The fourth chemical class of cannabinergic compounds, the aminoalkylindoles (AAIs), were initially developed at Sterling Winthrop as potential
Pravadoline R-(+)-WlN 55,212-2
Fig. 5. Representative aminoalkylindoles.
Pravadoline R-(+)-WlN 55,212-2
nonulcerogenic analogs of nonsteroidal anti-inflammatory drugs (NSAIDs) (64) and bear no structural relationship to the cannabinoids (e.g., 48,49, Fig. 5). These analogs also exhibited antinociceptive properties that eventually were attributed to their interactions with the cannabinoid receptors (65,66). The most widely studied compound of this series is WIN55,212-2 (49), a potent CBi and CB2 agonist with a slight preference for CB2. Cannabinergic activity resides principally with only one optical antipode and is more potent than A9-THC in several pharmacological and behavioral assays. WIN55,212-2 has played an important role in the identification and characterization of cannabinoid receptors and their associated functions and is now in standard use as a CB1/CB2 radioligand.
Aminoalkylindoles are generally synthesized by following either Method A or Method B as depicted in Scheme 12 (65,66). An appropriately functionalized indole was aroylated in the first step followed by N-alkylation (Method A). Alternatively, N-alkylation could be accomplished first, followed by aroylation of the resulting indole derivative at C3 (Method B). For acid-sensitive analogs, EtAlCl2 is used in place of AlCl3 in Friedel-Crafts acylation.
WIN55,212-2 (49) has been synthesized as shown in Scheme 13 (65). Swern oxidation of 50 afforded the very sensitive a-amino ketone 51. Reductive cyclization of 51 with Raney-Ni provided the key intermediate 52. The enan-tiomers of 52 were obtained by crystallization with (+)- and (-)-dibenzoyltar-taric acid (DBT). Hydrazine 54 obtained from enantiomerically pure 53 was then converted to enamino ketone 55, which when refluxed in the presence of acetic acid yielded (tf)-(+)-WIN55,212-2 (49) in 44% yield.
[3H]WIN55,212-2 has served as an important probe in the discovery of cannabinoid receptors. It was synthesized by catalytic tritium dehalogenation of oh oh
Scheme 11. Stereoselective synthesis of conformationally restricted hybrid cannabinoids (47). Reagents and conditions: (a) (i) LDA, THF, -78°C, 30 min, (ii) 41, -78°C, 30 min, (iii) AcOH, 68%; (b) NaBH4, MeOH, room temperature, 5 min, 97%; (c) TFA, CHCl3, 0.02 M, 0°C, 3.5 h, 76%; (d) Dess-Martin periodinane, CH2Cl2, 0°C to room temperature, 2.5 h, 69%; (e) Ph3PCH2OMe+Cl", Na tert-amylate, PhH, room temperature, 15 min, 88%; (f) wet Cl3CCO2H, CH2Cl2, room temperature, 45 min; (g) (i) K2CO3, MeOH, room temperature, 2 h, (ii) NaBH4, room temperature, 15 min, 91% from 46; (h) NaBH4, 6 mol % Pd(PPh3)4, THF, room temperature, 23 h, 94%; (i) triethylamine trihydrofluoride, Et3N, CH2Cl2, room temperature, 18 h, 96%; (j) 8 mol % Lindlar's catalyst, quinoline, 1 atm H2, PhH, room temperature, 2 h, 91%; (k) PhSSPh, PhH, hv, room temperature, 22 h, 64%.
(6_E)-1-[4-(1,1-Dimethylheptyl)-2-hydroxy-6-prop-2-en-yloxyphenyl]-3-hydroxy-10-ferf-butyldimethylsilyloxy-7-methyldec-6-en-8-yn-1-one (43). To a solution of diisopropylamine (1.80 mL, 1.30 g, 12.8 mmol) in 24 mL of THF at -78°C was added ra-BuLi (5.45 mL, 2.37 M in hexanes, 12.9 mmol). After 20 min, a solution of ketone 41 (1.97 g, 6.20 mmol) in 8 mL of THF at -78°C was added via cannula. After 30 min, a solution of aldehyde 42 (1.53 g, 5.75 mmol) in 8 mL of THF at -78°C was added via cannula. After 30 min, the reaction mixture was quenched with acetic acid (1.40 mL, 1.47 g, 24.5 mmol) warmed to room temperature and diluted with ether and water. The aqueous phase was extracted thrice with ether, and the combined organic extracts were washed with brine and dried (MgSO4). Purification by flash column chromatography on silica gel (2.5% to 5% to 10% EtOAc in hexanes) gave ketone 43 (2.28 g, 68% yield) as a pale yellow oil: Rf = 0.27 (EtOAc:hexanes 10:90).
Tricyclic C9 Alcohol 44. To a solution of diol (2.00 g, 3.41 mmol) derived from NaBH4reduction of 43 in 175 mL of CHCl3 at 0°C was added TFA (290 ||L, 429 mg, 3.76 mmol). After 3.5 h, the reaction mixture was quenched with saturated aqueous NaHCO3 and diluted with water. The aqueous phase was extracted twice with CH2Cl2, and the combined organic extracts were washed with brine and dried (MgSO4). Purification by flash column chromatography on silica gel (5% to 10% to 20% EtOAc in hexanes) gave alcohol 44 (1.48 g, 76% yield) as a colorless oil as a 1:1 mixture of diastereomers: Rf = 0.29 and 0.21 (EtOAc:hexanes 20:80).
Alkyne 35. To a solution of phenol obtained after deallylation of 47 (159 mg, 0.29 mmol) in 15 mL of CH2Cl2 at room temperature was added triethylamine trihydrofluoride (500 ||L, 495 mg, 3.07 mmol) and triethylamine (50 ||L, 36 mmol, 0.36 mmol). The reaction mixture was stirred at room temperature for 18 h and diluted with water. The aqueous phase was extracted four times with CH2Cl2, and the combined organic extracts were washed with brine and dried (MgSO4). Purification by flash column chromatography on silica gel (3% to 4% to 5% EtOH in CH2Cl2) gave 35 (120 mg, 96% yield) as a white foam: MP 87-88°C; Rf = 0.18 (EtOH:CH2Cl2 5:95).
(Z)-Alkene 36. To a solution of quinoline (31 mg, 0.24 mmol) in 5 mL of benzene at room temperature was added Lindlar catalyst (27 mg, 2.5% on CaCO3, 6.3 |mol) and a solution of alkyne 35 (35 mg, 0.082 mmol) in 20 mL of benzene. The static nitrogen atmosphere was replaced by hydrogen from a double balloon, and the reaction mixture was stirred at room temperature for 2 h, filtered through celite, and concentrated. Purification by flash column chro-matography on silica gel (2% to 3% EtOH in CH2Cl2) gave (Z)-alkene 36 (32 mg, 91% yield) as a white foam: MP 68-69°C; Rf = 0.26 (EtOH: CH2Cl2 6:94).
(E)-Alkene 37. A solution of (Z)-alkene 36 (33 mg, 0.077 mmol) and diphenyl disulfide (4 mg, 0.02 mmol) in 12 mL of benzene was irradiated with a 50 W incandescent flood lamp at room temperature for 12 h, at which time additional diphenyl disulfide (3 mg, 0.01 mmol) was added. Irradiation was continued for 10 h, and the reaction mixture was concentrated. Purification by flash column chromatography on silica gel (30% to 50% EtOAc in hexanes) gave alcohol 37 (21 mg, 64% yield) as a white foam: MP 82-83°C; Rf = 0.14 (EtOAc:hexanes 50:50).
54 55 49
54 55 49
Scheme 13 (Bottom of facing page). Synthesis of WIN55,212-2 (65). Reagents and conditions: (a) (i) TFAA, DMSO, CH2Cl2, -78°C, (ii) Et3N; (b) H2, Ra-Ni, EtOAc; (c) dibenzoyl tartaric acid, MeOH; (d) NaNO2, HCl; (e) LiAlH4, THF; (f) 1-naphthoylace-tone, C6H5CH3, H+, reflux; (g) AcOH, reflux.
(R)-(+)-3,4-Dihydro-3-[(4-morpholinyl)methyl]-2H-l,4-benzoxazine (53). Many initial attempts to resolve either enantiomer of 52 as the dibenzoyltartaric acid salts crystallizing from acetone/hexane mixtures only resulted in modest enantiomeric enhancements in ratios of approximately 45:55. Resolution was ultimately achieved on these mixtures by crystallization from CH3OH. Thus, 23.5 g of the (-)-dibenzoyltartrate salt of 52 was recrystallized twice from CH3OH to afford an off-white solid containing enan-tiomerically pure (R)-(+)-53 by gas chromatographic (GC) analysis. Characterization revealed this salt to be a 2:l complex with 53:dibenzoyltartaric acid, respectively. The free base of (R)-(+)-53 was made by partitioning the salt between EtOAc and saturated aqueous Na2CO3. An analytical sample was prepared by recrystallization from EtOAc/hexane: MP 94-96°C, [aL = -28.0° (c = 1, CHCk).
l-Amino-3,4-dihydro-3-[(4-morpholinyl)methyl]-2H-1,4-benzoxazine (54). To a mechanically stirred solution of 55.6 g (0.24 mol) of 53 in 1 L of 2 N HCl at 0°C was added over 10 min to an aqueous solution of 18 g (0.26 mol) of NaNO2. The mixture was stirred for 1 h at 0°C, then diluted with 1 L of H2O and 1 L of EtOAc, and was made neutral by the cautious addition of solid NaHCO3. The organic phase was washed with 1 L of saturated aqueous NaCl, dried over MgSO4, and filtered. Concentration under reduced pressure afforded nitrosamine, which was reduced directly as follows. To 1.5 L of THF under N2 at 0°C in a 3-L, three-neck flask fitted with a mechanical stirrer was cautiously (Caution!! see Note 3) added ~16 g (0.4 mol) of powdered LiAlH4. The nitrosamine was added as a solution in ~200 mL of THF. After the addition was complete, the mixture was refluxed for 1 h. The reaction mixture was then cooled in an ice bath, and ~60 mL of saturated aqueous Na2SO4 was cautiously added dropwise. The resulting thick suspension was stirred for a few minutes and then filtered through a plug of celite, and the filter cake was washed thoroughly with EtOAc. The filtrate was concentrated under reduced pressure to afford 58.4 g of the hydrazine 54.
WIN55, 212-2 (49). A solution of 5.8 g of 54, (0.026 mol) of 1-napthoylacetone and 0.5 g of pyridinium 3-nitrobenzenesufonic acid in 300 mL of H2O was refluxed for 3 h using a Dean-Stark water trap connected to a reflux condenser. The cooled solution was filtered and concentrated under reduced pressure. The concentrate was dissolved in 250 mL of AcOH and refluxed for 1 h. The cooled solution was then concentrated under reduced pressure, and the residue was partitioned between H2O and EtOAc. Concentrated aqueous NH4OH was added until the aqueous phase was alkaline. The organics were dried over MgSO4, filtered, and concentrated under reduced pressure. Chromatography followed by crystallization from CH3OH/Et2O afforded 49 in 51% yield from 54: MP 256-259°C.
Scheme 14. Synthesis of [3H]WIN55,212-2 (67). Reagents and conditions: (a) 10% Pd/C, Et3N, tritium gas.
[3H]WIN55,212-2. A solution of 25 mg (0.044 mmol) of precursor 56 in 2 mL of ethanol with 25 mg of 10% Pd/C and 0.025 mL of triethylamine was vigorously stirred with 80 Ci of tritium gas at ambient temperature and atmospheric pressure for 4 h. After this time the catalyst was filtered, labile tritium was removed by several evaporations of methanol, and the crude product (1960 mCi) was dissolved in 20 mL of ethanol. Purification was accomplished on two 500-mm silica gel plates eluted with hexane:ethyl acetate 50:50 to afford 988 mCi (a 37% radiochemical yield based on precursor 56) of product that was found to be >98% radiochemically pure and to coelute with authentic WIN55,212-2 (49) both on silica gel TLC (hexane:ethyl acetate 50:50) and reverse-phase HPLC (water:acetonitrile 35:65). The specific activity of product [3H]WIN55,212-2 was measured to be 60 Ci/mmol by UV assay (where E246 = 20,958) and the UV of product was superimposable on that of unlabeled compound. A proton-decoupled tritium NMR (CDCl3) of product 49 showed two peaks at 7.49 and 7.98 ppm.
56, as shown in Scheme 14 (67). The starting compound 56 was prepared analogously to the synthesis of 49.
In 1996, the Sterling Winthrop and Makriyannis laboratories further explored structural requirements at the N-1 position of aminoalkylindoles by synthesizing novel analogs in which the aminoalkyl chain of the indole ring is attached to a heterocyclic amine through a C-C bond (68). These analogs are generally more potent than the C-N analogs and exhibit more favorable physic-ochemical properties. Potency was optimum for N-methylpiperidinyl-2-methyl substitution at the N-1 position (57, 58, Fig. 6), while activity resided predominantly in the ^-enantiomer.
AM1241 (58, Fig. 6), a highly CB2-selective and potent agonist, was recently developed by Makriyannis. Design of this molecule incorporated N-methylpiperidinyl-2-methyl substituent at the N-1 position and a novel 2-iodo-5-nitrobenzoyl group at C-3.
Fig. 6. C-Attached aminoalkylindoles.
A standard approach utilized for the synthesis of these C-attached AAIs involves aroylation of indole using MeMgX and ArCOCl followed by N-alky-lation using 1-methyl-2-chloromethylpiperidine. The racemic compounds were then resolved either by forming diasteromeric salts with chiral acids or by HPLC using a semi-preparative Chiracel AD column using ethanol:hexane 20:80.
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