Isolation and Characterization of New Cannabis Constituents from a High Potency Variety

Abstract

Phytochemical investigation of a high potency variety of Cannabis sativa L. resulted in the isolation of six new metabolites, (±)-6,7-trans-epoxycannabigerolic acid (2), (±)-6,7-cis-epoxycannabigerolic acid (3), (±)-6,7-cis-epoxycannabigerol (4), (±)-6,7-trans-epoxycannabigerol (5), 5′-methyl-4-pentylbiphenyl-2,2′,6-triol (7), and 7-methoxycannabispirone (8), along with seven known compounds namely, cannabigerolic acid (1), 5′-methoxycannabigerolic acid (6), cannabispirone (9), β-cannabispiranol (10), dehydrocannabifuran (11), cannflavin B (12) and cannabigerol (13). The antimicrobial as well as the antileishmanial activities were investigated.

Introduction

Cannabis sativa L. (Cannabaceae), one of the oldest plants known in medicine, is the most widely used illicit drug in the world today. A total of almost 500 natural constituents have been isolated and/or identified from Cannabis [1], with Δ⁹-THC as the main biologically active component [2]. The availability of high potency marijuana on the illicit market with unprecedented Δ⁹-THC concentrations (> 20% by dry weight) [3] has renewed our interest in the discovery of new constituents from cannabis. We herein report the isolation and structure elucidation of six new metabolites (2, 3, 4, 5, 7 and 8) and seven known compounds (1, 6 and 9–13). This is the first report of the full NMR data for 1, 6 and 11. The antimicrobial and antileishmanial activities of the isolates are also reported.

Materials and Methods

General experimental procedures

¹H-NMR (400 MHz), ¹³C-NMR (100 MHz) and 2D-NMR spectra were recorded using the residual solvent signal as an internal standard on a Varian AS 400. IR spectra were measured on a Bruker Tensor 27. UV spectra were obtained on a Varian Cary 50 Bio UV-Visible spectrophotometer. Optical rotation was measured on an Autoplot IV automatic polarimeter. High resolution mass spectra were measured using a Bruker BioApex. HPLC was performed on a Waters Delta Prep 4000 Preparative Chromatography System connected to a Waters 486 Tunable Absorbance detector (206 nm) using a Phenomenex Luna C18 column (250×21.2 mm, 5 μm, 100 Å). Flash silica gel (J.T. Baker, 40–63 μm, 60 Å), C18 silica gel (Fluka, 40–63 μm, 60 Å) and Sephadex LH 20 (Fluka) were used for column chromatography. GC-MS analyses were carried out on a ThermoQuest Trace 2000 GC, equipped with a single split/splitless capillary injector, a ThermoQuest AS2000 autosampler and a Phenomenex ZB-5 column (30 m×0.25 mm×0.25 μm), interfaced to a ThermoQuest-Finnigan Trace MS quadrupole ion trap detector. The injector temperature was 250 °C and 1 μL injections were performed in the splitless mode, with the splitless time set at 60 s, the split flow set at 50 mL/min and the septum purge valve set to close 60 s after the injection occurred. The oven temperature was raised from 70 to 270 °C (hold 20 min) at a rate of 5 °C/min, for a total run time of 60 min; the transfer line temperature was 250 °C. Helium was used as the carrier gas at a constant pressure of 20 psi. The mass spectrometer was operated in the electron impact mode (EI⁺) and scanned from 40 to 800 amu at 1 scan/s, with an ionizing voltage of 70 eV and an emission current of 350μA. Data was recorded using an IBM Netfinity 3000 workstation with Microsoft Windows NT 4.0 operating system and Xcalibur (Version 1.2) data acquisition and analysis software.

Plant material

C. sativa plants were grown from high potency Mexican seeds (variety code CHPF-01). The seeds and plants were authenticated by Dr. Suman Chandra, The University of Mississippi, and the specimen (S1310V1) was deposited at the Coy Waller Complex, The University of Mississippi. Whole buds of mature female plants were harvested, air-dried, packed in barrels (# 1196) and stored at low temperature (−24 °C). The THC, CBG and CBD contents in the plant material determined by GC/FID analysis were 9.89 %, 0.42% and 0.25 %, respectively.

Extraction and isolation

The plant material (9.0 kg) was sequentially extracted with hexanes (2×60 L), CH₂Cl₂ (2×24 L), EtOAc (2×20 L), EtOH (2×20 L), EtOH/H₂O (36 L, 1: 1) and H₂O (40 L) at room temperature. The extracts were evaporated under reduced pressure at 40 °C to afford hexanes (1.48 kg), CH₂Cl₂ (0.15 kg), EtOAc (0.13 kg), EtOH (0.09 kg), EtOH/H₂O (0.77 kg) and H₂O (0.54 kg) extracts for a total extract of 3.16 kg (35.1%, w/w). A portion of the hexanes extract (40 g) was chromatographed on flash silica gel (1.2 kg, 10×50 cm) eluting with n-hexane. Fractions with Rf close to that of Δ⁹-THC according to silica gel TLC (n-hexane/EtOAc, 9: 1) were combined and purified by flash silica chromatography and Sephadex LH-20 (n-hexane as eluent), followed by final purification by preparative C18 HPLC (MeCN, 25 mL/min) to afford 4 (3.3 mg, rt = 9.5 min), 5 (9.0 mg, rt = 9.0 min), 11 (2.6 mg, rt = 5.3 min.) and 13 (45 mg, rt = 2.5 min). Portions of the CH₂Cl₂, EtOAc and EtOH extracts were combined (232.0 g) since they showed similar TLC profiles (EtOAc/n-hexane, 4: 6), and the resulting extract was subjected to VLC over silica gel (6 kg, 15×90 cm) eluting with EtOAc/n-hexane [0:100, 10: 90, 20: 80, 30: 70, 40: 60, 50: 50, 75: 25, 100:0 (2 L of each mixture)] followed by EtOH (4 L), yielding 9 fractions (I – IX). Fraction III (1.70 g) was chromatographed on silica gel (50 g, 2.0×50 cm, n-hexane/EtOAc, 100:0 to 85:15), yielding 36 fractions (III_1–36, 100 mL each). Fraction III_10–14 (35 mg) was purified by C18 HPLC (MeCN/H₂O, 95:5, 25 mL/min) to afford 6 (3.0 mg, rt = 7.4 min) and 8 (7.4 mg, rt = 8.8 min). Fraction III_15–30 (526 mg) afforded 1 (382 mg) upon precipitation from n-hexane/EtOAc. Fraction III_32–36 (130 mg) was purified by C18 HPLC (MeOH/H₂O, 65: 35, 25 mL/min) to give 2 (13.4 mg, rt = 9.4), 3 (4.0 mg, rt = 10.8 min) and 7 (2.4 mg, rt = 5.1 min). Fraction VII (6.70 g) was subjected to silica gel chromatography (400 g, 7.0×80 cm, n-hexane/EtOAc, 80:20) yielding 13 fractions (VII_1–13, 250 mL each). Fraction VII_9–10 (41 mg) was purified by C18 HPLC (MeOH/H₂O, 75: 25) to afford 9 (5.3 mg, rt = 15.3 min) and 10 (5.0 mg, rt = 4.0 min). Fraction VII₁₁ (2.78 g) was purified by Sephadex LH-20 (50 g, 2.0 × 50 cm, MeOH) followed by C18 flash chromatography (MeOH/H₂O, 8: 2) to afford 12 (264.1 mg).

Isolates

Cannabigerolic acid (1): White amorphous powder; UV (EtOH): λ_max = 255, 299 nm; IR (neat): ν_max = 3390, 1650 cm⁻¹; ¹H- and ¹³C-NMR, see Table 1 and Table 2; HR-ESI-MS (negative ion mode): m/z = 359.2214 [M − H]⁻ (calcd. for C₂₂H₃₁O₄:359.2222). (±)-6,7-trans-Epoxycannabigerolic acid (2): Yellow oil; UV (MeOH): λ_max = 215, 260, 300 (sh) nm; ${[\alpha ]}_{\mathrm{D}}^{25}$: 0 (c 1.2, MeOH); IR (neat): ν_max = 3402, 1650 cm⁻¹; ¹H- and ¹³C-NMR, see Table 1 and Table 2; HR-ESI-MS (positive ion mode): m/z = 399.2156 [M + Na]⁺ (calcd. for C₂₂H₃₂O₅Na: 399.2147).

Table 1.

¹H-NMR data for 1–7 and 13 (400 MHz, δ in ppm, J in Hz)

Position	1a	2a	1b	3b	4b	5b	6b	13b	7b
1	1.59 s	1.53 s	1.52 s	1.57 s	1.57 s	1.57 s	1.55 s	1.60 s	–
3	5.06 t (6.4)	5.06 t (6.4)	5.03 t (6.4)	5.08 t (6.4)	5.08 t (6.4)	5.07 t (6.4)	5.06 (m)	5.07 m	6.45 s
4	2.12 m	2.10 m	1.91 t (6.8)	2.13 m	2.13 m	2.11 m	2.03 m	2.09 m
5	2.22 m	1.49 m	2.10 t (7.2)	1.53 m	1.53 m	1.53 m	1.94 m	2.09 m	6.45 s
6	–	–	–	–	–	–	–	–	–
7	5.28 t (6.8)	3.62 t (5.2)	5.17 t (6.8)	3.88 t (5.2)	3.88 t (5.2)	3.87 t (5.2)	5.18 t (6.6)	5.29 m	–
8	3.43 d (6.8)	2.68 dd (5.2, 16.4) 2.33 dd (8.4, 16.8)	3.19 d (6.8)	2.45 m	2.45 m	2.43 m	3.32 d (7.6)	3.41 d (7.0)	–
9	1.67 s	1.61 s	1.59 s	1.66 s	1.66 s	1.65 s	1.63 s	1.69 s	–
10	1.82 s	1.04 s	1.71 s	1.32 s	1.32 s	1.32 s	1.76 s	1.82 s	–
2′	–	–	–	–	6.20 s	6.20 s	–	6.26 s	–
3′	–	–	–	–	–	–	–	–	6.99 d (8.2)
4′	6.28 s	6.18 s	6.25 s	6.30 s	6.30 s	6.28 s	6.31 s	6.26 s	7.15 dd (2.0, 8.2)
6′	–	–	–	–	–	–	–	–	7.03 d (2.0)
7′	–	–	–	–	–	–	–	–	2.30 s
1″	2.88 t (7.2)	2.86 t (7.2)	2.80 t (7.2)	2.90 t (7.2)	2.90 t (7.2)	2.84 t (7.2)	2.88 t (7.6)	2.45 t (7.5)	2.52 t (5.6)
2″	1.60 m	1.45 m	1.60 m	1.58 m	1.58 m	1.57 m	1.58 m	1.56 q(7.8)	1.59 m
3″	1.35 m	1.23 m	1.28 m	1.26 m	1.26 m	1.26 m	1.35 m	1.33 m	1.33 m
4″	1.35 m	1.23 m	1.28 m	1.26 m	1.26 m	1.26 m	1.35 m	1.33 m	1.33 m
5″	0.91 t (7.2)	0.83 t (6.4)	0.85 t (6.8)	0.88 t (6.4)	0.88 t (6.4)	0.88 t (6.4)	0.89 t (6.4)	0.90 t (6.9)	0.89 t (6.4)
OCH3	–	–	–	–	–	–	3.86 s	–	–

^*Assignments confirmed by DEPT-135, gHMQC, gCOSY, and gHMBC experiments.

^aIn DMSO-d6.

^bIn CDCl₃.

Table 2.

¹³C-NMR data for 1–7 and 13 (100 MHz, δ in ppm)

Position	1a	2a	1b	3b	4b	5b	6b	13b	7b
1	17.9	18.1	17.9	17.7	17.8	17.8	17.9	17.6	117.3
2	132.1	131.4	131.0	132.8	132.8	132.2	131.3	132.0	154.1
3	124.1	125.2	124.6	123.4	123.4	124.2	124.7	123.8	108.4
4	26.8	21.6	26.7	21.9	21.9	21.9	26.1	26.4	146.6
5	40.0	38.8	40.0	37.1	36.9	36.9	40.0	39.7	108.4
6	138.7	78.9	133.9	81.9	81.4	78.4	135.3	138.0	154.1
7	121.7	66.9	123.1	67.2	67.3	68.3	122.4	121.8	–
8	22.3	26.7	22.4	26.0	26.0	26.0	22.1	22.5	–
9	25.9	26.2	25.9	25.8	25.8	25.9	25.9	25.6	–
10	16.4	18.1	16.3	17.7	17.8	17.8	16.3	16.1	–
1′	163.9	151.4	163.3	153.0	152.8	154.5	163.0	154.8	152.5
2′	103.4	106.1	103.6	105.0	110.6	109.6	104.0	108.4	117.3
3′	147.7	138.8	145.1	145.1	143.3	143.3	147.5	142.7	132.0
4′	111.5	107.1	110.3	108.4	108.4	107.5	106.2	108.4	131.6
5′	160.7	156.0	160.0	153.8	152.8	153.6	162.3	154.8	132.1
6′	112.1	116.1	112.6	110.9	110.6	110.6	115.2	110.7	116.4
7′	–	–	–	–	–	–	–	–	20.7
1″	36.8	36.4	36.2	35.2	36.8	36.9	37.4	35.5	36.1
2″	31.7	31.1	31.6	31.2	31.3	31.0	31.9	30.8	30.8
3″	32.2	31.8	32.0	32.1	31.8	31.7	32.3	31.5	31.7
4″	22.8	22.6	22.0	22.7	22.7	22.7	22.7	22.2	22.8
5″	14.3	14.5	14.3	14.2	14.3	14.2	14.3	14.0	14.2
COOH	176.6	170.0	174.3	176.4	–	–	176.6	–	–
OMe	–	–	–	–	–	–	55.7	–	–

^*Assignments confirmed by DEPT-135, gHMQC, gCOSY and gHMBC experiments.

^aIn DMSO-d₆.

^bIn CDCl₃.

(±)-6,7-cis-Epoxycannabigerolic acid (3): Yellow oil; UV (MeOH): λ_max = 215, 260, 300 (sh) nm; ${[\alpha ]}_{\mathrm{D}}^{25}$: 0 (c 1.2, MeOH); IR (neat): ν_max = 3402, 1650 cm⁻¹; ¹H- and ¹³C-NMR, see Table 1 and Table 2; HR-ESI-MS (positive ion mode): m/z = 399.2194 [M + Na]⁺ (calcd. for C₂₂H₃₂O₅Na: 399.2147) and HR-ESI-MS (negative ion mode): m/z = 375.2116 [M − H]⁻ (calcd. for C₂₂H₃₁O₅: 375.2172).

(±)-6,7-cis-Epoxycannabigerol (4): Yellow oil; UV (MeOH): λ_max = 215, 260, 300 (sh) nm; ${[\alpha ]}_{\mathrm{D}}^{25}$: 0 (c 1.2, MeOH); IR (neat): ν_max = 3402, 1610 cm⁻¹; ¹H- and ¹³C-NMR, see Table 1 and Table 2; HR-ESI-MS (positive ion mode): m/z = 333.2493 [M + H]⁺ (calcd. for C₂₁H₃₃O₃:333.2431).

(±)-6,7-trans-Epoxycannabigerol (5): Yellow oil; UV (MeOH): λ_max = 215, 260, 300 (sh) nm; ${[\alpha ]}_{\mathrm{D}}^{25}$: 0 (c 1.2, MeOH); IR (neat): ν_max = 3402, 1610 cm⁻¹; ¹H- and ¹³C-NMR, see Table 1 and Table 2; HR-ESI-MS (positive ion mode): m/z = 333.2486 [M + H]⁺ (calcd. for C₂₁H₃₃O₃:333.2431).

5′-Methoxycannabigerolic acid (6): Yellow oil; UV (EtOH): λ_max = 221, 262, 300 nm; IR (neat): ν_max = 3400, 1650 cm⁻¹; ¹H-and ¹³C-NMR, see Table 1 and Table 2; HR-ESI-MS (positive ion mode): m/z = 375.2540 [M + H]⁺ (calcd. for C₂₃H₃₅O₄: 375.2535).

5′-Methyl-4-pentylbiphenyl-2,2′,6-triol (7): Yellow oil; UV (EtOH): λ_max = 210, 282 nm; IR (neat): ν_max = 3390, 2910, 1615 cm⁻¹; ¹H- and ¹³C-NMR, see Table 1 and Table 2 HR-ESI-MS (positive ion mode): m/z = 287.1612 [M+H]⁺ (calcd. for C₁₈H₂₃O₃:287.1647). 7-Methoxycannabispirone (8): White needles; m.p.123–124 °C; UV (EtOH): λ_max = 224, 275 nm; IR (neat): ν_max=3370, 1710, 1600 cm⁻¹; ¹H- and ¹³C-NMR, see Table 3; HR-ESI-MS (positive ion mode): m/z=261.1434 [M+H]⁺ (calcd. for C₁₆H₂₁O₃:261.1491).

Table 3.

¹H-NMR and ¹³C-NMR data for 8, 9 and 11 (CDCl₃, δ in ppm, J in Hz)

Position	8		9		11
	δH	δC	δH	δC	δH	δC
1	–	48.2	–	47.8	–	154.3
2	2.22 t (7.2)	35.5	2.21 t (7.2)	35.6	7.06 bs	110.5
3	2.93 t (7.2)	31.2	2.93 t (7.2)	31.2	–	134.8
4	6.37 bs	101.0	6.35 bs	102.0	6.66 bs	103.5
5	–	160.8	–	160.5	–	145.0
6	6.28 bs	97.4	6.15 bs	101.0	–	114.5
7	–	157.5	–	153.6	–	138.7
8	–	125.8	–	126.9	–	155.6
9	–	145.7	–	146.5	–	117.1
10	3.75 s	55.6	3.72 s	55.6	7.04 d (8.0)	127.0
11	3.78 s	55.1	–	–	7.13 d (8.0)	121.2
12	–	–	–	–	–	128.3
13	–	–	–	–	2.59 s	15.2
14	–	–	–	–	–	150.2
15	–	–	–	–	5.23 bs 5.64 bs	110.7
16	–	–	–	–	2.31 s	22.7
1′	–	–	–	–	2.73 (7.2)	36.3
2′	2.40 m 2.53 dt (6.4, 13.2)	39.2	2.42 m 2.53 dt (6.4, 13.2)	39.2	1.55 m	29.9
3′	1.82 dd (2.8, 6.4) 2.63 dt (4.6, 13.2)	34.5	1.82 dd (2.8, 6.4) 2.67 dt (4.6, 13.2)	34.5	1.25 m	31.5
4′	–	213.3	–	214.5	1.25 m	22.7
5′	1.81 dd (2.8, 6.4) 2.63 dt (4.6, 13.2)	34.5	1.82 dd (2.8, 6.4) 2.67 dt (4.6, 13.2)	34.5	0.88 t (6.8)	14.2
6′	2.40 m 2.53 dt (6.4, 13.2)	39.2	2.42 m 2.53 dt (6.4, 13.2)	39.2	–	–

^*Assignments confirmed by DEPT-135, gHMQC, gCOSY and gHMBC experiments.

Biological activity

The isolated compounds were tested in vitro against a culture of Leishmania donovani (L. Rivas, Centro de Investigaciones Biologocas CSIC. Madrid, Spain) promastigotes, using pentamidine (Sigma) and amphotericin B (Sigma) as positive controls (IC₅₀=0.15 and 0.9 ng/mL, resectively) [4]. Their antimicrobial activity against Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Cryptococcus neoformans, Mycobacterium intracellulare and Aspergillus fumigates (all from ATCC) [5] as well as the cytotoxicity [6] against Vero cells (African green monkey kidney fibroblast; ATCC) were also tested.

GC-MS trimethylsilyl derivatization

Dried samples (ca. 100μg) were mixed with pyridine (5 μL, silylation grade, Pierce) and BSTFA [N,O-bis(trimethylsilyl)trifluoroacetamide] (100μL, 98+%, Acros Organics), followed by heating at 75 °C for 1 h. After cooling to room temperature, CH₂Cl₂ (0.9 mL) was added to the reaction mixture and the solution analyzed by GC-MS.

Results and Discussion

Compound 1 was isolated as a white amorphous powder. Its HR-ESI-MS displayed a pseudomolecular ion at m/z = 359.2214 [M − H]⁻, indicating the molecular formula C₂₂H₃₂O₄. The IR spectrum of 1 showed the presence of hydroxy groups (3390 cm⁻¹) and a chelated carboxyl group (1650 cm⁻¹). The ¹H-NMR spectrum of 1 in DMSO-d₆ (Table 1) showed three methyl singlets at δ_H = 1.59 (H-1), 1.67 (H-9) and 1.82 (H-10), three methylenes at δ_H = 2.12 (m, H-4), 2.22 (m, H-5) and 3.43 (d, H-8) and two olefinic protons at δ_H = 5.06 (t, H-3) and 5.28 (t, H-7), attributed to a geranyl substituent. It also displayed an aromatic proton at δ_H = 6.28 (s, H-4′) corresponding to δ_C = 111.5 (C-4′) in the HMQC spectrum. The ¹³C-NMR, DEPT-135 and HMQC spectra showed 22 resonances, including 4 methyl, 7 methylene, 3 methine and 8 quaternary carbons. Two of these resonances were assigned to aromatic carbons bearing hydroxy groups at δ_C = 160.7 (C-5′) and 163.9 (C-1′), while a carboxyl group at δ_C = 176.6 (COOH) was attached to C-2′ (δ_C = 103.4). The structure was further confirmed by GC-MS: 1 spontaneously decarboxylated on injection to give cannabigerol (13) ([M]⁺ = 316). The trimethylsilyl derivative of 1 ([M]⁺ = 576) confirmed the HR-ESI-MS result and the presence of two phenolic and one carboxyl groups. The ¹H-NMR and IR data of 1 were similar to those previously reported for cannabigerolic acid [7], however, this is the first report of the ¹³C-NMR, DEPT, 2D-NMR and HR-ESI-MS data for 1.

Compound 2 was isolated as a yellow, optically inactive oil. Its molecular formula, C₂₂H₃₂O₅, was derived from HR-ESI-MS (m/z = 399.2156 [M + Na]⁺, 775.4364 [2M+ Na]⁺). The IR spectrum of 2 diplayed hydroxy groups at 3402 cm⁻¹ and a chelated carboxyl group at 1650 cm⁻¹. The ¹H- and ¹³C-NMR, DEPT and HMQC data of 2 in DMSO-d₆ (Table 1 and Table 2) were similar to those of 1 except for the presence of a 6,7-epoxy group [δ_H = 3.62 (t, J = 5.2 Hz, H-7); δ_C = 66.9 (C-7), 78.9 (C-6)] instead of the 6,7-double bond [δ_H = 5.28 (t, J = 6.8 Hz, H-7); δ_C = 121.7 (C-7), 138.7 (C-6)]. The 6,7-epoxy position was determined by the HMBC correlations of H-7 with C-5, C-6, C-10, C-8 and C-6′, and H₃–10 with C-5, C-6 and C-7 (Fig. 1). The structure was further confirmed by GC-MS: the trimethylsilylderivative of 2 ([M]⁺=592) confirmed the HR-ESI-MS result and the presence of two phenolic and one carboxyl groups. The lack of a NOESY correlation between H-7 and H₃–10 indicated a 6,7-trans-configuration, while lack of any optical rotation points to a racemic mixture of enantiomers (2a and 2b). Thus, 2 is (±)-6,7-trans-epoxycannabigerolic acid. Compound 3 was isolated as an optically inactive yellow oil. The HR-ESI-MS of 3 afforded an [M + Na]⁺ ion at m/z = 399.2194 implying a molecular formula C₂₂H₃₂O₅. The ¹H-, ¹³C- and 2D-NMR data of 3 (Table 1 and Table 2) are similar to those of 2 except for the downfield shift of C-6 (+ 3 ppm) [8] and the ROESY correlation between H-7 and H₃–10 (Fig. 1), these findings indicated the 6,7-cis configuration (3a and 3b). Therefore, 3 is (±)- 6,7-cis-epoxycannabigerolic acid.

Fig. 1.

HMBC correlations of 2 and 3 (→) and key ROESY correlations of 3 (↔).

Compound 4 was obtained as an optically inactive oil, with a molecular formula C₂₁H₃₂O₃ based on HR-ESI-MS (m/z = 333.2493 [M + H]⁺). GC-MS analysis (rt = 38.84 min) displayed a base peak at m/z = 193. The IR, ¹H- and ¹³C-NMR data of 4 in CDCl₃ were similar to those of cannabigerol (13) [9] (Table 1 and Table 2) except for the presence of a 6,7-epoxy group [δ_H = 3.88 (t, J = 5.2 Hz, H-7); δ_C = 67.3 (C-7), 81.4 (C-6)], while the ROESY correlation between H-7 and H₃–10, established 4 as (±)-6,7-cis-epoxycannabigerol.

Compound 5 was also isolated as an optically inactive oil and its molecular formula was determined as C₂₁H₃₂O₃ by HR-ESI-MS and ¹³C-NMR spectroscopy. GC-MS analysis of 5 (rt = 38.68 min) revealed a base peak at m/z = 193, while the NMR data were almost identical to those of 4 except for an upfield shift of C-6 (Table 2) and the absence of ROESY correlation between H-7 and H₃–10. Based on the above, 5 was elucidated as (±)-6,7-trans-epoxycannabigerol.

Compound 6 was isolated as a yellow oil. On the basis of its HR-ESI-MS at m/z = 375.2540 [M + H]⁺ and ¹³C-NMR spectroscopic data, the molecular formula was established as C₂₃H₃₄O₄. The structure was determined by comparing its ¹H- and ¹³C-NMR data in CDCl₃ (Table 1 and Table 2)with 1. Compound 6 contained an additional methoxy group [δ_H = 3.86 (s, OMe); δ_C = 55.7 (OMe)] instead of a hydroxy group. The location of the methoxy group was determined to be at C-5′ from HMBC correlations (OMe/C-5′; OMe/C-6′; H-4′/OMe), establishing 6 as 5′-methoxycannabigerolic acid. Although 6 is a known cannabis constituent [10], this is the first report of its full NMR assignments.

Compound 7 was isolated as a yellow oil. It gave a molecular formula of C₁₈H₂₂O₃ based on HR-ESI-MS (m/z= 287.1612 [M+H]⁺, 285.1581 [M − H]⁻), GC-MS ([M]⁺ 286) and ¹³C-NMR data. The IR absorption bands at 3390, 1615, 1242 and 1035 cm⁻¹ indicated the presence of hydroxy and benzene ring functionalities. The ¹H-NMR (Table 1) and COSY spectra of 7 indicated two aromatic ring systems, 1,2,4,6-tetrasubstituted ring A with two magnetically equivalent protons [δ_H = 6.45 (2H, s, H-3 and H-5)] and an ABX spin system for ring B [δ_H = 6.99 (1H, d, J = 8.2 Hz, H-3′), 7.15 (1H, dd, J = 2.0, 8.2 Hz, H-4′), 7.03 (1H, d, J = 2.0 Hz, H-6′)]. The ¹H-NMR also showed the presence of one aromatic methyl singlet at δ_H = 2.30 (H-7′) and an n-pentyl moiety. The ¹³C-NMR (Table 2), DEPT-135 and HMQC spectra revealed the presence of 18 carbon resonances, including 2 methyl, 4 methylene, 5 sp² methine, 3 oxyaryl and 4 quaternary carbons. The ¹H- and ¹³C-NMR data of ring A are suggestive of a phenyl substituted olivetol (biphenyl) [11], and together with HMBC correlations (Fig. 2) [H₃ –7′/C-4′, C-6′; H-3′/C-2′, C-1′] indicates that 7 is 5′-methyl-4-pentylbiphenyl-2,2′,6-triol.

Fig. 2.

Key HMBC correlations of 6.

Compound 8 was obtained as white needles. HR-ESI-MS at m/z = 261.1434 [M + H]⁺ and ¹³C-NMR established the molecular formula as C₁₆H₂₀O₃. GC-MS analysis of 8 showed a base peak at m/z = 203 and two other characteristic ions at m/z = 189 and 175, indicating that 8 is a spiroindane derivative [12]. ¹H- and ¹³C-NMR data of 8 were similar to those reported for cannabispirone 9 (Table 2) [13], [14], except for a methoxy instead of hydroxy group at C-7, establishing 8 as 7-methoxycannabispirone. The structure was confirmed by DEPT-135, COSY, HMQC and HMBC analysis. Although 8 is a known synthetic product [14], [15], this is the first report of its isolation from a natural source.

Compounds 9–13 were identified as cannabispirone [12], β-cannabispiranol [15], dehydrocannabifuran [16], [17], cannflavin B [9] and cannabigerol [9] by comparing their spectroscopic data with reported values. However, this is the first report for the ¹³C-NMR assignments for dehydrocannabifuran (11) (Table 3).

Compound 13 exhibited selective antimicrobial activity against Mycobacterium intracellulare with an IC₅₀ value of 15.0 μg/mL. Compounds 1 and 12 displayed moderate antileishmanial activity with IC₅₀ values of 12.0 and 5.0 μg/mL, respectively. All isolates lacked cytotoxicity against Vero cells (African green monkey kidney fibroblast).

Abbreviations

CBG

cannabigerol

CBD

cannabidiol

FID

flame ionization detector

Δ⁹-THC

Δ⁹-tetrahydrocannabinol

VLC

vacuum liquid chromatography