Justicia procumbens, have attracted considerable attention due to their pharmacological activities, including antitumor, anti-hepatitic, cytotoxic, anti-microbial, and anti-virus properties. Preparative high-speed counter-current chromatography (HSCCC) was successfully applied to the isolation and purification of four lignans (justicidin B (1), justicidin A (2), 6'-hydroxyjusticidin C (3) and lignan J1 (4)) from J. procumbens using stepwise elution with a pair of two-phase solvent systems composed of n-hexane–ethyl acetate–methanol–water at  and . The preparative HSCCC separation was performed on 300 mg of crude sample yielding compounds 1 (19.7 mg), 2 (9.86 mg), 3 (11.26 mg), and 4 (2.54 mg) in a one-step separation, with purities over 95% as determined by HPLC. The structures of these compounds were identified by MS, 1H-NMR and 13C-NMR. This is the first report on the application of HSCCC to the efficient separation of lignans from J. procumbens.Lignans, which are recognized as main constituents in  Justicia procumbens (Acanthaceae), known as “Juechuang” in Chinese, is one of the most popular traditional Chinese medicines (TCMs) used to treat fever, pain, and cancer [Jian-er syrup, a Chinese herbal medicine compound preparation [d cancer ,2,3. Itsd cancer ,5,6,7. Tparation . Moreoveparation .J. procumbens were identified as arylnaphthalide and diarylbutane lignans, and their glycosides [In the past few years, the bioactive principles isolated from ycosides ,11,12. Tycosides , anti-heycosides , cytotoxycosides , anti-miycosides , and antycosides . This mo1), justicidin A (2), 6'-hydroxyjusticidin C (3) and lignan J1 (4)   from a cJ. procumbens was first analyzed by HPLC. Different elution modes, flow rates, detection wavelengths and column temperatures were screened. The target components were satisfactorily separated with methanol-water  as the solvent system, when the flow rate, column temperature and detection wavelength were set at 0.8 mL/min, 25 °C and 254 nm. The HPLC chromatogram of the crude extract was as shown in The crude ethanol extract from KD values of the target compound  [K1/K2, where K1 > K2) between two components should be greater than 1.5; (iii) relatively short settling time (<20 s) to make sure the retention of stationary phase; (iv) higher retention of the stationary phase provides better peak resolution; (v) sufficient sample solubility; (vi) no sample decomposition [The suitable two-phase solvent system, selected according to the golden rules proposed by Ito, plays a vital role in the HSCCC separation . An idea5 and 2) ; (ii) toposition .KD values of the lignans 1, 2, 3 and 4 in different solvent systems composed of n-hexane-ethyl acetate-methanol-water at various volume ratios by HPLC analysis. KD values, expressed as the peak area of the target lignans in the upper phase divided by that in the lower phase were summarized in Thus, the preliminary studies were carried out to examine the KD values (compounds 1–4) between 0.5 and 2 at the same time. In other words, the four target compounds could not be separated using a single solvent system [KD values [As shown in t system ,27. To oD values . This meD values .KD values of compounds 1 and 2 between 0.5 and 2, were suitable for the separation of 1 and 2. When the two-phase solvent system 1.3:1:1.3:1 with separation factor α = 1.59 was used, separation of compounds 1 and 2 were achieved with satisfactory resolution and their retention time were shorter than that of the 1.4:1:1.4:1 solvent system. Similarly 2.5:1:2.5:1 were selected for the separation of 3 and 4.The two-phase solvent system at volume ratio of 1.3:1:1.3:1 or 1.4:1:1.4:1, with the 1 and 2 were eluted out, which was then followed by the mobile phase of the second solvent system with volume ratio of 2.5:1:2.5:1 until compounds 3 and 4 were eluted.According to previous literatures and results, we decided to combine the selected solvent systems (1.3:1:1.3:1 and 2.5:1:2.5:1) in one single run. The stepwise elution mode, which provided satisfactory resolution of these four target lignans, involved two steps: the crude sample was first eluted with the solvent system at volume ratio of 1.3:1:1.3:1 until compounds Under the optimized stepwise elution mode, the crude sample (300 mg) was dissolved in the 1.3:1:1.3:1 system (10 mL) for the preparative separation by HSCCC with the following condition: rotation speed, 1000 rpm (as recommended by the manufacturer); column temperature, 25 °C; flow rate, 3.0 mL/min; detection, 254 nm. As shown in 1–4 separated by HSCCC were dried under vacuum at 50 °C, redissolved in methanol and detected under the same HPLC conditions for the crude sample. The chromatograms were shown in 1–4 in 1 (19.7 mg), 2 (9.86 mg), 3 (11.26 mg), and 4 (2.54 mg) with the purities all over 95%. Since the contents of compounds 1–4 in the extract were 7.0%, 3.6%, 4.2% and 0.93% respectively as determined by HPLC, their recoveries were 93.8%, 91.3%, 89.4%, and 91.0%, respectively.The target compounds J. procumbens were purchased from Jinhua Pharmaceutical co., LTD. Analytical-grade methanol, ethanol, ethyl acetate, and n-hexane were purchased from Sinopharm Chemical Reagent Co., Ltd . Reverse osmosis Milli-Q water (18 MΩ)  was used for all solutions and dilutions. Methanol (chromatographic grade) used for HPLC analyses was purchased from Merck . Raw material samples of HSCCC was performed using a model TBE-300C high-speed counter-current chromatography instrument . The apparatus, with a maximum rotational speed of 1000 rpm, was equipped with three polytetrafluoroethylene preparative coils  and a 20 mL sample loop. The β values of the multilayer coil varied from 0.5 at internal terminal to 0.8 at the external terminal. Solvents were delivered by a TBP 5002 (Tauto Biotech Co. Ltd) pump. The UV-absorbance of the effluent was measured by a UV 1001 detector  at 254 nm. A DC-0506 constant-temperature circulating implement (Tauto Biotech Co. Ltd) was used to control the separation temperature. And a N2000 data analysis system  was employed for data collection and analysis.1H-NMR and 13C-NMR spectra were measured on an AVANCE 500 MHz NMR spectrometer with TMS as internal standard at 25 °C .The analytical HPLC system was a Waters Alliance 2695 equipped with a Waters model 2998 diode array detector and Waters Empower System . ESI-MS analyses were performed using a Thermo TSQ Quantum Access spectrometer with an ESI interface . J.procumbens were homogenized with a TCM grinder . The powder (1000 g) was extracted with 10 L 95% ethanol twice (each time for 3 h at the room temperature). Then the extract was filtered and concentrated under reduced pressure at 50 °C, yielding a crude sample (30 g) stored at 4 °C for the subsequent HSCCC separation.The dried herbs of KD) of each target compound in different two-phases solvent systems were measured by HPLC as follows: a small amount (1.5 mg) of crude sample was added into a 2.0 mL Eppendorf tube containing 1.5 mL pre-equilibrated two-phase solvent system. After shaking, the mixture was divided into two phases by centrifugation at 3000 rpm for 3 min. Then, an aliquot of each phase (10 μL) was analyzed by HPLC. The KD values were defined as the ratio of the peak area of the compound in the upper phase divided by that in the lower phase  was used for the purification of the compounds 1 and 2. The other solvent system consisting of n-hexane–ethyl acetate–methanol–water  was used for the purification of the compounds 3 and 4. The solvent systems were thoroughly mixed, vented in a separation funnel at room temperature, whose upper phase was used as a stationary phase and the lower phase as a mobile phase. Then the two phases were separated and degassed for 10 min before use. The sample solution was prepared by dissolving 300 mg of the crude sample in 10 mL solvent mixture consisting of equal volumes of both upper and lower phases.In this study, the solvent system consisting of In each separation, the coiled column of TBE-300C (320 mL) was entirely filled with the upper phase as stationary phase at a rate of 30 mL/min. Then, the apparatus was rotated at 1000 rpm, while the lower phase (mobile phase) was pumped into the column in a “head to tail” mode at a flow rate of 10 mL/min. After the mobile phase front was emerged and hydrodynamic equilibrium was established in the column, the flow rate was reduced to 3 mL/min. The sample solution  was then injected into the column through the injection valve. All through the experiment, the separation temperature was controlled at 25 °C. The effluent of the column was continuously monitored with a UV detector at 254 nm. Peak fractions were collected manually according to the elution chromatogram. After the separation was completed, the solvents in the column were pushed out .The peak fractions were evaporated under reduced pressure at 50 °C, and the residues were dissolved in methanol for HPLC analysis. The analyses were performed on a Waters Alliance 2695 liquid chromatography system, equipped with a quaternary solvent delivery system, an autosampler, and a 2998 diode array detector. A YMC-Pack ODS-A column  at 25 °C was used for all the analyses. Methanol-water was used as the mobile phase in gradient elution mode . The flow rate was 0.8 mL/min and the effluent was monitored on-line at 254 nm.1H-NMR and 13C-NMR data.The structural identification of each peak fractions separated by HSCCC was identified according to the analyses of ESI-MS, 1. White power, ESI-MS (m/z): 365.1 (M+H)+, 387.1 (M+Na)+; The molecular formula was C21H16O6. 1H-NMR (CDCl3) δ (ppm): 7.72 , 7.20 , 7.12 , 6.98 , 6.87 , 6.85 , 6.11 , 6.07 , 5.40 , 4.07 , 3.83 ; 13C-NMR (CDCl3) δ (ppm): 170.0 (C-11), 151.8 (C-7), 150.1 (C-6), 147.6 (C-4'), 147.6 (C-3'), 139.7 (C-1), 139.6 (C-3), 133.2 (C-10), 128.9 (C-9), 128.4 (C-1'), 123.5 (C-6'), 118.5 (C-2), 118.3 (C-4), 110.6 (C-2'), 108.3 (C-8), 106.0 (C-5), 105.9 (C-5'), 101.3 (3'-OCH2O-4'), 68.1 (C-12), 56.1 (OCH3-6), 55.9 (OCH3-7). After comparison with the data given in reference [Compound eference , it was 2. White power, ESI-MS (m/z): 395.1 (M+H)+, 417.1 (M+Na)+; The molecular formula was C22H18O7. 1H-NMR (CDCl3) δ (ppm): 7.55 , 7.07 , 6.97 , 6.83 , 6.80 , 6.10 , 6.06 , 5.56 , 4.14 , 4.08 , 3.82 ; 13C-NMR (CDCl3) δ (ppm): 169.6 (C-11), 151.6 (C-6), 150.3 (C-7), 147.8 (C-4'), 147.5 (C-4), 147.4 (C-3'), 134.4 (C-9), 130.6 (C-3), 128.5 (C-1), 126.0 (C-1'), 124.5 (C-6'), 123.6 (C-10), 119.3 (C-2), 110.8 (C-2'), 108.2 (C-5'), 106.2 (C-8), 101.2 (3'-OCH2O-4'), 100.6 (C-5), 66.7 (C-12), 59.7 (OCH3-4), 56.2 (OCH3-6), 55.9 (OCH3-7). After comparison with the data given in reference [Compound 3. Light yellow powder, ESI-MS (m/z): 433.0 (M+Na)+; The molecular formula was C22H18O8. 1H-NMR (CDCl3) δ (ppm): 7.71 , 6.96–7.02 , 6.82 , 6.81 , 6.11 , 6.08 , 5.15 , 4.39 , 4.07 , 3.85 ; 13C-NMR (CDCl3) δ (ppm): 169.3 (C-11), 155.5 (C-6), 152.4 (C-7), 149.8 (C-4'), 148.3 (C-4), 147.5 (C-3'), 139.0 (C-9), 133.5 (C-3), 129.7 (C-1), 126.5 (C-1'), 123.7 (C-6'), 123.0 (C-10), 109.8 (C-2), 109.4 (C-2'), 109.0 (C-5'), 104.2 (C-8), 102.3 (3'-OCH2O-4'), 101.4 (C-5), 68.9 (C-12), 63.6 (OCH3-4), 56.1 (OCH3-6), 55.9 (OCH3-7). After comparison with the data given in reference [Compound eference , it was 4. White powder, ESI-MS (m/z): 379.0 (M+H)+; The molecular formula was C21H14O7. 1H-NMR (CDCl3) δ (ppm): 7.72 , 7.03 , 6.96 , 6.76 , 6.73 , 6.09 , 6.07 , 5.12 , 4.32 ; 13C-NMR (CDCl3) δ (ppm): 169.1 (C-11), 150.8 (C-4), 148.2 (C-3'), 147.6 (C-4'), 139.4 (C-2), 138.4 (C-6), 138.3 (C-7), 135.2 (C-9), 129.6 (C-1), 129.4 (C-1'), 127.5 (C-10), 125.4 (C-6'), 122.9 (C-3), 121.7 (C-5), 109.8 (C-2'), 109.0 (C-5'), 101.8 (6-OCH2O-7), 101.4 (3'-OCH2O-4'), 100.2 (C-8), 68.9 (C-12), 63.6 (OCH3-4). After comparison with the data given in reference [J1.Compound J. procumbens by a stepwise HSCCC method has been implemented for the first time. The stepwise elution mode consisted of a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at different ratios. The purity of the fractions was over 95% in a one-step separation, demonstrating that stepwise HSCCC is an efficient technique to isolate and purify bioactive compounds with large difference of KD values from TCMs. HSCCC could be an important tool for the modernization of TCMs and providing a solution for the sample availability of these pharmacologically important natural products. The efficient preparative separation of four lignans from


H-indol-3-yl)ethylthiourea derivatives were prepared by condensation of 2-(1H-indol-3-yl)ethanamine with appropriate aryl/alkylisothiocyanates in anhydrous media. The structures of the newly synthesized compounds were confirmed by spectroscopic analysis and the molecular structures of 8 and 28 were confirmed by X-ray crystallography. All obtained compounds were tested for antimicrobial activity against Gram-positive cocci, Gram-negative rods and for antifungal activity. Microbiological evaluation was carried out over 20 standard strains and 30 hospital strains. Compound 6 showed significant inhibition against Gram-positive cocci and had inhibitory effect on the S. aureus topoisomerase IV decatenation activity and S. aureus DNA gyrase supercoiling activity. Compounds were tested for cytotoxicity and antiviral activity against a large panel of DNA and RNA viruses, including HIV-1 and other several important human pathogens. Interestingly, derivative 8 showed potent activity against HIV-1 wild type and variants bearing clinically relevant mutations. Newly synthesized tryptamine derivatives showed also a wide spectrum activity, proving to be active against positive- and negative-sense RNA viruses.A series of 2-(1 Indole derivatives have attracted a great deal of attention due to their wide therapeutic applications . TryptamIndole-derived azonine compounds, such as pericine, are CNS-active alkaloids, ligands of opioid receptors. Gliotoxin and its analogues act as antimicrobials and have antiproliferative activity in lymphosarcoma cells. Indometacin is a non-steroidal anti-inflammatory agent that reduces fever, pain or swelling. Current research on panobinostat is focused on its antiproliferative effects in various cancer cell lines .In order to search for potent inhibitors of hepatitis B virus (HBV) and hepatitis C virus (HCV), a series of N-substituted tryptamine analogues were synthesized ,5. What The thiourea moiety is an important synthon responsible for numerous biological activities, such as antimicrobial ,22,23 anH-indol-3-yl)ethanamine was condensed with the corresponding isothiocyanate  rods the MIC value ranged from 400 to 6.25 μg/mL and the average value was 25 μg/mL.Preliminary tests by disc-diffusion method indicated antimicrobial activity against standard Gram-positive cocci. The next step was estimation of MIC values for not only standard but also hospital strains. The study was carried out over 20 standard, 20 hospital of 25 μg/mL . For theStaphylococcus aureus (MSSA) for the derivatives 6 and 14 varied in the range 25–6.25 μg/mL. MRSA strains were also susceptible to these compounds, and the MIC value range changed from 25 to 6.25 μg/mL. The comparison between the chemical structure and the biological activity led to the conclusion that thiourea-derived tryptamines with halogen substituents at the ring are more potent antimicrobials than alkylthioureas. Analysis of a position and a type of an electronegative element at a benzene ring showed that chlorine at the position C-3 promotes antimicrobial activity (derivatives 6 and 14). Substitution of the benzene ring with a second chlorine atom in the 4-position increases antimicrobial activity (compound 6). The introduction of another substituent in the 4-position  causes the disappearance of antimicrobial activity . Other halogens (F and Br) in position 3 do not affect antimicrobial activity. On the other hand, the substitution of the benzene ring with bromine at position 2 implies the moderate antimicrobial activity, as in the previous derivatives, only for Gram-positive strains (compound 15). The methallyl indole derivative 19 expressed a moderate antibacterial activity. These results are in the general agreement with our previous research concerning the activity of heterocyclic thiourea compounds −: 324.4279, found: 324.4298.1-(2-(1H-Indol-3-yl)ethyl)-3-(4-methylphenyl)thiourea (3). This compound was synthesized as described previously −: 363.2921, found: 363.2943.1-(2-(1H-Indol-3-yl)ethyl)-3-(3-bromophenyl)thiourea (7). Yield 79%. m.p. 152–154 °C. 1H-NMR (DMSO-d6) δ (ppm): 3.09 ; 3.97 ; 6.06 ; 6.81 ; 7.00 ; 7.05 ; 7.08 ; 7.13 ; 7.20 ; 7.29 ; 7.36 ; 7.40 ; 7.57–7.60 ; 8.03 . 13C-NMR (DMSO-d6) δ (ppm): 24.3 (CH2), 44.5 (CH2), 111.4 (C), 111.5 (CH), 118.3 (CH), 118.5 (CH), 121.0 (CH), 121.1 (CH), 121.3 (CH), 122.8 (CH), 124.9 (CH), 126.3 (C), 127.2 (C), 130.4 (C), 136.3 (C), 141.2 (CH), 180.1 (C). HRMS (ESI) calc. for C17H16BrN3S [M − H]−: 373.2980, found: 373.2978.1-(2-(1H-Indol-3-yl)ethyl)-3-(4-bromophenyl)thiourea (8). Yield 81%. m.p. 156–158 °C. 1H-NMR (DMSO-d6) δ (ppm): 3.09 ; 3.95 ; 6.24 ; 6.71 ; 6.97 ; 7.11 ; 7.17–7.29 ; 7.39 ; 7.56 ; 7.62 ; 8.01 . 13C-NMR (DMSO-d6) δ (ppm): 24.4 (CH2), 44.5 (CH2), 111.4 (C), 111.5 (CH), 115.8 (CH), 118.2 , 118.5 (CH), 121.0 (CH), 122.8(CH), 124.7 (CH), 127.2 (CH), 131.3, 136.3 (C), 138.7, 180.1 (C). HRMS (ESI) calc. for C17H16BrN3S [M − H]−: 373.2980, found: 373.2978. Crystal data: crystal system monoclinic, space group Cc, unit cell dimensions a = 35.17(2), b = 10.863(4), c = 8.798(3) Å, β = 100.44(5) °, V = 3306(3) Å3; Z = 8, dc = 1.504 g/cm3, μ = 2.613 mm−1, F(000) = 1520. A crystal of dimensions 0.20 × 0.15 × 0.04 mm was used for intensity measurements. Within the θ range 2.98–26.31° , 9510 reflections were collected. The 4871 unique reflections [R(int) = 0.0699] were used for the refinement of 397 parameters. Final R indices on F2 for 2599 observed reflections [I > 2σ(I)] were: R1 = 0.0615, wR2 = 0.0825, goodness-of-fit = 0.941, Δρmax/min = 0.57/−0.44 e Å−3.1-(2-(1H-Indol-3-yl)ethyl)-3-cyclohexylthiourea (9). This compound was synthesized as described previously −: 312.3924, found: 312.3921.1-(2-(1H-Indol-3-yl)ethyl)-3-(3-fluorophenyl)thiourea (12). Yield 88%. m.p. 162–164 °C. 1H-NMR (DMSO-d6) δ (ppm): 3.10 ; 3.98 ; 6.13 ; 6.65 ; 6.87 ; 6.98 ; 7.05–7.23 ; 7.37 ; 7.49 ; 7.58–7.60 ; 8.01 . 13C-NMR (DMSO-d6) δ (ppm): 24.4 (CH2), 44.5 (CH2), 109.2 (C), 110.3 , 111.4 , 111.5 (CH), 118.3 (CH), 118.5 , 121.0 (CH), 122.8 (CH), 127.2 (CH), 130.1 , 136.3 (C), 141.3 , 160.3 , 163.5 (CH), 180.1 (C). HRMS (ESI) calc. for C17H16FN3S [M − H]−: 312.3924, found: 312.3921.1-(2-(1H-Indol-3-yl)ethyl)-3-(2-chlorophenyl)thiourea (13). Yield 79%. m.p. 170–172 °C. 1H-NMR (DMSO-d6) δ (ppm): 2.97 ; 3.75 ; 6.98 ; 7.07 ; 7.16–7.35 ; 7.47 ; 7.63 ; 7.95 ; 9.16 ; 10.83 . 13C-NMR (DMSO-d6) δ (ppm): 24.5 (CH2), 44.7 (CH2), 111.3 (C), 111.5 (CH), 118.2 (CH), 118.5 (CH), 120.9 (CH), 122.7 (CH), 126.9 (CH), 127.1 (CH), 127.2 (CH), 128.9 (C), 129.1 (C), 129.4(C), 136.0(C), 136.2 (CH), 181.1 (C). HRMS (ESI) calc. for C17H16ClN3S [M − H]−: 328.8470, found: 328.8467.1-(2-(1H-Indol-3-yl)ethyl)-3-(3-chlorophenyl)thiourea (14). Yield 85%. m.p. 169–171 °C. 1H-NMR (DMSO-d6) δ (ppm): 2.98 ; 3.77 ; 6.98 ; 7.04–7.14 ; 7.18 ; 7.23–7.36 ; 7.63 ; 7.91 ; 9.65 ; 10.84 . 13C-NMR (DMSO-d6) δ (ppm): 24.4 (CH2), 44.5 (CH2), 111.4 (C), 111.5 (CH), 118.2 , 118.5 (CH), 120.96 (CH), 122.0 (CH), 122.8 (CH), 123.4 (CH), 127.2 (C), 130.1 (C), 132.6 (C), 136.3 (C), 140.9 (CH), 180.1 (C). HRMS (ESI) calc. for C17H16ClN3S [M − H]−: 328.8470, found: 328.8468.1-(2-(1H-Indol-3-yl)ethyl)-3-(2-bromophenyl)thiourea (15). Yield 83%. m.p. 150–152 °C. 1H-NMR (DMSO-d6) δ (ppm): 2.98 ; 3.75 ; 6.98 ; 7.07 ; 7.12–7.17 ; 7.32–7.38 ; 7.57 ; 7.62–7.66 ; 7.91 ; 9.11 ; 10.83 . 13C-NMR (DMSO-d6) δ (ppm): 24.5 (CH2), 44.7 (CH2), 111.3 (C), 111.5 (CH), 118.2 (CH), 118.5 (CH), 120.2 (CH), 120.9 (CH), 122.8 (CH), 127.2 (CH), 127.4 (CH), 127.7 (C), 129.7 (C), 132.5 (C), 136.2 (C), 137.4 (CH), 181.1 (C). HRMS (ESI) calc. for C17H16BrN3S [M − H]−: 373.2980, found: 373.2978.1-(2-(1H-Indol-3-yl)ethyl)-3-phenethylthiourea (16). This compound was synthesized as described previously −: 272.3964, found: 272.3486.1-(2-(1H-Indol-3-yl)ethyl)-3-ethoxycarbonylthiourea (20). This compound was synthesized as described previously −: 420.2985, found: 420.2987.1-(2-(1H-Indol-3-yl)ethyl)-3-ethylthiourea (22). This compound was synthesized as described previously −: 342.8736, found: 342.8769.1-(2-(1H-Indol-3-yl)ethyl)-3-(3-(trifluoromethyl)phenyl)thiourea (24). Yield 92%. m.p. 169–171 °C. 1H-NMR (DMSO-d6) δ (ppm): 2.99 ; 3.79 ; 6.98 ; 7.08 ; 7.18 ; 7.35 ; 7.40 ; 7.50 ; 7.64 ; 7.98 ; 7.98 ; 9.79 ; 10.85 . 13C-NMR (DMSO-d6) δ (ppm): 24.4 (CH2), 44.4 (CH2), 79.2 (C), 111.4 (C), 111.4 (CH), 118.2 (CH), 118.4 (CH), 118.6 (CH), 121.0 , 122.8 , 125.8 , 126.1 (CH), 127.2 (CH), 128.8 (C), 129.5 , 136.3 (C), 140.4, 180.4 (C). HRMS (ESI) calc. for C18H16F3N3S [M − H]−: 362.3999, found: 362.3940.1-(2-(1H-Indol-3-yl)ethyl)-3-methylthiourea (25). This compound was synthesized as described previously −: 342.8736, found: 342.8772.1-(2-(1H-Indol-3-yl)ethyl)-3-(4-butyl-2-methylphenyl)thiourea (27). Yield 77%. m.p. 172–174 °C. 1H-NMR (DMSO-d6) δ (ppm): 0.8 ; 1.25–1.37 ; 1.49–1.59 ; 2.11 ; 2.49 ; 2.93 ; 3.69 ; 6.94–7.11 ; 7.25 ; 7.33 ; 7.63 ; 9.01 ; 10.79 . 13C-NMR (DMSO-d6) δ (ppm): 13.8 (CH3), 17.6 (CH2), 21.8 (CH2), 24.8 (CH3), 33.0 (CH2), 34.3 (CH2), 40.3 (CH2), 111.3 (C), 111.6 (CH), 118.1 (CH), 118.5 (CH), 120.9 , 122.6 (C), 126.2 (C), 127.3 (CH), 127.6 (CH), 130.4 (C), 134.5 (C), 136.2 (C), 140.6, 180.8 (C). HRMS (ESI) calc. for C22H27N3S [M − H]−: 364.5348, found: 364.5360.1-(2-(1H-Indol-3-yl)ethyl)-3-(3-chloro-4-fluorophenyl)thiourea (28). Yield 91%. m.p. 162–164 °C. 1H-NMR (DMSO-d6) δ (ppm): 2.98 ; 3.76 ; 6.98 ; 7.08 ; 7.18 ; 7.24–7.30 ; 7.34 ; 7.62 ; 7.72 ; 7.87 ; 9.60 ; 10.84 . 13C-NMR (DMSO-d6) δ (ppm): 24.4 (CH2), 44.5 (CH2), 111.4 (C), 111.5 (CH), 116.6 , 118.2 (CH), 118.4 , 121.0 (CH), 122.8 (CH), 123.6 (CH), 124.9 , 127.2 (C), 136.3 (C), 136.5 , 152.2 (C), 155.5 , 180.4 (C). HRMS (ESI) calc. for C17H15ClFN3S [M − H]−: 346.8375, found: 346.8378. Crystal data: crystal system triclinic, space group P-1, unit cell dimensions a = 8.592(2) Å, b = 12.4914(3) Å, c = 15.309(3) Å, α = 85.32(2)°, β = 81.17(2)°, γ = 88.87(2)°, V = 1678.1(6) Å3; Z = 4, dc = 1.428 g/cm3, μ = 0.377 mm−1, F(000) = 720. A crystal of dimensions 0.40 × 0.20 × 0.03 mm was used for intensity measurements. Within the θ range 2.04–28.54° , reflections collected 22058. The 5859 unique reflections [R(int) = 0.0360] were used for the refinement of 427 parameters. Final R indices on F2 for 5698 observed reflections [I > 2σ(I)] were: R1 = 0.0483, wR2 = 0.1019, goodness-of-fit 1.018, Δρmax/min = 0.76/−0.80 e Å−3.Staphylococcus aureus ATCC 4163, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 29213, Staphylococcus aureus ATCC 6538,Staphylococcus epidermidis ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778, Enterococcus hirae ATCC 10541, Micrococcus luteus ATCC 9341, Micrococcus luteus ATCC 10240 and Gram-negative rods: Escherichia coli ATCC 10538, Escherichia coli ATCC 25922, Escherichia coli NCTC 8196, Proteus vulgaris NCTC 4635, Pseudomonas aeruginosa ATCC 15442, Pseudomonas aeruginosa NCTC 6749, Pseudomonas aeruginosa ATCC 27853, Bordetella bronchiseptica ATCC 4617. Antifungal activity was tested against yeasts: Candida albicans ATCC 10231, Candida albicans ATCC 90028, Candida parapsilosis ATCC 220191. Microorganisms used in this study were obtained from the collection of the Department of Pharmaceutical Microbiology, Medical University of Warsaw, Poland.The antibacterial activity of compounds was tested against a series of Gram-positive bacteria: 4 CFU/mL (colony forming units per mL), except the final inoculum for E. hirae ATCC 10541, which was 105 CFU/mL. Minimal inhibitory concentrations were read off after 18 h of incubation at 35 °C.Antimicrobial activity was examined by the disc diffusion and Minimal Inhibitory Concentration (MIC) method under standard conditions, using Mueller–Hinton II agar medium  for bacteria and RPMI agar with 2% glucose  for yeasts, according to CLSI (previously NCCLS) guidelines . SolutioS. aureus topoisomerase IV decatenation assay was performed with 200 ng of kinetoplast DNA  as a substrate. S. aureus DNA gyrase supercoiling assay was performed with 0.5 µg of relaxed pBR322 DNA  as a substrate. Enzymes activity was detected by incubation for 30 min at 37 °C in a total reaction volume of 30 µL and in the presence of different concentrations of tested compounds and the reference ciprofloxacin at a concentration of 32 µg/mL. The reactions were terminated by adding an equal volume of STEB buffer , followed by extraction with 1 volume of chloroform/isoamyl alcohol (24:1). Then, 20 µL of the aqueous phase of each sample was loaded onto a 1% agarose gel. Following electrophoresis, gels were stained with ethidium bromide, visualized under UV light in a transilluminator  and analyzed by the Image Lab 6.0 software .Bos taurus]; baby hamster kidney (BHK-21) [ATCC CCL 10 (C-13) Mesocricetus auratus] and monkey kidney (Vero-76) [ATCC CRL 1587 Cercopithecus aethiops]. Human Immunodeficiency Virus type-1 (HIV-1) IIIB laboratory strain was obtained from the supernatant of the persistently infected H9/IIIB cells (NIH 1983). Viruses representative of ssRNA+ were: (i) Flaviviridae: yellow fever virus (YFV) [strain 17-D vaccine (Stamaril Pasteur J07B01)], bovine viral diarrhoea virus (BVDV) [strain NADL (ATCC VR-534)]; (ii) Picornaviridae: enterovirus B , and enterovirus C . Viruses representative of ssRNA- were: (iii) Pneumoviridae: human respiratory syncytial virus (RSV) [strain A2 (ATCC VR-1540)]; (iv) Rhabdoviridae: vesicular stomatitis virus (VSV) [lab strain Indiana (ATCC VR 1540)]. The virus representative of dsRNA was: (v) Reoviridae: reovirus type-1 (Reo-1) . DNA virus representatives were: v) Poxviridae: vaccinia virus (VV) [vaccine strain Elstree-Lister (ATCC VR-1549)]; (vi) Herpesviridae: human herpes 1 (HSV-1) [strain KOS (ATCC VR-1493)]. Mutants carrying NNRTI mutations used: Y181C mutant (NIH-N119) of HIV-1 derives from an AZT-sensitive clinical isolate passaged initially in CEM and then in MT-4 cells, in the presence of nevirapine (up to 10 µM); K103N + Y181C mutant (NIH A17) derives from an IIIB strain passaged in H9 cells in the presence of BI-RG 587 (up to 1 µM); K103R + V179D + P225H mutant (EFVR) derives from an IIIB strain passaged in MT-4 cells in the presence of efavirenz (up to 2 µM). N119, A17 and EFVR stock solutions had titres of 1.2 × 108, 2.1 × 107, and 4.0 × 107 CCID50/mL, respectively. Mutants carrying NRTI mutations used: AZTR strain ; MDR strain .Cell lines were purchased from American Type Culture Collection . Cell lines supporting the multiplication of RNA and DNA viruses were the following: CD4+ human T-cells containing an integrated HTLV-1 genome (MT-4); Madin Darby Bovine Kidney (MDBK)  were seeded at an initial density of 1 × 10R, AZTR, MDR) was based on inhibition of virus-induced cytopathogenicity in exponentially growing MT-4 cell acutely infected with a multiplicity of infection (m.o.i.) of 0.01. Briefly, 50 μL of RPMI containing 1 × 104 MT-4 cells were added to each well of flat-bottom microtitre trays, containing 50 μL of RPMI without or with serial dilutions of extracts or fractions. Then, 20 μL of a HIV-1 suspension containing 100 CCID50 were added. After 4-days incubation at 37 °C, cell viability was determined by the MTT method.Activity against HIV-1 wt and mutant strains  were determined by linear regression analysis. Efavirenz, 2′-C-methylguanosine, 2′-C-methylcytidine, 6-azauridine, mycophenolic acid and acycloguanosine were used as reference inhibitors.Compound’s activity against YFV and Reo-1 was based on inhibition of virus-induced cytopathogenicity in BHK-21 cells acutely infected with a m.o.i. of 0.01. Compound’s activity against BVDV was based on inhibition of virus-induced cytopathogenicity in MDBK cells acutely infected at a m.o.i. of 0.01. After a 3 or 4-days incubation at 37 °C, cell viability was determined by the MTT method, as described earlier . Compoun


Eranthis cilicica was performed as part of our continuous study on the plants of the family Ranunculaceae, which resulted in the isolation of eleven new cycloartane glycosides (1–11) and one new oleanane glycoside (13), together with one known oleanane glycoside (12). The structures of the new compounds were determined by extensive spectroscopic analysis, including two-dimensional (2D) NMR, and enzymatic hydrolysis followed by either X-ray crystallographic or chromatographic analysis. The aglycone (1a) of 2 and its C-23 epimer (8a), and the oleanane glycosides (12 and 13) showed cytotoxic activity against HL-60 leukemia cells with IC50 values ranging from 10.6 μM to 101.6 μM. HL-60 cells were much more sensitive to 8a (IC50 14.8 μM) than 1a (IC50 101.1 μM), indicating that the C-23 configuration is associated with the cytotoxicity of these cycloartane derivatives. Compound 12 was revealed so as to partially induce apoptotic cell death in HL-60 cells, as was evident from morphology of HL-60 cells treated with 12.Phytochemical analysis of the tubers of  Ranunculaceae, such as the Adonis -α-l-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid by direct comparison with an authentic sample isolated from Anemone coronaria +) data and 13C-NMR spectrum with 36 carbon signals ; four tertiary methyl groups at δH 1.42, 1.36, 1.29, and 1.03 (each s); a secondary methyl group at δH 0.97 ; and an anomeric proton δH 4.94 . Enzymatic hydrolysis of 1 using naringinase gave a new triterpene aglycone (1a: C30H46O5) as colorless needles and d-glucose as a carbohydrate moiety. Based on X-ray crystallographic analysis, the structure of 1a was unambiguously established as -16β,23:23,26:24,25-triepoxy-9,19-cycloartane-3β,28-diol (d-glucopyranosyl group to the C-3 hydroxy group of the aglycone in 1 was ascertained by long-range correlations between the anomeric proton (H-1′) at δH 4.94 and the C-3 carbon of the aglycone at δC 88.6 in the 1H-detected heteronuclear multiple-bond connectivity (HMBC) spectrum of 1. Thus, 1 was established as -16β,23:23,26:24,25-triepoxy-28-hydroxy-9,19-cycloartan-3β-yl β-d-glucopyranoside.Compound  signals . The IR ,28-diol . Linkage2 had the molecular formula C42H66O15, as determined by HR-ESI-TOF-MS (m/z 833.4297 [M + Na]+) and 13C-NMR  data. The molecular formula was larger than that of 1 by C6H10O5, which corresponded to one hexosyl unit. The 1H-NMR spectrum of 2 showed signals due to two anomeric protons at δH 5.20  and 4.87 , as well as a cyclopropane methylene group at δH 0.47 and 0.20 , four tertiary methyl groups at δH 1.42, 1.35, 1.27, and 1.02 (each s), a secondary methyl group at δH 0.97 . Enzymatic hydrolysis of 2 with naringinase gave 1a and d-glucose. The 1H- and 13C-NMR signals of the diglycoside moiety, which were assigned based on 1H-1H correlation spectroscopy (COSY) and 1H-detected heteronuclear multiple coherence (HMQC) spectra, indicated the presence of a terminal β-d-glucopyranosyl unit  and a C-4 glycosylated β-d-glucopyranosyl unit  in the molecule of 2 . In the HMBC spectrum of 2, long-range correlations were observed between H-1′′ of Glc (II) and C-3′ of Glc (I), and between H-1′ of Glc (I) and C-3 of the aglycone moiety at δC 88.7. The molecular formula of 2 was assigned as -16β,23:23,26:24,25-triepoxy-28-hydroxy-9,19-cylcoartan-3β-yl O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside.Compound 3 and 4, which had the same molecular formula as 2 (C42H66O15), gave 1a and d-glucose upon enzymatic hydrolysis. The 1H- and 13C-NMR spectra of 3 and 4 suggested that they were constitutional isomers of 2 based on the linkage position of the terminal d-glucosyl unit (Glc (II)) at the inner d-glucosyl unit (Glc (I)) in the diglucosyl residue. In the HMBC spectrum of 3, H-1′′ of Glc (II) at δH 5.28  showed a long-range correlation with C-3′ of Glc (I) at δC 88.7, and H-1′ of Glc (I) at δH 4.86 , in turn, showed a long-range correlation with C-3 of the aglycone at δC 88.6. On the other hand, HMBC correlations were observed between H-1′′ of Glc (II) at δH 5.28  and C-6′ of Glc (I) at δC 70.2, and between H-1′ of Glc (I) at δH 4.86  and C-3 of the aglycone at δC 88.4 in 4. Compounds 3 and 4 were established as O-β-d-glucopyranosyl-(1→3)-β-d-glucopyranoside and O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside of -16β,23:23,26:24,25-triepoxy-28-hydroxy-9,19-cycloartan-3β-yl, respectively.Compounds 5 (C48H76O20) also yielded 1a and d-glucose upon enzymatic hydrolysis. The 1H- and 13C-NMR spectra of 5 showed signals for two terminal β-d-glucopyranosyl units  and a C-4 and C-6 diglycosylated β-d-glucopyranosyl unit  -β-d-glucopyranoside.Compound 7 (CH2)] . In the 6 and 7 had the molecular formula C42H64O15 and C42H66O14, respectively, based on HR-ESI-TOF-MS and 13C-NMR data. The 1H- and 13C-NMR spectral properties of 6 were essentially analogous to those of 2; however, the hydroxymethyl signals at δH 4.02 and 3.94  and δC 63.4 assignable to H2-28 and C-28 in 2 were replaced by those due to an aldehyde group at δH 10.14 (s) and δC 210.2 in 6. Treatment of 6 with NaBH4 in EtOH afforded 2. On the other hand, comparison of the 1H- and 13C-NMR spectra of 7 with those 4 revealed that the H2-28/C-28 hydroxymethyl proton and carbon signals observed for 4 disappeared in the case of 7. Instead, the signals from a tertiary methyl group were detected at δH 0.80 (s) and δC 19.7 in the spectrum of 7. The methyl proton signal showed HMBC correlations with the C-8 (δC 47.5), C-13 (δC 46.3), C-14 (δC 44.7) and C-15 (δC 44.4) carbon signals. Compounds 6 and 7 were determined to be -16β,23:23,26:24,25-triepoxy-28-oxo-9,19-cycloartan-3β-yl O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside and -16β,23:23,26:24,25-triepoxy-9,19-cycloartan-3β-yl O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside.Compounds 8 had the same molecular formula as 2 (C42H66O15), and its 1H- and 13C-NMR spectral features were closely related to those of 2, except for the signals from the ring E and F parts. Enzymatic hydrolysis of 8 gave an aglycone  and d-glucose. The phase-sensitive NOE correlation spectroscopy (PHNOESY) spectrum of 8a showed NOE correlations between H-16 (δH 4.80) and H-22 α (δH 1.56)/H-26a (δH 3.82), H-20 (δH 1.85) and H-24 (δH 3.69), and between H-22β(δH 2.16) and H-24, indicating that 8a was a new aglycone, the C-23 epimer of 1a. Compound 8 was assigned as -16β,23:23,26:24,25-triepoxy-28-hydroxy-9,19-cycloartan-3β-yl O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside.Compound 9 and 10 had the same molecular formula C42H66O15, and their 1H- and 13C-NMR spectra were suggestive of cycloartane diglucosides closely related to 8. Indeed, enzymatic hydrolysis of 9 and 10 gave 8a and d-glucose. Assignments of the 1H- and 13C-NMR signals from the sugar moieties of 9 and 10, which were established by 1H-1H COSY and HMQC spectral analysis, implied that the diglucoside sequences of 9 and 10 corresponded to those of 3 and 4, respectively. Furthermore, HMBC correlations were observed from H-1′′ of Glc (II) at δH 5.31  to C-3′ of Glc (I) at δC 88.9, and from H-1′ at δH 4.89  to C-3 of the aglycone at δC 88.7 in 9, and from H-1′′ of Glc (II) at δH 5.16  to C-6′ of Glc (I) at δC 70.3, and from H-1′ at δH 4.89  to C-3 of the aglycone at δC 88.4 in 10. Compounds 9 and 10 were formulated as O-β-d-glucopyranosyl-(1→3)-β-d-glucopyranoside and O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside of -16β,23:23,26:24,25-triepoxy-28-hydroxy-9,19-cylcoartan-3β-yl, respectively.Compounds 11 (C42H66O14) bore close similarity to 10 in terms of the 1H- and 13C-NMR spectral features. However, the hydroxymethyl signals at δH 3.98 and 3.89  and δC 63.2 assignable to H2-28 and C-28 in 10 were replaced by the signals due to a methyl group at δH 0.79 (s) and δC 19.6 in 11. In addition, the methyl proton signal showed HMBC correlations with the C-8 (δC 47.6), C-13 (δC 46.3), C-14 (δC 44.5), and C-15 (δC 43.8) carbon signals. Compound 11 was established to be -16β,23:23,26:24,25-triepoxy-9,19-cycloartan-3β-yl O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside.Compound 13 had the molecular formula C53H86O22, as revealed by HR-ESI-TOF-MS (m/z 1075.5710 [M + H]+) and 13C-NMR  data. The molecular formula was larger than that of 12 by C6H10O5, which corresponded to one hexosyl unit. The 1H-NMR spectrum of 13 displayed signals for four anomeric protons at δH 6.02 (br s), 5.29 , 5.06  and 4.92 , as well as the signals for six tertiary methyl groups at δH 1.25, 1.05, 1.02, 1.00, 0.94 and 0.92 (each s), and an olefinic proton at δH 5.46 , suggesting that this compound was a tetraglycoside of an oleanoic acid derivative. Acid hydrolysis of 13 with 1M HCl in dioxane-H2O (1:1) gave 23-hydroxyolean-12-en-28-oic acid (hederagenin) as the aglycone -α-l-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid.Compound 2 and 8), and the aglycone (1a) of 2 and its C-23 epimer (8a), as well as oleanane glycosides (12 and 13) were evaluated for their cytotoxic activity against HL-60 cells using a modified 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assay  was much more potent than that of 1a (IC50 101.1 ± 0.44 μM), indicating that the C-23 configuration is associated with the cytotoxicity of these cycloartane derivatives. On the other hand, 2 and 8 were not cytotoxic to HL-60 cells at sample concentrations up to 200 μM. Compounds 12 and 13 were cytotoxic to HL-60 cells, with IC50 values of 10.6 ± 0.40 μM and 10.8 ± 0.53 μM, respectively. After HL-60 cells were exposed to 12 at a sample concentration of 20 μM for 72 h, they were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and observed under a fluorescence microscope. The cells showed nuclear chromatin condensation and nuclear disassembly, illustrated in 12 partially induced apoptotic cell death in HL-60 cells. We previously reported that the slight differences in the structure of cycloartane glycosides effected on cytotoxic activity . In thisT) assay . EtoposiThe instruments, experimental conditions, and plant material used (except for those mentioned below) were the same as those described in previous papers ,19. MeltEranthis cilicica tubers (1.3 kg) was subjected to Diaion HP-20 CC D25 −46.0 . HR-ESI-TOF-MS m/z: 671.3794 [M + Na]+ . IR νmax (film) cm−1: 3388 (OH), 2928 and 2870 (CH). 1H-NMR spectral data for 1 are provided in the 13C-NMR, see Compound 1 Compound 1 (4.5 mg) was treated with naringinase  in a mixture of HOAc/KOAc buffer  and EtOH (2 mL) at room temperature for 72 h. The reaction mixture was purified by silica gel CC eluted with CHCl3-MeOH (22:1) followed by MeOH to give 1a (2.4 mg) and a sugar fraction (0.6 mg). The sugar fraction was analyzed by HPLC under the following conditions: Capcell Pak NH2 UG80 column ; mobile phase of MeCN-H2O (7:3); detection by refractive index and optical rotation, and at a flow rate of 1.0 mL/min. d-glucose was identified by comparing its retention time and optical rotation with those of an authentic sample; tR 11.62 min .Enzymatic Hydrolysis of 1a: Colorless needles from MeOH-MeCN (1:1). mp 281–285 °C. [α]D24 −48.0 . HR-ESI-TOF-MS m/z: 509.3255 [M + Na]+ . IR νmax (film) cm−1: 3476 (OH), 2953, 2925 and 2868 (CH). 1H-NMR spectral data for 1a are provided in the 13C-NMR, see Compound 1a Monoclinic, space group C2, unit cell dimension a = 32.945(2) Å, b = 6.9250(2) Å, c = 13.5690(6), β = 111.270(2)°, V = 2884.8(2) Å3, Z = 4; T = 296 K, dcalc = 1.204 Mg/m3; μ  = 0.084 mm−1; R [I > 2σ(I)] = 0.0370, wR [I > 2σ(I)] = 0.1054, R [for all data] = 0.0409, wR [for all data] = 0.1081. Crystallographic data of 1a have been deposited with the Cambridge Crystallographic Data Centre (CCDC) as supplementary publication no. CCDC 1877743. Copies of the data can be obtained free of charge from the CCDC via http://beta-www.ccdc.cam.ac.uk.X-Ray Crystallography of 2: Amorphous solid. [α]D26 −34.0 . HR-ESI-TOF-MS m/z: 833.4297 [M + Na]+ . IR νmax (film) cm−1: 3397 (OH), 2930 and 2870 (CH). 1H-NMR spectral data for 2 are provided in the 13C-NMR, see Compound 3: Amorphous solid. [α]D24 −48.0 . HR-ESI-TOF-MS m/z: 833.4359 [M + Na]+ . IR νmax (film) cm−1: 3450 (OH), 2932 and 2872 (CH). 1H-NMR spectral data for 3 are provided in the 13C-NMR, see Compound 4: Amorphous solid. [α]D26 −44.0 . HR-ESI-TOF-MS m/z: 833.4340 [M + Na]+ . IR νmax (film) cm−1: 3408 (OH), 2933 and 2871 (CH). 1H-NMR spectral data for 4 are provided in the 13C-NMR, see Compound 5: Amorphous solid. [α]D28 −16.0 . HR-ESI-TOF-MS m/z: 973.5001 [M + H]+ . IR νmax (film) cm−1: 3363 (OH), 2932 and 2872 (CH). 1H-NMR spectral data for 5 are provided in the 13C-NMR, see Compound 2–5 Compounds 2 (54.1 mg), 3 (2.5 mg), 4 (5.1 mg), and 5 (4.8 mg) were independently subjected to enzymatic hydrolysis with naringinase as described for 1 to give 1a  and a sugar fraction . HPLC analysis of the sugar fraction under the same conditions as in the case of that of 1 showed the presence of d-glucose.Enzymatic Hydrolysis of 6: Amorphous solid. [α]D24 −84.0 . HR-ESI-TOF-MS m/z: 831.4100 [M + Na]+ . IR νmax (film) cm−1: 3387 (OH), 2925 and 2870 (CH), 1712 (C=O). 1H-NMR spectral data for 6 are provided in the 13C-NMR, see Compound 2 from 6 Compound 6 (1.7 mg) was dissolved in NaBH4 (2.2 mg) ethanolic solution (1 mL) and it was stirred at room temperature for 5 h. After Me2CO was added to the reaction mixture, the solvent was removed under reduced pressure. The residue was suspended with H2O (3 mL) and extracted with EtOAc (3 mL × 3). The EtOAc extract was chromatographed on silica gel eluted with CHCl3-MeOH-H2O (30:10:1) to yield 2 (1.2 mg).Preparation of 7: Amorphous solid. [α]D25 −70.0 . HR-ESI-TOF-MS m/z: 817.4393 [M + Na]+ . IR νmax (film) cm−1: 3418 (OH), 2931 and 2868 (CH). 1H-NMR spectral data for 7 are provided in the 13C-NMR, see Compound 8: Amorphous solid. [α]D26 −50.0 . HR-ESI-TOF-MS m/z: 833.4359 [M + Na]+ . IR νmax (film) cm−1: 3348 (OH), 2926 and 2867 (CH). 1H-NMR spectral data for 8 are provided in the 13C-NMR, see Compound 8a: Amorphous solid. [α]D24 −58.0 . HR-ESI-TOF-MS m/z: 487.3423 [M + H]+ . IR νmax (film) cm−1: 3442 (OH), 2955, 2927 and 2870 (CH). 1H-NMR spectral data for 8a are provided in the 13C-NMR, see Compound 9: Amorphous solid. [α]D26 −36.0 . HR-ESI-TOF-MS m/z: 833.4276 [M + Na]+ . IR νmax (film) cm−1: 3347 (OH), 2919 and 2871 (CH). 1H-NMR spectral data for 9 are provided in the 13C-NMR, see Compound 10: Amorphous solid. [α]D26 −72.0 . HR-ESI-TOF-MS m/z: 833.4313 [M + Na]+ . IR νmax (film) cm−1: 3388 (OH), 2933 and 2872 (CH). 1H-NMR spectral data for 10 are provided in the 13C-NMR, see Compound 8–10 Compounds 8 (31.3 mg), 9 (1.9 mg), and 10 (5.3 mg) were independently subjected to enzymatic hydrolysis with naringinase as described for 1 to give 8a  and a sugar fraction . HPLC analysis of the sugar fraction under the same conditions as in the case of that of 1 showed the presence of d-glucose.Enzymatic Hydrolysis of 11: Amorphous solid. [α]D26 −62.0 . HR-ESI-TOF-MS m/z: 795.4583 [M + H]+ . IR νmax (film) cm−1: 3387 (OH), 2927 and 2871 (CH). 1H-NMR spectral data for 11 are provided in the 13C-NMR, see Compound 13: Amorphous solid. [α]D27 +6.0 . HR-ESI-TOF-MS m/z: 1075.5710 [M + H]+ . IR νmax (film) cm−1: 3376 (OH), 2926 and 2858 (CH), 1696 (C=O). 1H-NMR (C5D5N) δH 6.02 , 5.46 , 5.29 , 5.06 , 4.92 , 4.48 , 4.46 , 4.36 , 4.34 , 4.24 , 4.18 , 4.10 , 4.07 , 3.86 , 3.26 , 1.54 , 1.25 , 1.05 , 1.02 , 1.00 , 0.94 , 0.92 . 13C-NMR (C5D5N) δC: 39.0, 26.3, 81.3, 43.5, 47.6, 18.2, 32.9, 39.8, 48.2, 36.9, 23.8, 122.6, 144.8, 42.2, 28.3, 23.7, 46.7, 42.0, 46.5, 30.9, 34.2, 32.0, 64.0, 13.9, 16.0, 17.5, 26.1, 180.1, 33.2, 23.8 (aglycone C-1–C-30), 104.7, 76.5, 74.2, 80.0, 65.5 (Ara C-1–C-5), 101.6, 71.2, 83.2, 72.6, 69.9, 18.5 (Rha C-1–C-6), 107.1, 73.3, 75.2, 70.0, 76.8, 62.1 , 106.5, 75.5, 78.3, 71.4, 78.5, 62.5 (Glc C-1–C-6).Compound 13 A solution of 13 (5.0 mg) in 1 M HCl  was heated at 95 °C for 1 h under an Ar atmosphere. After the reaction mixture was diluted with H2O (2 mL), it was extracted with Et2O (5 mL × 2). The Et2O extract was chromatographed on silica gel eluted with CHCl3-MeOH (19:1) to give hederagenin (2.6 mg). The H2O residue was neutralized using an Amberlite IRA-93ZU  column and passed through a Sep-Pak C18 cartridge  eluted with H2O-MeOH (3:2) to give a sugar fraction (1.8 mg). HPLC analysis of the sugar fraction under the same conditions as in the case of that of 1  showed the presence of l-arabinose, d-galactose, d-glucose, and l-rhamnose. tR (min): 7.08 , 8.14 , 12.86 , and 13.17 .Acid Hydrolysis of v/v) FBS supplemented with l-glutamine, penicillin G sodium salt (100 units/mL), and streptomycin sulfate (100 μg/mL). HL-60 (4 × 104 cells/mL) cells were continuously treated with each compound for 72 h, and cell growth was measured using an MTT reduction assay as previously described [50), was calculated from the dose response curve.HL-60 cells were maintained in an RPMI-1640 medium. The cell media contained heat-inactivated 10% (escribed . Data re5 cells/mL) were plated on coverslips in 96-well plates. After 24 h, HL-60 cells were treated with either 20 μM of 12 or 17 μM of etoposide for 72 h. The cells were fixed with 1% glutaraldehyde for 30 min at room temperature before staining with DAPI (0.5 μg/mL in H2O). They were observed under a CKX41 fluoroscence microscope .The cells (1 × 10E. cilicica tubers gave eleven new cycloartane glycosides (1–11) and one new oleanane glycoside (13), together with one known oleanane glycosides (12). The structures of the new compounds were established by extensive spectroscopic analysis, including 2D NMR, and enzymatic hydrolysis, followed by either X-ray crystallographic or chromatographic analysis. The new cycloartane glycosides 2 and 8, the aglycone 1a and its C-23 epimer 8a, and oleanane-type triterpene glycosides 12 and 13 were evaluated for their cytotoxic activity against HL-60 leukemia cells. The cytotoxic activity of 8a was much more potent than that of 1a, indicating that the C-23 configuration is associated with the cytotoxicity of these cycloartane derivatives. This is the first report of structure–activity relationships of cycloartane-type triterpenoids on C-23 epimer. Compounds 12 and 13 were moderately cytotoxic to HL-60 cells, and 12 partially induced apoptotic cell death in HL-60 cells.Further phytochemical examination of


The identification of these impurities should be useful for quality control and the validation of the analytical method in the manufacture of linagliptin.Linagliptin, a xanthine derivative, is a highly potent, selective, long-acting and orally bioavailable DPP-4 inhibitor for the treatment of type 2 diabetes. During the process development of linagliptin, five new process-related impurities were detected by high performance liquid chromatography (HPLC). All these impurities were identified, synthesized, and subsequently characterized by their respective spectral data (MS, HRMS,  The presence of impurities in a drug substance can have a significant impact on the quality and safety of the drug product . Accordi1, R)-8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-1H-purine-2,6-dione [Linagliptin 1, , a highl7) with 2-chloroacetonitrile (8) in the presence of hydrogen chloride afforded 2-(chloromethyl)-4-methylqu inazoline  which condensed with 8-bromo-7-(but-2-yn-1-yl)-3-methyl-1H-purine-2,6-dione (10) in the presence of sodium carbonate as a basic reagent, giving 8-bromo-7-(but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-1H-puri ne-2,6-dione . Subsequently, the condensation of 11 with (R)-2-(piperidin-3-yl)isoindoline-1,3-dione D-(−)-tartaric acid (12) using diisopropylethylamine as a basic reagent provided (R)-7-(but-2-yn-1-yl)-8-pipe ridin-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-1H-purine-2,6-dione . Compound 13 finally converted to the desired linagliptin (1) in 81.9% yield via aminolysis using ethanolamine as the aminolysis agent.In the industrial manufacturing process  of linagHowever, the literature search did not reveal much work on the impurity research of linagliptin. The impurity profile of linagliptin in this synthetic route is different from the earlier reported study ,6, whichHence, a comprehensive study was undertaken to identify and synthesize the impurities in a sample of linagliptin as described in this article. The study will help to understand the formation of the impurities in linagliptin synthesis and provide a clue on how to obtain a pure drug substance.During the process development of linagliptin, five new process-related impurities were detected by high performance liquid chromatography (HPLC) , varying13 using 10 equivalents of ethanolamine [2 is the by-product in this step, formed from incomplete aminolysis. Impurity 2 was further hydrolyzed to afford impurity 3 due to water/tetrahydrofuran (THF) as a solvent. The formed linagliptin moiety (1) reacted with starting material 13 or with 2, providing impurity 4. During the condensation of 11 with 12 to afford 13, the intermediate 11 cannot be converted completely and may remain in the intermediate 13. Unfortunately, the small amount of 11 remaining in the intermediate 13 reacted with ethanolamine in the next aminolysis process, which explains the formation of impurity 5. Similarly, during the condensation of 10 with 9 to afford 11, the intermediate 10 cannot be converted completely and may remain in the intermediate 11. Subsequently, 10 remaining in the intermediate 11 took part in the condensation reaction with 12 and in the aminolysis reaction in the next two steps, which finally leads to the formation of impurity 6  to obtain a homogeneous system and a smaller inventory ratio of ethanolamine was used for more moderate aminolysis conditions (Compound nditions .2 showed a molecular ion peak at m/z 686.23 (M + Na)+ in positive ion mode, indicating the mass of this compound to be 663. The 1H-NMR of this compound revealed three additional D2O exchangeable signals at 8.35, 6.95 and 3.70, indicating the two amide N-H protons and one O-H proton. Additional signals observed in the region of 8.04–7.47 indicated eight aromatic C-H protons. In the IR spectrum, observed bands at 3473 and 3263 cm−1 indicated the amide N-H and O-H stretching. The 13C-NMR revealed 35 carbon atoms and further Distortionless Enhancement by Polarization Transfer (DEPT) exhibited eight secondary carbon atoms. Based on all the spectral data , the structure of impurity 2 was confirmed as (R)-N1-(1-(7-(but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)piperidin-3-yl)-N2-(2-hydroxyethyl)phthalamide.The mass spectrum of 3 was prepared by the alkali hydrolysis of 13 with phase transfer catalyst + in positive ion mode and m/z 619.31 (M − H)− in negative ion mode, indicating the mass of this compound to be 620. The 1H-NMR of this impurity revealed two D2O exchangeable signals at 12.86 and 8.35, indicating one acid O-H proton and one amide N-H proton, and an additional eight proton signals at 7.41–8.25 corresponding to aromatic protons. Interestingly, we found the methyl group (CH3) in quinazoline at δ 2.89 ppm is acidic at a certain degree, which explains the proton exchange of the CH3 group with D2O which takes place sometimes. The proton exchange’s resulting compound  as (R)-2-((1-(7-(but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)piperidin-3-yl)carbamoyl)benzoic acid.The mass spectrum of und 3-d,  also exp2O added . The IR 4 with the low yield of 1.5% directly via 13 reacting with the linagliptin moiety (1). Instead, we have exploited a different concept for the synthesis of compound 4 starting from phthaloyl dichloride (14). However, 12% of the single-benzoyl chloride compound 15 and 11% of the cyclization product 13 would be generated meanwhile due to the steric hindrance from linagliptin, which influenced the purity of 4. We reduced them to below 0.3% and finally got compound 4 with a high purity of 99.15% by washing the reaction mass with CH2Cl2 and aqueous sodium hydroxide solution instead of the column chromatography (We can only obtain compound tography .4 exhibited a molecular ion peak at m/z 1075.24 (M + H)+, 1097.22 (M + Na)+ in positive ion mode, indicating the mass of this compound to be 1074. The molecular formula of this compound was C58H58N16O6 as confirmed by the high resolution mass spectrum (HRMS). In the IR spectrum the observed band at 3243 cm−1 indicated the amide N-H. The 13C-NMR showing 29 carbon atoms indicated the symmetry in the structure of compound 4. Based on all the spectral data , the structure of impurity 4 was confirmed as N,N-bis((R)-1-(7-(but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)piperidin-3-yl)phthalamide.The mass spectrum of 5 was independently provided via the condensation of compound 11 with ethanolamine. In order to prepare 5 in good yield, we replaced the solvent of tetrahydrofuran/water with toluene to obtain a homogeneous system and a more violent reaction temperature. The increasing inventory ratio of ethanolamine also contributed to the high yield of 94% (Compound d of 94% .5 showed a molecular ion peak at m/z 456.51 (M + Na)+ in positive ion mode, indicating the mass of this compound to be 433. The 1H-NMR of this compound revealed two D2O exchangeable signals at 7.23 and 4.78, indicating the N-H and O-H protons, and an additional four proton signals at 7.62–8.25 corresponding to aromatic protons. In the IR spectrum the observed bands at 3446 and 3364 cm−1 indicated the N-H and O-H stretching. The 13C-NMR revealed 22 carbon atoms and DEPT showed four secondary carbon atoms. Based on all the spectral data , the structure of impurity 5 was confirmed as 7-(but-2-yn-1-yl)-8-((2-hydroxyethyl)amino)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-1H-purine-2,6-dione.The mass spectrum of 6 was independently synthesized, starting from compound 10, following a synthetic process analogous to that of linagliptin. Differently, we have exploited a one-pot process for the synthesis of compound 6 instead of the two-step operation in greater yield (Compound er yield .6 showed a molecular ion peak at m/z 317.14 (M + H)+ in positive ion mode, indicating the mass of this compound to be 316, which is 156 amu less than that of linagliptin (1). The 1H-NMR of this compound revealed two D2O exchangeable signals at 10.94 and 8.27, indicating the imide N-H proton and N-H2 proton, and no additional aromatic protons were observed. In the IR spectrum the observed band at 3021 cm−1 indicated the imide N-H stretching and double peaks of 3115 and 3074 cm−1, corresponding to primary amine N-H stretching. The 13C-NMR revealed 15 carbon atoms and DEPT showed five secondary carbon atoms. Impurity 6 was characterized by all respective spectral data  as (R)-8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-1H-purine-2,6-dione.The mass spectrum of 11 , 13  and 1  were prepared according to the literature procedure [The compound rocedure . Other m1H- and 13C-NMR spectra were recorded on a Bruker Arance III 400 MHz spectrometer. The solvents used were DMSO-d6 or CDCl3. The 1H-NMR chemical shift values were reported as δ ppm relative to tetramethylsilane (TMS) and the 13C-NMR chemical shift values were reported on δ ppm relative to DMSO-d6 or CDCl3. DEPT spectra revealed the presence of methyl and methine groups as positive peaks and methylene as negative peaks. The IR spectra were recorded in the solid state as KBr dispersion using a NICOLET 670 FT-IR spectrophotometer. Mass spectra and high resolution mass spectrum were recorded on Agilent 6120B series single quadrupole LC-MS and Q-Tof micro YA019 instrument. Melting points were measured on a WRS-1B apparatus. The specific rotation was calculated from an optical rotation measurement performed on the Autopol IV, serial number 80799  at the wavelength of 589 nm (D line of a sodium lamp), at 20 °C.The 2, 3, 4, 5 and 0.15 μg/mL or 0.03% for impurity 6. Limit of Quantity (LOQ) was 0.25 μg/mL or 0.05% for impurities 2, 3, 4, 5 and 0.33 μg/mL or 0.07% for impurity 6.The HPLC analyses were recorded on a Dionex UItiMate 3000 HPLC instrument using Agilent Eclipse XDB C18 column  in a thermostated column heater at 55 °C. The mobile phases consisting of A  and B (acetonitrile) were used with the gradient mode at the flow rate of 1.5 mL/min. The UV detection at 225 nm was used. Initial gradient starts with 5% of B and at 18 min it was set to 40%. The ratio had been set to 70% at 30 min and at 30.1 min it was 5%, which continued up to 35 min. The samples were diluted in acetonitrile with a concentration of 0.5 mg/mL. The injection volume was 5 µL. Limit of Detection (LOD) was 0.10 μg/mL or 0.02% for impurities This HPLC method further subjected for LC-MS. Samples were run in Electro-Spray Ionization positive mode (ESI+) and 12 L/min nebulizer gas flow rate. The fragmentor voltage was 70 V and capillary voltage was maintained at 3.0 kV. The drying gas temperature was at 350 °C.1H-NMR, 13C-NMR, MS, HRMS) of the compounds 2–6 as well as the HPLC chromatogram of the compounds 1–6 are provided in the The detailed spectral data -3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)piperidin-3-yl)-N2-(2-hydroxyethyl)phthalamide ((R)-N2). To a stirred solution of compound 13  in CH2Cl2 (50.0 mL) was added ethanolamine  and maintained the reaction mass at room temperature for 24 h. Then added 15 mL H2O stirring for 30 min to dilute ethanolamine. The isolated solid was collected by filtration and washed with 10 mL H2O and 20 mL CH2Cl2. Then dried to yield 2 as a light yellow solid , HPLC purity 97.52%; m.p. 189–192 °C; c = 1 g/100 mL, DMSO); IR (KBr) νmax 3473, 3263 ; 3062 (aromatic C-Hν); 2937, 2850 (C-Hν); 2230 (C≡Cν); 1698, 1662 (C=Oν); 1522 ; 1440, 1400 (C-Hδ); 1228 (C-Oν); 763 (aromatic C-Hγ) cm−1; 1H-NMR  δ 8.35 , 8.04 , 7.89 , 7.82–7.76 , 7.64–7.52 , 7.47 , 6.95 , 5.56 , 4.89 , 4.31 , 3.78 , 3.70 , 3.60 , 3.52 , 3.40 , 3.23 , 2.91 , 2.17–2.10 , 1.92 , 1.75 ; 13C-NMR  δ 169.29, 168.75, 168.46, 161.44, 156.37, 153.80, 151.41, 149.51, 148.03, 136.58, 136.51, 134.51, 129.80, 129.67, 128.33, 128.26, 128.02, 127.57, 126.13, 122.94, 103.90, 81.74, 74.21, 60.22, 53.84, 50.47, 46.13, 46.08, 42.55, 35.90, 29.90, 29.59, 23.53, 22.01, 3.55; HRMS (ESI) m/z 664.3019 .(R)-2-((1-(7-(But-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)piperidin-3-yl)carbamoyl)benzoic acid (3). To a stirred solution of compound 13  in CH2Cl2 (50.0 mL) was added aqueous sodium hydroxide solution (1.0 g of NaOH in 50.0 mL of H2O) and tetra-n-butylammonium bromide . Then maintained the reaction mass at rt for 72 h. Then water phase was collected and washed with CH2Cl2 (25.0 mL) for three times. To the stirred water phase was added 1 mol/L aq. HCl (25.0 mL) and maintained at rt for 30 min. The isolated solid was collected by filtration and washed with water (20.0 mL) and CH2Cl2 (20.0 mL). Then dried to yield 3 as a light yellow solid , HPLC purity 97.34%; m.p. 144–148 °C;c = 1 g/100 mL, DMSO); IR (KBr) νmax: 3527 (amide N-Hν); 3255 (acid O-Hν); 3068 (aromatic C-Hν); 2944, 2856 (C-Hν); 2224 (C≡Cν); 1700, 1654 (C=Oν); 1573, 1519 ; 1440, 1400 (C-Hδ); 763 (aromatic C-Hγ) cm−1; 1H-NMR  δ 12.86, 8.35 , 8.25 , 7.96–7.89 , 7.85–7.78 , 7.68 , 7.59 , 7.51 , 7.41 , 5.34 , 4.92 , 4.10–4.01 , 3.82 , 3.63 , 3.40 , 3.14–2.98 , 2.89 , 2.02–1.86 , 1.76 , 1.59 ; 13C-NMR  δ 169.44, 169.28, 168.79, 161.43, 156.46, 153.79, 151.40, 149.50, 148.06, 137.82, 134.51, 133.49, 130.68, 129.64, 129.51, 128.35, 128.32, 127.57, 126.12, 122.93, 103.88, 81.70, 74.26, 53.98, 50.43, 46.06, 46.01, 35.92, 29.91, 29.56, 23.56, 22.01, 3.56; HRMS (ESI) m/z 621.2599 .N,N-bis((R)-1-(7-(But-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)piperidin-3-yl)phthalamide (4). Phthaloyl dichloride  was added slowly to a stirred solution of 1  and triethylamine  in CH2Cl2 (50.0 mL) at rt and refluxed for 24 h. Then the mixture was cooled to rt and 1 mol/L aq. NaOH (30 mL) was added, and maintained for 2 h. The isolated solid was collected by filtration and washed with water (20.0 mL), CH2Cl2 (20.0 mL) and methanol (20.0 mL). Then dried to yield 4 as a light yellow solid , HPLC purity 99.15%; c = 1 g/100 mL, CHCl3); IR (KBr) νmax 3243 (amide N-Hν); 3068 (aromatic C-Hν); 2945, 2858 (C-Hν); 2224 (C≡Cν); 1700, 1662 (C=Oν); 1634, 1563, 1519 ; 1440, 1400 (C-Hδ); 763 (aromatic C-Hγ) cm−1; 1H-NMR  δ 8.37 , 8.22 , 7.92–7.85 , 7.79 , 7.67–7.62 , 7.50 , 5.30 , 4.90 , 4.10–4.00 , 3.81 , 3.65 , 3.36 , 3.15–2.99 , 2.88 , 1.98–1.84 , 1.74 , 1.65–1.55 ; 13C-NMR  δ 168.68, 168.52, 161.01, 155.73, 154.36, 151.71, 149.89, 147.29, 135.04, 133.23, 130.18, 128.80, 128.38, 126.71, 124.83, 123.10, 104.60, 81.53, 73.01, 53.57, 51.11, 46.32, 45.83, 35.63, 29.54, 28.76, 21.74, 21.67, 3.60; HRMS (ESI) m/z 1075.4800 .7-(But-2-yn-1-yl)-8-((2-hydroxyethyl)amino)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-1H-purine-2,6-dione (5). Ethanolamine  was added to a stirred solution of 11  in toluene (32.0 mL) at reflux temperature and maintained for 2 h. Then the mixture was cooled to rt and 20 mL H2O was added, and stirred for 30 min. The isolated solid was collected by filtration and washed with toluene (10.0 mL). Then dried to yield 5 as a white solid , HPLC purity 99.61%; m.p. 238–239 °C; IR (KBr) νmax 3446, 3364 ; 2936, 2867 (C-Hν); 1702, 1652 (C=Oν); 1619, 1581, 1540 ; 1448, 1397 (C-Hδ); 1225 (C-Oν); 764 (aromatic C-Hγ) cm−1; 1H-NMR  δ 8.25 , 7.95–7.88 , 7.82 , 7.72–7.62 , 7.23 , 5.31 , 4.89 , 4.78 , 3.61 , 3.46 , 3.39 , 2.89 , 1.77 ; 13C-NMR  δ 169.19, 161.68, 154.38, 152.99, 151.50, 149.54, 149.52, 134.45, 128.31, 127.51, 126.11, 122.92, 101.31, 81.06, 74.27, 60.20, 45.89, 45.56, 33.13, 29.80, 21.99, 3.57; HRMS (ESI) m/z 434.1937 .(R)-8-(3-Aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-1H-purine-2,6-dione (6). Diisopropylethylamine  was added to a stirred solution of 10  and 12  in NMP (50 mL) and maintained the reaction mass at 100 °C for 13 h. Then ethanolamine  was added at 65 °C and stirred for 4 h at 65 °C. The isolated solid was collected by filtration and washed with NMP (30 mL). Then dried to yield 6 as a off-white solid , HPLC purity 99.36%; m.p. 299–302 °C; c = 1 g/100 mL, DMSO); IR (KBr) νmax 3115, 3074 (primary amine N-Hν); 3020 (imide N-Hν); 2947, 2793 (C-Hν); 2242 (C≡Cν); 1706 (C=Oν); 1660 ; 1610, 1519 ; 1445, 1384 (C-Hδ) cm−1; 1H-NMR  δ 10.94 , 8.27 , 4.92 , 3.65 , 3.48–3.40 , 3.33 , 3.15 , 2.00 , 1.96–1.85 , 1.81 , 1.67 ; 13C-NMR  δ 154.97, 153.99, 150.76, 148.37, 103.93, 81.20, 73.75, 51.43, 50.33, 46.09, 35.16, 28.49, 27.27, 21.85, 3.14; HRMS (ESI) m/z 317.1714 .1H-NMR, 13C-NMR (DEPT) and IR techniques.Five new process-related impurities detected by HPLC varying from 0.15% to 0.5% were identified, synthesized, and subsequently characterized by HPLC, MS, HRMS, 3, 4 and 5 could be reduced to below the identification threshold of 0.1% by salifying linagliptin with hydrochloric acid while impurities 2 and 6 could be controlled within 0.1% by recrystallization from toluene. The detection limit of impurities 2, 3, 4 and 5 was 0.10 μg/mL or 0.02% while the detection limit of impurity 6 was 0.15 μg/mL or 0.03%. Our efforts to synthesize and characterize them effectively prove to be valuable when it comes to complying with the regulatory norms as well as assessing the quality of linagliptin.We have developed appropriate in-process checks and strategies in order to control these impurities within the acceptable level. For example, impurities


The structures of compounds 1–5 were determined by spectroscopic analyses, especially 2D NMR techniques. The structure of compound 5 was further confirmed by X-ray crystallographic analysis. Compounds 1 and 2 have a 3,4-{1'-[(Z)-2''-methoxy-2''-oxoethylidene]}-pentano-3- skeleton. Three novel C19 homolignans, taiwankadsurins D ( Kadsura belongs to the family Schisandraceae and it is only distributed in eastern and southern Asia + at m/z 611.2107 in the HRESIMS. The UV absorption bands at 212, 259 and 292 nm suggested that 1 possessed a biphenyl chromophore. The IR absorption indicated the presence of hydroxyl  and carbonyl  groups. The 13C-NMR spectroscopic data and DEPT analysis revealed that compound 3 contains 30 carbons, including ten quaternary sp2 carbons , two ester carbons (δC 168.7 and 172.4), two quaternary sp3 oxygen-bearing carbons (δC 73.8 and 76.6), two oxygen-bearing methylene carbons (δC 72.4 and 101.5), two sp2 methine carbons (δC 102.5 and 111.0), two sp3 methine carbons (δC 42.4 and 44.1), two oxygen-bearing sp3 methine carbons (δC 83.9 and 86.6), and eight methyl groups . The HMBC correlations of H-11/C-9, C-10, C-12, C-13, C-15; H-9/C-10, C-15; H-4/C-2, C-3, C-5, C-6, C-16; H-6/C-5, C-16; Me-17/C-6, C-7, C-8; Me-18/C-7, C-8, C-9 implied that compound 3 indeed possessed a schizandrin type dibenzocyclooctadiene system + in HRESIMS. The UV spectrum showed absorptions at λmax 255, 220 nm and IR bands at νmax 3,510, 1,735, 1,725 cm−1 suggested that compound 4 contained phenyl, benzoyl, α,β-unsaturated ketone and hydroxyl functionalities. The 1H and 13C-NMR spectroscopic data (4 possessed a substituted cyclohex-2-enone moiety and a spirodihydrobenzofuran ring in a homolignan skeleton similar to kadsuphilol G , where P = (oF2 + 2 cF2)/3). Copies of the deposited crystal data (CCDC 829589) can be obtained, free of charge, from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 (0) 1223 336033 or E-Mail: deposit@ccdc.cam.ac.uk.SHELX-86  and fullKadsura philippinensis has resulted in isolation of five new lignans 1–5, including three novel C19 homolignans, designated taiwankadsurins D, E and F. Their structures have been established by spectroscopic analyses, especially 2D NMR techniques. In addition, the structure of compound 5 was further confirmed by X-ray crystallographic analysis.Phytochemical investigation of the aerial part of Taiwanese


The structures were elucidated by means of extensive NMR and MS analysis. The antifouling activity against larval settlement of the barnacle Balanus albicostatus were evaluated using capsaicin as a positive control. Compounds 1-3 and 5 showed more potent antifouling activity than capsaicin. Compound 5, which exhibited almost the same antifouling activity as starting material, showed better stability than starting material. These compounds all showed antifouling activity in a non-toxic way against larval settlement of the barnacle B. albicostatus. Analysis of structure-activity relationships (SAR) demonstrated that the substituents on the C-15 and C-16 position of pimarane diterpenoid were responsible for the antifouling activity.Five new pimarane diterpenoids  Compound 2 was synthesized like compound 1 but isolated by chromatography (petroleum ether-acetone 13:1) as colorless oil (13% yield). ESIMS m/z: 349.5 [M + 1]+; 1H-NMR (CDCl3): δH 5.18 , 4.16 , 3.89 , 3.62 , 0.82 , 0.76 , 0.72 , 0.66 , 1.98 ; 13C-NMR (CDCl3) δC 39.2 (C-1), 19.0 (C-2), 42.1 (C-3), 33.3 (C-4), 54.9 (C-5), 22.7 (C-6), 36.3 (C-7), 139.9 (C-8), 50.3 (C-9), 38.5 (C-10), 18.8 (C-11), 32.1 (C-12), 37.3 (C-13), 126.9 (C-14), 76.2 (C-15), 66.5 (C-16), 23.4 (C-17), 33.8 (C-18), 22.1 (C-19), 15.1 (C-20), 21.1 (CH3 of COCH3), 171.4 (C=O).15S,-16-Dimethoxy-ent-8(14)-pimarane (3). The starting material  in THF (1.5 mL) was treated with sodium hydride , and the reaction mixture was stirred at room temperature for 3 h. Then, methyl iodide  was added into the mixture with continuous stirring at room temperature for 8 h. After that, the mixture was poured into saturated aqueous NH4Cl solution and then extracted with ethyl acetate three times. The total extract was washed with saturated aqueous NaCl solution, dried with Na2SO4 and evaporated. The residue was chromatographed over silica gel (elution with 25:1 petroleum ether- acetone) yielded compound 3 as a colorless oil (93% yield). ESIMS m/z: 357.4 [M + Na]+; 1H-NMR (CDCl3): δH 5.26 , 3.49 , 3.31 , 3.12 , 0.81 , 0.80 , 0.77 , 0.74 , 3.39 , 3.28 ; 13C-NMR (CDCl3): δC 38.2 (C-1), 18.0 (C-2), 41.2 (C-3), 32.3 (C-4), 53.9 (C-5), 21.8 (C-6), 35.2 (C-7), 135.9 (C-8), 49.4 (C-9), 37.6 (C-10), 17.7 (C-11), 30.9 (C-12), 36.5 (C-13), 128.3 (C-14), 86.1 (C-15), 73.5 (C-16), 22.5 (C-17), 32.7 (C-18), 21.1 (C-19), 14.1 (C-20), 59.5 (OCH3), 58.0 (OCH3).15S-16-di-o-Methanesulfonyl-ent-8(14)-pimarane (4). The starting material  in anhydrous pyridine (0.2 mL) was treated with methanesulfonyl chloride , and the reaction mixture was stirred at 0 °C for 10min. Then, a solution of NaOH  in water (0.2 mL) plus DMSO (0.13 mL) was added into the mixture over 5 min with continuous stirring at 0 °C for 10 min. After this time, the mixture was poured into ice-water and then extracted with aether. The total extract was washed with saturated aqueous NaCl solution, dried with Na2SO4 and evaporated. Column chromatography on silica gel (petroleum ether-acetone 10:1) gave the monosubstitution production (20% yield), and column chromatography on silica gel (petroleum ether-acetone 20:1) gave compound 4 as a white powder (37% yield). ESIMS m/z: 485.3 [M + Na]+; 1H-NMR (CDCl3): δH 5.22 , 4.86 , 4.50 , 4.32 , 1.01 , 0.88 , 0.84 , 0.81 , 3.09 , 3.09 ;13C-NMR (CDCl3): δC 38.2 (C-1), 17.9 (C-2), 41.0 (C-3), 32.3 (C-4), 53.8 (C-5), 21.5 (C-6), 36.9 (C-7), 139.7 (C-8), 49.2 (C-9), 38.2 (C-10), 17.5 (C-11), 30.6 (C-12), 37.6 (C-13), 125.0 (C-14), 85.3 (C-15), 67.0 (C-16), 23.1 (C-17), 32.7 (C-18), 21.1 (C-19), 14.0 (C-20), 36.2 (SO2CH3), 35.0 (SO2CH3).15S,-16-ent-Epoxypimarane (5). The monosubstitution production  in THF (1.5 mL) was treated with sodium ethoxide , and the reaction mixture was stirred at room temperature for 90 min. The mixture was poured into ice-water, then extracted with saturated aqueous NH4Cl solution three times, and extracted with aether three times. The total extract was washed with saturated aqueous NaCl solution, dried with Na2SO4 and evaporated. Column chromatography on silica gel (petroleum ether-acetone 60:1) gave compound 5 as colorless oil (36% yield). ESIMS m/z: 289.8 [M + 1]+; 1H-NMR (CDCl3): δH 5.03 , 2.79 , 2.56 , 2.51 , 0.88 , 0.87 , 0.85 , 0.80 ; 13C-NMR (CDCl3): δC 41.2 (C-1), 18.2 (C-2), 41.5 (C-3), 33.2 (C-4), 53.8 (C-5), 21.5 (C-6), 35.0 (C-7), 139.3 (C-8), 49.7 (C-9), 38.5 (C-10), 18.0 (C-11), 32.7 (C-12), 37.1 (C-13), 123.9 (C-14), 32.3 (C-15), 57.8 (C-16), 24.0 (C-17), 33.4 (C-18), 21.1 (C-19), 13.7 (C-20).B. albicostatus adults were collected from the intertidal zone in Xiamen, China. Adults released the stage I and II nauplius upon immersion in seawater after drying for 12 h [Chaetoceros muelleri at a concentration of 2.0 × 105 cells·mL-1. Each day seawater and diet were renewed. After 5~6 days, larvae reached the cyprid stage [et al. with a minor modification [2Cl2, and aliquots of the solution were added to the glass dishes and air-dried. To each glass dish were added 30 cyprids and 10 mL FSW. There were three replicates for the FSW control and each of the compound concentrations. The dishes were incubated in the dark at 25 °C for 48 h. After that, the numbers of larvae which settled, died or did not settle in each replicate were enumerated under a stereomicroscope. In addition, the stability of diterpenoid was demonstrated by means of their 2-month exposure in FSW. Each diterpene was applied to the dish, and then FSW was poured on the dish. Three replicates were set up for each of the experiment. After 2-month exposure in FSW, these compounds were dissolved in CH2Cl2, which was then evaporated. By comparison of these compounds, no differences were found in weight before and after 2-month exposure in FSW. These indicated that all of compounds were difficult to be dissolved in the FSW, and these compounds might be bound to the surface of the petri dish. Furthermore, the stabilities of these compounds were tested using the method of TLC, which were identified by comparison of the spots and Rf value of these compounds before and after 2-month exposure in FSW. Compound showing the same spots and Rf value as those before were regarded as stable. Compounds that showed the different spots and Rf values from those before were regarded as degraded. One-way ANOVA followed by a Dunnett's test for multiple comparisons of treatment means with a control, was used for the settlement or mortality percentages analysis. The significance level was set as 5%. The EC50 (the concentration that reduces the settlement rate by 50% relative to the control) and LC50  both with a 95% confidence interval were estimated using the Spearman-Karber method [for 12 h . Naupilafication . Tested r method ,25.B. albicostatus. These diterpenoids, which significantly reduced larval settlement at 0.1-1 µg/cm2 (P < 0.001), exhibited barnacle antifouling activities with EC50 values ranging from 0.04 to 7.47 µg/cm2. Compound 5, which exhibited almost the same antifouling activity as the starting material, showed better stability than the latter, indicating that compound 5 has better commercial potential than the starting material. Moreover, the SAR demonstrated that the functional groups on the C-15 and C-16 position of pimarane diterpenoid were responsible for the antifouling activity.Five new pimarane diterpenoids were synthesized and evaluated against settlement of cyprid larvae of the barnacle


The synthesis of novel fluorescent N(9)-alkylated 2-amino-6-triazolylpurine and 7-deazapurine derivatives is described. A new C(2)-regioselectivity in the nucleophilic aromatic substitution reactions of 9-alkylated-2,6-diazidopurines and 7-deazapurines with secondary amines has been disclosed. The obtained intermediates, 9-alkylated-2-amino-6-azido-(7-deaza)purines, were transformed into the title compounds by CuAAC reaction. The designed compounds belong to the push–pull systems and possess promising fluorescence properties with quantum yields in the range from 28% to 60% in acetonitrile solution. Due to electron-withdrawing properties of purine and 7-deazapurine heterocycles, which were additionally extended by triazole moieties, the compounds with electron-donating groups showed intramolecular charge transfer character (ICT/TICT) of the excited states which was proved by solvatochromic dynamics and supported by DFT calculations. In the 7-deazapurine series this led to increased fluorescence quantum yield (74%) in THF solution. The compounds exhibit low cytotoxicity and as such are useful for the cell labelling studies in the future. The imidine) . This pa[A and B  18–19].–19.d]pyrand B . Therefore, it comes as no surprise that also 7-deazapurine derivatives containing heterocyclic substituents at various attachment points –27 have E  and F + calcd for C12H17Cl2N4, 287.0825; found, 287.0826.Azidation: NaN3  was added to a solution of 9-alkyl-2,6-dichloro-9H-purine  in acetone (50 mL) and stirred for 14 h at 50 °C, protected from the daylight. Then, the reaction mixture was evaporated and suspended in water (30 mL). The resulting precipitate was filtered and dried in vacuum.2,6-Diazido-9-heptyl-9H-purine (2a): Colorless solid; reaction time – 14 h; yield 8.4 g, 93%. IR (KBr) ν (cm−1): 2932, 2858, 2170, 2123; 1H NMR  δ 7.87 ), 4.15 , 1.93–1.77 , 1.39–1.15 , 0.84  ppm; 13C NMR  δ 155.9, 154.1, 153.7, 143.7, 121.5, 44.2, 31.6, 29.8, 28.7, 26.6, 22.6, 14.1 ppm; HRMS–ESI (m/z): [M + H]+ calcd for C12H17N10, 301.1632; found, 301.1646.Synthesis of 9-alkyl-6-azido-2-pyrrolidino-9H-purine or 9-alkyl-6-azido-2-piperidino-9H-purine derivatives 6a,b: 9-Alkyl-2,6-diazido-9H-purine 2  was dissolved in DMF (30 mL), pyrrolidine or piperidine  was added and the reaction mixture was stirred isolated from the daylight at 30 °C for 5 h. After that, the mixture was evaporated and silica gel column chromatography (DCM/MeCN 50:1) was used to provide the desired product.6-Azido-9-heptyl-2-pyrrolidino-9H-purine (6a): Slightly brown solid, reaction time – 4 h; yield 0.88 g, 51%. IR (KBr) ν (cm−1): 3062, 2927, 2858, 2148, 2122, 1570, 1254; 1H NMR  δ 7.56 ), 4.03 , 3.62–3.54 , 2.00–1.92 , 1.83 , 1.37–1.16 , 0.85  ppm; 13C NMR  δ 157.4, 154.8, 152.4, 140.3, 117.0, 47.0 (2C)*, 43.4, 31.7, 29.6, 28.8, 26.6, 25.6 (2C)*, 22.7, 14.1 ppm; HRMS–ESI (m/z): [M + H]+ calcd for C16H25N8, 329.2197; found, 329.2195.Synthesis of 9-alkyl-2-pyrrolidino-6-purine derivatives 7a–f and 9-alkyl-2-piperidino-6-purine derivatives 8a–f : Alkyne (1.2 equiv) and 10% AcOH water solution (1 mL) were added to a solution of compound 6a  in THF (7 mL). The flask was isolated from daylight and CuSO4∙5H2O (10 mol %) and sodium ascorbate (20 mol %) were added. The reaction mixture was heated for 20 h at 50 °C. The reaction mixture was cooled to ambient temperature and the precipitated product (bright yellow/green in color) was filtered. The product on the filter was washed with water (5 mL) and MTBE (3 × 5 mL). Then the product was transferred into a flask and dissolved in CHCl3 (7 mL). H2S gas was bubbled through the latter solution until dark brown/black suspension appeared. The resulting mixture was filtered through celite, the filtrate was evaporated under reduced pressure and dried in vacuo. Products can be further purified by silica gel column chromatography, if required.9-Heptyl-6--2-pyrrolidino-9H-purine (7a): Slightly yellow solid, yield 134 mg, 51%. IR (KBr) ν (cm−1): 2952, 2924, 2857, 1622, 1540; 1H NMR  δ 9.36 ), 8.29 ), 8.01 , 7.50 , 7.40 , 4.15 , 3.71–3.55 , 2.08–1.93 , 1.88 , 1.40–1.16 , 0.84  ppm; 13C NMR  δ 156.5, 156.3, 146.4, 143.9, 143.7, 129.6, 128.7, 128.2, 125.5, 120.0, 114.4, 46.6, 42.8, 30.7, 28.4, 27.7, 25.6, 24.7, 21.6, 13.4 ppm; HRMS–ESI (m/z): [M + Na]+ calcd for C24H30N8Na, 453.2478; found, 453.2477.Synthesis of 2-dialkylamino-9-methyl-6--7-deazapurines 10a–f, 11a–f : A mixture of azide 3  and secondary amine (1.2 mmol) in CH3CN (2 mL) was protected from daylight and stirred for 8–24 h at 40 °C. After completion of the SNAr reaction (TLC control), the reaction mixture was cooled to rt and the corresponding alkyne (0.52 mmol), DIPEA , AcOH  and CuI  were added. The reaction mixture was stirred under argon atmosphere at rt for 8–10 h (TLC control). Then the reaction mixture was poured into aqueous 10% ammonia solution (25 mL), stirred for 10 minutes and extracted with CHCl3 (3 × 20 mL). The combined organic layers were washed with water (2 × 30 mL), dried over anhyd Na2SO4 and filtered. After removal of the solvent in vacuum, the crude products were purified by silica gel column chromatography (CHCl3/EtOAc 6:1) to afford compounds 10a–f, 11a–f.ribed in , a typic6--9-methyl-2-pyrrolidino-7-deazapurine (10b): Yellow solid, mp 258 °C dec, yield 99 mg, 66%. 1H NMR  δ 2.01–2.11 , 3.65–3.72 , 3.74 , 3.88 , 6.88 , 7.02 , 7.08 , 7.92 , 8.79 ; 13C NMR  δ 25.6, 30.7, 46.9, 55.3, 99.5, 101.6, 114.3, 116.2, 123.1, 126.7, 127.3, 146.7, 147.0, 156.6, 156.8, 159.8; HRMS–ESI (m/z): [M + H]+ calcd for C20H22N7O, 376.1880; found, 376.1887.The initial molecular geometries were generated by using a molecular mechanics method  and systematic conformational analysis as implemented in Avogadro 1.1.1 software. The minimum energy conformers found by molecular mechanics were further optimized with the Gaussian 09 program package  by the m2 at 37 °C. The cell number and viability of the cells were determined by applying the trypan blue exclusion method and Alamar Blue assay, respectively. Following the separated incubation of MCF-7 and MCF-10A cells (7.0 × 104 cells per well) in a culture medium for 24 h at 37 °C containing 5% CO2, each of the culture media were replaced with 500 μL of cell culture medium containing the compound, and then further cultured for an additional 1 h. Those cells were washed twice with 1× PBS and fixed to the membrane using 4% paraformaldehyde in 5.0 mM sodium phosphate buffer  for 10 min. The fluorescence images of cancer cells were acquired with a fluorescence microscope . Light from an Hg lamp (100 W) passed separately through filter (365 ± 20 nm) before it was used to excite the compound. The emission filters used in this study are long pass filters, with cut-on wavelengths at 420 nm .The Human breast adenocarcinoma cell line MCF-7, and normal mammary epithelial cell line MCF10A were ordered from Food Industry Research and Development Institute (Taiwan). MCF-7 cells were cultured in RPMI medium supplemented with fetal bovine serum (10%), antibiotic–antimycotic (1.0%). MCF-10A cells were cultured in α-MEM supplemented with FBS (10%), and antibiotic–antimycotic (1.0%). All cells were cultured in an environment equilibrated with 5% COFile 11H and 13C NMR spectra.Full experimental procedures, emission spectra, DSC data, and copies of


Calyciphylline Q (3) is the first calyciphylline A derivative possessing a double bond between C-18 and C-19. Their structures and relative configurations were elucidated on the basis of spectroscopic methods, especially 2D NMR techniques. Compounds 1, 2, 8 and 9 exhibited cytotoxic activity against P-388 cells with IC50 values of 5.7, 6.5, 10.3 and 13.8 µM, respectively. Compounds 1 and 2 also showed cytotoxic activity against SGC-7901 cells with IC50 values of 22.4 and 25.6 µM.Five new alkaloids, daphnicyclidins M and N (compounds  Daphniphyllum are mainly distributed in southeast of Asia and are well known for producing highly polycyclic and structurally diverse alkaloids, which have drawn a great deal of attention from the biogenetic and synthetic points of view  and HMBC correlation of H-16 to C-14, and H3-23 to C-22. The relative configuration of 1 was elucidated by NOESY correlations as depicted in a computer-generated three-dimensional drawing, as shown in Four partial structures: 3-21 to H-4 and H-4 to H-6 indicated that H-4 and H-6 were in the β-orientation. The α-orientation of H-18 was deduced from the NOESY correlations of H-18 to H-7a and H-7b to H-4. Thus, it was clear that the ring B took a boat conformation. In consideration of biosynthetic pathway + in the ESIMS. The molecular formula C23H27NO3 of 3 was established by HRESIMS at m/z 388.1890 , corresponding to 11 degrees of unsaturation. The 13C-NMR , one methine  and one double bond carbon  were assigned to those bearing a nitrogen.Calyciphylline Q , b  and c (C-16 to C-17) as shown in 3-20 to C-2, C-18 and C-19. A ketone carbonyl at C-1 was revealed from HMBC cross-peaks of H-2 and H-3a to C-1. The HMBC correlations from H3-21 to C-4, C-5 and C-6, from H-3a, H-7a and H-12a to C-5 indicated the connectivities of C-21 to C-4 and C-6 via C-5. The connectivity of fragment b and c through C-10 and the presence of ring F were suggested by the HMBC correlations from H-11a, H2-16 and H-17a to C-10, from H2-11 and H-17b to C-9, and from H2-16 and H-17a to C-15. The linkages of C-13 to C-1, C-5 and C-9 through C-8 were confirmed by the HMBC correlations of H2-13 to C-5, H3-21 to C-5 and C-8, and H-13a to C-1 and C-9. The ring E was elucidated on the basis of the HMBC correlations of H-13a to C-15, H2-16 to C-15 and C-14 and H-17a to C-15. The methoxycarbonyl group at C-14 was deduced from a comprehensive analysis of the chemical shifts  and HMBC correlation of H-13b to C-22, H2-16 to C-14, and H3-23 to C-22. The relative configuration of 3 was elucidated by NOESY spectrum as shown in 3-21 to H-3b, H-4 and H-6 indicated that H-3b, H-4, H-6 and CH3-21 were all on the same side, and assumed to be in β-orientation just the same as those of daphniyunnine A + in the ESIMS, and the molecular formula was established as C21H27NO3 by HRESIMS at m/z 342.2068 , corresponding to nine degrees of unsaturation. The 13C-NMR  and two methylenes  were ascribed to those bearing an oxidative nitrogen. Comparison of the NMR  spectra of 4 with those of daphlongamine E + in the ESIMS, and the molecular formula was determined as C21H29NO4 by HRESIMS at m/z 360.2172 , with 8 degrees of unsaturation. The 13C-NMR  and two methylenes  were ascribed to those bearing an oxidative nitrogen. Since three out of eight degrees of unsaturation have been accounted for, 5 was inferred to possess five rings. A comparison of the 13C chemical shifts of C-4 (δC = 87.9), C-7 (δC = 67.4) and C-19 (δC = 66.1) in 5 with those of daphniyunnine B (longeracinphyllin B) indicated the presence of an N-oxide group attached to those C-atoms +; positive HRESIMS [M+Na]+m/z 418.1632 .Daphnicyclidin N (2): amorphous light yellow powder;  −65.5 ; UV (MeOH) λmax (log ε) 281 (1.14), 204 (5.00) nm; IR (KBr) νmax 3425, 2920, 1686, 1643, 1616, 1525, 1456, 1385, 1126, 991 cm−1; 1H-NMR and 13C-NMR, see m/z 448 [M+Na]+; positive HRESIMS [M+Na]+m/z 448.1738 .Calyciphylline Q (3): light yellow oil;  −16.8 ; UV (MeOH) λmax (log ε) 280 (4.96) nm; IR (KBr) νmax 2925, 1709, 1685, 1615, 1440, 1378, 1096 cm−1; 1H-NMR and 13C-NMR, see m/z 388 [M+Na]+; positive HRESIMS [M+Na]+m/z 388.1890 .Calyciphylline R (4): light yellow oil;  −44.7 ; UV (MeOH) λmax (log ε) 270.5 (4.23), 204.5 (4.88) nm; IR (KBr) νmax 2920, 1745, 1695, 1575, 1445, 1380 cm−1; 1H-NMR and 13C-NMR, see m/z 342 [M+H]+; positive HRESIMS [M+H]+m/z 342.2068 .Calyciphylline S (5): light yellow oil;  −81.2 ; UV (MeOH) λmax (log ε) 247.5 (4.54) nm; IR (KBr) νmax 3425, 2920, 1701, 1675, 1611, 1438, 1380, 1226, 995, 565 cm−1; 1H-NMR and 13C-NMR, see m/z 360 [M+H]+; positive HRESIMS m/z [M+H]+ 360.2172 .1–9 against mouse lymphocytic leukemia P-388 cells, human lung carcinoma A-549 cells, human promyelocytic leukemia HL-60 cells and human gastric carcinoma SGC-7901 cells was evaluated by 3--2,5-diphenyltetrazolium bromide (MTT) colorimetric assay method in vitro. All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL benzyl penicillin, and 100 U/mL streptomycin at 37 C in a humidified atmosphere with 5% CO2. The logarithmic phase cells were seeded on 96-well plates at the concentration of 4 × 103 cell/mL and incubated with various concentrations  of test compounds in triples wells for 48 h, and cisplatin was used as positive control. After that, 20 μL MTT (5 mg/mL) was added to each well, and incubated for another 4 h. The water-insoluble dark blue formazan crystals formed during MTT cleavage in actively metabolizing cells were dissolved in DMSO. The optical density of each well was measured with a Bio-Rad 680 microplate reader at 570 nm. Cytotoxicity was expressed as the concentration of drug inhibiting cell growth by 50% (IC50).The cytotoxicity of compounds Daphniphyllum macropodum, lead to the isolation of five new Daphniphyllum alkaloids 1–5, along with four known ones 6–9. Their structures and relative configurations were elucidated on the basis of spectroscopic methods, especially 2D NMR techniques. All of the compounds were tested for cytotoxic activity against P-388, A-549, HL-60 and SGC-7901 cell lines. P-388 cells were sensitive to compounds 1, 2, 8 and 9, which exhibited selective cytotoxic activity with IC50 values of 5.7, 6.5, 10.3 and 13.8 µM, respectively. Interestingly, compounds 1 and 2 also showed a moderate cytotoxic activity against SGC-7901 cells with IC50 values of 22.4 and 25.6 µM. These preliminary results suggested that the cytotoxicity of these compounds appeared to be structure dependent, indicating that Daphniphyllum alkaloids of the 1 and 2 structural type possessed the potential for further investigation.Phytochemical investigation of the stem bark of