80 Acta Chim. Slov. 2005, 52, 80-85 Short Communication Synthesis, Crystal Structural and Pharmacological Study of Af-Cyclopropylmehtyl-7a- [ (R) -l-hydroxyl-l-methyl-3- (thien- 2-yl)propyl]-6,14-endoethanotetrahydronooripavine He Liu,* Bo-hua Zhong, Chun-he Liu, Bo Wu, and Ze-Hui Gong No. 7 Department, Institute of Pharmacology and Toxicology Academy, Beijing 100850, P. R. China Abstract Received 27-09-2004 Ar-Cyclopropylmethyl-7a-[(K)-l-hydroxyl-l-methyl-3-(thien-2-yl)propyl])-6,14-endoethanotetrahydrooripavine (thienorphine, I), has been synthesized and evaluated for its in vivo analgesic activities. Thienorphine was structur-ally characterized by infrared (IR), NMR spectra, FAB-MS and X-ray diffraction. The crystal structure indicates that thienorphine maintained the main rigid structure of morphine and contains a C6-C14 enthano bridge. The C7 substituent is l-hydroxyl-l-methyl-3-(thien-2-yl)propyl group adopting R-configuration. The cyclopropylmethyl group is located at the equatorial position as expected. The packing diagram of thienorphine showed the pres-ence of the intramolecular and intermolecular O-H* • »O hydrogen bond linking the molecules into an infinite quasi-one-dimensional chain structure. In vivo pharmacological study thienorphine exhibited excellent analgesic activity. Key words: crystal structure, oripavine derivative, analgesic Introduction Opioid analogues stili remain important drugs for the relief of severe pain and morphine is stili the drug of choice in such situations. For many years, the search for new opioid derivatives that act on the CNS and have pain-relieving properties and devoid of undesired side effects, such addiction, has been the goal of a large number of scientists.1'2 The abuse of cocaine and other stimulant drugs is becoming a significant social and pub-lic health concern in the world.3 Consequently, a wide variety of modifications of the well-known alkaloids morphine, codeine and oripavine have been described.4 The svnthesis and pharmacological of 6,14-endoeth-anomorphinan derivatives have been extensively studied. The tvpical examples of the pharmacological active compounds reported in the literature such as buprenorphine (Temgesic),5 etorphine(Immobilon)6 and dihydroetorphine.7 These kinds of compounds are characterized by a 6,14-endoethano bridge and a lipophilic substituted in position 7 a of the C-ring. We have engaged in the synthesis and biological activity study of oripavine derivative for many years, and have found Ar-Cyclopropylmethyl-7a-[l-(i?)-l-hy-droxy-l-methyl-3-(thien-2-yl)propyl]-6,14-endoethan-otetrahvdrooripavine (thienorphine, I, in chart 1), is a very potent oripavine derivative with mixed agonist and antagonist opiate receptor activities. Thienorphine showed very good analgesic activity in mouse using the acetic acid writhing model, mouse heat radiant tail-flick assay, and mouse heat plate test. \ // C HO O OCH3 Chart 1. Thienorphine, I. Results and discussion Description of the crystal structure The X-ray ORTEP structure of I with atomic labeling is shown in Figure 1. The crystal structure of I maintains the main rigid structure of morphine as described in the literature, such as morphine,8 3-methoxyetorphine,9 and buprenorphine.10 A rigid pentacyclic structure consisting of a benzene ring A, partially unsaturated six-membered ring B and cyclohex-ane ring D, a piperidine ring E, a dihydrofuran ring C and the C6-C14 ethano bridge. Rings A, B and D are the phenanthrene ring system that has little conformational flexibility. The shape of the title compound likes a three-dimensional “T” with rings A, B and C forming a near perfect vertical plane and rings E and D forming a more distorted horizontal plane. The piperidine ring E is in the chair conformation and the D ring is boat with the atoms 6 and 14 fore and aft. The new ethano Liu et al. Synthesis, X-Ray, and Analgesic Activity of Thienorphine Acta Chim. Slov. 2005, 52, 80-85 81 Table 1. Analgesic activity of I and buprenorphine. Test model Compound ED50(mg/kg) S.C. P.O. Rat acetic acid buprenorphine 0.02 0.37 writhing I 0.08 0.64 Rat hot plate test buprenorphine I 1.01 0.57 12.52 3.10 Rat tail-flick model buprenorphine 8.75 –a I 1.75 2.61 a Maximal analgesic efficacy < 30%. Figure 1. ORTEP view of the thienorphine with 50% thermal ellipsoid probability. Figure 2. Packing diagram of the thienorphine showing hydrogen bonds. bridge the original boat-shape ring D formed the bicycle[2,2,2]octane cage. The l-hydroxyl-l-methyl-3-(thien-2-yl)propyl group on C7 position adaptedi?-con-figuration. The Grignard reaction shows a remarkably high degree of stereoselectivity and strictly obeyed the Cram’s rules(Scheme l),11 as a result the i?-configura-tion was the almost šole product. Since the cyclopro-pylmethyl group on N is large than an electron pair or a protonated electron pair, it is predominantly equatorial (as shown), the stereostructrue would be important in determining relative agonist potencies as discussed in the literature.12 The hydroxyl group 0(4) is included by forming an intramolecular hydrogen bond with methyl ether oxygen 0(3), and the distance betvveen 0(4) and 0(3) is 2.586 A with the H—O separation is 1.870 A, falling in to the normal range of the O—O separation for hydrogen bonding,13 the bond angle is 145.15°. As shown in Figure 2, an infinite quasi-one-di-mensional chain structure was formed through O—H-O intermolecular hydrogen bonds in which the O atoms of the hydroxyl group links another hydroxyl group of benzene ring in the adjacent molecule. The O—O separation is 2.733 A with the O—H separation is 1.914 A, the bond angles are 176.67°. Liu et al. Synthesis, X-Ray, and Analgesic Activity of Thienorphine 82 Acta Chim. Slov. 2005, 52, 80-85 Scheme 1. Synthesis of Thionorphine I. Reagents and conditions: a) 2-(thien-2-yl)ethylmagnesium bromide, anhydrous Et20. b) CNBr, CH2C12. c) KOH, diethylene glycol. d) Cyclopropylmethyl bromide, NaHC03, DMF. Analgesic activity of thienorphine The results in Table 1 clearly show that compound 1 and buprenorphine displayed definite analgesic activ-ity. Buprenorphine is a potent opioid analgesic that is being developed as a treatment for the opiate abuse and dependence.514 Compared with the analgesic activity of two compounds, I exhibited higher analgesic activity in rat hot plate test model and rat tail-flick model. It was 2 or 4 time more potent than buprenorphine. Experimental General Ali the reagents for syntheses were commercially available and used without further purification or puri-fied by standard methods prior to use. Melting points were determined using a RY-1 apparatus and are uncor-rected. JH NMR spectra were recorded on JNM-ECA-400 400 MHz instrument in the solvent indicated below. Chemical shift values are reported in parts per million (ppm) relative to that for tetramethylsilane used as an internal reference standard. Mass spectra were obtained from Micromass ZabSpec and API3000 instruments. Elemental analysis was carried at the CarloErba-1106. IR spectra were measured on a FT-IR 170SX (Nicolet) spectrometer with KBr pellets. Synthesis Thienorphine I was prepared by a modified meth-od as described in the literature.15 According to Scheme 1, 7a-acetyl-6,14-endoethanotetrahydrothebaine 1 was coupled with Grignard reagent, 2-(thiophen-2-yl)ethylmagnesium bromide, to give 7ct-[(i?)-l-Hydroxy-l-methyl-3-(thien-2-yl)propyl]-6,14-endoethanoetrahy-drothebaine 2. This Grignard reaction shows a remark-ably high degree of stereoselectivity. The R isomer was the almost šole product, whereas the 5 isomer was not afforded. A rnkture of the intermediate 2 and cyanogens bromide in dried methylene chloride was refluxed to obtain Ar-Cyano-7a-[(i?)-l-hydroxy-l-methyl-3-(thien-2-yl)propyl]-6,14-endoethanotetrahydronorthebaine 3. 7a-[l-(i?)-l-Hydroxy-l-methyl-3-(thien-2-yl)propyl]-6,14-endoethanotetrahydronororipavine 4 was obtained by treating 3 with KOH in diethylene glycol at 205-210 °C. A mixture of 4 (7.0 g, 0.015 mol), cyclopropyl-methyl bromide (4.1 g, 0.030 mol) and dried sodium bicarbonate (4.0 g, 0.048 mol) in Ar^V-dimethylforma-mide (200 mL) was vigorously stirred at 70-80 °C under nitrogen gas for 16 h. The mixture was filtered. The filtrate was evaporated to dryness. The residue was dis-solved with CH2C12, dried (Na2S04) and evaporated to obtain a crude product. The crude product was purified by chromatography on silica gel column, eluting with a CH2Cl2/MeOH mkture (20:1) to give white solid, then recrystallized from methanol to give colorless crystal 1 (3.8 g). Yield: 48.4%, mp 170-172 °C. IR (KBr): 3406, 3224, 2989, 2926, 1634, and 1609. JH NMR (CDC13) 5 8.98 (1H, s, Ar-OH), 7.29 (1H, d, / 5.0Hz, ArH), 6.93 (1H, m, ArH), 6.85 (1H, m, ArH), 6.73 (1H, d, / 8Hz, ArH), 6.55 (1H, d, / 8 Hz, ArH), 4.61 (1H, s, OH), 4.39 (1H, s,), 3.41 (3H, s, -OCH3), 2.85-2.98 (4H, m), 2.58-2.76 (2H, m), 2.10-2.27 (4H, m), 1.80-2.05 (4H, m), 1.66-1.76 (2H, m, CH2), 1.45-1.55 (2H, m, CH2), 1.30 (3H, s, -CH3), 1.18-1.24 (1H, m, CH), 1.07 (1H, m, CH), 0.75 (1H, m, Cprop-CH), 0.58 (1H, m), 0.44 (2H, m, Cprop-CH2), 0.08 (2H, m, Cprop-CH2). 13C NMR (CDC13) 5 3.48, 3.97, 9.39, 17.7, 23.2, 23.9, 29.8, 31.6, 35.6, 35.9, 43.5, 43.6, 45.7, 47.2, 52.8, 58.3, 59.8, 75.8 (C16, C19), 80.5, 97.4, 116.5, 117.4, 119.5, 122.7, 123.9, 126.7, 128.1, 132.2, 145.5, 146.0. ESI-MS: 522.1(M+1). Anal. Calcd For C31H39N04S: C 71.37, H 7.53, N 2.68. Found: C 71.17, H 7.63, N 2.46. Liu et al. Synthesis, X-Ray, and Analgesic Activity of Thienorphine Acta Chim. Slov. 2005, 52, 80-85 83 Table 2. Crystallographic data and mary for thienorphine. structure refinement sum- Empirical formula Molecular weight Measured temperature Crystal size (mm3) Crystal system Space group Unit celi dimensions C31H39NO4S 521.69 293(2) K 0.32x0.25x0.20 Orthorhombic P2,2,2, a = 11.304(4) A b = 11.481(4) A c = 20.534(5) A 2664.8(15) 4 1.300 0.159 1120 1.98 - 26.46° 99.5 % -10/14, -14/11, -20/25 15140 / 5477 339 0.0368 R\= 0.0467, wR2 = 0.1045 1.021 0.305 and -0.265 Table 3. Atomic coordination and equivalent isotropic displace-ment parameters for thienorphine. x y z U(eq) Volume K (A3) Z Aa,cd(g cm-3) fl (mm"1) F (000) 9 range(°) Completeness to 0 h/k/l Reflection collected/unique Parameters refined Rint Finalifindices [/>2o(/)] Goodness of fit Residual electron densities (e A"3) X-Ray diffraction analysis Single-crystal X-ray diffraction measurement was carried out on a Bruker Smart 1000 CCD diffractom-eter. The determination of unit celi parameters and data collections was performed with Mo Ka radiation (X = 0.71073 A) and unit celi dimensions were obtained with least-squares refinements. The structure was solved by direct methods with SHELXL-97 program16 and aH data were corrected by using semi-empirical absorption corrections (SADABS) method. Ali the other non-hydrogen atoms were located in successive difference Fourier syntheses. The final refinement was carried out by full matrix least-squares methods with anisotropic thermal parameters for non-hydrogen atoms on F2. The hydrogen atoms were added theoretically, and riding on the concerned atoms and refined with fixed thermal factors. Further details of the structure analyses are given in Table 2. Positional parameters and atomic coordinates are given in Table 3, whereas bond distances and angles are listed in Table 4, respectively. Further details of the crystal structure investigation are available from the Cambridge Crystallographic Center with quotation number CCDC 212447.17 S(l) N(l) O(l) 0(2) 0(3) 0(4) C(l) C(2) C(3) C(4) C(5) C(6) C(7) C(8) C(9) C(10) C(ll) C(12) C(13) C(14) C(15) C(16) C(17) C(18) C(19) C(20) C(21) C(22) C(23) C(24) C(25) C(26) C(27) C(28) C(29) C(30) C(31) 3333(1) 2308(2) 8083(2) 6899(1) 7317(1) 6244(2) 5117(2) 6256(3) 6930(2) 6361(2) 6007(2) 6281(2) 5192(2) 4095(2) 3182(2) 3461(2) 4557(2) 5190(2) 4819(2) 4313(2) 3892(2) 2722(2) 5245(2) 6412(2) 5355(2) 4235(3) 3866(4) 3098(3) 2029(2) 1536(3) 2103(4) 5755(3) 1110(2) 641(2) 613(3) -522(3) 8431(2) 7240(1) 4666(2) 5641(2) 5603(2) 4582(2) 4414(2) 4453(3) 4885(2) 5317(2) 5363(2) 5398(2) 4364(2) 4343(2) 3861(2) 3789(2) 3835(2) 4518(2) 5071(2) 5140(2) 3923(2) 6088(2) 5854(2) 2978(2) 3261(2) 3765(2) 3850(3) 5069(4) 5784(3) 5489(2) 6438(4) 7404(4) 2498(3) 4534(3) 3330(3) 2583(3) 3062(3) 4718(3) 4767(1) 6872(1) 8244(1) 6998(1) 5631(1) 4523(1) 8622(1) 8706(1) 8193(1) 7590(1) 6485(1) 6035(1) 5561(1) 5925(1) 7089(1) 7831(1) 8017(1) 7534(1) 6829(1) 6662(1) 6688(1) 7006(1) 6804(1) 6440(1) 4881(1) 4464(1) 4232(2) 4662(1) 4993(1) 5300(2) 5220(3) 4916(1) 7137(2) 7113(2) 7721(2) 7455(2) 5933(1) 120(1) 36(1) 51(1) 35(1) 41(1) 55(1) 45(1) 45(1) 36(1) 31(1) 30(1) 30(1) 28(1) 29(1) 31(1) 38(1) 36(1) 30(1) 27(1) 27(1) 34(1) 40(1) 31(1) 33(1) 37(1) 49(1) 79(1) 55(1) 47(1) 79(1) 101(2) 53(1) 51(1) 52(1) 58(1) 64(1) 52(1) Pharmacology Experiments were performed according to three methods: rat acetic acid writhing model, rat hot plate test model and rat tail-flick model as described in the literature,17 which are the sensitive and predictive ani-mal models for analgesic drugs as shown by the good correlation betvveen values obtained in rats and analge- Liu et al. Synthesis, X-Ray, and Analgesic Activity of Thienorphine 84 Acta Chim. Slov. 2005, 52, 80–85 Table 4. Bond lengths(A), angles(°) of the thienorphine. Bond lengths: S(l)-C(25) 1.684(5) 0(3)-C(6) 1.456(3) C(4)-C(12) 1.370(3) S(l)-C(22) 1.706(3) C(l)-C(2) 1.390(4) C(27)-C(28) 1.481(4) N(l)-C(27) 1.467(3) C(2)-C(3) 1.392(4) C(ll)-C(12) 1.377(3) N(l)-C(9) 1.479(3) C(6)-C(18) 1.522(3) 0(4)-C(19) 1.452(3) 0(1)-C(3) 1.359(3) C(6)-C(7) 1.569(3) C(l)-C(ll) 1.397(3) 0(2)-C(4) 1.386(3) N(l)-C(16) 1.468(3) C(3)-C(4) 1.396(3) 0(2)-C(5) 1.477(3) C(8)-C(14) 1.534(3) 0(3)-C(31) 1.413(3) C(9)-C(10) 1.556(3) Bond angles: C(25)-S(l)-C(22) 92.9(2) N(l)-C(9)-C(14) 108.10(18) C(17)-C(14)-C(9) 112.61(18) C(27)-N(l)-C(16) 108.8(2) C(12)-C(13)-C(15) 113.99(18) C(13)-C(14)-C(9) 105.48(18) C(27)-N(l)-C(9) 115.8(2) C(15)-C(13)-C(5) 111.74(19) C(16)-C(15)-C(13) 113.1(2) C(16)-N(l)-C(9) 111.29(19) C(12)-C(13)-C(14) 105.55(18) N(l)-C(16)-C(15) 111.5(2) C(4)-0(2)-C(5) 107.15(17) C(15)-C(13)-C(14) 110.21(18) C(14)-C(17)-C(18) 110.08(18) C(12)-C(4)-0(2) 113.50(19) C(20)-C(19)-C(7) 112.1(2) C(6)-C(18)-C(17) 110.77(19) 0(2)-C(5)-C(6) 114.51(18) C(5)-C(13)-C(14) 113.09(18) 0(4)-C(19)-C(26) 108.0(2) 0(2)-C(5)-C(13) 107.26(16) C(8)-C(14)-C(17) 105.35(19) O(4)-C(19)-C(20) 104.8(2) C(6)-C(5)-C(13) 107.56(18) C(8)-C(14)-C(13) 108.65(18) C(26)-C(19)-C(20) 109.4(2) C(8)-C(7)-C(19) 112.02(19) C(17)-C(14)-C(13) 109.92(18) 0(4)-C(19)-C(7) 108.5(2) C(19)-C(7)-C(6) 118.03(19) C(8)-C(14)-C(9) 114.80(19) C(26)-C(19)-C(7) 113.6(2) sic doses in humans. As regards the experiments carried out in vivo, test compounds were administered as S.C. and P.O. methods. The experiments were performed on Wistar rats (180-200 g) of both sexes, kept at ambient temperature on a 12 h light/dark schedule, with free access to food and water before the experiments. The experimental groups consisted of 5-8 animals each. Statistical analysis used Kruskal-Wallis/Mann-Whit-ney. Differences in pre-and post-drug latencies were analyzed by the Wilcoxon test. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 30271492). References 1. a) J. W. Lewis, M. J. Readhead, A. C. B. Smith, /. Med. Chem. 1973, 16, 9-14. b) D. M. Zimmerman, J. D. Leander, /. Med. Chem. 1999, 33, 895-902. c) D. W. Raisch, Ann. Pharmacother 2002, 36, 312-321. 2. R. K. Portnoy, In Proceedings of the 7th World Congress on Pain, Progress in Pain Research and Management; G. F. Crebhart, D. F. Hammond, F. S. Jensen, Eds. IASP, Seattle, WA, 1994, 29, pp 595. 3. H. B. Crome, Drug Alcohol Depend. 1999, 55, 247-263. 4. a) K. W. Bentley Central Analgetics, D. Fednicer, Eds. John Wiley & Sons: New York, 1982. b) G. R. Fenz, S. M. Evans, D. E. Walters, A. J. Hopfinger, D. F. Hammond. Opiates; Academic Press: Orlando, 1986. c) A. F. Casy, R. F Parfitt, Opioid Analgesics: Chemistry and Receptors; Plenum Press: New York, 1986. d) J. Marton, Cs. Simon, S. Hosztafi, Z. Szabo, A. Marki, A. Borsodi, S. Makleit, Bioorg. Med. Chem. 1997, 5, 369-382. e) S. M. Husbands, J. W. Fewis,/. Med. Chem. 2000, 43, 139-141. 5. a) R. C. Heel, R. N. Brogden, F. M. Speight, G. S. Avery, Drugs 1979, 17, 81-86. b) P. J. Hoskin, G. W. Hanks, Drugs 1991, 41, 326-338. 6. A. E. Fakemori, G. Hayashi, S. E. Smits, Eur. J. Pharmacol. 1972, 20, 85-91. 7. D. X. Wang, X. Q. Fu, B. Y. Qin, /. Pharm. Pharmacol. 1995, 47, 669-676. 8. F. Gylbert,^4cto Crystallogr. B 1973, 29, 1630-1635. 9. K. W. Van den Hende, N. R. Nelson, /. Am. Chem. Soc. 1967, 89, 2901-2905. 10. a) B. Kratochvil, M. Husak, P. Bulej, A. Jegorov, Collect. Czech Chem. Comm. 1994, 59, 2472-2479. b) J. Marton, Z. Szabó, S. Garadnay, S. Miklós, S. Makeit, Tetrahedron 1998, 54, 9143-9152. 11. a) D. J. Cram, F. A. A. Elhafez,/. Am. Chem. Soc. 1952, Liu et al. Synthesis, X-Ray, and Analgesic Activity of Thienorphine Acta Chim. Slov. 2005, 52, 80–85 85 74, 5828-5835. b) A. Mengle, O. Reiser, Chem. Rev. 1999, 99, 1191-1224. 12. a) G. H. Loew, D. S. Berkowitz,/. Med. Chem. 1975, 18, 656-662. b) G. H. Loew, D. S. Berkovvitz,/. Med. Chem. 1979, 22, 603-607. 13. G. R. Desiraju,^4cc. Chem. Res.1991, 24, 290-296. 14. S. M. Husbands, S. W. Breeden, K. Grivas, J. W. Lewis, Bioorg. Med. Chem Lett. 1999, 9, 831-834. 15. a) K. W. Bentley, D. G. Hardy, B. Meek, /. Am. Chem. Soc. 1967, 89, 3273-3280. b) K. W. Bentley, D. G. Hardy, /. Am. Chem. Soc. 1967, 89, 3281-3292. c) K. W. Bentley, D. G. Hardy, H. P. Crocker, D. I. Haddlesey, P. A. Mayor, /. Am. Chem. Soc. 1967, 89, 3312-3321. d) J. Marton, Z. Szabo, S. Garadnay, S. Mikos, S. Makleit, Tetrahedron 1998, 54, 9143-9152. 16. G. M. Sheldrick, SHELXS-97, Program for X-ray Crystal Structure Solution, Göttingen University, Germany 1997. G. M. Sheldrick, SHELXL-97, Program for X-ray Crystal Structure Refinement, Göttingen University, Germany 1997. 17. Ouotation number CCDC 212447 at www.ccdc.cam. ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033; email: deposit@ccdc.cam.ac.uk]. 18. a) R. T. Li, J. C. Cai, X .C. Tang, M. S. Cai, Arch. Pharm. Pharm. Med. Chem. 1999, 332, 179. b) E. Elisabetsky, T. A. Amador, R. R. Albuquerque, D. S. Nunes, A. C. T. Carvalho,/. Ethnopharmacol. 1995, 48, 77. c) J. E. Davies, D. N. Kellet, J. C. Pennington,^4rc/z. Int. Pharmacodyn. Ther. 1976, 221, 21 A. Povzetek Sintetizirali smo “thienorphine” (I) ter in vivo testirali njegove analgetske lastnosti. Strukturo I smo določili na osnovi IR in NMR analiz in rentgenske difrakcijske analize. Liu et al. Synthesis, X-Ray, and Analgesic Activity of Thienorphine