2017 Volume 65 Issue 10 Pages 920-929
Buprenorphine shows strong analgesic effects on moderate to severe pain. Although buprenorphine can be used more safely than other opioid analgesics, it has room for improvement in clinical utility. Investigation of compounds structurally related to buprenorphine should be an approach to obtain novel analgesics with safer and improved profiles compared to buprenorphine. In the course of our previous studies, we observed that derivatives obtained by cyclizing C-homomorphinans were structurally related to buprenorphine. Hence, we synthesized cyclized C-homomorphinan derivatives with various oxygen functionalities on the side chains and evaluated their in vitro pharmacological profiles for the opioid receptors. Among the tested compounds, methyl ketone 2a with an N-methyl group showed full agonistic activities for the μ and the δ receptors and partial agonistic activity for the κ receptor. These properties were similar to those of norbuprenorphine, a major metabolite of buprenorphine, which reportedly contributes to the antinociceptive effect of buprenorphine. From these results, we concluded that cyclized C-homomorphinan would be a possible lead compound to obtain novel analgesics with buprenorphine-like properties.
Opioid analgesics are prescribed to ameliorate moderate to severe cancer and non-cancer pain. Among these analgesics, buprenorphine has many unique characteristics. In contrast to other strong opioids such as morphine, oxycodone, and fentanyl, buprenorphine is a partial agonist for the μ opioid receptor with low efficacy in in vitro assays.1,2) Despite its low efficacy, the analgesic effect of buprenorphine was 25 times as strong as that of morphine.3) Interestingly, the use of opioid analgesics like morphine for treating neuropathic pain is generally controversial but buprenorphine was reported to be effective in releasing neuropathic pain.4–7) Norbuprenorphine, a major metabolite of buprenorphine, was a partial agonist for the μ receptor with high efficacy and a full agonist for the δ receptor.2) The plasma concentration of norbuprenorphine reportedly reached levels that were comparable to or higher than that of buprenorphine.8) Therefore, norbuprenorphine was suggested to contribute to the overall pharmacological effects of buprenorphine including its strong antinociceptive effects.
Buprenorphine can be used safely in elderly patients or renal impairment patients in contrast to other opioid analgesics.9–13) In addition, although respiratory depression, which sometimes causes fatal outcomes, is one of representative side effects of opioid analgesics, the respiratory depression of buprenorphine showed a ceiling effect.14) While buprenorphine has the aforementioned advantages, there still is room for improvement in clinical utility due to the limitations of this drug. For example, since buprenorphine is metabolized by CYP3A4, a CYP isozyme involved in metabolism of a wide variety of medicines, buprenorphine occasionally received the effects of a drug–drug interaction with some medicines such as anti-human immunodeficiency virus (HIV) drugs (protease inhibitors),15) benzodiazepines,16) and azole antifungals.17,18) Additionally, buprenorphine is hardly administered orally because it extensively suffers from a first pass effect.19,20) Indeed, buprenorphine is developed as suppository or injectable, sublingual, transdermal formulation. To obtain an improved analgesic, one which retains the advantages and overcomes the shortcomings of buprenorphine, an investigation of compounds structurally related to buprenorphine may be an efficient strategy.
We have previously reported the synthesis and activity of opioid ligands with a C-homomorphinan skeleton.21) In the course of this study, we noticed that cyclization between the 15-hydroxyl group and 6-carbonyl group of C-homomorphinans would afford compounds 1–3 whose structures resemble that of buprenorphine. The side chain of 1e is located in a space similar to that of buprenorphine. This observation encouraged us to seek the compounds showing buprenorphine-like pharmacological properties (Figs. 1, 2). Herein, we report the synthesis of cyclized C-homomorphinan derivatives 1a–j, 2a and 3a, and the evaluation of their in vitro pharmacological profiles.
CPM=cyclopropylmethyl.
As buprenorphine has a tertiary alcohol moiety on the side chain, we designed and synthesized cyclized C-homomorphinan derivatives with various oxygen functional groups (compounds 1a–i, Chart 1). Compound 4, which was derived from naltrexone by our previously reported methods,22) was converted to enones 6 and 7 by Horner–Wadsworth–Emmons reaction. The treatment of 6 and 7 with zinc/acetic acid cleaved the ether bridge and the subsequent oxy-Michael addition occurred to give ketones 1a and b. Secondary alcohols 1c and c′ were synthesized from 9, which was obtained from 3-protected C-homomorphinan 5 by a similar procedure. Reduction of the ketone 9 gave a diastereomeric mixture of secondary alcohols 10 and 10′, which were successfully separated by column chromatography. Deprotection of the benzyl groups gave the desired compounds 1c and c′. The treatment of 1a or 9 with suitable Grignard reagents provided tertiary alcohols. While the reaction with tert-butyl magnesium bromide gave the diastereomeric mixture of the alcohols 1e and e′, the other reactions afforded the single diastereomers.23) The less polar 1e and the more polar 1e′ were successfully separated by column chromatography.
Reagents and conditions: (a) BnBr, K2CO3, DMF, r.t.; (b) LiCl, dimethyl (2-oxopropyl)phosphonate, DIPEA, THF, 65°C; (c) diethyl (2-oxo-2-phenylethyl)phosphonate, 8 M KOH aq., EtOH, r.t.; (d) Zn, AcOH, reflux, 7–54% (2 or 3 steps); (e) NaBH4, MeOH, 0°C, 38–41%; (f) H2, Pd(OH)2, MeOH, r.t., 33–79%; (g) RMgX, THF, r.t., 11d: 54% (22% recovery of 9), 11e: 38%, 11e′: 52%, 11f: quant., 1g: 43%, 1h; 19%, 11i: 97%; (h) H2, Pd(OH)2, MeOH, r.t., 1d: 66%, 1e: 23%, 1e′: 16%, 1f: 53%, 1i: 66%.
To investigate the role of the oxygen functional groups, we synthesized the derivative without these functionalities (1j, Chart 2). Dehydration of compound 1i under acidic conditions gave an inseparable mixture (5 : 1) of olefins 12. Hydrogenation of 12 under acidic condition gave diastereomeric mixture of compounds 1j.
Reagents and conditions: (a) 2 M HCl aq., THF, 60°C, 65%; (b) H2, Pd/C, MeSO3H, MeOH, r.t., 49%.
As N-substituents were known to have a strong impact on the affinity and activity of the compounds for the opioid receptors, we synthesized N-methyl and N-phenethyl derivatives of the methyl ketone (Chart 3). N-Methyl compound 2a was obtained from 1a by removal of the cyclopropylmethyl group under the standard conditions and subsequent reductive amination.24,25) N-Phenethyl compound 3a was synthesized from 3-protected C-homomorphinan 13 by removal of the cyclopropylmethyl group, phenethylation of the nitrogen atom of 15, and subsequent deprotection of the phenolic group.
Reagents and conditions: (a) TrocCl, K2CO3, (CHCl2)2, 150°C, (b) Zn, AcOH, r.t., 59% (2 steps); (c) (HCHO)n, NaBH(OAc)3, AcOH, (CH2Cl)2, r.t., 6% (3 steps); (d) phenethyl bromide, K2CO3, DMF, r.t.; (e) pyridinium chloride, 180°C, 16% (2 steps).
The binding properties of the synthesized compounds for the opioid receptors were evaluated by competitive binding assays (Table 1). Although the methyl ketone 1a bound to the δ and κ receptors with Ki values comparable to those of buprenorphine, its affinity for the μ receptor was ca. 3-fold lower. The phenyl ketone 1b exhibited lower binding affinities for the μ and the κ receptors than did 1a, which suggested that compact substituents are preferred over bulky ones. While the less polar secondary alcohol 1c showed affinities for all the three receptor types comparable to the values of ketone 1a, the other diastereomer 1c′ showed much lower affinity for the δ receptor. This result may imply that the binding to the δ receptor required a polar interaction between the δ receptor and the oxygenated functionality such as the ketone or the hydroxyl group in the ligand. The lower binding affinity of 1c′ for the δ receptor may result from an inappropriate spatial position of the hydroxyl group in 1c′, which led to decreased interaction between 1c′ and the receptor. Although tertiary alcohol 1d bearing the smallest substituent bound as strongly as methyl ketone 1a to the μ and κ receptors, the affinity for the δ receptor was drastically diminished. The tertiary alcohols with bulkier substituents (1e, e′, f–i) had stronger binding affinities toward the δ receptor than 1d did. These outcomes suggested that hydrophobic interactions are important for obtaining sufficient binding interactions to the δ receptor. Concerning the tertiary alcohol, less polar 1e again had superior binding affinity as compared to more polar 1e′ for all three receptors. Although the binding affinities of tertiary alcohols 1d–h for the μ receptor were lower than those of buprenorphine and norbuprenorphine, these alcohols bound to the δ and κ receptors with affinities similar to those of buprenorphine and norbuprenorphine; the Ki values for the δ receptor were in the lower nanomolar to subnanomolar range, while the Ki values for the κ receptor were in the subnanomolar range. The binding affinities of these tertiary alcohols to the μ and κ receptors were comparable or somewhat inferior to those of ketone 1a. In the case of 1j without an oxygen functional group, the affinities to the three receptor types decreased drastically. Therefore, polar interaction appeared to be indispensable for the binding. Conversion of the cyclopropylmethyl group on the nitrogen (1a) to the methyl group (2a) resulted in dramatically reduced binding affinities for all the three receptors, which was in accordance with the usual trend.26–28) N-Phenethyl derivative 3a bound to the μ and the δ receptors more strongly than N-methyl derivative 2a, which also corresponds to the generally observed trend.28–30)
 |
a) Binding assays were carried out in duplicate using human μ, δ, or κ recombinant cell (Chinese hamster ovary (CHO) cell) membranes. b) [3H] DAMGO was used. c) [3H] DPDPE was used. d) [3H] U-69,593 was used. e) Ref. 2. DAMGO: [D-Ala2, NMePhe4, Gly5-ol]enkephalin, DPDPE: [D-Pen2, D-Pen5]enkephalin.
The functional activities for the opioid receptors of the synthesized compounds were assessed by guanosine 5′-O-(3-[35S]thiotriphosphate) ([35S]GTPγS) binding assays (Table 2). The methyl ketone 1a was a partial agonist for the μ and κ receptors and a full agonist for the δ receptor. A bulkier phenyl ketone 1b decreased the efficacy and the potency for the δ and κ receptors. Secondary alcohols 1c and c′ were partial agonists for the δ and κ receptors with low efficacy. The less polar diastereomer 1c, which had stronger binding affinities for the opioid receptors, was a more potent or comparable agonist than the more polar diastereomer 1c′. The agonistic activities of almost all the tertiary alcohols 1d–i, especially toward the δ and κ receptors were lower than those of ketone 1a. Compound 1d showed no agonistic activities for the δ and κ receptors and partial agonistic activity for the μ receptor with low efficacy. Compound 1e′, the more polar diastereomer with the buprenorphine-like substituent, was also less effective. On the other hand, 1e, the less polar counterpart of 1e′, had partial agonist activities toward all the three receptor types and was relatively selective for the μ receptor. In contrast, 1e with a short and bulky t-butyl group was relatively selective for the μ receptor, while 1i bearing a longer n-propyl substituent was relatively selective to the κ receptor. In accordance with this tendency, 1f with a short and bulky substituent, and with very low agonistic activity, was selective toward the μ receptor whereas 1h with a long substituent was relatively selective to the κ receptor. Contrary to our expectation, the efficacy of 1j lacking an oxygen functional group showed sufficient agonistic activities. It is not clear now why 1j was more potent toward the δ receptor than 1i despite the fact that 1j bound much less weakly to the δ receptor than 1i (Table 1). N-Methyl ketone 2a possessed similar profiles to norbuprenorphine—it was a full agonist for the μ receptor, a partial agonist for the κ receptor and full agonist for the δ receptor with lower potency. Ketone 3a with an N-phenethyl group was a partial agonist for the μ and δ receptors and more potent than 1a at the μ receptor. However, this compound showed no agonistic activity toward the κ receptor.
 |
a) [35S]GTPγS binding assays were carried out in duplicate using human μ, δ, or κ expressed CHO cells. b) Emax was calculated as the % of the response obtained with DAMGO. c) Emax was calculated as the % of the response obtained with DPDPE. d) Emax was calculated as the % of the response obtained with U-69593. e) Ref. 2. f) Not determined. g) Percentage stimulation relative to the reference compound at 10 µM.
In the course of this study, we found that compound 2a possessed similar functional profiles to norbuprenorphine. Kuhlman et al. reported that the mean steady-state plasma concentration of norbuprenorphine exceeded that of buprenorphine after daily administration of buprenorphine to humans.8) Norbuprenorphine did have analgesic effects, and it is suggested that a portion of the entire pharmacological effects of buprenorphine, including antinociception, is brought about by its metabolite, norbuprenorphine.2) Although further structure–activity relationship studies and in vivo experiments are required, compound 2a may be expected to be a starting point for developing useful analgesics that overcomes some limitations of buprenorphine.
We synthesized cyclized C-homomorphinan derivatives bearing various oxygen functional groups. Although it was difficult to define the general relationship between the structures of the cyclized C-homomorphinan derivatives and the in vitro pharmacological profiles toward the opioid receptors, some derivatives showed moderate agonist activities to all three receptor types. Among them, compound 2a revealed similar in vitro profiles to norbuprenorphine, which is a major metabolite of buprenorphine. Further modification of compound 2a may provide an analgesic with fewer side effects, which shows improved profiles as compared to buprenorphine.
All reagents and solvents were obtained from commercial suppliers and were used without further purification. Melting points were determined on a Yanako MP-500P melting point apparatus and were uncorrected. IR spectra were recorded on a JASCO FT/IR-460Plus. NMR spectra were recorded on an Agilent Technologies VXR-400NMR for 1H- and 13C-NMR. Chemical shifts were reported as δ values (ppm) referenced to tetramethylsilane. MS were obtained on a JMS-AX505HA, JMS-700 MStation, or JMS-100LP instrument by applying an electrospray ionization (ESI) method. Elemental analyses were determined with a Yanako MT-5 and JM10 for carbon, hydrogen, and nitrogen. The progress of the reaction was determined on Merck Silica Gel Art. 5715 (TLC) visualized by exposure to UV light or with sodium phosphomolybdate solution (sodium phosphomolybdate (9.68 g) in distillated water (400 mL), sulfuric acid (20 mL), and 85% phosphoric acid (6 mL)). Column chromatographies were carried out using Kanto Silica Gel 60 N (60–230 µm), Fuji Silysia CHROMATOREX® PSQ 60B (60 µm), or Fuji Silysia CHROMATOREX® NH-DM2035 (60 µm). The reactions were performed under argon atmosphere unless otherwise noted.
(6R,6aS,11aR)-1-(14-(Cyclopropylmethyl)-2-hydroxy-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-10-yl)propan-2-one (1a)Anhydrous lithium chloride (0.19 g, 4.5 mmol), N,N-diisopropylethylamine (DIPEA) (0.95 mL, 5.6 mmol) and dimethyl (2-oxopropyl)phosphonate (0.39 mL, 2.2 mmol) were added to tetrahydrofuran (THF) (4.0 mL) at 0°C and the solution was stirred at the same temperature for 15 min. Then a solution of compound 4 (0.19 g, 29 mmol) in THF (4.0 mL) was added and the reaction mixture was stirred at 65°C for 25 h. After cooling to ambient temperature, saturated aqueous sodium hydrogen carbonate was added and extracted with diethyl ether. The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. The residue containing compound 6 was used for the next reaction without further purification. To a solution of the residue in acetic acid (6.0 mL) was added zinc powder (1.9 g, 29 mmol) and the reaction mixture was refluxed for 14.5 h. After cooling to ambient temperature, the resulting mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure. To the residue was added 4 M aqueous sodium hydroxide and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated under reduced pressure. Column chromatography (chloroform–methanol=100 : 0 to 20 : 1) gave the title compound (0.11 g, 54% yield for 2 steps) as a yellow oil.
1H-NMR (CDCl3, 400 MHz) δ: 0.07–0.14 (m, 2H), 0.42–0.55 (m, 2H), 0.87–0.99 (m, 1H), 1.18–1.26 (m, 1H), 1.40–1.58 (m, 5H), 1.66–1.71 (m, 1H), 1.83 (d, J=12.7 Hz, 1H), 2.09–2.15 (m, 2H), 2.21 (s, 3H), 2.37–2.56 (m, 3H), 2.59–2.64 (m, 1H), 2.68–2.77 (m, 1H), 2.98 (s, 2H), 3.00–3.08 (m, 1H), 3.41–3.45 (m, 1H), 6.63 (s, 1H), 6.66 (d, J=2.6 Hz, 1H), 6.94 (d, J=7.9 Hz, 1H). An exchangeable OH proton was not observed. High resolution (HR)-MS (ESI): Calcd for C24H32NO3 [M+H]+: 382.2382. Found: 382.2381. IR (neat, cm−1): 3584, 2925, 1706, 1611, 1579, 1453, 1362, 1300, 1223, 1161, 1074, 1049.
1a·(−)-camphorsulfonate (CSA): Anal. Calcd for C24H31NO3·CSA·1.4H2O: C, 63.90; H, 7.86; N, 2.19. Found: C, 63.84; H, 7.84; N, 2.47.
(6R,6aS,10R,11aR)-2-(14-(Cyclopropylmethyl)-2-hydroxy-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-10-yl)-1-phenylethanone (1b)To a solution of compound 4 (22 mg, 0.065 mmol) in ethanol (0.11 mL) were added distilled water (18 µL), 12 M aqueous potassium hydroxide (16 µL, 0.20 mmol) and diethyl (2-oxo-2-phenylethyl)phosphonate (25 mg, 0.098 mmol) and the mixture was stirred at ambient temperature for 2 d. Then the reaction mixture was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated under reduced pressure. Purification with preparative TLC (chloroform–methanol=50 : 1) gave the compound 7 (28 mg, 97% yield) as a brown oil. To a solution of compound 7 in acetic acid (0.66 mL) was added zinc powder (0.22 g, 3.3 mmol) and the reaction mixture was stirred at 70°C for 2 h. After cooling to ambient temperature, the resulting mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure. To the residue was added 4 M aqueous sodium hydroxide at 0°C and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated under reduced pressure. Purification with preparative TLC (chloroform–methanol=50 : 1) gave the title compound (2.0 mg, 7% yield for 2 steps) as a colorless oil and recovered compound 7 (2.7 mg, 9%).
1H-NMR (CDCl3, 400 MHz) δ: 0.06–0.17 (m, 2H), 0.41–0.56 (m, 2H), 0.83–1.03 (m, 1H), 1.14–1.29 (m, 1H), 1.35–1.58 (m, 5H), 1.63–1.78 (m, 2H), 1.97–2.16 (m, 2H), 2.16–2.36 (m, 3H), 2.44–2.63 (m, 1H), 2.63–2.86 (m, 3H), 3.00–3.15 (m, 1H), 3.49 (br s, 1H), 6.65 (s, 1H), 6.66 (d, J=6.7 Hz, 1H), 6.93 (d, J=8.7 Hz, 1H), 7.13–7.19 (m, 3H), 7.22–7.29 (m, 2H). An exchangeable OH proton was not observed. HR-MS (ESI): Calcd for C29H34NO3 [M+H]+: 444.2539. Found: 444.2531. IR (neat, cm−1): 2917, 2849, 1733, 1611, 1454, 1260, 1032.
1b·(−)-CSA: Anal. Calcd for C29H33NO3·CSA·1.5H2O: C, 66.64; H, 7.46; N, 1.99. Found: C, 66.70; H, 7.71; N, 2.05.
(6R,6aS,10R,11aR)-14-(Cyclopropylmethyl)-10-(2-hydroxypropyl)-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-2-ol (Less Polar Diastereomer 1c and More Polar Diastereomer 1c′)To a solution of compound 4 (0.17 g, 0.49 mmol) in N,N-dimethylformamide (DMF) (2.0 mL) were added benzyl bromide (0.063 mL, 0.53 mmol) and potassium carbonate (0.17 g, 1.2 mmol), and the reaction mixture was stirred at ambient temperature for 11 h. Then saturated aqueous sodium hydrogen carbonate was added and extracted with diethyl ether. The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. Column chromatography (n-hexane–ethyl acetate=5 : 1 to 1 : 1) gave compound 5. Compound 9 was prepared from compound 5 according to the procedure used to prepare compound 1a (77 mg, 33% yield for 3 steps, a yellow oil). To a solution of compound 9 (0.20 g, 0.42 mmol) in methanol (5.0 mL) was added sodium borohydride (39 mg, 1.0 mmol) at 0°C and the reaction mixture was stirred at the same temperature for 1 h. Then saturated aqueous sodium hydrogen carbonate was added and stirred for 30 min. Then the resulting mixture was extracted with chloroform. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated under reduced pressure. Roughly purification of the obtained crude product by column chromatography (chloroform–methanol=20 : 1) gave compound 10 (81 mg, a pale yellow oil) and compound 10ʹ (75 mg, a pale yellow oil).
Under a hydrogen atmosphere, each solution of compound 10 (20 mg) or 10′ (25 mg) in methanol (1.0 mL) was stirred in the presence of palladium hydroxide (12 mg or 15 mg, respectively) at ambient temperature for 24 h. After filtration through a pad of Celite, the filtrate was concentrated under reduced pressure. Purification with preparative TLC (ammonia-saturated chloroform–methanol=50 : 1) gave the title compound 1c (13 mg, calculated yield of 79%) as a pale yellow oil and the title compound 1c′ (7 mg, calculated yield of 33%) as a pale yellow oil.
1c: 1H-NMR (CDCl3, 400 MHz) δ: 0.06–0.17 (m, 2H), 0.42–0.53 (m, 2H), 0.79–0.97 (m, 2H), 1.21–1.36 (m, 6H), 1.36–1.58 (m, 5H), 1.62–1.79 (m, 2H), 1.88–1.99 (m, 2H), 2.30 (d, J=12.4 Hz, 2H), 2.36–2.53 (m, 1H), 2.62–2.83 (m, 1H), 3.01–3.18 (m, 1H), 3.38 (br s, 1H), 4.26–4.40 (m, 1H), 6.61–6.68 (m, 2H), 6.94 (d, J=8.1 Hz, 1H). Exchangeable two OH protons were not observed. HR-MS (ESI): Calcd for C24H34NO3 [M+H]+: 384.2539. Found: 384.2528.
1c·(−)-CSA: Anal. Calcd for C24H33NO3·CSA·3.3H2O: C, 60.47; H, 8.30; N, 2.07. Found: C, 60.35; H, 8.07; N, 2.06.
1c′: 1H-NMR (CDCl3, 400 MHz) δ: 0.08–0.15 (m, 2H), 0.42–0.53 (m, 2H), 0.84–0.94 (m, 1H), 1.18–1.23 (m, 1H), 1.20 (d, J=6.2 Hz, 3H), 1.24–1.31 (m, 1H), 1.31–1.47 (m, 1H), 1.47–1.58 (m, 3H), 1.67 (d, J=12.1 Hz, 1H), 1.81 (d, J=13.2 Hz, 1H), 1.93–2.08 (m, 2H), 2.16–2.32 (m, 3H), 2.40–2.54 (m, 2H), 2.61 (d, J=9.3 Hz, 1H), 2.76 (dd, J=6.5, 18.3 Hz, 1H) 3.04 (d, J=18.1 Hz, 1H), 3.42 (d, J=6.3 Hz, 1H), 4.21–4.32 (m, 1H), 6.59 (d, J=2.5 Hz, 1H), 6.65 (dd, J=2.6, 8.2 Hz, 1H), 6.92 (d, J=8.3 Hz, 1H). Exchangeable two OH protons were not observed. HR-MS (ESI): Calcd for C24H34NO3 [M+H]+: 384.2539. Found: 384.2544. IR (neat, cm−1): 3315, 2926, 1612, 1455, 1327, 1295, 1133, 1045.
1c′·(−)-CSA: Anal. Calcd for C24H33NO3·CSA·2.7H2O: C, 61.46; H, 8.25; N, 2.11. Found: C, 61.45; H, 8.03; N, 1.87.
(6R,6aS,10R,11aR)-14-(Cyclopropylmethyl)-10-(2-hydroxy-2-methylpropyl)-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-2-ol (1d)To a solution of compound 9 (0.11 g, 0.23 mmol) in THF (3.0 mL) was added methyl magnesium iodide (1.0 M in diethyl ether, 1.1 mL, 1.1 mmol) at 0°C and the solution was stirred at ambient temperature for 12 h. Then saturated aqueous ammonium chloride was added at 0°C and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated under reduced pressure. Purification with preparative TLC (chloroform–methanol=20 : 1) gave compound the tertiary alcohol 11d (59 mg, 54%) as a pale yellow oil with a starting material 9 (22% recovery). Compound 1d was prepared from the obtained alcohol 11d according to the procedure used to prepare compound 1c.
Yield: 66%. A pale yellow oil. 1H-NMR (CDCl3, 400 MHz) δ: 0.51–0.63 (m, 2H), 0.71–0.81 (m, 2H), 1.20–1.84 (m, 9H), 1.25 (s, 3H), 1.26 (s, 3H), 1.97–2.12 (m, 3H), 2.12–2.27 (m, 1H), 2.42–2.54 (m, 1H), 2.87–3.05 (m, 2H), 3.13–3.35 (m, 2H), 3.61–3.85 (m, 2H), 4.03–4.13 (m, 1H), 6.60–6.65 (m, 1H), 6.87–6.95 (m, 2H), 7.54 (br s, 1H). HR-MS (ESI): Calcd for C25H36NO3 [M+H]+: 398.2695. Found: 398.2681. IR (neat, cm−1): 3315, 2925, 1611, 1452, 1098.
1d·(−)-CSA: Anal. Calcd for C25H35NO3·CSA·1.8H2O: C, 63.47; H, 8.31; N, 2.11. Found: C, 63.51; H, 8.19; N, 2.04.
(6R,6aS,10R,11aR)-14-(Cyclopropylmethyl)-10-(2-hydroxy-2,3,3-trimethylbutyl)-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-2-ol (1e and 1e′)Compound 1e and 1e′ was prepared from compound 9 according to the procedure used to prepare compound 1d.
Compound 1e Yield: 9%. A colorless oil. 1H-NMR (CDCl3, 400 MHz) δ: 0.07 (s, 9H), 0.41–0.49 (m, 2H), 0.79–1.00 (m, 2H), 1.18–1.35 (m, 7H), 1.35–1.58 (m, 4H), 1.58–1.76 (m, 2H), 1.87–2.00 (m, 2H), 2.24–2.46 (m, 3H), 2.58–2.75 (m, 1H), 3.04 (d, J=18.2 Hz, 1H), 3.28–3.37 (m, 1H), 3.62–3.68 (m, 1H), 4.26–4.43 (m, 1H), 6.60–6.65 (m, 2H), 6.95 (d, J=8.5 Hz, 1H). Exchangeable two OH protons were not observed. HR-MS (ESI): Calcd for C28H42NO3 [M+H]+: 440.3165. Found: 440.3147. IR (neat, cm−1): 3282, 2922, 1613, 1405, 1053.
1e·(−)-CSA: Anal. Calcd for C28H41NO3·CSA·H2O·0.4CHCl3: C, 62.52; H, 8.12; N, 1.90. Found: C, 62.27; H, 7.98; N, 1.89.
Compound 1e′ Yield: 8%. A colorless oil. 1H-NMR (CDCl3, 400 MHz) δ: 0.02–0.18 (m, 5H), 0.43–0.61 (m, 2H), 0.77–1.05 (m, 3H), 1.05–1.66 (m, 12H), 1.66–1.89 (m, 1H), 1.95–2.37 (m, 5H), 2.37–2.93 (m, 3H), 2.93–3.18 (m, 2H), 3.36–3.79 (m, 3H), 6.58–6.72 (m, 2H), 6.85–6.96 (m, 1H). Exchangeable two OH protons were not observed. HR-MS (ESI): Calcd for C28H42NO3 [M+H]+: 440.3165. Found: 440.3147. IR (neat, cm−1): 3458, 2923, 1710, 1611, 1577, 1455, 1376, 1297, 1021.
1e′·(−)-CSA: Anal. Calcd for C28H41NO3·CSA·1.1H2O·0.4CHCl3: C, 62.37; H, 8.12; N, 1.89. Found: C, 62.16; H, 7.87; N, 2.26.
(6R,6aS,10R,11aR)-14-(Cyclopropylmethyl)-10-(2-hydroxy-2-phenylpropyl)-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-2-ol (1f)Compound 1f was prepared from compound 9 according to the procedure used to prepare compound 1d.
Yield: 53%. A colorless oil. 1H-NMR (CDCl3, 400 MHz) δ: 0.04–0.16 (m, 2H), 0.40–0.52 (m, 2H), 0.84–0.98 (m, 1H), 1.05 (d, J=9.5 Hz, 1H), 1.20 (d, J=6.9 Hz, 3H), 1.23–1.29 (m, 1H), 1.31–1.65 (m, 5H), 1.90 (d, J=12.2 Hz, 1H), 2.03–2.23 (m, 4H), 2.41–2.67 (m, 3H), 2.69–2.82 (m, 1H), 2.84–3.10 (m, 2H), 3.37–3.47 (m, 1H), 6.42 (d, J=2.5 Hz, 1H), 6.62 (dd, J=2.5, 8.2 Hz, 1H), 6.90 (d, J=8.3 Hz, 1H), 7.14–7.30 (m, 5H). Exchangeable two OH protons were not observed. HR-MS (ESI): Calcd for C30H38NO3 [M+H]+: 460.2852. Found: 460.2871. IR (neat, cm−1): 2924, 1611, 1579, 1494, 1452, 1363, 1299, 1244, 1074, 1049.
1f·(−)-CSA: Anal. Calcd for C30H37NO3·CSA·0.3H2O: C, 68.90; H, 7.75; N, 2.01. Found: C, 69.01; H, 7.98; N, 1.99.
(6R,6aS,10R,11aR)-14-(Cyclopropylmethyl)-10-(2-hydroxy-2-methyl-3-phenylpropyl)-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-2-ol (1g)Compound 1g was prepared from compound 1a according to the procedure used to prepare compound 1d except for the final step (the debenzylation).
Yield: 43%. A yellow oil. 1H-NMR (CDCl3, 400 MHz) δ: 0.05–0.21 (m, 2H), 0.43–0.56 (m, 2H), 0.79–1.02 (m, 1H), 1.10–1.35 (m, 2H), 1.17 (s, 3H), 1.35–1.57 (m, 5H), 1.82 (d, J=12.1 Hz, 1H), 2.02–2.16 (m, 2H), 2.07 (d, J=12.1 Hz, 1H), 2.16–2.25 (m, 1H), 2.25–2.37 (m, 1H), 2.37–2.57 (m, 1H), 2.46 (d, J=12.7 Hz, 1H), 2.57–2.69 (m, 1H), 2.75 (d, J=12.7 Hz, 1H), 2.69–2.85 (m, 1H), 2.94–3.14 (m, 1H), 3.14–3.30 (m, 1H), 3.33–3.53 (m, 1H), 6.58 (d, J=2.6 Hz, 1H), 6.63 (dd, J=8.2, 2.6 Hz, 1H), 6.94 (d, J=8.2 Hz, 1H), 7.20–7.33 (m, 5H). Exchangeable two OH protons were not observed. HR-MS (ESI): Calcd for C31H40NO3 [M+H]+: 474.3008. Found: 474.2990.
1g·(−)-CSA: Anal. Calcd for C31H39NO3·CSA·2.5H2O·0.3CHCl3: C, 63.05; H, 1.78; N, 7.73. Found: C, 62.85; H, 7.68; N, 1.54.
(6R,6aS,10R,11aR)-14-(Cyclopropylmethyl)-10-(2-hydroxy-2-methyl-4-phenylbutyl)-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-2-ol (1h)Compound 1h was prepared from compound 1a according to the procedure used to prepare compound 1d except for the final step (the debenzylation).
Yield: 19%. A brown oil. 1H-NMR (CDCl3, 400 MHz) δ: 0.04–0.09 (m, 2H), 0.39–0.47 (m, 2H), 0.84–0.93 (m, 1H), 1.14–1.56 (m, 6H), 1.26 (s, 3H), 1.74 (d, J=12.0 Hz, 1H), 1.78–1.83 (m, 2H), 1.94 (d, J=14.6 Hz, 1H), 2.05–2.20 (m, 2H), 2.26–2.50 (m, 5H), 2.59 (dd, J=12.0, 3.8 Hz, 1H), 2.63–2.80 (m, 3H), 3.00 (d, J=18.2 Hz, 1H), 3.34 (d, J=6.3 Hz, 1H), 4.51 (br s, 1H), 6.57 (d, J=4.1 Hz, 1H), 6.62 (dd, J=4.1, 1.3 Hz, 1H), 6.93 (d, J=4.1 Hz, 1H), 7.14–7.28 (m, 5H). An exchangeable OH proton was not observed. HR-MS (ESI): Calcd for C32H42NO3 [M+H]+: 488.3165. Found: 488.3158. IR (neat, cm−1): 3303, 2925, 1495, 1454, 1361, 1295, 1216, 747, 699.
1h·(−)-CSA: Anal. Calcd for C32H41NO3·CSA·1.8H2O: C, 67.05; H, 8.12; N, 1.86. Found: C, 67.00; H, 8.08; N, 1.67.
(6R,6aS,10R,11aR)-14-(Cyclopropylmethyl)-10-(2-hydroxy-2-methylpentyl)-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-2-ol (1i)Compound 1i was prepared from compound 9 according to the procedure used to prepare compound 1d.
Yield: 64%. A colorless oil. 1H-NMR (CDCl3, 400 MHz) δ: 0.02–0.10 (m, 2H), 0.36–0.47 (m, 2H), 0.79–0.94 (m, 4H), 1.21 (d, J=11.3 Hz, 1H), 1.33–1.54 (m, 12H), 1.72 (d, J=12.1 Hz, 1H), 1.89 (d, J=14.6 Hz, 1H), 2.04–2.22 (m, 3H), 2.23–2.51 (m, 4H), 2.57–2.64 (m, 1H), 2.66–2.75 (m, 1H), 2.96–3.06 (m, 1H), 3.35 (d, J=5.5 Hz, 1H), 6.59 (d, J=2.5 Hz, 1H), 6.64 (dd, J=2.5, 8.2 Hz, 1H), 6.91 (d, J=8.2 Hz, 1H). Exchangeable two OH protons were not observed. HR-MS (ESI): Calcd for C27H40NO3 [M+H]+: 426.3008. Found: 426.3001. IR (neat, cm−1): 3465, 2927, 1610, 1492, 1455, 1381, 1234, 1026.
1i·(−)-CSA: Anal. Calcd for C27H39NO3·CSA·0.3CHCl3·H2O: C, 62.95; H, 8.12; N, 1.97. Found: C, 62.97; H, 8.19; N, 2.19.
(6R,6aS,10S,11aR)-14-(Cyclopropylmethyl)-10-(2-methylpent-2-en-1-yl)-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-2-ol (12) (5 : 1 Mixture of Geometric Isomers)To a solution of compound 1i (26 mg, 0.060 mmol) in THF (0.50 mL) was added 2 M hydrochloric acid (1.0 mL), and stirred at 60°C for 21.5 h. After cooling to 0°C, potassium carbonate was added and extracted with chloroform–ethanol=3 : 1. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated under reduced pressure. Purification with preparative TLC (chloroform–methanol=20 : 1) gave the title compound (5 : 1 mixture of geometric isomers, 16 mg, 65%) as a colorless oil.
Major geometric isomer: 1H-NMR (CDCl3, 400 MHz) δ: 0.07–0.17 (m, 2H), 0.44–0.52 (m, 2H), 0.88–0.99 (m, 4H), 1.12–1.19 (m, 1H), 1.32–1.61 (m, 6H), 1.69 (s, 3H), 1.78 (s, 1H), 1.84 (d, J=12.1 Hz, 1H), 1.96–2.09 (m, 2H), 2.19–2.30 (m, 3H), 2.38–2.71 (m, 4H), 2.74–2.86 (m, 1H), 3.05 (d, J=18.2 Hz, 1H), 3.44 (d, J=6.0 Hz, 1H), 5.20 (t, J=6.9 Hz, 1H), 6.60–6.67 (m, 2H), 6.88–6.94 (m, 1H). An exchangeable OH proton was not observed. HR-MS (ESI): Calcd for C27H38NO2 [M+H]+: 408.2903. Found: 408.2902. IR (neat, cm−1): 3435, 2928, 2116, 1643, 1455, 1328, 1300, 1242, 1148, 1074, 994, 753.
(6R,6aS,10S,11aR)-14-(Cyclopropylmethyl)-10-(2-methylpentyl)-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-2-ol (1j)Under a hydrogen atmosphere, a solution of geometric mixture of compound 12 (38 mg, 0.093 mmol) and methanesulfonic acid (MSA) (7.2 µL, 0.11 mmol) in methanol (4.0 mL) was stirred in the presence of 10% palladium on carbon (20 mg) at ambient temperature for 8 h. After filtration through a pad of Celite, the filtrate was concentrated under reduced pressure. Then saturated aqueous potassium carbonate was added and the mixture was extracted with chloroform–ethanol=3 : 1. The combined organic layer was washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. Column chromatography (chloroform–methanol containing 10% ammonia=10 : 1) gave the title compound as a pale yellow oil. 1H-NMR (CDCl3, 400 MHz) δ: 0.07–0.18 (m, 2H), 0.42–0.50 (m, 2H), 0.84–0.93 (m, 7H), 1.08–1.91 (m, 15H), 2.18–2.82 (m, 7H), 3.04 (d, J=18.1 Hz, 1H), 3.43 (d, J=6.0 Hz, 1H), 6.62–6.65 (m, 2H), 6.92 (d, J=8.9 Hz, 1H). An exchangeable OH proton was not observed. HR-MS (ESI): Calcd for C27H40NO2 [M+H]+: 410.3059. Found: 410.3067. IR (neat, cm−1): 2925, 1612, 1455, 1214, 993, 754.
1j·(−)-CSA: Anal. Calcd for C27H39NO2·CSA·1.2H2O: C, 66.98; H, 8.72; N, 2.11. Found: C, 66.91; H, 8.63; N, 2.08.
(6R,6aS,10R,11aR)-1-(2-Hydroxy-14-methyl-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-10-yl)propan-2-one (2a)To a solution of compound 1a (87 mg, 0.23 mmol) in 1,1,2,2-tetrachloroethane (1.0 mL) were added 2,2,2-trichloroethyl chloroformate (0.13 mL, 0.92 mmol) and potassium carbonate (0.13 g, 0.92 mmol) and stirred at 150°C for 17 h. After cooling to 0°C, 2 M hydrochloric acid was added and extracted with chloroform. The combined organic layer was washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The residue was used for the next reaction without further purification. To a solution of the residue in acetic acid (2.0 mL) was added zinc powder (0.49 g, 7.6 mmol) and stirred at ambient temperature for 12.5 h. After filtration through a pad of Celite, the filtrate was concentrated under reduced pressure. Then the residue was taken up in 4 M aqueous sodium hydroxide and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated under reduced pressure. Column chromatography (ammonia-saturated chloroform–methanol=100 : 1 to 50 : 1) gave compound 14 as a pale yellow solid. To a solution of compound 14 in 1,2-dichloroethane (2.5 mL) were added paraformaldehyde (0.17 g, 5.6 mmol), acetic acid (64 µL, 1.1 mmol) and sodium triacetoxyborohydride (0.29 g, 1.4 mmol), and stirred at ambient temperature for 13 h. Then saturated aqueous sodium hydrogen carbonate was added and extracted with chloroform. The combined organic layer was washed with brine, dried over sodium sulfate and concentrated under reduced pressure. Purification with preparative TLC (chloroform–10% ammonia methanol solution=10 : 1) gave the title compound (4.8 mg, 6% for 3 steps) as a colorless oil.
1H-NMR (CDCl3, 400 MHz) δ: 1.22–1.32 (m, 1H), 1.37–1.59 (m, 5H), 1.69–1.76 (m, 1H), 1.83 (d, J=12.8 Hz, 1H), 2.11–2.26 (m, 2H), 2.20 (s, 3H), 2.32–2.53 (m, 2H), 2.38 (s, 3H), 2.72 (dd, J=6.2, 18.2 Hz, 1H), 2.95–3.20 (m, 4H), 6.61–6.68 (m, 2H), 6.97 (d, J=8.2 Hz, 1H). An exchangeable OH proton was not observed. HR-MS (ESI): Calcd for C21H28N1O3 [M+H]+: 342.2069. Found: 342.2061. IR (neat, cm−1): 3583, 2925, 1705, 1612, 1451, 1360, 1299, 1243, 1158.
2a·(−)-CSA: Anal. Calcd for C21H27NO3·CSA·3.0H2O: C, 59.31; H, 7.87; N, 2.23. Found: C, 59.21; H, 7.73; N, 2.22.
(6R,6aS,10R,11aR)-1-(2-Methoxy-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-10-yl)propan-2-one (15)Compound 15 was prepared from compound 13 according to the procedure used to prepare compound 2a except for the final step (the reductive ammination was not carried out).
Yield: 59%. A brown oil. 1H-NMR (CDCl3, 400 MHz) δ: 1.16–1.23 (m, 1H), 1.26–1.38 (m, 1H), 1.43–1.60 (m, 3H), 1.65–1.72 (m, 1H), 1.78–1.88 (m, 2H), 2.23–2.39 (m, 1H), 2.27 (s, 3H), 2.42–2.66 (m, 3H), 2.90–3.06 (m, 3H), 3.19–3.29 (m, 2H), 3.80 (s, 3H), 6.64 (d, J=2.7 Hz, 1H), 6.72 (dd, J=2.7, 8.4 Hz, 1H), 7.05 (d, J=8.4 Hz, 1H). An exchangeable NH proton was not observed. HR-MS (ESI): Calcd for C21H28N1O3 [M+H]+: 342.2069. Found: 342.2059. IR (neat, cm−1): 2934, 1706, 1611, 1492, 1432, 1302, 1221, 1162.
(6R,6aS,10R,11aR)-1-(2-Hydroxy-14-phenethyl-6,7,8,9,10,11-hexahydro-5H-6,11a-(epiminoethano)-6a,10-epoxycyclohepta[a]naphthalen-10-yl)propan-2-one (3a)To a solution of compound 15 (75 mg, 0.22 mmol) in DMF (1.0 mL) were added (2-bromoethyl)benzene (89 µL, 0.66 mmol) and potassium carbonate (0.15 g, 1.1 mmol), and stirred at ambient temperature for 1 h. Then distilled water was added, and extracted with diethyl ether. The combined organic layer was washed with brine, dried over sodium sulfate and concentrated under reduced pressure. Column chromatography (chloroform 100%) gave the tertiary amine. A mixture of pyridinium chloride (3.0 g, 26 mmol) and the obtained tertiary amine was stirred at 180°C for 7 h. After cooling to ambient temperature, saturated aqueous sodium hydrogen carbonate was added and extracted with chloroform–ethanol=3 : 1. The combined organic layer was washed with brine, dried over sodium sulfate and concentrated under reduced pressure. Purification with preparative TLC (saturated ammonia-chloroform–methanol=50 : 1) gave the title compound (15 mg, 16% yield) as a pale yellow amorphous solid.
1H-NMR (CDCl3, 400 MHz) δ: 1.18–1.28 (m, 2H), 1.34–1.59 (m, 4H), 1.62–1.71 (m, 1H), 1.85 (d, J=12.7 Hz, 1H), 2.08–2.23 (m, 2H), 2.21 (s, 3H), 2.47 (d, J=12.7 Hz, 1H), 2.54–2.61 (m, 1H), 2.67–2.89 (m, 5H), 2.96–3.02 (m, 2H), 3.08–3.20 (m, 1H), 3.27–3.35 (m, 1H), 6.65 (d, J=2.5 Hz, 1H), 6.69 (dd, J=8.3, 2.5 Hz, 1H), 6.94 (d, J=8.3 Hz, 1H), 7.15–7.21 (m, 3H), 7.23–7.30 (m, 2H). An exchangeable OH proton was not observed. HR-MS (ESI): Calcd for C28H34N1O3 [M+H]+: 432.2539. Found: 432.2546. IR (film, cm−1): 3584, 3282, 2924, 1704, 1610, 1450, 1362.
3a·(−)-CSA: Anal. Calcd for C28H33NO3·CSA·1.3H2O: C, 66.41; H, 7.57; N, 2.04. Found: C, 66.51; H, 7.84; N, 2.20.
Binding AssaysThe human μ, δ, or κ opioid receptor recombinant CHO cell pellets were resuspended in 50 mM Tris–HCl buffer containing 5 mM MgCl2, 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), pH 7.4. The cell suspensions were disrupted by use of a glass-teflon homogeniser and centrifuged at 48000×g for 15 min. The supernatant was discarded and the pellets were resuspended in buffer at a concentration of 5 mg protein/mL and stored at −80°C until further use. Human μ, δ, or κ opioid receptor recombinant CHO cell membranes were incubated for 2 h at 25°C in 0.25 mL of the buffer containing with various concentrations of the tested compound, 2 nM [3H] DAMGO, [3H] DPDPE, or [3H] U69,593 (PerkinElmer, Inc.), respectively. The incubation was terminated by collecting membranes on Filtermat B filter (PerkinElmer, Inc.) using a FilterMate™ harvester (PerkinElmer, Inc.). The filters were then washed three times with 50 mM Tris–HCl buffer, pH 7.4. Then, MeltiLex scintillant (PerkinElmer, Inc.) was melted onto the dried filters. Radioactivity was determined by a MicroBeta scintillation counter (PerkinElmer, Inc.). Nonspecific binding was measured in the presence of 1 µM unlabeled DAMGO, DPDPE, or U-69,593 (PerkinElmer, Inc.). Ki values were calculated by Prism software (version 5.0).
[35S]GTPγS Binding AssaysHuman μ, δ, or κ opioid receptor recombinant CHO cell membranes were incubated for 2 h at 25°C in 0.25 mL of 50 mM Tris–HCl buffer containing 5 mM MgCl2, 1 mM EGTA, 100 mM NaCl, pH 7.4 with various concentrations of the tested compound, 30 µM guanosine 5′-diphosphate (GDP) and 0.1 nM [35S] GTPγS (PerkinElmer, Inc.). The incubation was terminated by collecting membranes on Filtermat B filter (PerkinElmer, Inc.) using a FilterMate™ harvester (PerkinElmer, Inc.). The filters were then washed three times with 50 mM Tris–HCl buffer, pH 7.4. Then, MeltiLex scintillant (PerkinElmer, Inc.) was melted onto the dried filters. Radioactivity was determined by a MicroBeta scintillation counter (PerkinElmer, Inc.). EC50 values were calculated by Prism software (version 5.0). Nonspecific binding was measured in the presence of 10 µM unlabeled GTPγS (PerkinElmer, Inc.). DAMGO, DPDPE, or U-69,593 (PerkinElmer, Inc.) was used as the standard μ, δ, or κ opioid receptor agonist, respectively.
We acknowledge the institute of Instrumental Analysis of Kitasato University, School of Pharmacy for its facilities.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
