN 55,212-2 for 6 h.

(74) To measure CB.sub.2 agonistic activity, HEK293T-CB.sub.2-CRE-luc cells were treated either with increasing concentrations of the test compounds or with WIN 55,212-2 (positive control for CB.sub.2), for 15 min and then with Forskolin (10 μM) during 6 h.

(75) For CB.sub.2 antagonistic activity in cells the potential repression of Forskolin-induced CRE-luc inhibition induced by the compounds was analyzed. As positive controls AM630 or SR144588 were used, two known CB.sub.2 antagonists.

(76) After six hours of stimulation the cells were lysed (in 25 mM Tris-phosphate pH 7.8, 8 mM MgCl.sub.2, 1 mM DTT, 1% Triton X-100, and 7% glycerol) and luciferase activity was measured using an Autolumat LB 9501 (Berthold Technologies, Bad Wildbad, Germany) following the instructions of the luciferase assay kit (Promega, Madison, Wis.). The background obtained with the lysis buffer was subtracted in each experimental value, and the specific transactivation expressed as fold induction over basal levels (CRE-Luc).

(77) Results

(78) CB.sub.1 Agonistic Activity

(79) HEK 293T-CB.sub.1 cells were transfected with the CRE-Luc plasmid and 24 h later stimulated with either Win-55,212-2 (1 μM, positive control) or the test compounds for six hours. The negative control (untreated cells, 0% activation) is not listed. It was found that none of the 5 compounds tested showed CB.sub.1 agonistic activity (FIGS. 6-10).

(80) CB.sub.1 Antagonistic Activity

(81) HEK 293T-CB.sub.1 cells were transfected with the CRE-Luc plasmid and 24 h later stimulated with either Win 55,212-2 (1 μM positive control) in the presence or the absence of the test compounds for six hours. The negative control (untreated cells, 0% activation) is not listed. It was found that all the compounds tested showed CB.sub.1 antagonistic activity (FIGS. 11-15), with (+)-CBD-HPE as the most potent CB.sub.1 antagonist (FIG. 15).

(82) CB.sub.2 Agonistic Activity

(83) HEK 293T-CB2 cells were transfected with the CRE-Luc plasmid and 24 h later stimulated with either forskolin (10 μM, positive control) in the absence or the presence of WIN 55,212-2 or the test compounds for six hours. The negative control (untreated cells, 0% activation) is not listed. (+)-CBD (FIG. 16) and (+)-CBD-HPE (FIG. 20) showed some CB.sub.2 agonistic activity at higher concentrations. The other compounds tested were negative for this activity (FIGS. 17-19).

(84) CB.sub.2 Antagonistic Activity

(85) The potential antagonistic activities of the compounds on the CB.sub.2 receptor were studied in the following. WIN 55,212-2 repression in Forskolin-induced CRE-Luc inhibition was prevented in the presence of either AM630 or SR144588, two known CB.sub.2 antagonists. The negative control (untreated cells, 0% activation) is not listed. None of the compound tested showed CB.sub.2 antagonistic activity (FIGS. 21-25).

(86) TABLE-US-00003 TABLE 3 CB.sub.1 and CB.sub.2 agonism/antagonism of the tested compounds MOLECULAR CB1 CB1 CB2 CB2 COMPOUND WEIGHT (MW) (agonism) (antagonism) (agonism) (antagonism) (+)-CBD 314.5 — + + — (+)-CBDV 286.4 — + — — (+)-CBD-ME 372.5 — + — — (+)-CBD-GE 402.5 — + — — (+)-CBD-HPE 444.6 — + (+) —

EXAMPLE 6

Bioactivity Study

(87) The test compounds were dissolved in DMSO (10 mg/ml stock solution) and diluted in media for the in the following described experiments.

(88) Experimental Procedure

(89) Cell cultures: Human primary monocytes were extracted from the whole blood of medically healthy volunteers, who provided written informed consent at the local blood bank (University Hospital of Freiburg, Germany), following a standardized protocol (gradient preparation, Lymphocytes separation medium, PAN Biotech, P04-60125, Aidenbach, Germany) using completely endotoxin-free cultivation. Using 50 ml tubes, 25 ml Pancoll was loaded with 25 ml of blood (buffy coats). The gradient was established by centrifugation at 1800 rpm, 20° C. for 40 min with slow acceleration and deceleration. Peripheral blood mononuclear cells in the interphase were carefully removed and re-suspended in 50 ml pre-warmed phosphate-buffered saline (PBS) (Pan Biotech, P04-36500), followed by centrifugation for 10 min at 1600 rpm and 20° C. The supernatant was discarded and the pellet washed in 50 ml PBS and centrifuged as described above. The pellet was then re-suspended in 50 ml RPMI-1640 low-endotoxin medium supplemented with 10% human serum (Hexcell, Berlin, Germany, SP2080). After counting the number of cells in a particle counter (Euro Diagnostics, Krefeld, Germany), cells were seeded in 24-well plates for enzyme linked immunosorbent assay (ELISA) (2.2 mio. cells/well) or 96-well plates at a density of 2×104 cells/well for cell viability testing, and incubated at 37° C. with 5% CO.sub.2. The medium and the non-adherent cells (lymphocytes) were removed and fresh RPMI-1640 medium containing 1% human serum added. Enriched monocytes were then ready to be used for the experiments. Primary human fibroblasts and HaCat keratinocytes were obtained from the Uniklinik Freiburg. All cells were maintained in supplemented DMEM (Invitrogen, Life-Technologies, Darmstadt, Germany) medium containing 10% FBS (Bio&Sell, Feucht, Germany) and 1% antibiotics penicillin/streptomycin (from Invitrogen, DMEM complete medium) at 37° C. in a humidified atmosphere of 5% CO.sub.2.

(90) Cell viability: Cells were incubated with the cannabinoids (3 doses, n=4) for 24 h. Cytotoxicity was analyzed by Alamar Blue staining (formazan). Then cells were washed once with 100 μl PBS, and 100 μl of medium-Alamar Blue-Mix (90% medium, 10% Alamar Blue, DAL1025, Thermo Fisher) was then added to each well. The plate was then incubated at 37° C. for 2 h in a humidified 5% CO.sub.2 atmosphere, and the color reaction determined using a 96-well plate reader (Berthold, Offenburg, Germany, excitation 544 nm, emission 590 nm).

(91) Determination of inflammatory molecules in fibroblasts: Primary human fibroblasts were cultivated as described above and seeded (500000 cells/well) in 24-well plates. Cells were incubated with IL-1beta (Roche, Mannheim, Germany, 10 U/ml) in absence and presence of the cannabinoids (5 doses, n=3) for 24 h. Unstimulated cells served as a negative control. 24 h after cell stimulation, supernatants were removed, centrifuged and investigated for IL-6, PGE2, and IL-8 concentrations by ELISAs according to the manufacturer's protocol (PGE2 from Cayman/Biomol, Hamburg, Germany; IL-6, and IL-8 from Immuno-tools, Frisoythe, Germany). The respective extinction is determined using a 96-well plate reader (Berthold, Offenburg, Germany).

(92) Determination of MMPs and TIMPs in keratinocytes: Keratinocytes (HaCat) were cultivated as described above. Cells were seeded in 24-well plates incubated with Poly I:C (InvivoGen, San Diego, Calif.; USA, 10 μg/ml) in absence and presence of the cannabinoids (5 doses, n=3) for 24 h. Unstimulated cells served as a negative control. 24 h after cell stimulation, supernatants were removed, centrifuged and investigated for MMP1, MMP9 and TIMP1 concentrations by ELISAs according to the manufacturer's protocol (from Biotechne, Wiesbaden, Germany). The respective extinction was determined using a 96-well plate reader (Berthold, Offenburg, Germany).

(93) Measurement of cytokines and PGE2 in primary human monocytes: Cells were incubated with LPS (Sigma-Aldrich, Taufkirchen, Germany, 10 ng/ml) for 24 h. The cannabinoids (5 doses) were added 30 min before LPS treatment. After 24 h, supernatants were removed, centrifuged and investigated for IL-1beta, IL-8, IL-6, TNFalpha, MMP9, isoprostane, and PGE2 concentrations in EIAs (PGE2 and isoprostane, from Cayman/Biomol, Hamburg, Germany) or ELISAs (IL-1beta, Hiss, Freiburg, Germany; TNFalpha, IL-6 and IL-8, ImmunoTools, Frysoithe, Germany, MMP9, Biotechne, Wiesbaden, Germany) using manufacturer's protocol. The respective extinction was determined using a 96-well plate reader (Berthold, Offenburg, Germany). Each dose was analyzed 4 times in two buffy coats from 2 different donors (2 buffy coats used from 2 different healthy blood donors with final n=4 values).

(94) Results

(95) Effects on Cell Viability in Human Monocytes

(96) Cytotoxicity assays were performed in primary human monocytes. (+)-CBD and (+)-CBDV affected cell viability starting in the doses of 25 μM and higher (FIGS. 26+27), the other three cannabinoids starting with 50 μM (FIGS. 28-30). To be able to compare the activity of the five cannabinoids, 25 μM were used as highest dose.

(97) Effects on LPS-Induced Inflammatory Parameters in Human Monocytes

(98) As shown in FIG. 31, (+)-CBD potently inhibited LPS-stimulated TNFalpha and PGE2 release starting at 0.1 μM (TNFalpha) and 1 μM (PGE2) with maximal effects using 25 μM, which showed an inhibition of around 80%. LPS-induced IL-6 was inhibited in the doses of 5 to 25 μM, IL-8 only in the dose of 25 μM, whereas LPS-mediated IL-1beta, MMP9 and isoprostane were not affected by (+)-CBD. The non-inhibiting effects on these 3 parameters suggest that the use of 25 μM is not killing the cells and thus affecting the other parameters.

(99) (+)-CBDV showed comparable effects to (+)-CBD besides a weaker effect on TNFalpha and a slight effect on IL-1beta in the highest dose (FIG. 32).

(100) (+)-CBD-ME only slightly inhibited LPS-induced IL-6 and TNFalpha with around 20%, but increased the chemokine IL-8 in the doses of 5-25 μM and IL-1beta in the dose of 25 μM (FIG. 33).

(101) As shown in FIG. 34, (+)-CBD-GE dose-dependently inhibited LPS-stimulated TNFalpha, IL-6, IL-1, isoprostane, and PGE2 release starting at 0.1 μM and maximal effects using 25 μM, which showed an inhibition of around 80% for IL-6 and PGE2 and approx. 40% for the other three parameters. LPS-induced IL-8 was not affected, whereas LPS-mediated MMP9 was strongly enhanced (20-60% more than LPS values) in the doses of 5-25 μM.

(102) (+)-CBD-HPE only slightly inhibited LPS-induced PGE2 in the doses of 5 to 25 μM and IL-6 in the doses of 10 and 25 μM. LPS-stimulated IL-8 and MMP9 were only slightly prevented in the dose of 25 μM, whereas LPS-mediated IL-1beta was potently increased (almost doubling the LPS effect) in the dose of 25 μM, whereas TNFalpha induced by LPS was not affected (FIG. 35).

(103) Effects on IL-1-Induced Inflammatory Parameters in Human Dermal Fibroblasts

(104) As shown in FIG. 36, (+)-CBD potently and dose-dependently inhibited IL-1-stimulated IL-6 and IL-8 release starting at 0.1 μM with maximal effects using 25 μM, showing an inhibition of more than 90%. IL-1-induced PGE2 was strongly inhibited in the doses of 10 to 25 μM (more than 80% inhibition).

(105) (+)-CBDV showed a comparable profile to (+)-CBD, but with a weaker activity on IL-6 and IL-8 which started using 5 μM. IL-1-induced PGE2 was strongly inhibited in the doses of 10 to 25 μM (more than 80% inhibition) (FIG. 37).

(106) (+)-CBD-ME slightly increased LPS-induced IL-6 and IL-8 and slightly decreased IL-1-mediated PGE2. The dose of 25 μM seems to be toxic in fibroblasts (FIG. 38).

(107) As shown in FIG. 39, (+)-CBD-GE dose-dependently inhibited all IL-1-stimulated parameters starting at 5 μM and maximal effects using 25 μM, which showed an inhibition of around 95% for IL-6 and IL-8 and approx. 80% for PGE2.

(108) (+)-CBD-HPE inhibited IL-1-induced IL-6 and IL-8 in the doses of 10 and 25 μM (95% inhibition) and PGE2 only in the dose of 25 μM (50% inhibition) (FIG. 40).

(109) Effects on Poly I:C-Induced Proteases in Human HaCat Keratinocytes

(110) As shown in FIG. 41, (+)-CBD slightly inhibited Poly I:C-stimulated MMP9 and TIMP1 starting at 5 μM with maximal effects using 25 μM, showing an inhibition of around 40% for MMP9 and 70% for TIMP1. Poly I:C-induced MMP1 was only slightly inhibited in the doses of 25 μM (more approx. 40% inhibition).

(111) (+)-CBDV showed slight inhibitory effects on Poly I:C-stimulated MMP9 and TIMP1 but enhanced Poly I:C-stimulated MMP1 in the dose of 25 μM (FIG. 42).

(112) (+)-CBD-ME decreased dose-dependently slightly Poly I:C-induced TIMP1 in the doses of 5 to 25 μM (40% inhibition) but enhanced Poly I:C-stimulated MMP9 and MMP1 (FIG. 43).

(113) As shown in FIG. 44, (+)-CBD-GE (25 μM) showed slight inhibitory effect on Poly I:C-stimulated MMP9 and TIMP1 (30-50% inhibition) but did not affect MMP1 induced by Poly 1:0.

(114) (+)-CBD-HPE showed inhibitory effects on all Poly I:C induced parameters with an inhibition of 40% for MMP9, 60% for TIMP1, and 20% of MMP1 at the dose of 25 μM (FIG. 45).

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