Elucidation of the Structure and Biological Activity of a New Sesquiterpene from Cistus creticus L. Konstantinos atzellis, Georgia Pagona, Apostolos Spyros, Costas Demetzos, aralambos E. Katerinopoulos Department of Chemistry, University of Crete, eraklion 71409, Crete, Greece, and School of Pharmacy, Department of Pharmaceutical Technology, Panepistimiopolis Zografou 15771, University of Athens, Athens, Greece. 9 13 4 5 3 4a 6 A B 2 8a 7 1 8 11 12 10
Composition of the Essential il from the Resin of C. creticus L. Components Rt % στο δείγµα ladano3.d01 1 2-methyl butanoic acid 5.674 0.13 2 Tricyclene 7.432 0.18 3 α-pinene 7.895 2.67 4 Camphene 8.410 1.39 5 Thuja-2,4(10)-diene 8.578 0.05 6 Benzaldehyde 8.684 0.05 7 β-pinene 9.463 0.60 8 Phenol 9.575 0.16 9 exanoic acid 10.200 0.04 10 α-phellandrene 10.565 0.12 11 α-terpinene 11.100 0.13 12 p-cymene 11.420 0.23 13 Limonene 11.638 0.35 14 γ-terpinene 12.983 0.25 15 Acetophenone 13.182 0.03 16 Terpinolene 14.353 0.19 17 Linalool 14.881 0.10 18 3,5-heptadien-2-one, 6-methyl 15.037 0.22 (E)- 19 [R]-(+)-Norinone 16.550 0.06 20 trans-pinocarveol 16.789 0.99 21 Camphor 16.939 0.16 22 3-Methylcamphenilol 17.192 0.47 23 Isoborneol 17.616 0.17 24 Borneol 18.147 2.07 25 4-Terpineol 18.653 1.02 26 p-cymen-8-ol 19.093 0.14 27 α-terpineol 19.462 4.55 28 Myrtenol 19.728 0.20 29 Verbenone 20.094 0.05 30 trans-carveol 20.636 0.13 31 1-Cyclohexene-1- carboxaldehyde, 4-(1- methylethyl)- 23.174 0.05 32 Bornyl acetate 23.778 0.10 33 Thymol 24.092 0.03 34 Carvacrol 24.577 1.03 35 Megastigmatrienenone 26.445 0.07 36 α-cubebene 26.821 0.10 37 Longicyclene 26.933 0.06 38 Copaene 28.054 0.64 39 β-boutbonene 28.435 0.14 Components Rt % στο δείγµα ladano3.d01 40 2-butanone,4-(2,2-dimethyl-6- methylenecyclo hecyl-) 28.892 0.12 41 Eugenol methyl ether 29.152 0.05 42 Isocaryohryllene 29.421 0.07 43 β-caryophyllene 30.033 1.10 44 α-guaiene 30.827 0.13 45 β-cubebene 31.328 0.12 46 α-caryophyllene 31.488 0.12 47 Arromadendrene 31.826 0.45 48 Drimane Sesquiterpene 32.634 18.59 49 Eremophilene 33.108 2.41 50 Ledene 33.420 1.11 51 α-muurolene 33.539 0.24 52 1,1,4α-trimethyl-5,6- dimethylenedecahydronaphthale ne 33.875 0.10 53 γ-cadinene 34.131 0.29 54 δ-cadinene 34.544 1.71 55 Cubenene 34.910 0.59 56 Cyclohexane,1-methyl-2,4- bis(1-methylethenyl)- (1.alpha,2.beta,4,beta) 35.094 0.23 57. Palustrol 36.355 0.14 58. Iasocaryorhyllene 36.613 0,15 59. Caryophyllene xide 36.946 0.36 60. Epiglobulol 37.432 1.67 61. Ledol 37.859 0.87 62. Cubenol 38.819 1.15 63. β-selinenol 39.708 0.48 64. 7[11]-Selinen-4α-ol 39.898 1.10 65. Viridiflorol 40.024 0.65 66. Sclareol xide (cisa/b) 48.356 0.42 67. β-selinene 51.565 0.75 68. Manoyl xide 52.401 10.10 69. Epimanoyl xide 53.262 14.69 70. Kaur-16-ene 53.842 0.21 71. Manool 54.228 0.13 72. unulane-16-dien-3-ol 55.287 0.75 73. (7α-Isopropenyl-4,5- dimethyloctahydroindene-4-yl) methanol 74. 4α-7,7,10αtetramethyldodecahydrobenzolf) chromen-3-ol 58.025 0.27 59.390 0.31 75. 3-xomanoyle xide 59.895 0.09 Total in the mixture 80.56%
Analysis of the components of Labdanum by Gas Chromatography Mass Spectrum of Major Component 204
3 4 4a 5 6 2 8a 7 1 8 12 13 11 cis-drima-7,9(11)-diene 9 10 trans-drima-7,9(11)-diene
3 4 5 23A + 23B trans πορεια 10 9 7 cis πορεια cis πορεια 6 12 + ίχνη 8 8 + ίχνη 12 SiMe 3 SiMe 3 ιαχωρισµός Separation of διαστερεοϊσοµερών diastereomers 14 13 18A + 18B 15A + 15B Br 19A + 19B Br 16A + 16B 20 17 1 2
10 eq EG, Ph t-buk, t-bu 1 eq PTSA 60 C, 4h, 93% MeI, C 6 6, 0 C,74% 3 4 5 N 2 N 2, DEG K, 200 C, 98% NaB 4, Et, PPTS -30 C, 2h, 88% Et: 2 9:1 reflux, 1.5h, 99% 10%Pd/C Et, 95% 9B 7 10%Pd/C Et, 3.5h 90%, (+8% of 12) 6 Jones 10 91%, (+1.5%of 8) 12 8
1 8a 4a 5 beta-equatorial alcohol 9B trans-attack to alpha face alpha-axial alcohol 9A cis-attack to beta face
SiMe 3 8 LDA, Me 3 SiCl 0 C, 79% 13 TBAF, MeI TF, 90% 15A - 15B PBP, Ac 2.5h, rt, 94% 33 C 3 Li TF/MPA 7:1-78 o C έως RT 24 h 47% Burgess reagent (Me 2 CNS 2 NEt 3 ) Et 3 N, TF reflux, 48h 17 C 2 DBU, toluene 3.5h, reflux, 93% 16A - 16B Br 2 (85%)
Comparing NMR data of the cis- and the trans-analog with those of Demedzos et al 13 CNMR C 1 C 2 C3 C 4 C 4a C 5 C 6 C 7 C 8 C 8a C 9 C 10 C 11 C 12 C 13 A.F. Barreto et al. trans-product 33.4 42.3 19.1 37.8 37.9 158.3 131.3 126.6 24.4 48.7 101.8 21.2 22.2 33.0 20.6. Akita et.al. trans-product 33.4 42.2 19.1 37.7 37.8 158.2 131.2 126.5 24.3 48.7 103.7 21.1 22.1 32.9 20.6 C. Demetzos et al. 37 32.2 22.7 39.6 39.1 146.5 144.5 117.7 29.7 39.8 106.2 21.1 15.7 14.1 21.6. E. Katerinopoulos et al, cis -product 34.2 43.7 18.7 37.5 37.8 150.2 131.7 125.2 32.2 49.8 104.9 21.5 23.6 33.0 20.4 1 Η NMR 7 (C) 9 (C 2 ) 9 (C 2 ) 10 (C 3 ) 11 (C 3 ) 12 (C 3 ) 13 (C 3 ) Polovnika et al. trans-product A.F. Barreto et al. trans-product. Akita et.al. trans-product 5.65 4.77 4.81 1.77 0.90 0.84 0.94 5.65 4.78 4.83 1.78 0.91 0.85 0.95 5.65 4.80 4.84 1.80 0.93 0.87 0.97 C. Demetzos et al. 5.37 4.64 4.69 1.7 0.85 0.75 1.5. E. Katerinopoulos et al cis -product 5.48 4.93 4.94 1.77 0.83 0.70 1.02
1 -NMR SPECTRUM 3 methyl groups : 1.73ppm (s), 0.92ppm (s), 0.79ppm (d, next to tertiary carbon) 3 olefinicη : 4.70ppm (d) 5.34ppm (bd) 1.099 1.125 1.151 1.396 1.404 1.420 1.447 1.434 1.502 1.455 1.527 1.663 1.556 1.739 0.929 0.810 0.796 1.772 1.797 1.844 1.872 1.911 1.938 1.979 2.153 2.011 2.202 3.00 3.04 1.22 1.87 1.37 1.05 2.00 3.06 1.10 0.90 2.36 1.43 1.96 4.0 3.0 2.0 1.0 0.0 4.710 4.701 2.08 5.0 5.354 5.342 0.96 ppm (t1)
13 C-NMR SPECTRUM 15 carbon atoms, 4 olefinic 150.309 146.394 117.893 108.251 38.715 37.716 32.293 31.507 39.653 39.429 31.392 29.259 21.092 20.773 15.761 150 100 50 ppm (t1)
DEPT 45 o ppm (f1) 100 DEPT SPECTRA 50 117.704 108.066 39.444 38.514 37.516 32.102 31.308 31.201 29.070 20.912 20.589 15.579
DEPT SPECTRA DEPT 90 o : C 117.704 38.505 37.504 ppm (f1) 100 50
DEPT SPECTRA DEPT 135 o : C 3 & C positive peaks, C 2 negative peaks. Peak at 108 ppm: exocyclic double bond! 117.704 108.065 39.450 38.521 37.527 32.108 31.318 31.205 29.540 29.077 20.920 20.595 15.583 ppm ( t1 ) 100 50
Initial Remarks Molecular Weight: 204 Molecular Formula: C 15 24 Degree of Unsaturation: 4 Primary Carbons: 3 Secondary Carbons: 6 Tertiary Carbons: 3 Quaternary Carbons: 3 Two double bonds, two rings, isopropenyl group? 3 4 4a 5 6 2 1 13 8a 8 12 7 9 10 11
- gcsy SPECTRUM 3 4 4a 5 6 2 1 13 8a 8 12 7 9 10 11 1 Me 2 8 4 Me 8' 7
- gcsy SPECTRUM Position Proton δ 1 (ppm) CSY 1 1 1.64 (m) 2,2, 13 2 3 2 1.49 (m) 1, 2, 3 2 1.39 (m) 1, 2, 3,3 3 1.76 (m) 3, 4 3 1.28 (brd, 12.9) 2,2, 3, 4,4 3 4 4a 5 6 4 1.90 (m) 3, 4 4 4 2.17 (m) 3,3, 4 5 5 5.33 (brd, 6.0) 6,6 6 1.98 (m) 5, 6, 7 6 6 1.84 (m) 5, 6, 7 2 1 13 8a 8 12 7 9 10 11 7 7 2.12 (brd, 13.0) 6, 6, 8 8 8 1.90 (brd, 13.0) 8 8 1.11 (dd, 13.0,13.0) 7, 8 10 10 1.72 (s) 11 11 11 4.69 (d,4.5) 10 13 13 0.78 (d,7.0) 1 Τα πρωτόνια που χαρακτηρίζονται π.χ. Η-8, δηλώνουν πρωτόνια ψευδοαξονικής διαµόρφωσης.
C- gmqc SPECTRUM Correlation of carbons and protons in each group 3 4 4a 5 6 2 1 13 8a 8 12 7 9 10 11
General Conclusions C- gmqc SPECTRUM Carbon δ 13 C (ppm) Proton δ 1 C-1 38.71 1 1.64 (m) C-2 31.40 C-3 29.26 2 2 3 3 1.49 (m) 1.39 (m) 1.76 (m) 1.28 (brd) C-4 32.30 4 2.17 (m) C-5 117.90 5 5.33 (brd) C-6 31.50 6 6 1.98 (m) 1.84 (m) C-7 37.71 7 2.12 (brd) C-8 39.65 8 8 1.90 (brd) 1.11 (dd) C-10 21.09 10 1.72 (s) C-11 108.26 11 4.69 (d) C-12 20.77 12 0.91 (s) C-13 15.76 13 0.78 (d)
- interaction through space NESΥ SPECTRUM 3 4 4a 5 6 2 1 13 8a 8 12 7 9 10 11 Me Me
Conclusions from NE interactions Me 2 1 4 Me 8 The resonance of axial Η-8 appears as a pseudotriplet (doublet of doublets) due to the J coupling with Η-8 και Η-7 ( 2 J 7,8 = 3 J 8,8 = 13z). These J values correspond to a trans-diaxial conformation for Η-8 and Η-7, while it suggests an equatorial conformation for the bulky isopropenyl group that is facing the same direction with Η-8. A strong ΝΟΕ observed between Η-12 και Η-13 confirms the proximity of the two methyl groups. Moreover, the proton Η-8 interacts strongly with the Η-12 a fact indicating that the Me-12 is axial. Finally, axial -7 interacts strongly with -1 that must be also axial, confirming that Me-13 is equatorial. 8' 7 All three groups adopt a syn conformation!
Comparing NMR Spectra values. 13 CNMR C 1 C 2 C3 C 4 C 4a C 5 C 6 C 7 C 8 C 8a C 9 C 10 C 11 C 12 C 13. Itokawa et al. 38.69 31.37 29.23 32.29 146.28 117.85 31.37 37.71 39.67 39.44 150.14 21.04 108.17 20.76 15.75 C. Demetzos et al α 39.10 22.70 21.60 32.20 144.50 117.70 29.70 37.00 39.60 39.80 146.50 21.10 106.20 15.70 14.10 Isolated Eremophilene 38.70 31.40 29.26 32.30 146.39 117.90 31.50 37.71 39.65 39.43 150.29 21.09 108.26 20.77 15.56 1 Η NMR. Itokawa et al. 5 (C) 11 (C 2 ) 11 (C 2 ) 10 (C 3 ) 12 (C 3 ) 13 (C 3 ) 5.37 (m) 4.70 (brs) 1.73 (s) 0.79 (d, 6.0) 0.92 (s) C. Demetzos et al α 5.37 4.64 4.69 1.7 0.85 0.75 Isolated Eremophilene 5.33 (brd, 6.0) 4.69 (d, 4.5) 1.72 (s) 0.78 (d, 7.0) 0.91 (s). [α] D +12.45 ο ± 0.55 (c=2.5, CCl3). [α] D -11.1ο (c=0.18, CCl3), Itokawa,. et al. Chem. Pharm. Bull. 1985, 33, 1148.
$ Konstantinos atzellis Georgia Pagona Apostolos Spyros Manolis Roussakis Ministry of Education, Greece Region of Crete University of Crete Kostas Demetzos Kostas Economakis