Fluorinated CBD compounds, compositions and uses thereof

Abstract

The present invention relates to fluorine substituted CBD compounds, compositions thereof and uses thereof for the preparation of medicaments.

Claims

1. A compound having the general formula (IV): ##STR00015## wherein is a single bond or a double bond; R.sub.1 is selected from straight or branched C.sub.1-C.sub.8 alkyl, straight or branched C.sub.2-C.sub.10 alkenyl, straight or branched C.sub.2-C.sub.10 alkynyl, C(O)R.sub.8, C(O)OR.sub.9 each optionally substituted by at least one F; R.sub.2 is selected from straight or branched C.sub.1-C.sub.8 alkyl, straight or branched C.sub.2-C.sub.10 alkenyl, straight or branched C.sub.2-C.sub.10 alkynyl, each optionally substituted by at least one F; R.sub.3 and R.sub.4 are each independently selected from H, straight or branched C.sub.1-C.sub.5 alkyl, OR.sub.10, C(O)R.sub.11, OC(O)R.sub.12; provided that at least one of R.sub.3 and R.sub.4 is different than H; R.sub.5 is selected from a straight or branched C.sub.5-C.sub.12 alkyl, a straight or branched C.sub.5-C.sub.9 alkoxy, a straight or branched C.sub.1-C.sub.7 ether, each being optionally substituted by at least one substituent selected from OH, NH.sub.3, straight or branched C.sub.1-C.sub.5 amine, halogen, phenyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl; R.sub.8, and R.sub.9 are independently selected from H, OH, straight or branched C.sub.1-C.sub.5 alkyl, straight or branched C.sub.1-C.sub.5 alkoxy, NH.sub.3, straight or branched C.sub.1-C.sub.5 amine; R.sub.10 is selected from H, a straight or branched C.sub.1-C.sub.5 alkyl; and R.sub.11 and R.sub.12 are independently selected from H, OH, straight or branched C.sub.1-C.sub.5 alkyl, straight or branched C.sub.1-C.sub.5 alkoxy, NH.sub.3, straight or branched C.sub.1-C.sub.5 amine; and R.sub.15 and R.sub.16 are each optionally selected from H and F; provided that at least one of R.sub.15 and R.sub.16 is F or at least one of R.sub.1 and R.sub.2 is substituted with F.

2. The compound of claim 1, wherein is a double bond.

3. The compound of claim 1, wherein R.sub.1 is straight or branched C.sub.1-C.sub.8 alkyl; R.sub.3 and R.sub.4 are each independently OR.sub.10; R.sub.10 is selected from H, a straight or branched C.sub.1-C.sub.5 alkyl.

4. The compound of claim 1, wherein R.sub.1 is straight or branched C.sub.1-C.sub.8 alkyl; and R.sub.3 and R.sub.4 are OH.

5. The compound of claim 1, wherein R.sub.5 is straight or branched C.sub.5-C.sub.12 alkyl.

6. The compound of claim 1, wherein at least one of R.sub.1 and R.sub.2 is substituted with F.

7. The compound of claim 1, wherein R.sub.1 is selected from straight or branched C.sub.1-C.sub.8 alkyl, straight or branched C.sub.2-C.sub.10 alkenyl, straight or branched C.sub.2-C.sub.10 alkynyl, each being substituted by F.

8. A pharmaceutical composition comprising at least one compound according to claim 1.

9. A pharmaceutical composition comprising at least one compound according to claim 2.

10. A pharmaceutical composition comprising at least one compound according to claim 3.

11. A pharmaceutical composition comprising at least one compound according to claim 4.

12. A pharmaceutical composition comprising at least one compound according to claim 5.

13. A pharmaceutical composition comprising at least one compound according to claim 6.

14. A pharmaceutical composition comprising at least one compound according to claim 7.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

(2) FIG. 1 shows the effects of HU-475 (a compound of the invention 1, 3 and 10 nmol, n=8-9 animals/group) and vehicle (n=7) microinjected into the dorsolateral periaqueductal gray of rats submitted to the elevated plus maze. Data expressed as meansSEM of the percentage of entries onto the open arms. * indicates significant difference from vehicle (p<0.05).

(3) FIG. 2 shows the effects of HU-475 (a compound of the invention 1, 3 and 10 nmol, n=8-9 animals/group) and entries onto vehicle (n=7) microinjected into the dorsolateral periaqueductal gray of rats submitted to the elevated plus maze. Data expressed as meansSEM of the percentage of time spent in the open arms. + indicates a trend (p<0.1) for a difference from vehicle.

(4) FIG. 3 shows the effects of HU-474 (a compound of the invention 1, 3 and 10 mg/kg n=6-8 animals/group) and vehicle (n=7) in mice tested in the elevated plus maze. Data expressed as meansSEM of the percentage of time in the open arms. * indicates significant difference from vehicle.

(5) FIG. 4 shows the effects of HU-474 (a compound of the invention 1, 3 and 10 mg/kg n=6-8 animals/group) and vehicle (n=7) in mice tested in the elevated plus maze. Data expressed as meansSEM of the percentage of entries onto the open arms.

(6) FIG. 5 shows the effects of HU-474 (a compound of the invention 1, 3 and 10 mg/kg n=6-8 animals/group) and vehicle (n=7) in mice immobility time (s) showed by mice tested in the FST. Data expressed as meansSEM. * indicates significant difference from vehicle.

(7) FIG. 6 shows the effects of HU-474 (3 and 10 mg/kg i.p.) in mice on the impairment of PPI induced by MK-801 (M0.5 mg/kg). Results are expressed as meansSEM. * indicates significant difference from vehicle-vehicle, # significant difference from vehicle-MK group.

DETAILED DESCRIPTION OF EMBODIMENTS

Example 1: Fluorination of Cannabidiol (HU-474)

(8) ##STR00012##

(9) To a solution of cannabidiol (942 mg, 3 mmol) in dry CH.sub.2Cl.sub.2 (42 mL) was added 1-fluoropyridinium triflate (742 mg, 3 mmol) and the reaction mixture was stirred at ambient temperature overnight. After dilution with CH.sub.2Cl.sub.2 the mixture was washed with saturated aqueous solution of NaHCO.sub.3. The organic layer was separated, dried over MgSO.sub.4 and evaporated. The oil obtained was chromatographed on a silica gel column (75 g). Elution with 2% ether in petroleum ether gave the compound 4-fluoro-cannabidiol (HU-474) as a solid (300 mg, 27%) m.p. 59-61 C. .sup.1H NMR (300 MHz, CDCl.sub.3) 6.17 (1H, s, arom.) 5.52 (1H, s), 4.56 (1H, s), 4.44 (1H, s), 3.92 (1H, s), 2.50 (2H, b), 2.19-2.05 (2H, b), 1.77 (3H, s), 0.86 (3H, t). MS, m/e=332 (M.sup.+).

Example 2: Fluorination of Cannabidiol Diacetate (HU-475)

(10) Step A

(11) ##STR00013##

(12) To a suspension of SeO.sub.2 (219 mg, 2 mmol) and t-BuOOH (2.8 mL, 70% in water) in CH.sub.2Cl.sub.2 (7 mL) was added a solution of cannabidiol diacetate (2 g, 5 mmol) in CH.sub.2Cl.sub.2 (10 mL). After stirring the mixture at ambient temperature overnight, it was washed with saturated aqueous solution of NaHCO.sub.3 followed by a saturated solution of NaHSO.sub.3. The organic layer was separated, dried over MgSO.sub.4, filtered and evaporated. The oil obtained was purified on a silica gel column (50 g). Elution with 13% ether in petroleum ether gave the required compound 10-hydroxy-cannabidiol diacetate (670 mg, 40%) as oil. .sup.1H NMR (300 MHz, CDCl.sub.3), 6.7 (2H, s), 5.17 (1H, s), 4.99, (1H, s), 4.87 (1H, s), 3.70-3.80 (2H m), 3.53-3.58 (1H, m), 2.52-2.57 (2H, t J=7.6 Hz), 2.39-2.48 (1H d J=5.1 Hz), 2.18 (6H, s), 2.06 (1H, s), 2.0 (1H, s), 1.58 (1H, s), 1.25-1.31 (4H, m), 0.86-0.90 (3H t, J=6.45 Hz).

(13) Step B

(14) ##STR00014##

(15) The alcohol (414 mg, 1 mmol) in dry CH.sub.2Cl.sub.2 (4 mL) was added under N.sub.2 atmosphere to an ice-cold solution of DAST (0.18 mL, 1.5 mmol). After 15 min. at 0 C. solid Na.sub.2CO.sub.3 (125 mg, 1 mmol) was added. The organic phase was then washed twice with cold 1 M aqueous Na.sub.2CO.sub.3 solution, followed by water. The organic layer was separated, dried over MgSO.sub.4, filtered and evaporated. The resulting crude material was purified on a silica gel column (20 g) using 10% ether in petroleum ether to provide the fluorinated product 10-fluoro-cannabidiol diacetate (HU-475) (77.5 mg, 18.6%). .sup.1H NMR (300 MHz, CDCl.sub.3) 6.74 (2H, s), 5.21 (1H, s), 5.01 (1H, s), 4.87 (1H, s), 4.60 (1H, s), 4.50 (1H, s), 3.6 (1H, b), 2.73 (1H, t), 2.57 (2H, t), 2.21 (6H, s), 2.08-1.59 (8H, ms), 1.32 (3H, s), 0.90 (3H, t). MS, m/e=416 (M.sup.+).

Example 3: In Vivo Effect of HU-474 in Mice and HU-475 in Rats

(16) 1. Animals

(17) Male Wistar rats (220-250 g) and Swiss mice (25-30 g) originated from the Central Animal Farm of the School of Medicine of Ribeiro Preto (FMRP-USP) were maintained in groups of five animals per box (413317 cm) in a temperature controlled room (242 C.) with a 1212 h light-dark cycle. They received water and food ad libitum throughout the study period.

(18) 2. Compositions

(19) HU-474 (1, 3 and 10 mg/kg) was administered intraperitoneally (IP) in mice at 10 mL/kg volume and HU-475 (1, 3 and 10 nmol) was injected intra-dlPAG in rats. Both drugs were dissolved in 2% Tween 80 in sterile saline.

(20) 3. Stereotaxic Surgery (HU-475)

(21) Rats were submitted to a stereotaxic surgery to unilaterally implant cannulae (9.0 mm, 0.6 mm OD) into the dlPAG (coordinates: lateral: 1.9 mm; depth: 4.3 mm; angle: 16 from lambda; Paxinos and Watson, 2005), fixed to the skull with acrylic cement (Campos & Guimares, 2008). The surgeries were performed under anesthesia with tribromoethanol 2.5% (10.0 mL/kg, IP) and immediately after the animals received Veterinary Pentabiotic (0.2 mL, intramuscular) and analgesic (Banamine, 1.0 mL/kg, subcutaneous) to prevent infections and decrease post-surgical pain. After surgery, animals underwent a recovery period of 5-7 days before the behavioral tests.

(22) 4. Microinjection (HU-475)

(23) Animals received unilateral microinjections of vehicle or HU-475 into the dlPAG before being submitted to the behavioral tests. To this aim, microneedles (10.0 mm, 0.3 mm OD), connected to a microsyringe (Hamilton, USA, 10 mL) through a segment of polyethylene (P10) were inserted into the guide cannulae. Solutions were injected with the help of an infusion pump (KD Scientific, USA). A 0.2 L solution volume was injected over 1 min. After the injections, the needles remained inserted in the cannulae for additional 30 seconds to prevent drug reflux (Campos & Guimares, 2008).

(24) 5. Apparatus

(25) 5.1 Elevated Plus-Maze (EPM) Rats

(26) The wood EPM used to perform the experiments was located in a sound attenuated and temperature controlled room (23 C.), with one incandescent light (40 W) placed 1.3 m away from the maze. The apparatus consists of two opposing open arms (5010 cm) without side walls, perpendicular to two enclosed arms (501040 cm), with a central platform common to all arms (1010 cm). The apparatus is elevated 50 cm above the ground and has an acrylic edge (1 cm) in the open arms to prevent animal falls. In this model, rodents naturally avoid the open arms, exploring more extensively the enclosed arms. Anxiolytic drugs increase the exploration in open arms without affecting the number of enclosed arms entries, which is usually used to assess general exploratory activity (File, 1992). Ten minutes after the last injection the animal was placed on the central platform of the maze with the head facing one of the enclosed arms. The test lasted for 5 min and was recorded. The animal behavior was analyzed with the help of the Anymaze Software (version 4.5, Stoelting). This software indicates the location of the animal in the EPM and automatically calculates the percentage of entries (Peo) and time spent in the open arms (Pto) and the number of entries in the enclosed arms (EA). Animals were only considered to enter an open or enclosed arm when 90% of their bodies were inside the region. All experiments were performed in the morning period (8 to 12 a.m.).

(27) 5.2 Elevated Plus-Maze (EPM) Mice

(28) Similar to 5.1 except that the each arm measured 305 cm.

(29) 5.3 Forced Swimming Test (FST) Mice

(30) Animals were individually submitted for 6 min of forced swimming in glass cylinders (height 25 cm, diameter 17 cm) containing 10 cm of water. The mice were videotaped and the immobility time (characterized by slow movements necessary to avoid drowning) was measured during the last 4-min period. The water was changed after each trial to maintain the temperature at 23-25 C. and to prevent the influence of alarm substances (Zanelati et al., 2010).

(31) 6. HistologyRats

(32) After the behavioral tests animals were anesthetized with chloral hydrate 4% (10 mL/kg) and perfused with saline 0.9%. Brains were removed and kept in formalin solution 10% for 3-7 days. Soon after, brains were cut into 50-m thick sections in a cryostat (Cryocut 1800). The injection sites were identified in diagrams from the Paxinos and Watson's atlas (Paxinos and Watson, 2005). Rats receiving injections outside the aimed area were included in a separate group (out group).

(33) 7. Statistical Analysis

(34) Results from HU-475 and HU-474 tests in the elevated plus maze were analyzed by Kruskal-Wallis followed by Mann-Whitney tests. Data from animals tested in the FST were analyzed by one-way ANOVA followed by Duncan test.

(35) Results.

(36) HU-475.

(37) The drug increased the percentage of entries (X.sup.2=9.66, DF=4, p<0.05, FIG. 1) and tended to do the same (X.sup.2=8.5, DF=4, p=0.075, FIG. 2) with the percentage of time spent into the open arms. No effect was found in the number of enclosed arm entries.

(38) FIG. 1 shows the effects of HU-475 (1, 3 and 10 nmol, n=8-9 animals/group) and vehicle (n=7) microinjected into the dorsolateral periaqueductal gray of rats submitted to the elevated plus maze. Results from animals that received the dose of 3 nmol outside the target region are shown in the OUT group (n=6). Data expressed as meansSEM of the percentage of entries onto the open arms. * indicates significant difference from vehicle (p<0.05). FIG. 2 shows the effects of HU-475 (1, 3 and 10 nmol, n=8-9 animals/group) and vehicle (n=7) microinjected into the dorsolateral periaqueductal gray of rats submitted to the elevated plus maze. Results from animals that received the dose of 3 nmol outside the target region are shown in the OUT group. Data expressed as meansSEM of the percentage of time spent in the open arms. + indicates a trend (p<0.1) for a difference from vehicle.

(39) HU-474.

(40) The drug increased the percentage of time spent in the open arms of the pEPM (X.sup.2=8.13, DF=3, p<0.05, FIG. 3. No effect was found in the percentage of entries onto these same arms (FIG. 4) and the number of enclosed arms entries. The drug also decreased immobility time in the FST (F3.2=4.06, p=0.019, FIG. 5) at the dose of 3 m/kg. The doses of 1 and 10 mg/kg were ineffective. FIG. 3 shows the effects of HU-474 (1, 3 and 10 mg/kg n=6-8 animals/group) and vehicle (n=7) in mice tested in the elevated plus maze. Data expressed as meansSEM of the percentage of time in the open arms. * indicates significant difference from vehicle. FIG. 4 shows the effects of HU-474 (1, 3 and 10 mg/kg n=6-8 animals/group) and vehicle (n=7) in mice tested in the elevated plus maze. Data expressed as meansSEM of the percentage of entries onto the open arms. FIG. 5 shows the effects of HU-474 (1, 3 and 10 mg/kg n=6-8 animals/group) and vehicle (n=7) in mice immobility time (s) showed by mice tested in the FST. Data expressed as meansSEM. * indicates significant difference from vehicle.

(41) Discussion (Comparative Results with CBD)

(42) Intra-dlPAG injection of HU-475 increased exploration of the open arms of the EPM without changing the number of enclosed arm entries. This indicates an anxiolytic-like effect (File, 1991) and was similar to that produced by CBD using the same paradigm, including a bell-shaped dose-response curve. However, the effective dose of CBD was 30 nmol (doses tested: 15, 30 and 60 nmol), same dose produced an anxiolytic-like effect in the Vogel punished licking test (Campos & Guimares, 2008). In this model, therefore, HU-475 was 10 times more potent that CBD.

(43) Systemic administration of HU-474 induced anxiolytic-like effects in mice tested the EPM with a characteristic bell-shaped dose-response curve. The effective dose was 3 mg/kg. In comparison with CBD, Onaivi et al. (1990) in a study conducted with a different mice strain (ICR), observed similar anxiolytic effects at the doses of 1 and 10 mg/kg i.p. (with the 10 mg/kg of CBD being more effective). HU-474 also decrease immobility time in mice tested in the forced swimming test, a model sensitive to antidepressant drugs. CBD also produced an antidepressant-like effect in Swiss mice tested in this model at the dose of 30 mg/kg i.p. (doses tested 3, 10, 30 and 100 mg/kg). Therefore, in this model HU-474 was 10 times more potent than CBD.

Example 4: Pre-Pulse Inhibition Test (HU-474)

(44) 1. Animals

(45) The experiments were performed using male C57BL/6J mice weighting 25-30 g. The animals were maintained throughout the experimental period under standard laboratory conditions with free access to water and food will be used.

(46) 2. Compositions

(47) HU-474 (3 and 10 mg/kg) was dissolved in 2% Tween 80 in sterile saline (vehicle). MK-801 (a NMDA antagonist, 0.5 mg/kg, Sigma, USA) was dissolved in saline. Drugs were administered intraperitoneally (ip) at 10 mL/kg volume.

(48) 3. Experimental Procedure

(49) The animals (n=9-11/group) received i.p. administration of vehicle or HU-474 (3 and 10 mg/kg) followed, 30 minutes later, by saline or MK-801 (0.5 mg/kg), resulting in the following experimental groups: vehicle+saline, HU 10+saline, vehicle+MK-801, HU 3+MK-801, HU 10+MK-801. The animals were submitted to PPI test 20 minutes after the last drug injection.

(50) 4. Pre-Pulse Inhibition (PPI)

(51) The PPI was carried out in three consecutive steps. The first consisted of an acclimation period during which no stimulus was presented. In the second step, called habituation, only the stimulus that triggers the startle (pulse) was presented. The step that assessed the inhibition of startle response pulse consisted of 64 random presentations of the different stimuli: (i) pulse (white noise) 105 dB at 20 ms, (II) pre-pulse (pure tone frequency of 7 kHz) 80, 85 and 90 dB at 10 ms, (III) followed by pre-pulse 100 ms interval between them and (IV) zero (no stimulus). During this session the stimuli are presented at regular intervals of 30 s, 8 presentations of each stimulus. The percentage of the PPI was expressed as the percent inhibition of startle amplitude in response to multiple presentations of the pulse preceded by pre-pulse (PP), depending on the amplitude of the response only to the pulse (P), which was obtained in the following formula: % PPI=100((PP/P)100). Using this formula 0% represents no difference between the amplitude of startle triggered only by the pulse or pulse preceded by the pre-pulse and therefore no pre-pulse inhibition. This transformation was performed in order to reduce the statistical variability attributable to differences between animals and represents a direct measure of pre-pulse inhibition (Issy et al., 2009).

(52) 5 Statistical Analysis

(53) The percentage of PPI was analyzed by repeated measures MANOVA with the treatment as the independent factor and the prepulse intensity (80, 85 and 90 dB) as repeated measure. Duncan's post hoc test (P<0.05) was used to identify differences revealed by significant MANOVA.

(54) Results

(55) The MANOVA revealed significant main effects of prepulse intensity (F.sub.2.70=23.53, P<0.05) and treatment (F.sub.4.35=45.42, P<0.05) but no interaction between prepulse intensity and treatment (F8.70=1.08, P>0.05). MK-801 promoted significant PPI disruption for all prepulse intensities tested (P<0.05, Duncan post-test). HU-474 (10 mg/kg) attenuated MK-801 PPI disruption in all prepulse intensities tested (P<0.05, FIG. 6). FIG. 6 shows the effects of HU-474 (3 and 10 mg/kg i.p.) in mice on the impairment of PPI induced by MK-801 (M0.5 mg/kg). Results show the percent inhibition of startle amplitude in response to multiple presentations of the pulse preceded by pre-pulse and are expressed as meansSEM. * indicates significant difference from vehicle-vehicle, # significant difference from vehicle-MK group.

(56) Discussion (Comparison with CBD Results)

(57) A single CBD administration (5 mg/kg, I.p.) attenuated PPI deficits caused by MK801 (0.3-1 mg/kg, i.p.) in Swiss mice (Long et al., 2006). Observe that in this case the effective CBD dose (the authors also tested 1 and 15 mg/kg) was lower than that observed in HU-474 (10 mg/kg).

(58) The above experimental procedure is also performed with 30 mg/kg dose of HU-474. HU-474 is also tested in dopamine-based models (hyperlocomotion induced by d-amphetamine). CBD effective doses are 30 and 60 mg/kg (Swiss mice). 30 mg/kg dose are able to attenuate the hyper-locomotion induced by MK801 (Moreira and Guimares, 2005).