Chemistry of Natural Compounds, Vol. 48, No. 6, January, 2013 [Russian original No. 6, November–December, 2012]
SEARCH FOR COMPOUNDS WITH ANTIVIRAL ACTIVITY )-CYTISINE DERIVATIVES AMONG SYNTHETIC (
I. P. Tsypysheva,1* A. V. Kovalcskaya,1 A. N. Lobov,1 V. V. Zarubaev,2 L. A. Karpinskaya,2 I. A. Petrenko,1 E. A. Nikolaeva,1 A. A. Shtro,2 and M. S. Yunusov1
UDC 547.94:834.2
The antiviral activity of several amides and thio- and carboxamides of ()-cytisine in addition to bromination and nitration products of its 2-pyridone core was studied. Compounds with a selectivity index close to 10 were found. Keywords: ()-cytisine, H1N1 flu virus. Some plants that exhibit antiviral properties are known to bear alkaloids. Thus, several genera of the family Fabaceae, e.g., Sophora, Acacia, and Caragana, yield extracts that are highly active against hepatitis C (HCV), hepatitis B (HBV), Herpes simplex 1 (HSV-1), and several other viruses [1–5]. The search for new antiviral agents should be conducted among representatives of the most common structural groups of quinolizidine alkaloids, including ()-cytisine, because plants of the family Fabaceae are the principal producers of them [6] and the aforementioned antiviral properties may be associated with them. The pharmacological profiles of ()-cytisine itself and its derivatives are very broad. Neuropharmacological, antiinflammatory, analeptic, and antidiabetic activity was established for them [7]. An example of a study of the antiviral properties of ()-cytisine itself and several of its synthetic derivatives was published [8]. We synthesized two groups of derivatives of ()-cytisine and N-methylcytisine that contained substituents on the secondary N atom and in the 2-pyridone core in order to discover compounds with antiviral activity and to establish the structure–activity relationship. The transformation products of the ()-cytisine secondary amine were amides of m-fluoro-, m-bromo-, and p-chlorobenzoic acids (1–3) that were prepared by the hydroxysuccinimide method as before [9]. The syntheses of thio- and carboxamides 4 and 5, substituted ureas 6 and 7, nitro derivatives 9 and 10, carbamide 12, and dibromo derivative 8 were described by us previously [10, 11]. Dinitro derivative 11 was synthesized by repeated nitration of 9. O
X
O
R2
13 5
N 8
N
R1
11
N
3
14
N H
6N
N
N
H
N
N
1 -6
O
7
N
R1
10
O
Me
O
O
8 - 12
1: R1 = 3-F-Ph, X = O; 2: R1 = 3-Br-Ph, X = O; 3: R1 = 4-Cl-Ph, X = O; 4: R1 = NH2, X = S 5: R1 = NH2, X = O; 6: R1 = NHPh, X = O; 8: R1 = R2 = Br; 9: R1 = NO2, R2 = H 10: R1 = H, R2 = NO2; 11: R1 = R2 = NO2; 12: R1 = NHCONHPh, R2 = H
Table 1 summarizes results from the study of the toxicity and antiviral activity of cytisine derivatives 1–12. According to the results, starting alkaloid ()-cytisine had low toxicity and antiviral activity. Transformation products of the ()-cytisine secondary amine, i.e., amides of m-fluoro-, m-bromo-, and p-chlorobenzoic acids 1–3, differed in toxicity and activity. This indicated that the nature of the corresponding acid and the halogen atom and its position on the aromatic ring affected the antiviral properties. 1) Institute of Organic Chemistry, Ufa Scientific Center, Russian Academy of Sciences, 450054, Ufa, Prosp. Oktyabrya, 71, e-mail:
[email protected]; 2) Research Institute of the Flu, Ministry of the Russian Federation for Health and Social Development, 197376 St. Petersburg, Ul. Prof. Popova, 15/17. Translated from Khimiya Prirodnykh Soedinenii, No. 6, November–December, 2012, pp. 920–923. Original article submitted April 13, 2012. 1042
0009-3130/13/4806-1042
©2013
Springer Science+Business Media New York
TABLE 1. Cytotoxic and Antiviral Properties of (–)-Cytisine Derivatives 1–12 Compound
CTD50, Pg/mL
ED50, Pg/mL
SI
Compound
CTD50, Pg/mL
ED50, Pg/mL
SI
Rimantadine Ribavirin (–)-Cytisine 1 2 3 4 5
60 > 1000 > 500 250 290 > 500 > 500 > 500
12 6.8 109 200 44 200 >500 >500
5 > 147 5 1 7 3 1 1
6 7 8 9 10 11 12
> 300 > 500 > 500 > 500 > 500 300 435
> 300 200 300 > 500 200 170 57
1 3 2 1 3 2 8
Thio- and carboxamides 4–6, including bimolecular product 7 and 8–11 that were substituted in the 2-pyridone part of the molecule, did not exhibit noticeable antiviral activity. The toxicity of these derivatives for MDCK cells was also low (Table 1). Compound 12 had more pronounced antiviral properties with selectivity index (SI) 8. This could be indicative of a relationship between the presence of an aryl-substituted carbamide group in the 3-position of the 2-pyridone core of N-methylcytisine and the manifestation of antiviral properties. In general, none of the studied compounds had an SI value of 10 and greater that is characteristic of potentially active etiotropic antiviral compounds although several of them (2 and 12) had values close to this. The results provided a basis for further targeted optimization of anti-flu drugs based on natural alkaloids, in particular ()-cytisine.
EXPERIMENTAL Melting points of crystalline compounds were determined on a Boetius apparatus (PHMK 05 VEB Wagetechnik Rapido, Radebeul). Optical rotation angles were measured on a Perkin–Elmer 341 LC polarimeter (Na lamp, 589 nm wavelength). The structures of the synthesized compounds were confirmed by PMR and 13C NMR spectroscopy and LC/MS. PMR and 13C NMR spectra were recorded relative to TMS internal standard on Bruker Avance III pulsed spectrometers at operating frequency 500.13 MHz ( 1 H) and 125.47 MHz ( 13C). Two-dimensional 1 H– 1 H COSY, 1H– 13C HSQC, 1H– 13C HMBC, and 1H–1H NOESY spectra were recorded using standard modes of multi-pulse sequences in the instrument software. Resonances of mixtures of E- and Z-conformers of 1–3 were fully assigned based on an analysis of their NMR spectra. High-resolution mass spectra were recoded in a Thermo Finnigan MAT95XP instrument (EI, 70 eV). We used virus strain A/California/07/09(H1N1)pdm09 that was obtained from the Centers for Disease Control and Prevention (CDC, Atlanta, USA). The reference drugs were rimantadine and ribavirin because of their moderate and high effectiveness. The experimental methods were described before [12, 13]. Canine kidney cells MDCK (Madin-Darby Canine Kidney cells) were inoculated into 96-well microplates (Orange Scientific No. 5530100) with 0.2 mL per well and cultivated at 37°C with 5% CO2 until a monolayer formed. A series of two-fold dilutions of each of the compounds from 1000 to 4 Pg/mL in MEM medium (Biolot, St. Petersburg) were prepared in order to determine the cytotoxicity. Cells were incubated in the presence of the dissolved compounds for 48 h at 36°C and 5% CO2, after which the degree of destruction of the cell monolayer was estimated using a micro-tetrazolium test (MTT). For this, cells rinsed of medium were incubated for 1 h with MTT solution (Calbiochem No. 475989, 0.5 Pg/mL) in normal saline. Wells were washed and filled with DMSO (0.1 mL), after which the optical density of the cells was measured on a Victor2 1420 microplate reader (Perkin–Elmer, Finland) at wavelength 535 nm. The concentration of the compound in the well for which 50% destruction of the cell monolayer (CTD50) occurred was calculated based on the results. Flu virus was cultivated in 10–11-day developing chicken embryo (DCE) by injecting into the allantoic cavity 1–10 ID50/0.2 mL of virus-containing fluid. Flu viruses were cultivated for 48 h at 36°C. Compounds dissolved in MEM medium (PAA, Austria) were added to MDCK cell culture and held in plates for 1 h at 36°C and 5% CO2. Cell culture was inoculated with 10-fold dilutions of virus from 101 to 106. Plates with virus and compounds were incubated in a CO2 incubator for 48 h at 36°C and 5% CO2. After the incubation was finished, culture fluid was transferred into the corresponding round-bottom plate wells and an equal amount of 1% suspension of chicken erythrocytes 1043
was added. The hemagglutination reaction was calculated after 40 min at room temperature. The virus titre was taken as the negative decimal logarithm of the maximum virus dilution capable of causing total agglutination of erythrocytes. The 50% inhibiting concentration (EC50) for each compound that halved virus production and the selectivity index (ratio of CTD50 and EC50) were calculated based on the results. )-Cytisine Amides 1–3. A mixture of the appropriate acid (0.95 mmol) and N-hydroxysuccinimide Synthesis of ( (1 mmol) in dioxane was treated with N,Nc-dicyclohexylcarbodiimide (1 mmol), stirred on a magnetic stirrer for 2 h at room temperature, treated with ()-cytisine (1 mmol), stirred at the same temperature until the reaction was finished (TLC monitoring), and filtered. The solid was rinsed with dioxane. The filtrate was concentrated. The residue was chromatographed over SiO2 with elution by benzene:EtOH (97:3) to afford 1–3 in 90–98% yields. Compound 1. Amorphous, [D]D20 78.0° (CHCl3), HR-MS (EI, m/z): 312.1269 [M]+; calcd for C18H17FN2O2, 312.1259. E-Conformer. PMR spectrum (CDCl3, G, ppm, J/Hz): 2.03 (1H, m, Hanti-8), 2.04 (1H, m, Hsyn-8), 2.53 (1H, m, H-9), 2.86 (1H, ddd, 2J = 13.2, 3J11E-9 = 2.2, 4J11E-10E = 1.0, HE-11), 3.12 (1H, m, H-7), 3.42 (1H, dd, 2J = 13.2, 3J13E-7 = 2.4, HE-13), 3.84 (1H, ddd, 2J = 15.7, 3J10E-9 = 6.7, 4J10E-11E= 1.0, HE-10), 3.93 (1H, ddt, 2J = 13.2, 3J13D-7 = 3.2, 4J13D-11D = 1.7, 4J 2 3 4 3 4 13D-8syn = 1.7, HD-13), 4.14 (1H, dt, J = 15.7, J10D-9 = 1.0, J10D-8anti = 1.0, HD-10), 6.11 (1H, dd, J3-4 = 9.1, J3-5 = 1.5, 3 4 2 3 3 H-5), 6.48 (1H, dd, J3-4 = 9.1, J3-5 = 1.5, H-3), 7.31 (1H, dd, J16-17 = 8.4, J16-18 = 1.5, H-16), 7.32 (1H, dd, J4-3 = 9.1, 3J 3 3 4 3 3 4-5 = 7.00, H-4), 7.44 (1H, ddd, J19-18 = 8.4, J19-20 = 7.6, J19-17 = 5.5, H-19), 7.75 (1H, ddt, J18-19 = 8.4, J18-17 = 8.4, 4J 4 3 3 4 18-16 = 2.7, J18-16 = 1.5, H-18), 7.89 (1H, ddd, J20-19 = 7.6, J20-18 = 1.5, J20-16 = 1.2, H-20). 13C NMR spectrum (CDCl , G, ppm, J/Hz): 26.16 (C-8), 27.44 (C-9), 34.98 (C-7), 48.07 (C-11), 48.94 (C-10), 52.81 3 (C-13), 105.41 (C-5), 116.94 (d, 2JC-F = 23.45, C-18), 117.94 (C-3), 121.07 (d, 2JC-F = 21.18, C-16), 125.85 (C-20), 129.13 (C-15), 130.30 (C-19), 138.86 (C-4), 148.25 (C-6), 163.34 (C-14), 163.51 (d, 1JC-F = 233.70, C-17), 163.53 (C-2). Z-Conformer. PMR spectrum (CDCl3, G, ppm, J/Hz): 2.03 (1H, m, Hanti-8), 2.06 (1H, m, Hsyn-8), 2.57 (1H, m, H-9), 2.92 (1H, dd, 2J = 13.2, 3J13E-7 = 2.4, HE-13), 3.12 (1H, m, H-7), 3.40 (1H, ddd, 2J = 13.2, 3J11E-9 = 2.2, 4J11E-10E = 1.0, HE-11), 3.89 (1H, ddd, 2J = 15.7, 3J10E-9 = 6.7, 4J10E-11E = 1.0, HE-10), 3.93 (1H, ddt, 2J = 13.2, 3J13D-7 = 3.2, 4J13D-11D = 1.7, 4J 2 3 4 4 2 13D-8syn = 1.7, HD-13), 4.04 (1H, ddt, J = 13.2, J11D-9 = 3.3, J11D-13D= 1.7, J11D-8syn = 1.7, HD-11), 4.22 (1H, dt, J = 15.7, 3J 4 2 3 4 4 10D-9 = 1.0, J10D-8anti = 1.0, HD-10), 4.67 (1H, ddt, J = 13.2, J13D-7 = 3.2, J13D-11D = 1.7, J13D-8syn = 1.7, HD-13), 6.11 3 4 3 4 3 (1H, dd, J3-4 = 9.1, J3-5 = 1.5, H-5), 6.44 (1H, dd, J3-4 = 9.1, J3-5 = 1.5, H-3), 7.27 (1H, dd, J4-3 = 9.1, 3J4-5 = 7.00, H-4), 7.28 (1H, dd, 2J16-17 = 8.4, 3J16-18 = 1.5, H-16), 7.44 (1H, ddd, 3J19-18 = 8.4, 3J19-20 = 7.6, 4J19-17 = 5.5, H-19), 7.77 (1H, ddt, 3J 3 4 4 3 3 4 18-19 = 8.4, J18-17 = 8.4, J18-16 = 2.7, J18-16 = 1.5, H-18), 7.87 (1H, ddd, J20-19 = 7.6, J20-18 = 1.5, J20-16 = 1.2, H-20). 13C NMR spectrum (CDCl , G, ppm, J/Hz): 26.09 (C-8), 27.58 (C-9), 34.98 (C-7), 48.94 (C-10), 49.08 (C-13), 48.07 3 (C-11), 106.32 (C-5), 116.91 (d, 2JC-F = 23.45, C-18), 117.39 (C-3), 121.01 (d, 2JC-F = 21.18, C-16), 125.81 (C-20), 129.21 (C-15), 130.36 (C-19), 139.38 (C-4), 148.43 (C-6), 163.34 (C-14), 163.51 (d, 1JC-F = 234.0, C-17), 163.55 (C-2). + Compound 2. Amorphous, [D]20 D 136.0° (CHCl3). HR-MS (EI, m/z): 372.0925 [M] ; calcd for C18H17BrN2O2, 372.0919. E-Conformer. PMR spectrum (CDCl3, G, ppm, J/Hz): 2.03 (1H, m, Hanti-8), 2.03 (1H, m, Hsyn-8), 2.53 (1H, m, H-9), 2.85 (1H, ddd, 2J = 13.2, 3J11E-9 = 2.2, 4J11E-10E = 1.0, HE-11), 3.12 (1H, m, H-7), 3.41 (1H, dd, 2J = 13.2, 3J13E-7 = 2.4, HE-13), 3.82 (1H, ddd, 2J = 15.7, 3J10E-9 = 6.7, 4J10E-11E = 1.0, HE-10), 3.93 (1H, ddt, 2J = 13.2, 3J13D-7 = 3.2, 4J13D-11D = 1.7, 4J 2 3 4 2 3 13D-8syn = 1.7, H D-13), 4.13 (1H, dt, J = 15.7, J10D-9 = 1.0, J10D-8anti = 1.0, H D-10), 4.78 (1H, ddt, J = 13.2, J11D-9 = 3.3, 4J 4 3 4 3 4 11D-13D = 1.7, J11D-8syn = 1.7, HD-11), 6.14 (1H, dd, J3-4 = 7.00, J3-5 = 1.5, H-5), 6.50 (1H, dd, J3-4 = 9.1, J3-5 = 1.5, 3 3 3 3 3 H-3), 7.33 (1H, dd, J19-18 = 8.2, J19-20 = 9.2, H-19), 7.33 (1H, dd, J4-3 = 9.1, J4-5 = 7.00, H-4), 7.71 (1H, ddd, J18-19 = 8.2, 4J 4 3 3 4 3 18-16 = 2.1, J18-20 = 1.5, H-18), 7.99 (1H, td, J20-19 = 9.2, J20-18 = 1.5, J20-16 = 1.5, H-20), 8.20 (1H, dd, J16-18 = 2.2, 3J 16-20 = 1.5, H-16). 13C NMR spectrum (CDCl , G, ppm): 26.09 (C-8), 27.42 (C-9), 34.98 (C-7), 48.04 (C-11), 49.01 (C-10), 52.81 3 (C-13), 105.82 (C-5), 117.74 (C-3), 122.56 (C-17), 128.61 (C-20), 129.09 (C-15), 130.19 (C-19), 132.83 (C-16), 136.78 (C-18), 139.11 (C-4), 148.36 (C-6), 163.32 (C-14), 163.46 (C-2). Z-Conformer. PMR spectrum (CDCl3, G, ppm, J/Hz): 2.03 (1H, m, Hanti-8), 2.03 (1H, m, Hsyn-8), 2.57 (1H, m, H-9), 2.91 (1H, dd, 2J = 13.2, 3J13E-7 = 2.4, HE-13), 3.12 (1H, m, H-7), 3.38 (1H, ddd, 2J = 13.2, 3J11E-9 = 2.2, 4J11E-10E = 1.0, HE-11), 3.88 (1H, ddd, 2J = 15.7, 3J10E-9 = 6.7, 4J10E-11E = 1.0, HE-10), 4.05 (1H, ddt, 2J = 13.2, 3J11D-9 = 3.3, 4J11D-13D = 1.7, 4J 2 3 4 2 3 11D-8syn = 1.7, HD-11), 4.21 (1H, dt, J = 15.7, J10D-9 = 1.0, J10D-8anti = 1.0, HD-10), 4.65 (1H, ddt, J = 13.2, J13D-7 = 3.2, 4J 4 3 4 3 4 13D-11D = 1.7, J13D-8syn = 1.7, H D-13), 6.13 (1H, dd, J3-4 = 7.00, J3-5 = 1.5, H-5), 6.45 (1H, dd, J3-4 = 9.1, J3-5 = 1.5, 3 3 3 4 4 H-3), 7.27 (1H, dd, J4-3 = 9.1, J4-5 = 7.00, H-4), 7.71 (1H, ddd, J18-19 = 8.2, J18-16 = 2.1, J18-20 = 1.5, H-18), 8.00 (1H, td, 3J 3 4 3 3 20-19 = 9.2, J20-18 = 1.5, J20-16 = 1.5, H-20), 8.18 (1H, dd, J16-18 = 2.2, J16-20 = 1.5, H-16). 1044
NMR spectrum (CDCl3, G, ppm): 25.97 (C-8), 27.51 (C-9), 34.39 (C-7), 48.89 (C-13), 49.01 (C-10), 51.54 (C-11), 106.60 (C-5), 117.20 (C-3), 122.22 (C-17), 128.58 (C-20), 129.03 (C-15), 129.79 (C-19), 132.81 (C-16), 136.81 (C-18), 139.52 (C-4), 48.58 (C-6), 163.38 (C-14), 163.65 (C-2). 20 72.5° (CHCl ). HR-MS (EI, m/z): 328.0925 [M]+; calcd for C H ClN O , Compound 3. Amorphous, [D]D 3 18 17 2 2 328.0918. E-Conformer. PMR spectrum (CDCl3, G, ppm, J/Hz): 2.02 (1H, m, Hanti-8), 2.05 (1H, m, Hsyn-8), 2.53 (1H, m, H-9), 2.86 (1H, ddd, 2J = 13.3, 3J11E-9 = 2.4, 4J11E-10E = 1.0, HE-11), 3.12 (1H, m, H-7), 3.42 (1H, dd, 2J = 13.3, 3J13E-7 = 2.0, HE-13), 3.84 (1H, ddd, 2J = 15.6, 3J10E-9 = 6.7, 4J10E-11E = 1.0, HE-10), 3.92 (1H, ddt, 2J = 13.3, 3J13D-7 = 3.4, 4J13D-11D = 1.7, 4J 2 3 4 2 3 13D-8syn = 1.7, H D-13), 4.13 (1H, dt, J = 15.6, J10D-9 = 1.0, J10D-8anti = 1.0, H D-10), 4.80 (1H, ddt, J = 13.3, J11D-9 = 3.4, 4J 4 3 4 3 4 11D-8syn = 1.7, J11D-13D = 1.7, HD-11), 6.10 (1H, dd, J5-4 = 6.9, J5-3 = 1.2, H-5), 6.48 (1H, dd, J3-4 = 9.0, J3-5 = 1.5, H-3), 7.27 (1H, dd, 3J4-3 = 9.0, 3J4-5 = 6.9, H-4), 7.44 (2H, d, 3J = 8.5, H-17, 19), 8.03 (2H, d, 3J = 8.5, H-16, 20). 13C NMR spectrum (CDCl , G, ppm): 26.17 (C-8), 27.44 (C-9), 34.99 (C-7), 48.08 (C-11), 48.93 (C-10), 52.81 3 (C-13), 105.40 (C-5), 117.97 (C-3), 125.56 (C-15), 129.00 (C-17), 131.43 (C-16), 138.84 (C-4), 140.53 (C-18), 148.41 (C-6), 163.34 (C-2), 163.78 (C-14). Z-Conformer. PMR spectrum (CDCl3, G, ppm, J/Hz): 2.03 (1H, m, Hanti-8), 2.05 (1H, m, Hsyn-8), 2.57 (1H, m, H-9), 2.91 (1H, dd, 2J = 13.3, 3J13E-7 = 2.0, HE -13), 3.12 (1H, m, H-7), 3.40 (1H, ddd, 2J = 13.3, 3J11E-9 = 2.2, 4J11E-10E = 1.0, HE-11), 3.88 (1H, ddd, 2J = 15.7, 3J10E-9 = 6.7, 4J10E-11E = 1.0, HE-10), 4.03 (1H, ddt, 2J = 13.3, 3J11D-9 = 3.4, 4J11D-8syn = 1.7, 4J 2 3 4 2 3 11D-13D = 1.7, HD-11), 4.22 (1H, dt, J = 15.7, J10D-9 = 1.0, J10D-8anti = 1.0, H D-10), 4.67 (1H, ddt, J = 13.3, J13D-7 = 3.4, 4J 4 3 4 3 4 13D-11D = 1.7, J13D-8syn = 1.7, HD-13), 6.11 (1H, dd, J5-4 = 6.9, J5-3 = 1.2, H-5), 6.11 (1H, dd, J5-4 = 6.9, J5-3 = 1.2, 3 4 3 3 3 H-5), 6.44 (1H, dd, J3-4 = 9.0, J3-5 = 1.5, H-3), 7.32 (1H, dd, J4-3 = 9.0, J4-5 = 6.9, H-4), 7.44 (2H, d, J = 8.5, H-17, 19), 8.00 (2H, d, 3J = 8.5, H-16, 20). 13C NMR spectrum (CDCl , G, ppm): 26.10 (C-8), 27.58 (C-9), 34.43 (C-7), 48.93 (C-10), 49.07 (C-13), 51.59 3 (C-11), 106.34 (C-5), 117.41 (C-3), 125.46 (C-15), 129.00 (C-17), 131.38 (C-16), 139.38 (C-4), 140.47 (C-18), 148.22 (C-6), 163.58 (C-2), 163.85 (C-14). 3,5-Dinitromethylcytisine (11). A solution of 3-nitro derivative 9 (1 g, 4 mmol) in conc. H2SO4 (5 mL) was treated with NaNO3 (1.3 g, 8 mmol), stirred on a magnetic stirrer at room temperature until the reaction was finished (TLC monitoring), poured onto ice, neutralized with crystalline Na2CO3, and extracted with EtOAc (5 u 10 mL). The extracts were combined and dried over Na2SO4. The organic layers were evaporated. The residue was chromatographed over SiO2 with elution by 20 349.2° (CHCl ). HR-MS (EI, m/z): benzene:MeOH (95:5) to afford 11 (0.82 g, 70%), mp 176–177°C (CHCl3), [D]D 3 294.0979 [M]+; calcd for C12H14N2O5, 294.0959. PMR spectrum (CDCl3, G, ppm, J/Hz): 1.86 (1H, dtt, 2J = 13.3, 3J8syn-7 = 3.4, 3 J8syn-9 = 3.4, 4J8syn-11D = 1.7, 4J 2 3 3 4 8syn-13D = 1.7, Hsyn-8), 1.91 (1H, dtd, J = 13.3, J8anti-7 = 3.2, J8anti-9 = 3.2, J8anti-10D = 1.1, Hanti-8), 2.17 (3H, s, H 3-14), 2.30 (1H, ddd, 2J = 11.2, 3J11E-9 = 2.0, 4J11E-10E = 1.1, HE-11), 2.43 (1H, dd, 2J = 11.6, 3J13E-7 = 2.8, HE-13), 2.57 (1H, m, H-9), 2.92 (1H, ddt, 2J = 11.2, 3J11D-9 = 3.3, 4J11D-13D = 1.7, 4J11D-8syn = 1.7, HD-11), 3.27 (1H, ddt, 2J = 11.6, 3J13D-7 = 3.2, 4J 4 2 3 4 13D-11D = 1.7, J13D-8syn = 1.7, HD-13), 4.09 (1H, ddd, J = 16.0, J10E-9 = 6.7, J10E-11E = 1.1, H E-10), 4.15 (1H, m, H-7), 2 3 4 4.19 (1H, dt, J = 16.0, J10D-9 = 1.0, J10D-8anti = 1.0, HD-10), 9.15 (1H, s, H-4). 13C NMR spectrum (CDCl , G, ppm): 24.44 (C-8), 26.95 (C-9), 32.42 (C-7), 45.86 (C-14), 53.30 (C-10), 59.82 3 (C-13), 61.66 (C-11), 127.79 (C-5), 132.95 (C-3), 133.78 (C-4), 153.86 (C-6), 160.84 (C-2). 13C
ACKNOWLEDGMENT The work was supported by the RFBR Grant No. 12-03-00724-a and a grant of the RF President for support of leading scientific schools NSh-7014.2012.3.
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