Endocannabinoids

In 1992 an arachidonic acid ethanolamide derivative (67, Fig. 8) isolated from porcine brain and characterized as an endogenous ligand for the cannabi-noid receptors was named anandamide (75). It was then followed by the discovery of two other endocannabinoids 2-AG (76-78) (68, Fig. 8) and 2-arachi-donoylglyceryl ether (79) (noladin ether, 69, Fig. 8). Anandamide is a highly

Scheme 15. Synthesis of AM281 (74). Reagents and conditions: (a) (i) LiHMDS, THF (ii) EtO2CCO2Et; (b) 2,4-dichlorophenylhydrazine hydrochloride, EtOH; (c) AcOH; (d) 4-aminomorpholine, LiHMDS, THF; (e) Bu6Sn2, Pd(PPh3)4, EtsN; (f) I2, CCI4.

N-(Morpholin-4-yl)-5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-zole-3-carboxamide (65). To a magnetically stirred solution of ester 64 (3.00 g, 6.60 mmol) and 4-aminomorpholine (772 ||L, 7.90 mmol) in dry tetrahydrofuran (25 mL) was added a 1.0 M solution of lithium bis(trimethylsilyl)amide in hexane (10.0 mL, 10.0 mmol). The resulting mixture was stirred at room temperature for 2 h and then quenched with saturated aqueous ammonium chloride and extracted with 3 x 50 mL of chloroform. The combined extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and evaporated. Purification by flash column chromatography on silica gel with chloroform:ethyl acetate 50:50 afforded amide 65 as a white solid (3.30

N-(Morphohn-4-yl)-1-(2,4-dichlorophenyl)-5-(4-tributyltinphenyl)-4-methyl-1H-pyra-

zole-3-carboxamide (66). To a magnetically stirred suspension of 65 (2.40 g, 4.70 mmol) in freshly distilled triethylamine (150 mL) was added bis(tributyltin) (3.16 mL, 6.25 mmol) followed by tetrakis(triphenylphosphine)palladium(0) (272 mg, 0.23 mmol) at room temperature under an argon atmosphere, and the reaction mixture was heated to reflux for 30

h. After cooling to room temperature, the precipitate was removed by filtration. The filtrate was then concentrated under reduced pressure and purified by flash column chromatogra-phy on silica gel with petroleum ether:acetone 80:20 to give the tributyltin derivative 66 (470 mg, 21% yield based on the recovery of starting material): MP 64-68°C.

N-(Morpholin-4-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-pyra-zole-3-carboxamide (61). To a magnetically stirred solution of organotin compound 66 (290 mg, 0.40 mmol) in carbon tetrachloride (15 mL) was added dropwise a 0.02 M solution of iodine in carbon tetrachloride (25 mL, 0.50 mmol) at room temperature. After TLC showed that the reaction was completed, carbon tetrachloride was removed under reduced pressure. The residue was purified by flash column chromatography on silica gel (petroleum ether:ethyl acetate 50:50) to afford the desired product 61 as a white solid (215 mg, 97% yield): MP 265-268°C (dec.).

Fig. 8. Endogenous cannabinoid receptor agonists.

70 71 72

ACPA AM881, Chloroanandamide AM356, (Rj-(+)-Methanandamide

Fig. 9. Head-group-modified analogs of anandamide.

70 71 72

ACPA AM881, Chloroanandamide AM356, (Rj-(+)-Methanandamide

Fig. 9. Head-group-modified analogs of anandamide.

lipophilic compound with four nonconjugated cis double bonds and is sensitive to both oxidation and hydrolysis. It was shown to bind to the CB1 receptor with moderate affinity (Ki = 61 nM), has low affinity for the CB2 receptor (Ki = 1930 nM), and behaves as a partial agonist in the biochemical and pharmacological tests used to characterize cannabinoid activity. Its role as a neurotransmitter or neuromodulator is supported by its pharmacological profile as well as by the biochemical mechanisms involved in its biosynthesis and bioinactivation.

The SAR studies of endocannabinoids have been reviewed (80-83). The chemical structure of anandamide can be divided into two major molecular fragments (67, Fig. 8): a polar ethanolamido head group and a hydrophobic arachidonoyl chain. The polar head group is comprised of a secondary amide functionality with an N-hydroxyalkyl substituent, while the hydrophobic fragment is a nonconjugated all-cis tetraolefinic chain and an «-pentyl tail reminiscent of the lipophilic side chain found in the classical cannabinoids.

Anandamide and its head-group-modified analogs (70-72, Fig. 9) were prepared by reaction of the appropriate amino alcohols or aminophenols with arachidonic acid chloride. Alternatively, direct transformation of the methyl ester of arachidonic acid to the corresponding amides was achieved by cyanide-catalyzed amidation (84).

The general procedure for the synthesis of head group modified analogs is as follows (85,86).

0-1860

Fig. 10. Tail-modified analogs of anandamide and methanandamide.

0-1860

Fig. 10. Tail-modified analogs of anandamide and methanandamide.

N-Arachidonoylamine analogs. A solution of arachidonic acid (200 mg, 0.66 mmol) and dry dimethylformamide (0.05 mL, 0.66 mmol) in 5 mL of dry benzene was cooled in an ice bath, and oxalyl chloride (0.12 mL, 1.32 mmol) was added dropwise under nitrogen. The reaction mixture was stirred at 25°C for an additional hour when 5 mL of anhydrous THF was added, and the mixture was cooled in an ice bath. Subsequently, a solution of the appropriate amino alcohol (10-fold excess) in 5 mL of anhydrous THF was added. After further stirring at room temperature for 15 min, the reaction mixture was diluted with chloroform (15 mL), washed successively with 10% HCl and NaOH solutions, and dried (MgSO4). The solvent was removed under vacuum, and the residue purified by column chromatography on silica gel.

Although there is very little structural similarity between the classical cannabinoids and anandamide, there is considerable evidence suggesting that these two classes of cannabimimetic agents bind similarly to the CBi-active site (87). It was thus assumed that incorporation of the 1',1'-dimethylalkyl side chain (from classical cannabinoids) in AEA structure would lead to potent analogs. Indeed, tail-chain-modified anandamide analogs (73,74, Fig. 10) bearing a dimethylalkyl chain exhibited marked increases in receptor affinities and in vivo potencies (88-90).

For the synthesis of tail-modified analogs, methyl 14-hydroxy-(all-ds)-5,8,11-tetradecatrienoate (78, Scheme 16) is the key intermediate. It can be synthesized either starting from arachidonic acid (86,91-94) or through a reaction sequence (95) depicted in Scheme 16. In this procedure, alcohol 78 was synthesized starting from hex-5-ynoic acid, which was converted into its methyl ester 75 by treatment with p-TSA in MeOH. The ester 75 was then coupled with 4-chloro-but-2-yn-1-ol (96) in the presence of CuI as a catalyst to give 10-hydroxy-deca-5,8-diynoic acid methyl ester 76. Bromination with CBr4/Ph3P gave the propargylic bromide, which was then coupled with but-3-yn-1-ol in the presence of CuI as a catalyst to provide the triynoic acid methyl ester 77 (97).

Scheme 16. Preparation of 14-hydroxy-(all-aX>-5,8,11-tetradecatrienoate (78)(95). Reagents and conditions: (a) CuI, Nal, K2CO3, 4-chlorobut-2-yn-1-ol, DMF, 18 h, 23°C, 87%; (b) CBr4, PPh3, CH2Cl2, -20 to 23°C, 1 h, 92%; (c) CuI, Nal, K2CO3, but-3-yn-1-ol, DMF, 18 h, 23°C, 85%; (d) P-2 Ni, ethanol, 3 h, 23°C, 50%.

Methyl hex-5-ynoate (75). A stirred solution of hex-5-ynoic acid (5 g, 44.6 mmol), p-TSA (58 mg, 0.3 mmol) in MeOH (8 mL) and CH2Q2 (17 mL) was refluxed for 24 h. The mixture was quenched with saturated aqueous NaHCO3 and the organic layer was separated. The aqueous layer was extracted with CH2Q2. The combined organic layers were dried over MgSO4 and evaporated under reduced pressure to yield the methyl ester 75 (5.46 g, 97%).

10-Hydroxydeca-5,8-diynoic acid methyl ester (76). A mixture of K2CO3 (5.94 g, 43 mmol), CuI (4.1 g, 22 mmol), 4-chlorobut-2-yn-1-ol (4.47 g, 43 mmol), NaI (6.44 g, 43 mmol), and methyl hex-5-ynoate (5.46 g, 43 mmol) in dimethyl formamide (DMF) (86 mL) was stirred overnight at 25°C. The mixture was diluted with ethyl acetate and filtered through a pad of celite. It was washed with saturated aqueous NH4Cl followed by brine. The solution was dried over MgSO4, and the solvent was evaporated under vacuum. The oily residue was dissolved in hexanes:ethyl acetate 50:50 and filtered through a pad of silica gel to provide a yellowish oil (7.2 g, 87%), which was used in the subsequent step without further purification since some of the diyne decomposed upon flash chromatography.

14-Hydroxytetradeca-5,8,11-triynoic acid methyl ester (77). To a stirred solution of diyne 76 (8.65 g, 44.6 mmol) and CBr4 (17.74 g, 53.5 mmol) in CH2Q2 (80 mL) cooled to -20°C, a solution of triphenylphosphine (14.6 g, 55.7 mmol) in CH2Q2 (40 mL) was added dropwise. After the addition, the cooling bath was removed and the mixture was stirred for an additional 1 h. Hexanes:ethyl acetate 80:20 was then added until triph-enylphosphine oxide precipitated. The mixture was filtered through a pad of silica gel to yield methyl 10-bromodeca-5,8-diynoate as a colorless oil (10.54 g, 92%). Attempts to further purify it by flash chromatography resulted in partial decomposition of the bromide. Hence, it was used as such in the subsequent reaction. A mixture of K2CO3 (9.67 g, 70 mmol), CuI (6.66 g, 35 mmol), but-3-yn-1-ol (5.3 mL, 70 mmol), NaI (10.50 g, 70 mmol) and methyl 10-bromodeca-5,8-diynoate (17.99 g, 70 mmol) in DMF (140

(continues on top of page 141)

mL) was stirred overnight at 25°C. The mixture was diluted with ethyl acetate and filtered through a pad of celite. It was washed with saturated aqueous NH4Q followed by brine. The solution was dried over MgSO4 and the solvent was evaporated under vacuum. The oily residue was dissolved in hexanes:ethyl acetate 50:50 and filtered through a pad of silica gel to provide compound 77 as a yellowish oil (14.63 g, 85%), which was used in the subsequent step without further purification. Attempts to purify 77 by flash chromatography resulted in partial decomposition of the triyne.

14-Hydroxytetradeca-(all-aX)-5,8,n-trienoic acid methyl ester (78). To a stirred solution of Ni(OAc)2 (14.93 g, 60 mmol) in EtOH (450 mL) was added ethylenedi-amine (4 mL, 60 mmol) followed by a 1 M solution of NaBH4 (60 mL). The mixture was stirred at 25°C for 0.5 h. The triyne 77 (6.6 g, 26.8 mmol) was added to the reaction mixture and a H2 atmosphere (balloon) was kept over the reaction mixture. It was stirred for 3 h at 25°C, and the solvent was removed in vacuo. The residue was dissolved in hexanes:ethyl acetate 50:50 and filtered through a pad of silica gel. Purification by flash chromatography (hexanes:ethyl acetate 65:35) provided the desired triene 78 as a colorless oil (3.38 g, 50%).

Partial reduction of the triyne 77 over nickel boride catalyst (98) provided the key intermediate 78.

The alcohol 78 was then converted to tail-modified analog 73, as shown in Scheme 17. Phosphonium iodide 79, obtained from 78, was treated with NaHMDS to give the ylide, which was then allowed to react with the aldehyde 80 in a Wittig reaction to give the ester derivative 81. Hydrolysis of the ester 81 with LiOH, followed by treatment with oxalyl chloride (85,88) and a suitable amine gave the tail-modified analog 73 (Scheme 17).

Was this article helpful?

0 0

Post a comment