The structures of these new compounds were determined through spectroscopic and MS analyses. Compounds 1, 4, 9, 12, and 15 exhibited inhibition  values ≤7.65 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1, 2, 4, 8 and 9 inhibited fMLP/CB-induced elastase release with IC50 values ≤8.17 µg/mL. This investigation reveals bioactive isolates  could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.Three new coumarin derivatives, 8-formylalloxanthoxyletin ( Zanthoxylum avicennae (Lam.) DC. (Rutaceae) is an evergreen shrub distributed in Vietnam, Philippines, southern China, and Taiwan + ion at m/z 309.0739 . The presence of carbonyl groups was revealed by the bands at 1693, 1729 cm−1 in the Infrared (IR) spectrum, and was confirmed by the resonances at δ 159.4, 185.7 in the 13C NMR spectrum. The 1H NMR spectrum of 1 showed the presence of a 2,2-dimethyl-2H-pyran moiety , a methoxy group [δ 3.92 ], a formyl group [δ 10.53 ], and the typical H-3 and H-4 protons of the coumarin nucleus . The 1H NMR spectrum of 1 was similar to those of alloxanthoxyletin (4) +  and supported by the 1H, 13C, and DEPT NMR data. The presence of carbonyl groups was revealed by the bands at 1695 and 1734 cm−1 in the IR spectrum, and was confirmed by the resonances at δ 160.0 and 199.8 in the 13C NMR spectrum. The 1H NMR spectrum of 2 displayed the presence of a 2,2-dimethyl-2H-pyran moiety , a methoxy group [δ 3.81 ], an (E)-3-oxobut-1-enyl group , and the typical H-3 and H-4 protons of the coumarin nucleus . The 1H NMR data of 2 were similar to 8-formylalloxanthoxyletin (1), except that the (E)-3-oxobut-1-enyl group at C-8 of 2 replaced 8-formyl group of 8-formylalloxanthoxyletin (1). This was supported by the NOESY correlations between H-1'' (δ 7.83) and H-4'' (δ 2.42) and OMe-7 (δ 3.81) and by the HMBC correlation between H-1'' (δ 7.83) and C-7 (δ 159.2), C-8 (δ 107.5), C-9 (δ 154.1), and C-3'' (δ 199.8) of 2. In addition, the (E)-configuration of 2 was established by the following evidences: (a) The larger coupling constant (J = 16.5 Hz) between H-1'' and H-2'' of 2; (b) The NOESY correlations were observed between H-1'' (δ 7.83) and H-4'' (δ 2.42) (E)-avicennone was elucidated as structure 2. This structure was confirmed by the 1H-1H COSY, NOESY (((δ 2.42) . Thus, -Avicennone (3) was isolated as amorphous powder. The molecular formula C19H18O5 was deduced from a sodium adduct ion at m/z 349.1054 [M + Na]+  in the HR-ESI mass spectrum. The presence of carbonyl groups was revealed by the bands at 1694, 1734 cm−1 in the IR spectrum. The 1H NMR spectrum of 3 illustrated the presence of a 2,2-dimethyl-2H-pyran moiety , a methoxy group [δ 3.72 ], an (Z)-3-oxobut-1-enyl group , and the typical H-3 and H-4 protons of the coumarin nucleus . The 1H and 13C NMR data of 3 were similar to those of (E)-avicennone (2), except that the (Z)-3-oxobut-1-enyl group  at C-8 of 3 replaced the (E)-3-oxobut-1-enyl group at C-8 of (E)-avicennone (2). This was supported by the coupling constant (J = 12.6 Hz) between H-1'' and H-2'' of 3 and by the NOESY correlations between H-1'' (δ 6.79) and H-2'' (δ 6.40). According to the above data, the structure of 3 was elucidated as (Z)-avicennone. This was supported by 1H–1H COSY and NOESY  with corresponding authentic samples or literature values, and this included nine coumarins, alloxanthoxyletin (4) [5) [6) [cis-avicennol methyl ether (7) [8) [9) [10) [11) [12) [c]phenanthridine derivatives, norchelerythrine (13)  and2•−) and hydrogen peroxide) produced by human neutrophils are involved in the pathogenesis of a variety of inflammatory diseases. The effects on neutrophil pro-inflammatory responses of compounds isolated from the stem bark of Z. avicennae were evaluated by suppressing fMet-Leu-Phe/cytochalasin B (fMLP/CB)-induced superoxide anion (O2•−) generation and elastase release by human neutrophils. The inhibitory activity data on neutrophil pro-inflammatory responses are summarized in 2•− generation and elastase release, respectively. From the results of our biological tests, the following conclusions can be drawn: (a) 8-Formylalloxanthoxyletin (1), alloxanthoxyletin (4), xanthoxyletin (9), aesculetin dimethyl ether (12), and γ-fagarine (15) exhibited potent inhibition (IC50 ≤ 7.65 µg/mL) of superoxide anion (O2•−) generation by human neutrophils in response to fMLP/CB; (b) 8-Formylalloxanthoxyletin (1), (E)-avicennone (2), alloxanthoxyletin (4), avicennin (8), and xanthoxyletin (9) exhibited potent inhibition (IC50 ≤ 8.17 µg/mL) against fMLP-induced elastase release; (c) Among the coumarins with a 2,2-dimethyl-2H-pyran moiety at C-6,7, xanthoxyletin (9) (with a 5-methoxy group) exhibited more effective inhibition than its analogue, luvangetin (10) (with a 8-methoxy group) against fMLP-induced O2•− generation and elastase release; (d) Among the coumarins (1–8) with a 7-methoxy group and a 2,2-dimethyl-2H-pyran moiety at C-5,6, 8-formylalloxanthoxyletin (1) (with a 8-formyl group) and alloxanthoxyletin (4) (without any substituted group at C-8) exhibited more effective inhibition than its analogues  against fMLP-induced O2•− generation and elastase release; (e) Alloxanthoxyletin (4) and xanthoxyletin (9) were the most effective among these compounds, with IC50 values of 1.47 ± 0.53 and 1.47 ± 0.41 µg/mL, respectively, against fMLP-induced superoxide anion generation; and (f) 8-Formylalloxanthoxyletin (1) exhibited the most effective among the isolates, with IC50 value of 2.59 ± 0.52 µg/mL, against fMLP-induced elastase release.Granule proteases  and reactive oxygen species (ROS) , hydrogen peroxide) and produced by human neutrophils contribute to the pathogenesis of inflammatory diseases. Inhibition of the inappropriate activation of neutrophils by drugs has been proposed as a way to ameliorate inflammatory diseases. Based on the results of our biological tests (9), alloxanthoxyletin (4), and 8-formylalloxanthoxyletin (1) were the most effective among these compounds, with IC50 values of 1.47 ± 0.41, 1.47 ± 0.53, and 4.60 ± 0.83 µg/mL, respectively, against fMLP-induced superoxide anion generation. 8-Formylalloxanthoxyletin (1), alloxanthoxyletin (4), and xanthoxyletin (9) exhibited the most effective among the isolates, with IC50 values of 2.59 ± 0.52, 3.43 ± 0.63, and 4.18 ± 0.73 µg/mL, respectively, against fMLP-induced elastase release. Our study suggests Z. avicennae and its isolates  could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases. More experiments should be performed to deduce the action modes of these compounds.Granule proteases  and reactive oxygen species (ROS)  by venipuncture, using a protocol (No. 102-1595A3) approved by the Institutional Review Board at Chang Gung Memorial Hospital. All donors gave written consent. The Medical Ethics Committee of Chang Gung Memorial Hospital approved this consent procedure.3. Ultraviolet (UV) spectra were obtained on a Jasco UV-240 spectrophotometer (Japan Spectroscopic Corporation). Infrared (IR) spectra (neat or KBr) were recorded on a Perkin Elmer 2000 FT-IR spectrometer . Nuclear magnetic resonance (NMR) spectra, including correlation spectroscopy (COSY), nuclear Overhauser effect spectrometry (NOESY), heteronuclear multiple-bond correlation (HMBC), and heteronuclear single-quantum coherence (HSQC) experiments, were acquired using Varian INOVA-500 or Varian VNMRS-600 spectrometer  operating at 500 or 600 MHz (1H) and 125 or 150 MHz (13C), respectively, with chemical shifts given in ppm (δ) using tetramethylsilane (TMS) as an internal standard. Electrospray ionisation (ESI) and high-resolution electrospray ionization (HRESI)-mass spectra were recorded on a Bruker APEX II  or a VG Platform Electrospray ESI/MS mass spectrometer . Silica gel  was used for column chromatography (CC). Silica gel 60 F-254  was used for thin-layer chromatography (TLC) and preparative thin-layer chromatography (PTLC).Melting points were determined on a Yanaco micro-melting point apparatus and were uncorrected. Optical rotations were measured using a Jasco DIP-370 polarimeter  in CHClZ. avicennae was collected from Yanpu, Pingtung County, Taiwan, in June 2006 and identified by Jih-Jung Chen. A voucher specimen (ZA-200606) was deposited in the Department of Pharmacy, Tajen University, Pingtung, Taiwan.The stem bark of Z. avicennae was pulverized and extracted three times with MeOH (40 L each) for 3 days. The MeOH extracts were concentrated under reduced pressure at 35 °C, and the residue (550 g) was partitioned between EtOAc and H2O (1:1). The EtOAc layer was concentrated to give a residue . The water layer was further extracted with n-BuOH, and the n-BuOH-soluble part  and the water-solubles  were separated. Fraction A (115 g) was chromatographed on silica gel , eluting with CH2Cl2, gradually increasing the polarity with MeOH to give 14 fractions: A1 , A2 , A3 , A4 , A5 , A6 , A7 , A8 , A9 , A10 , A11 , A12 , A13 , A14 . Fraction A2 (11.8 g) was chromatographed further on silica gel  eluting with n-hexane/EtOAc (3:1–0:1) to give 14 fractions . Part (112 mg) of fraction A2-13 was purified by preparative TLC  to afford 12 (3.8 mg) (Rf = 0.70). Part (96 mg) of fraction A2-14 was purified by preparative TLC  to obtain 15 (3.3 mg) (Rf = 0.60). Fraction A4 (9.1 g) was chromatographed further on silica gel  eluting with n-hexane/EtOAc (3:1–0:1) to give 13 fractions . Fraction A4-1 (273 mg) was purified by CC  to afford 11 subfractions . Fraction A4-1-9 (33 mg) was purified by preparative TLC  to give 11 (3.8 mg) (Rf = 0.50). Part (140 mg) of fraction A4-7 was purified by preparative TLC  to obtain 7 (3.7 mg) (Rf = 0.47). Part (148 mg) of fraction A4-9 was purified by preparative TLC  to yield 1 (3.5 mg) (Rf = 0.24). Fraction A5 (8.8 g) was chromatographed further on silica gel  eluting with n-hexane/EtOAc (10:1–0:1) to give 21 fractions . Part (81 mg) of fraction A5-8 was purified by preparative TLC  to afford 16 (6.0 mg) (Rf = 0.71). Part (72 mg) of fraction A5-11 was purified by preparative TLC  to yield 9 (20.1 mg) (Rf = 0.33). Fraction A5-14 (225 mg) was purified by CC  to afford 5 subfractions . Fraction A5-14-2 (28 mg) was purified by preparative TLC  to give 4 (10.2 mg) (Rf = 0.35). Fraction A5-14-3 (67 mg) was purified by preparative TLC  to obtain 6 (40.1 mg) (Rf = 0.30). Part (118 mg) of fraction A5-17 was purified by preparative TLC  to afford 10 (77 mg) (Rf = 0.40). Fraction A6 (10.5 g) was chromatographed further on silica gel  eluting with n-hexane/acetone (2:1–0:1) to give 11 fractions . Part (125 mg) of fraction A6-1 was purified by preparative TLC  to obtain 2 (3.9 mg) (Rf = 0.87) and 3 (2.8 mg) (Rf = 0.88). Fraction A7 (9.2 g) was chromatographed further on silica gel  eluting with n-hexane/EtOAc (6:1–0:1) to give 17 fractions . Part (136 mg) of fraction A7-7 was purified by preparative TLC  to obtain mixture of 17 and 18 (14.1 mg) (Rf = 0.50). Part (135 mg) of fraction A7-8 was purified by preparative TLC  to yield 8 (10.2 mg) (Rf = 0.46). Part (125 mg) of fraction A7-9 was purified by preparative TLC  to give 5 (3.5 mg) (Rf = 0.79). Part (95 mg) of fraction A7-13 was purified by preparative TLC  to yield 13 (4.3 mg) (Rf = 0.48). Part (118 mg) of fraction A7-15 was purified by preparative TLC  to give 14 (5.3 mg) (Rf = 0.62).The dried stem bark (8.75 kg) of max (log ε) = 224 (4.39), 261 (4.15), 281 (4.11) nm. IR (KBr): υmax = 1729 (C=O), 1693 (C=O) cm−1. 1H NMR : δ = 1.53 , 3.92 , 5.70 , 6.32 , 6.64 , 8.00 , 10.53  : δ = 28.5 (Me-2' × 2), 63.6 (OMe), 79.7 (C-2'), 105.5 (C-10), 110.7 (C-6), 110.7 (C-8), 113.5 (C-3), 115.4 (C-4'), 129.3 (C-3'), 137.8 (C-4), 155.3 (C-5), 157.5 (C-9), 159.4 (C-2), 160.2 (C-7), 185.7 (CHO)  (Amorphous powder. UV (MeOH): λ s, CHO) . 13C NMR.7 (CHO) . ESI-MS:M + Na]+ . HR-ESI-09.0739) .max (log ε) = 227 (4.00), 281 (4.16) nm. IR (neat): υmax = 1734 (C=O), 1695 (C=O) cm−1. 1H NMR : δ = 1.51 , 2.42 , 3.81 , 5.70 , 6.30 , 6.59 , 7.32 , 7.83 , 8.02  : δ = 28.2 (Me-2' × 2), 29.7 (C-4''), 62.4 (OMe), 78.7 (C-2'), 106.3 (C-10), 107.5 (C-8), 110.8 (C-6), 113.2 (C-3), 116.1 (C-4'), 129.5 (C-3'), 130.3 (C-2''), 131.6 (C-1''), 138.2 (C-4), 149.7 (C-5), 154.1 (C-9), 159.2 (C-7), 160.0 (C-2), 199.8 (C-3'')  (Amorphous powder. UV (MeOH): λHz, H-4) . 13C NMR (C-3'') . ESI-MS:M + Na]+ . HR-ESI-49.1052) .max (log ε) = 226 (4.01), 280 (4.14) nm. IR (KBr): υmax = 1734 (C=O), 1694 (C=O) cm−1. 1H NMR : δ = 1.49 , 2.23 , 3.72 , 5.64 , 6.23 , 6.40 , 6.56 , 6.79 , 7.99  : δ = 28.2 (Me-2' × 2), 29.7 (C-4''), 62.4 (OMe), 77.8 (C-2'), 105.9 (C-10), 107.9 (C-8), 110.3 (C-6), 113.0 (C-3), 116.2 (C-4'), 128.6 (C-1''), 129.1 (C-3'), 132.3 (C-2''), 138.2 (C-4), 148.9 (C-5), 152.6 (C-9), 156.9 (C-7), 159.8 (C-2), 200.6 (C-3'')  (Amorphous powder. UV (MeOH): λHz, H-4) . 13C NMR (C-3'') . ESI-MS:M + Na]+ . HR-ESI-49.1052) .The effect of the isolated compounds on neutrophil pro-inflammatory response was evaluated by monitoring the inhibition of superoxide anion generation and elastase release in fMLP/CB-activated human neutrophils in a concentration-dependent manner. The purity of the tested compounds was >98% as identified by NMR and MS.2+)-free HBSS buffer at pH 7.4 and were maintained at 4 °C prior to use.Human neutrophils from venous blood of healthy, adult volunteers (20–28 years old) were isolated using a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes . Purifiec [c and 1 mM Ca2+, neutrophils (6 × 105/mL) were equilibrated at 37 °C for 2 min and incubated with different concentrations (10–0.01 μg/mL) of compounds or DMSO (as control) for 5 min. Cells were incubated with cytochalasin B (1 μg/mL) for 3 min prior to the activation with 100 nM formyl-l-methionyl-l-leucyl-lphenylalanine for 10 min. Changes in absorbance with the reduction of ferricytochrome c at 550 nm were continuously monitored in a double-beam, six-cell positioner spectrophotometer with constant stirring . Calculations were based on differences in the reactions with and without SOD (100 U/mL) divided by the extinction coefficient for the reduction of ferricytochrome c (ε = 21.1/mM/10 mm).The assay for measurement of superoxide anion generation was based on the SOD-inhibitable reduction of ferricytochrome c ,20. In bp-nitroanilide as the elastase substrate. Briefly, after supplementation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM), neutrophils (6 × 105/mL) were equilibrated at 37 °C for 2 min and incubated with compounds for 5 min. Cells were stimulated with fMLP (100 nM)/CB (0.5 μg/mL), and changes in absorbance at 405 nm were monitored continuously in order to assay elastase release. The results were expressed as the percent of elastase release in the fMLP/CB-activated, drug-free control system.Degranulation of azurophilic granules was determined by measuring elastase release as described previously ,20. Expet-test. A probability of 0.05 or less was considered significant. The software SigmaPlot was used for the statistical analysis.Results are expressed as the mean ± standard error of the mean (SEM), and comparisons were made using Student’s 1–3), were isolated from the stem bark of Z. avicennae. The structures of these compounds were established on the basis of spectroscopic data. Reactive oxygen species (ROS) , hydrogen peroxide) and granule proteases  produced by human neutrophils contribute to the pathogenesis of inflammatory diseases. The effects on neutrophil pro-inflammatory responses of isolates were evaluated by suppressing fMLP/CB-induced O2•− generation and elastase release by human neutrophils. The results of anti-inflammatory experiments indicate that compounds 1, 2, 4, 9, 11, and 15 can significantly inhibit fMLP-induced O2•− generation and/or elastase release. 8-Formylalloxanthoxyletin (1), alloxanthoxyletin (4), and xanthoxyletin (9) were the most effective among the isolated compounds, with IC50 values of 4.60 ± 0.83/2.59 ± 0.52, 1.47 ± 0.53/3.43 ± 0.63, and 1.47 ± 0.41/4.18 ± 0.73 µg/mL, respectively, against fMLP-induced O2•− generation and elastase release. Our study suggests Z. avicennae and its isolates  are worthy of further biomedical investigation and could be expectantly developed as potential candidates for the treatment or prevention of various inflammatory diseases.Eighteen compounds, including three new coumarins (


AbstractAcari: Hydrachnidia) from streams in South Korea are presented. Two species are described as new to science: Torrenticolaneodentiferasp. n. (Torrenticolidae) and Atractidesermilovisp. n. (Hygrobatidae). Five species are reported as first records for Korea: Wandesia (Wandesia) reducta Tuzovskij, 1987, Wandesia (Wandesia) cf.rara Tuzovskij, 1990, Sperchon (Sperchon) orientalis Tuzovskij, 1990, Feltria (Feltria) kuluensis Tuzovskij, 1988 and Atractides (Atractides) constrictus . The latter species is redescribed and elevated to species rank based on new material from the Russian Far East.New records of water mites ( Recently, the senior author  publishePageBreakThe water mites of this study mainly were collected by the senior author during his trip in South Korea in May, 2013. The paper aims to describe this material in order to prepare the way towards the production of an identification key of this important group of freshwater invertebrates.National Institute of Biological Resources, Korea (NIBR); material from the Russian Far East is deposited in the research collections of the Institute of Biology and Soil Science, Vladivostok, Russia (IBSS).Water mite were collected by hand netting, sorted on the spot from the living material, fixed in Koenike-fluid and dissected as described elsewhere e.g., . HolotypAtractides and a detailed methodological introduction, see In the section ‘Material examined’ collecting site abbreviations derive from the geographical database Pešić. The composition of the material is given as: males/females/deutonymphs. All measurements are given in μm. For a detailed description and discussion of the characteristics of the genus  = first acetabulumAc-1,  = above sea levelasl,  = first coxaeCx-I,  = coxoglandularia of fourth coxaeCxgl-4,  = dorsoglandulariaDgl-1-4,  = dorsal lengthdL,  = heightH,  = lengthL,  = lateroglandulariaLgl-1-4,  = Leg 1, sixth segment (tarsus)I-L-6,  = medial lengthmL,  = number of specimens examinedn,  = National ParkNP,  = palp, first segmentP-1,  = preocularePreoc.;  = pregenital scleritepregen,  = postocularePostoc.;  = proximal large ventral seta at I-L-5S-1,  = distal large ventral seta at I-L-5S-2,  = ventroglandularia 1Vgl-1,  = ventral lengthvL,  = widthW.The following abbreviations are used: Taxon classificationAnimaliaTrombidiformesHydryphantidaeTuzovskij, 1987Wandesiareducta37°17.081'N, 128°15.389'E, 25.v.2013 Pešić & Karanović 0/1/0 (mounted).SOUTH KOREA: CR22 Gangwon Province, Chiaksan NP, Silim town, stream shaded, substrate: stones, gravels; Wandesiareducta Tuzovskij, 1987. This species was described by The single female from this study matches the general morphology of Far East of Russia . New forTaxon classificationAnimaliaTrombidiformesHydryphantidaeTuzovskij, 1990Wandesiarara36°26.499'N, 129°08.114'E, 23.v.2013 Pešić & Karanović 0/1/0 (mounted).SOUTH KOREA: CR16 Gyeongsangbuk Province, Juwangsan NP, Woroe-ri, Cheong song-eup, Dalgikpo, waterfall, Idiosoma L 1800, coxae and genital field: Fig. Gnathosoma vL 189, chelicera Fig.  total L Legs: setation as given in Fig. Wandesiarara Tuzovskij, 1990 a species described from the Primory Territory in the Russian Far East on the basis of a single deutonymph .SOUTH KOREA: CR20 Chungcheongbuk Province, Mt. Vorak, Deokjusanseong, stream, Sperchonorientalis Tuzovskij, 1990, a species described from the Primory Territory in the Russian Far East : one female, CR22 Gangwon Province, Chiaksan NP, Silim town, stream shaded, stones, gravels, 37°17.081'N, 128°15.389'E, 25.v.2013 Pešić & Karanović, dissected and slide mounted.Holotype male (NIBR), dissected and slide mounted, SOUTH KOREA: CR21 Chungcheongbuk Province, Woraksan NP, Jungseonam, River exposed to sunlight, sand, gravel substrate, Idiosoma dimensions relatively large (L 580-630); dorsal shield with colour pattern as illustrated in Fig. General features — Idiosoma elongated; dorsal shield with colour pattern as illustrated in Fig. Male. Genital field subrectangular; ejaculatory complex conventional in shape Rivobatesmicroepimeratus36°51.705'N, 128°06.030'E, 25.v.2013 Pešić & Karanović 0/1/0 (mounted).SOUTH KOREA: CR20 Chungcheongbuk Province, Mt. Vorak, Deokjusanseong, stream, Hygrobatesmicroepimeratus  a species described from the Primory Territory in the Russian Far East  and Hygrobatestaniguchii Imamura, 1954 . In the original description Hygrobatesdiversiporus), gave particular weight to the smaller dimensions of coxae which occupy one third of venter. According to Hygrobatestaniguchii differs from Hygrobatesmicroepimeratus in larger coxae. Hygrobatesezoensis differs in P-2 with a more pronounced and acute ventrodistal projection and stouter P-3 stat. n.Megapusnodipalpisconstrictus37°17.081'N, 128°15.389'E, 25.v.2013 Pešić & Karanović 5/0/0 (1/0/0 mounted). RUSSIA: Primory Territory, Khasansky District, “Kedrovaya Pad National Nature Biosphere Reserve”, Sea of Japan basin, Kedrovaya River , 43°06.056'N; 131°33.310'E; 27.vi.1993 Tiunova 4/15/0 (2/2/0 mounted); Primory Territory, Partizansky District, Partizanskay River basin, Tigrovaya River , 43°11.401'N; 133°12.660'E, 12.vi.2010 Semenchenko & Sidorov 2/3/2 (1/1/2 mounted).SOUTH KOREA: CR22 Gangwon Province, Chiaksan NP, Silim town, stream , Adults. Integument striated, muscle insertions unsclerotized. Setae Dgl-1, Dgl-3, Dgl-4 and Lgl-2 longer than other dorsal setae; Preoc. and Postoc. without glandularia  — Idiosoma L/W 600/538 (560/476); coxal field: L 369 (336); Cx-III W 409 (383); Cx-I+II mL 138 (135), lL 263 (238); genital field L/W 129 (109)/ 157 (122), L Ac 1-3: 40-42 (37-42), 51-52 (39-40), 72 (50-51); ejaculatory complex L 127 (106).Palp: Total L 317 (275), dL/H: P-1, 29/34 ; P-2, 77/55 (65/56); P-3, 75/48 (56/46); P-4, 97/29 (93/38); P-5, 39/15 (32/16); L P-2/P-4 ratio 0.79 (0.69). Gnathosoma vL 153 (127).Legs: I-L-5 dL 218 (174), vL 142 (151), dL/vL ratio 1.53 (1.44), maximum H 59 (49), dL/maximum H 3.7 (4.45), S-1 L 105 (84), L/W ratio 8.5 (12), S-2 L 74 (73), L/W ratio 5.4 (9.1), distance S-1-2, 24 (16), L ratio S-1/2, 1.42 (1.15); I-L-6 dL 139 (120); central H 21 (20), dL/central H ratio 6.7 (6); dL I-L-5/6 ratio 1.57 (1.45).Female  — Idiosoma L/W 765/730 (730/600); coxal field: L 405 (369); Cx-III W 402 (468); Cx-I+II mL 138 (139), lL 290 (264); genital field L/W 162 (142)/197 (174), genital plate L 137 (118); pregen W 65 (62); L Ac 1-3: 50 (47), 56 (50), 54 (51).Palp: Total L 400 (368), dL/H: P-1, 38/51 (43/29); P-2, 89/76 (72/44); P-3, 116/56 (105/43); P-4, 124/35 (116/29); P-5, 33/19 (32/16); L P-2/P-4 ratio 0.72 (0.62). Gnathosoma vL 160 (147).PageBreakPageBreakPageBreakPageBreakL/W ratio 7.5 (8.2), distance S-1-2, 28 (24), L ratio S-1/2, 1.29 (1.32); I-L-6 dL 179 (156); central H 22 (20), dL/central H ratio 8.1 (7.8); dL I-L-5/6 ratio 1.36 (1.42).Legs: I-L-5 dL 248 (221), vL 170 (156), dL/vL ratio 1.46 (1.42), maximum H 79 (56), dL/maximum H 3.1 (3.9), S-1 L 124 (108), L/W ratio 12.4 (13.5), S-2 L 97 (82), Deutonymph — Idiosoma L/W 323/237); coxal field: L 173; Cx-III W 191; Cx-I+II mL 62, lL 119; genital plate L/W 49/28; L Ac 1-2: 19, 17.Palp: Total L 165, dL/H: P-1, 18/18; P-2, 37/30; P-3, 43/22; P-4, 49/21; P-5, 18/10; L P-2/P-4 ratio 0.75. Gnathosoma vL 81.Legs: I-L-5 dL 96, vL 76, dL/vL ratio 1.3, maximum H 31, dL/maximum H 1.5, S-1 L 47, L/W ratio 11.8, S-2 L 46, L/W ratio 9.2, distance S-1-2, 2.5, L ratio S-1/2, 1.02; I-L-6 dL 70; central H 16, dL/central H ratio 4.4; dL I-L-5/6 ratio 1.36.Atractidesconstrictus was originally described by Atractidesnodipalpis. In the original description Atractidesnodipalpis, as from the latter species, Atractidesconstrictus can easily be distinguished by the fused Vgl-1 and -2. Males examined from Korea show a general conformity with material from the Far East of Russia. Differences are found in a more enlarged Ac-3, a larger S-1/2 interspace and I-L-6 relatively longer compared to I-L-5 in specimens from Korea.Far East of Russia Megapusgracilis36°40.996'N, 129°25.201'E, 24.v.2013 Pešić & Karanović 0/1/0; CR18 Gyeongsangbuk Province, Haenggok-ri, river, exposed to sunlight, 36°57.182'N, 129°17.670'E, 24.v.2013 Pešić & Karanović 0/1/0.SOUTH KOREA: CR17 Gyeongsangbuk Province, Hupo-Myeon, shadded stream, Far East of Russia , dissected and slide mounted, SOUTH KOREA: CR19 Chungcheongbuk Province, Sobaeksan NP, shaded stream, PageBreaktrodistal protrusion of P-2 conus- shaped; S-1 distally truncated, S-2 thicker and shorter, small setal interspace (8 μm): I-L-6 short (L I-L-5/6 ratio 1.59) and stout (L/H ratio 5.0).. Median suture line relatively Cx-I+II long (> 100 μm); acetabula large (maximum diameter > 50 μm) in triangular position; venPageBreakIntegument striated, muscle insertions unsclerotized. Coxal field: caudal margin Cx-I straight, apodemes of Cx-II directed laterally. Genital field: anterior margin of primary sclerotization slightly concave, but secondary sclerotization forming narrow semicircular border, posterior margin slightly indented, Ac in triangular position Fig. . ExcretoMeasurements — Idiosoma L/W 550/425; coxal field: L 319; Cx-III W 366; Cx-I+II mL 121, lL 231; genital field L/W 112/120, L Ac 1-3: 34, 42, 51-55.Palp: Total L 289, dL/H, dL/H ratio: P-1, 31/25, 1.25; P-2, 66/51, 1.3; P-3, 62/44, 1.39; P-4, 96/26, 3.67; P-5, 34/13, 2.5; L P-2/P-4 ratio 0.96.Legs: I-L-5 dL 171, vL 132, dL/vL ratio 1.3, maximum H 48, dL/maximum H 3.59, S-1 L 77, L/W ratio 8.4, S-2 L 65, L/W ratio 6.0, distance S-1-2, 8, L ratio S-1/2, 1.19; I-L-6 dL 108, central H 22, dL/central H ratio 5.0; dL I-L-5/6 ratio 1.59.Female: unknown.Named after Dr Sergey Ermilov , for his contribution to the taxonomy of oribatid mites.Atractidessamsoni  in the small S-1/2 interspace, I-L-6 stocky, postgenital area with smooth excretory pore and unfused Vgl-1/2 and a palp with a conus shaped ventrodistal protrusion in male. The latter species can be distinguished by the shorter medial suture line of Cx-I, smaller acetabula, more slender S-1 and -2, and I-L-6 only weakly curved and longer , a species similar in the shape of palp , a larger Ac (maximum diameter > 39 μm), and I-L-6 relatively short compared to I-L-5 (L I-L-5/6 ratio 1.4-1.6), differ in wider setal interspace on I-L-5, I-L-6 more slender and more narrow centrally, P-4 ventral setae inserted more closely to each other and genital field deeply indented both anteriorly and posteriorly.The new species resembles nger see . Males oA permanent sandy/bouldary stream, shaded by riparian vegetation Fig. .Korea, only known from the locus typicus.Taxon classificationAnimaliaTrombidiformesFeltriidaeTuzovskij, 1988FeltriakuluensisPageBreak36°26.499'N, 129°08.114'E, 23.v.2013 Pešić & Karanović 1/0/0 (mounted).SOUTH KOREA: CR16 Gyeongsangbuk Province, Juwangsan NP, Woroe-ri, Cheong song-eup, Dalgikpo, waterfall, Feltriakuluensis Tuzovskij, 1988, a species described from the Magadan region in the Russian Far East  and Dgl-4 on paired, transverse, laterally pointed posterodorsal platelets (fig. 7-1, in Feltriaminuta differs in relatively more slender palp, P-4 with both ventral setae on slightly elevated, parallel longitudinal extensions, and male IV-L-6 relatively more shorter with ventrolateral extension bearing one fine seta and two enlarged, transparent setae, adpressed to each other and directed ventrally (The single male from this study matches the general morphology of entrally .Feltriaishikariensis Imamura, 1954 from Kangreung, Korea. This species was described orginally by Feltriarotunda based on three specimens (probably, by mistake assigned to a male, see figs 48–49 in Feltriaishikariensis from Hokkaido, Japan. Feltriaishikariensis clearly differs from the species illustrated by Feltriakuluensis in all abovementioned characters, it is very likely that the specimens attributed to Feltriaishikariensis refer to Feltriakuluensis.PageBreakPageBreakFeltriaminuta Koenike, 1892. As mentioned by Feltriakuluensis.In the same paper, Far East of Russia . New for


Wuchereria bancrofti, Brugia malayi and B. timori, is a debilitating disease with an adverse social and economic impact. The infection remains unabated in spite of treatment with existing antifilarial drugs diethylcarbamazine (DEC) and ivermectin which are chiefly microfilaricides. There is therefore, need for macrofilaricides, embryostatic agents and better microfilaricides. In the present study we explored the antifilarial potential of crude extract and its molecular fractions of the plant Taxodium distichum using in vitro assay systems and rodent models of B. malayi infection.Lymphatic filariasis caused by T. distichum was solvent fractionated and sub-fractionated. Four molecules, 3-Acetoxylabda-8(20), 13-diene-15-oic acid (K001), Beta-sitosterol (K002), labda-8(20),13-diene-15-oic acid (K003) and Metasequoic acid A (K004) were isolated from the fractions and their structure determined by spectroscopic analysis. The extract, subfractions and molecules were evaluated for antifilarial activity against B. malayi by 3--2,5 diphenyltetrazolium bromide (MTT) reduction and motility assays in vitro and in two animal models, Meriones unguiculatus and Mastomys coucha, harbouring B. malayi infection.Ethanolic extract (A001) of aerial parts of in vitro. The diterpenoid K003 produced 100 % reduction in motility of both mf and adult worms and > 80 % inhibition in MTT reduction potential of adult female worms. In B. malayi-M. unguiculatus model, A001 killed all the adult worms in > 80 % of infected animals. K003 was embryostatic (> 95 %) in this model. In the B. malayi-M. coucha model, K003 killed ~54 % of adult worms  and rendered > 36 % female worms sterile; it also stopped any further rise in microfilaraemia after day 42 post-initiation of treatment.A001 was effective in killing microfilariae (mf) and adult worms T. distichum possesses potent antifilarial activity and the active principle was localised to K003 which showed significant macrofilaricidal activity and late suppression of peripheral microfilaraemia and some embryostatic activity. These findings indicate that labdane diterpenoid molecule(s) may provide valuable leads for design and development of new macrofilaricidal agent(s). To the best of our knowledge, this is the first report on antifilarial efficacy of products from the plant T. distichum.Ethanolic extract of aerial parts of the plant  Wuchereria bancrofti, Brugia malayi and B. timori, is one of the neglected tropical diseases and is recognised as the world’s most disabling and disfiguring parasitic disease with an adverse social and economic impact. Around 1.4 billion people in 73 countries worldwide which includes 553 million in India alone, are at risk of the infection + 345.5847.Compound 2 K002 : white needles, molecular formula C29H50O. 1H NMR : δ1.8, 1.9 (H-1), 1.4, 1.9 (H-2), 3.5 (H-3), 2.3 (H-4), 5.35, 5.37 (H-6), 2.0, 1.85 (H-7), 1.5, 2.0 (H-8), 0.94 (H-9), 1.5 (H-11), 2.0-2.1 (H-12), 1.05 (H-14), 1.25, 1.6 (H-15), 1.8 (H-16), 1.08 (H-17), 0.69 (H-18), 0.94 (H-19), 1.35 (H-20), 0.85 (H-21), 1.3 (H-22), 1.56 (H-23), 0.9 (H-24), 1.7, 1.3,1.2 (H-25), 1.02 (H-26), 1.1 (H-27), 1.2 (H-28), 0.88 (H-29) and 13C NMR : 37.6 (C-1), 32.3 (C-2), 72.2 (C-3), 42.6 (C-4), 141.1 (C-5), 122.1 (C-6), 32.0 (C-7), 32.3 (C-8), 50.5 (C-9), 36.9 (C-10), 21.4 (C-11), 40.1 (C-12), 42.6 (C-13), 56.4 (C-14), 23.4 (C-15), 28.6 (C-16), 57.1 (C-17), 12.2 (C-18), 19.1 (C-19), 36.5 (C-20), 19.4 (C-21), 34.3 (C-22), 24.7 (C-23), 46.2 (C-24), 29.5 (C-25), 19.8 (C-26), 20.2 (C-27), 26.4 (C-28), 12.3 (C-29). [M + H]+ 414.3418.Compound 3 K003 (labda-8(20),13-diene-15-oic acid: purity: > 95 %): Pale yellow oil, molecular formula C20H32O2. 1H NMR : δ 1.4, 1.1 (H-1), 1.5-1.6 (H-2), 1.1, 1.2 (H-3), 0.9 (H-5), 1.4, 1.6 (H-6), 1.9-2.3 (H-7), 1.4 (H-9), 1.3-1.6 (H-11), 1.5-2.4, 2.0 (H-12), 5.86 (H-14), 2.2 (H-16), 4.85, 4.45 (H-17), 0.82 (H-18), 0.77 (H-19), 0.63 (H-20) and 13C NMR : 38.7 (C-1), 19.8 (C-2), 42.5 (C-3), 33.8 (C-4), 55.7 (C-5), 24.8 (C-6), 39.2 (C-7), 148.3 (C-8), 56.6 (C-9), 40.0 (C-10), 21.9 (C-11), 40.4 (C-12), 164.1 (C-13), 115.6 (C-14), 173.1 (C-15), 19.4 (C-16), 106.9 (C-17), 33.8 (C-18), 22.0 (C-19), 14.7 (C-20). [M + H]+ 305.2917.Compound 4 K004 (Metasequoic acid A: purity: > 95 %): Pale yellow oil, molecular formula C20H30O2. 1H NMR : δ 1.55 (H-1), 1.6 (H-2), 0.45 (H-3), 1.50-1.51 (H-5), 1.3, 1.9 (H-6), 2.0-2.4 (H-7), 1.53-1.54 (H-9), 0.8 (H-11), 2.0-2.4 (H-12), 5.68 (H-14), 2.17 (H-16), 4.89, 4.54 (H-17), 1.02 (H-18), 0.66 (H-19), 0.51 (H-20) and 13C NMR : 35.0 (C-1), 21.36 (C-2), 18.25 (C-3), 20.03 (C-4), 50.5 (C-5), 26.2 (C-6), 38.4 (C-7), 148.7 (C-8), 55.9 (C-9), 39.1 (C-10), 22.1 (C-11), 40.5 (C-12), 164.2 (C-13), 115.3 (C-14), 172.6 (C-15), 19.6 (C-16), 107.8 (C-17), 28.6 (C-18), 18.28 (C-19), 13.8 (C-20). [M + H]+ 303.2429.1H and 13C NMR values for these compounds were similar to those reported in literature [The terature –29.in vitro activity of ethanolic extract (A001) of aerial parts of the plant, its fractions, subfractions and compounds are shown in Table . A001 was found to be more effective in killing mf (LC100: 3.91 μg/ml) than adult worms (LC100: 15.63 μg/ml) and the IC50 values for the respective parasite stages were found to be 1.95 and 10.00 μg/ml. A001 exerted 47 % inhibition in MTT-reduction assay over the DMSO control. Of the four fractions F001 was more effective in killing (100 % killing) mf (LC100: 7.83 μg/ml) requiring four times lesser concentration than needed for killing adult worms (LC100: 31.25 μg/ml); the fraction exerted 80 % inhibition in MTT reduction potential of the adult parasites. Of the six subfractions, SF2 killed both adult worms and mf (LC100: 7.83 μg/ml) and exerted 78 % inhibition in MTT-reduction assay. SF1 was also active but it was more microfilaricidal (LC100: 7.83 μg/ml) than macrofilaricidal (LC100: 31.25 μg/ml); the IC50 value of both the subfractions, SF1 and SF2 against mf was < 3.95 μg/ml. SF4 was less effective on motility of mf (LC100: 62.5 μg/ml) and adult worms (LC100: 125 μg/ml) but it caused 84 % inhibition in MTT reduction assay. The 2 diterpenoid compounds K003 and K004 isolated from SF2 killed mf (LC100: 31.25 μg/ml) and adult worms (LC100: 125 μg/ml); both compounds produced > 80 % inhibition in MTT-reduction assay.Results of tract A00 of aeria100 for mf and adult worms were 2.5 μM and 5 μM, respectively).DEC-C required 500 μM (mf) to 1000 μM (adult worms) to kill the parasite stages whereas ivermectin required much lower concentrations to kill both the parasite stages . A001  exhibited remarkable adulticidal activity; five of the six treated animals showed 100 % macrofilaricidal  activity while the sixth animal showed motile and active adult parasites and 28.57 % of the female parasites showed non-viable mf or dead/distorted embryonic stages in uterus. One-way ANOVA and Tukey’s multiple comparison tests showed that compound K003 (100 mg/kg) exerted remarkable embryostatic activity  = 88.56, P < 0.001; Table M. unguiculatus model.Table t = 18.92, P < 0.01; Table F =358.04, P < 0.01; Table The reference drug ivermectin did not affect mf in p.c. (data not shown) but produced mild but not statistically significant effect on adult worms. The drug also produced significant embryostatic effect  is presented in Fig. t = 4.82, P = 0.0004) and equaled the levels in DEC treated group . The trend in post-treatment microfilaraemia . More than 36 % of the surviving female parasites were found sterile although this was not statistically significant (P > 0.05) when compared with the sterile female parasites of control animals . Further, one-way ANOVA analysis revealed that K003 was superior to DEC with respect to macrofilaricidal efficacy as evidenced by lesser worm recovery from K003 treated animals .Antifilarial efficacy of K003 and reference drug DEC in mia Fig.  was asceq = 7.28, P = 00074) than from the control animals.DEC-C (50 mg/kg) which is chiefly a microfilaricide, caused > 80 % reduction in microfilarial count on day 7/8 p.i.t. which gradually increased and relapsed by day 49 p.i.t.; the count further increased rapidly and crossed the pretreatment level by day 56 p.i.t. Fig. . DEC alsB. malayi-M. coucha model, K003 produced significant macrofilaricidal activity and rendered more than 36 % female worms sterile; it also stopped any further rise in microfilaraemia after day 42 p.i.t.Microfilaraemia in control animals was progressively higher than the pretreatment level and never equalled the 0 day level. About 28 % of female worms recovered from the control animals were found to be sterilised. None of the animals showed any signs of toxicity as inferred from cage-side observations of gross clinical signs. In summary, in T. distichum, we found that the antifilarial activity was localised to labdane diterpenoids K003 and K004 of the subfractions SF2. Although there are reports that diterpenoids show antibacterial, antifungal, anti-protozoal and anti-inflammatory [T. distichum.In the present bioassay-guided fractionation of the ethanolic extract from the aerial parts of the conifer ammatory , 31 actiT. distichum that preferentially target the adult worms and embryogenesis in the female worms. This is considered as a superior approach for control of filariasis (WHO recommends this approach for new drug discovery) since killing/interfering with embryogenesis of even one female adult worm will stop production of millions of mf that circulate in the blood and this may effectively interrupt or reduce the transmission of the infection. Unfortunately, the currently available antifilarial drugs are largely microfilaricides with little effect, if any, on adult worms.In line with our earlier studies on a number of plant products , 9, 11, in vitro. However, the IC50 of pure compounds was higher than that of crude extract. At present the reasons for this differences in the activity between ethanolic extract and the compound isolated from it are not clear, but it appears that greater activity in the crude extract could be due to additive or synergistic effects of K003 and K004 in it; other substance(s) such as flavonoids in the ethanolic extract might have also contributed to this activity since we have recently shown that some flavonoids possess antifilarial activity [M. unguiculatus model (primary model), the ethanolic extract (A001) showed a remarkable macrofilaricidal activity while the purified compound K003 of the extract exerted embryostatic activity only with mild macrofilaricidal activity. Conversely, in the M. coucha model (secondary model), the compound exhibited significant macrofilaricidal activity but only mild  embryostatic activity. However, M. coucha model exhibits close similarity to human infection in several respects such as the course of infection [In the present study, we found micro- and macrofilaricidal activity in the crude extract and purified compounds K003 and K004 activity . In the nfection , pathogenfection , 33, immnfection  and respnfection –38. The M. coucha model, microfilaraemia stopped increasing after day 42 p.i.t. with K003, and by day 80 p.i.t., the levels were significantly lower than those in untreated control and equaled the levels in DEC treated group. Summing it up, the overall microfilaraemia was the highest in control group and the least in DEC group with K003 taking the intermediate position , 13-diene-15-oic acid (K001), Beta-sitosterol (K002), labda-8(20),13-diene-15-oic acid (K003) and Metasequoic acid A (K004) were isolated. The structures of the compounds were elucidated by spectroscopic analysis. A001 and labdane diterpenoid compounds K003 and K004 showed micro- and macrofilaricidal activity in vitro. In the M. unguiculatus model, A001 killed all the adult worms in > 80 % of infected animals; the active principle was localised to labdane diterpenoid compound K003 which showed remarkable embryostatic activity in this model. In M. coucha model the compound K003 exerted significant macrofilaricidal activity with late suppression of peripheral microfilaraemia and some embryostatic activity. Thus, in conclusion the findings indicate that labdane diterpenoid molecule(s) may provide valuable lead for design and development of new macrofilaricidal agent(s). Future studies will include chemical modification of K003 and K004 to maximise the antifilarial activity; regulatory toxicity studies will be carried out on the molecules maximised for activity.Ethanolic extract A001 prepared from aerial parts of the plant


AbstractCastoponera Deeleman-Reinhold, 2001, Castoponerachristaesp. n., is described here. The species is closely related to Castoponeralecythus Deeleman-Reinhold, 2001, but can be distinguished by the structures of the male palp and the female genitalia.A new species of the genus  Castoponera Deeleman-Reinhold, 2001 is endemic to Southeast Asia, and is currently comprised of three species , and the paratypes in FRCS and the Museum of Nature and Human Activities, Hyogo, Japan (MNHAH).The holotype designated here is deposited in the PageBreakTaxon classificationAnimaliaAraneaeCorinnidaeYamasakisp. n.http://zoobank.org/F527D81F-E1D1-4FC9-A0C2-69C5333D96BAHolotype male , Sungai Liku (Liku River), 4°14'N, 114°03'E, Lambir Hills National Park, Sarawak, Borneo, 29-II-2009, T. Itioka leg. Paratypes: 1 female , same locality as the holotype, 22-VIII-2007, Y. Hashimoto & T. Endo leg.; 1 male , same locality as the holotype, 31-III-2014, F. Hyodo leg.Castoponerachristae sp. n. is distinguishable from Castoponeraciliata  and Castoponerascotopoda by the long embolus . Total length 7.4/8.5. Carapace length 3.07/4.20; width 1.87/2.43; height 0.97/1.20. Clypeus height 0.28/0.37. Eye size: ALE, approximately 0.06 mm in diameter. Pedicel wrapped in tube-like sclerite extending from abdomen , Danum Valley Field Centre, 4°58'N, 117°48'E, Sabah, Borneo, 18-XII-2006, Y. Hashimoto leg.; 1 female , same locality, 9-I-2008, Y. Hashimoto & T. Endo leg.; 1 female, Tawau Hills Park, 4°23'N, 117°53'E, Sabah, Borneo, 17-XI-2009, T. Yamasaki leg.; 1 male, Poring Hot Spring, Kinabalu Park, 6°02'E, 116°42'E, Sabah, Borneo, 12-XI-2010, T. Yamasaki leg.1 male . Total length 7.4/8.7. Carapace length 3.13/3.45; width 1.87/2.13; height 0.98/1.05. Clypeus height 0.26/0.28. Eye size: ALE, approximately 0.05 mm in diameter. Pedicel wrapped in tube-like sclerite extending from abdomen (Figs Figs . Carapacmen Figs . Abdomenmen Figs .Male palp Figs , 32–33. Leg spination. Femur I 1-1-1d, 1pl; tibia I 2-2-2v; metatarsus I 2-2v; femur II 1-1-1d, 1pl; tibia II 1-2-2v; metatarsus II 2-2v; femur III 1-1-1d, 1-1pl, 1-1rl; tibia III 1d, 1-2v, 1-1pl, 1-1rl; metatarsus III 2-2v, 1-1pl, 1-1rl, with 5 distal spines; femur IV 1-1-1d, 1-1pl, 1rl; tibia IV 1d, 1-1-2v, 1-1pl, 1-1rl; metatarsus IV 2-1v, 1-1pl, 1-1rl, with 5 distal spines.PageBreakwith plumose setae, some white and some light brown, and posterior surface covered with long setae; thick white plumose setae forming following markings: transverse band on anterior dorsum, transverse band encircling middle part, large patch on posterior dorsum; posterior end bearing tuft of long white plumose setae.Coloration and setation Figs . CarapacPageBreakPageBreakFigs . Almost Female genitalia Figs , 34–35. Leg spination. Femur I 1-1-1d, 1-1pl; tibia I 1-2-2v; metatarsus I 2-2v; femur II 1-1d, 1pl; tibia II 2-2-2v; metatarsus II 2-2v; femur III 1-1d, 1pl, 1rl; tibia III 1d, 1-2v, 1-1pl, 1-1rl; metatarsus III 2-2v; 1-1pl, 1-1rl, with 5 distal spines; femur IV 1-1-1d, 1-1pl, 1-1rl; tibia IV 1d, 1-1-2v, 1-1pl, 1-1rl; metatarsus IV 2-2v, 1-1pl, 1-1rl, with 5 distal spines.Coloration and setation Figs . Almost 2°02S, 113°40'E, SE PageBreakKalimantan (Danum Valley Feild Centre, Sabah ; Tawau Hlimantan ; Aranio limantan .Castoponeralecythus. For the comparison with Castoponerachristae sp. n., see Diagnosis and Remarks in Castoponerachristae sp. n.We examined 1 male and 1 female collected from the type locality, and these specimens agreed with the original description of Castianeirinae are considered to be myrmecomorphies (Castoponerachristae sp. n. or Castoponeralecythus occur sympatrically with Ponerinae ants such as Diacamma Mayr, Odontoponera Mayr and Leptogenys Roger. These ants might be the suitable models of Batesian mimicry for Castoponerachristae sp. n. and Castoponeralecythus because they are common and abundant in the forest floor, and have a sting. Castoponerachristae sp. n. and Castoponeralecythus are especially similar to Diacamma spp. in the coloration and setation of the abdomen (Figs Diacamma ants.The members of morphies . In the men Figs . The tra


In initial structure activity relationships study, the 1-methylbenzimidazole ring was replaced by the isosteric heterocycles benzimidazole, benzoxazole, and benzothiazole and the position of the hydroxamic acid substituent on the phenyl ring was varied. Whereas compounds bearing a para substituted hydroxamic acid (9a-d) were active HDAC inhibitors, the meta substituted analogues (8a-d) were appreciably inactive. Compounds 9a-d selectively inhibited HDAC6 (IC50 = 0.1–1.0μM) over HDAC1 (IC50 = 0.9–6μM) and moreover, also selectively inhibited the growth of lung cancer cells vs. patient matched normal cells. The compounds induce a cell cycle arrest in the S-phase while induction of apoptosis is neglible as compared to controls. Molecular modeling studies uncovered that the MM-GBSA energy for interaction of 9a-d with HDAC6 was higher than for HDAC1 providing structural rationale for the HDAC6 selectivity.We have screened our compound collection in an established cell based assay that measures the derepression of an epigenetically silenced transgene, the locus derepression assay. The screen led to the identification of 4-[4-(1-methylbenzimidazol-2-yl)piperazin-1-yl]sulfonylbenzenecarbohydroxamic acid ( Aberrant regulation of gene expression is at the basis of several human diseases including many forms of cancer. This is partly the result of deregulation of DNA methylation or the post translational modification of histone tails. The cellular enzymes that mediate these modifications include DNA-methyltranferases (DNMTs), the recently described TET proteins that hydroxylate methylated DNA, histone acetyl transferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs), histone demethylases (HDMs) and histone kinases. Together, these enzymes influence transcription by maintaining tissue–specific epigenetic and transcriptional patterns as well as by acting as co-regulators for transcription factors. In disease, this regulation of the transcriptional process can be altered as a consequence of changes in the expression or function of epigenetic enzymes ,2. The m+ for their activity. Most inhibitors of Class I, II and IV HDACs thus act by chelating active site zinc ..5a,b,d w3a,b,d were obtained from 2-chlorobenzazoles 1a,b,d by nucleophilic substitution of the chlorine atom with ethyl N-piperazinecarboxylate 2. Subsequent removal of the ethylcarboxylate moiety with aqu. 40% HBr at 70°C gave the benzazol-2-yl piperazines 5a,b,d, in good yields. 2-Piperazin-1-yl-benzoxazole 5c was synthesized by the treatment of 2-chlorobenzoxazole 1c with piperazine hydrate 4 at 0°C in CH2Cl2 sulfonyl-benzoic acid (6a): 2-(Piperazin-1-yl)benzimidazole hydrobromide 5a (0.200 g. 1.06 mM), triethylamine  and 3-chlorosulfonylbenzoic acid  was reacted for overnight at RT to give colourless crystals . Compound 6a was recrystalissed from EtOH . IR : 2994 (-OH), 1648 (C = O), 1613, 1560 , 1344 (-SO2-). 1H NMR : 2.98–3.12 , 3.55–3.66 , 6.87–6.92 , 7.13–7.18 , 7.73–781 , 7.98–8.02 , 8.21–8.24 .3-[4-(1-Methylbenzimidazol-2-yl)piperazin-1-sulfonyl]-benzoic acid (6b): 1-Methyl-2-(piperazin-1-yl)benzimidazole 5b (0.300 g. 1.38 mM), triethylamine  and 3-chlorosulfonylbenzoic acid  was reacted for 23 h at RT to give colourless crystals . Compound 6b was recrystallised from EtOH (m. pt. 291–293°C). IR : 2855 (-OH), 1714 (C = O), 1616, 1637 , 1349 (-SO2-). 1H NMR : 3.14 , 3.33 , 3.51 , 7.05–7.09 , 7.29–7.39 , 7.79–7.86, , 8.02–8.06 , 8.24–8.29 .3-(4-Benzoxazol-2-yl-piperazin-1-sulfonyl)-benzoic acid (6c): 2-(Piperazin-1-yl)benzoxazole 5c , triethylamine  and 3-chlorosulfonylbenzoic acid  was reacted for 23 h at RT to give colourless crystals . Compound 6c was recrystallized from EtOH : 2867 (-OH), 1685 (C = O), 1640, 1581 , 1348 (-SO2-). 1H NMR : 3.00–3.11 , 3.69 , 6.95–7.03 , 7.08–7.15 , 7.23–7.27 , 7.34–7.37 , 7.73–7.8 , 7.95–7.99 , 8.19–8.23 .3-(4-Benzothiazol-2-yl-piperazin-1-sulfonyl)-benzoic acid (6d): 2-(Piperazin-1-yl)benzothiazole 5d (0.300 g. 1.37 mM), triethylamine  and 3-chlorosulfonylbenzoic acid  was reacted overnight at RT to give colourless crystals . Compound 6d was recrystallized from EtOH . IR : 3441 (OH), 1704 (C = O), 1592, 1536 , 1350 (-SO2-). 1H NMR : 3.06 , 3.66 , 7.01–7.09 , 7.21–7.29 , 7.43 , 7.65–7.75 , 7.87 , 8.18–8.22 .4-[4(1H-Benzimidazol-2-yl)-piperazin-1-sulfonyl]-benzoic acid (7a): 2-(Piperazin-1-yl)benzimidazole hydrobromide 5a , triethylamine  and 4-chlorosulfonylbenzoic acid  was reacted overnight at RT to give colourless crystals . Compound 7a was recrystallized from EtOH (m. pt. > 350°C). IR : 2894 (-OH), 1638 (C = O), 1610 . 1H NMR : 2.98–3.12 , 3.55–3.66 , 6.87–6.92 , 7.11–7.16 , 7.87 , 8.52 .4-[4-(1-Methylbenzimidazol-2-yl)piperazin-1-yl]sulfonylbenzoic acid (7b): 1-Methyl-2-(piperazin-1-yl)benzimidazole 5b (0.300 g. 1.38 mM), triethylamine  and 4-chlorosulfonylbenzoic acid  was reacted for 3 days at RT to give colourless crystals . Compound 7b was recrystallised from EtOH . IR : 2867 (-OH), 1704 (C = O), 1614, 1586 , 1354 (-SO2-), 1H NMR : 3.14 , 3.32 , 3.51 , 7.04–7.09 , 7.29–7.39 , 7.90 , 8.19 .4-(4-Benzoxazol-2-yl-piperazin-1-sulfonyl)-benzoic acid (7c): 2-(Piperazin-1-yl)benzoxazole 5c (0.200 g. 1.48 mM), triethylamine  and 4-chlorosulfonylbenzoic acid  was reacted overnight at RT to give colourless crystals . Compound 7c was recrystallized from EtOH (m. pt. > 350°C). IR : 2917 (-OH), 1689 (C = O), 1582 , 1346 (-SO2-). 1H NMR : 3.09 , 3.69 , 6.96–7.03 , 7.08–7.16 , 7.25 , 7.36 , 7.86 , 8.14 .4-(4-Benzothiazol-2-yl-piperazin-1-sulfonyl)-benzoic acid (7d): 2-(Piperazin-1-yl)benzothiazole 5d (0.300 g. 1.38 mM), triethylamine  and 4-chlorosulfonylbenzoic acid  was reacted for 23 h at RT to give colourless crystals . Compound 7d was recrystallized from EtOH (m. pt. 325–327°C). IR : 2992 (-OH), 1689 (C = O), 1593 , 1352 (-SO2-). 1H NMR : 3.09 , 3.66 , 7.02–7.10 , 7.21–7.29 , 7.43 , 7.74 , 7.87 , 8.5 .8a-d and 9a-d a solutions of acids 6a-d and 7a-d were prepared to which is added a solution of hydroxylamine.For the synthesis of compounds N-methylmorpholine  with the mixture stirred for a further 1 h and filtered. The reaction was controlled with TLC (PE/EtAc 3:7).To a solution carboxylic acid derivatives 6a-d and 7a-d  in 30 mL dry THF at 0°C is added ethylchloroformate , To a solution of potassium hydroxide  in 25 mL dry EtOH was cooled to 0°C and hydroxylamine HCl  was added in small portions After the addition the mixture was stirred for a further 1 h at 0°C and the precipitated KCl was filtered.The prepared acid anhydride in A was added dropwise to the freshly prepared hydroxylamine solution in EtOH at 0°C. The reaction was stirred further for 1 h and then at room temperature till the reaction declared complete after TLC control (PE/EtAc 3:7). After complete reaction (1 h.), the mixture was evaporated to dryness and the residue taken up in cooled distilled water and stirred for 30 min., the precipitate filtered under suction and washed with petrolether. The products were dried in high vacuum at 60°C.3-[4(1H-Benzimidazol-2-yl)-piperazin-1-sulfonyl]-N-hydroxy-benzamid (8a): Benzoic acid derivative 6a , N-methylmorpholine , ethylchloroformate , KOH  and hydroxylamine HCl  was reacted to give colourless crystals  of compound 8a (m. pt. 349–350°C). IR : 3423 (-OH), 2982–3068 (-NH), 1613 (C = O), 1344 (-SO2) 1H NMR : 3.08 , 3,62 , 6,95 , 7.19 , 7.74–7.82 , 8.00–8.04 , 8.21–8.24 , 10.40 , 11.2 . Anal. Calcd. for C18H19N5O4S (401.44): C, 53.85; H, 4.77; N, 17.45. Found: C, 54.20; H, 5.01; N, 17.32.3-[4-(1-Methyl-Benzimidazol-2-yl)-piperazin-1-sulfonyl]-N-hydroxy-benzamid (8b): Benzoic acid derivative 6b , N-methylmorpholine , ethylchloroformate , KOH  and hydroxylamine HCl  was reacted to give colourless crystals  of compound 8b (m. pt. 167–169°C). IR : 3432–3349 (-OH), 2854–3068 (-NH), 1615 (C = O), 1597 , 1349 (-SO2-). 1H NMR : 3.13 , 3.34 , 3.51 , 7.05–7.09 , 7.29–7.40 , 7.73–7.95 , 8.02–8.12 , 8.23–8.29 , 9.25 , 11.55 . Anal. Calcd. for C19H21N5O4S (415.47): C, 54.93; H, 5.09; N, 16.86. Found: C, 55.20; H, 5.30; N, 16.90.3-(4-Benzoxazol-2-yl-piperazin-1-sulfonyl)-N-hydroxy-benzamid (8c): Benzoic acid derivative 6c , N-methylmorpholine , ethylchloroformate , KOH ) and hydroxylamine HCl  was reacted to give colourless crystals  of compound 8c (m. pt. 257–262°C). IR : 1580 , 1348 (-SO2-). 1H NMR : 3.09 , 3.70 , 6.96–7.03 , 7.08–7.16 , 7.24–7.27 , 7.34–7.38 , 7.69–7.81 , 7.87–8.08 , 8.20–8.25 , 9.23 , 11.50 . Anal. Calcd. for C18H18N4O5S (402.42): C, 53.72; H, 4.51; N, 13.92. Found: C, 53.79; H, 4.57; N, 13.89.3-(4-Benzothiazol-2-yl-piperazin-1-sulfonyl)-N-hydroxy-benzamid (8d): Benzoic acid derivative 6d , N-methylmorpholine , ethylchloroformate , KOH  and hydroxylamine HCl  was reacted to give colourless crystals  of compound 8d (m. pt. 207–210°C). IR : 2949–3298 , 1653 (C = O), 1591, 1532 , 1350 (-SO2-). 1H NMR : 3.08 , 3:67 , 7.01–7.09 , 7.21–7.29 , 7.72–7.76 , 7.41–7.45 , 7.87–7.91 , 8.04–8.08 , 8.85–9.65 , 11.12–11.62 . Anal. Calcd. for C18H18N4O4S2 (418.49): C, 51.66; H, 4.34; N, 13.39. Found: C, 51.72; H, 4.32; N, 13.26.4-[4(1H-Benzimidazol-2-yl)-pipreazin-1-sulfonyl]-N-hydroxy-benzamid (9a): Benzimidazol derivate 7a , N-methylmorpholine , ethylchloroformate , KOH  and hydroxylamine HCl  was reacted to give colourless crystals  of compound 9a . IR : 2859-3077, 1644 (C = O), 1611 (C = N), 1360 (-SO2-). ESI-MS (m/z): 402.14 [M+1]. 1H NMR : 3.15 , 3.35 , 6.90–6.94, 7.17–7.22 , 7.90 , 8.15 , 9.23 , 11.50 . Anal. Calcd. for C18H19N5O4S (401.44): C, 53.85; H, 4.77; N, 17.45. Found: C, 54.07; H, 4.91; N, 17.48.4-[4-(1-Methyl-Benzimidazol-2-yl)-piperazin-1-sulfonyl]-N-hydroxy-benzamid (9b): Benzoic acid derivative 7b , N-methylmorpholine , ethylchloroformate , KOH  and hydroxylamine HCl  was reacted to give colourless crystals  of compound 9b (m. pt. 269–271°C). IR : 3379 (-OH), 2855–3188 (-NH), 1615 (C = O), 1353 (-SO2). 1H NMR : 3.14 , 3.33 , 3.52 , 6.98–7.14 , 7.25–7.45 , 7.84–7.93 , 7.98–8.02 , 8.17–8.21 , 9.29 , 11.50 . Anal. Calcd. for C19H21N5O4S (415.47): C, 54.93; H, 5.09; N, 16.86. Found: C, 55.18; H, 5.27; N, 16.95.4-(4-Benzoxazol-2-yl-piperazin-1-sulfonyl)-N-hydroxy-benzamid (9c): Benzoic acid derivative 7c , N-methylmorpholine , ethylchloroformate , KOH  and hydroxylamine HCl  was reacted to give colourless crystals  of compound 9c (m. pt. 335–338°C). IR : 3400 (-OH), 2869–3094 (-NH), 1689 (C = O), 1582 (C = N), 1346 (-SO2-). 1H NMR : 3,10 , 3.70 , 7.00 , 7.13 , 7.26 , 7.36 , 7.81–7.89 , 7.97 , 8.15 , 9.25 , 11.45 . Anal. Calcd. for C18H18N4O5S (402.42): C, 53.72; H, 4.51; N, 13.92. Found: C, 53.83; H, 4.55; N, 13.91.4-(4-Benzothiazol-2-yl-piperazin-1-sulfonyl)-N-hydroxy-benzamid (9d): Benzoic acid derivative 7d , N-methylmorpholine , ethyl chloroformate , KOH  and hydroxylamine HCl  was reacted to give colourless crystals  of compound 9d . IR : 2858–3046 , 1689/1639 (C = O), 1593 (C = N),1543 (C = C),1350 (-SO2-) 1H NMR : 3.09 , 3.66 , 7.06 , 7.26 , 7.43 , 7.74 , 7.83–7.87 , 7.97 , 8.15 , 10.40 , 11.20 . Anal. Calcd. for C18H18N4O4S2 (418.49): C, 51.66; H, 4.34; N, 13.39. Found: C, 51.79; H, 4.39; N, 13.40.LDR cells were maintained in DMEM with L-glutamine and high glucose, 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, 1% v/v penicillin-streptomycin and 10% heat inactivated FBS. Cells were seeded on glass bottom chambers at low-medium density and allowed to attach overnight. Compounds were then added for 24h at the indicated dose and cells were imaged under a fluorescent microscope equipped with a camera using a 20 or 40X objective. All images shown were exposed for identical time.et al., [3H]acetate-prelabeled chicken reticulocyte histones as substrate. 50 μL of mouse HDAC1 were incubated with different concentrations of compounds for 10 min on ice and 10 μL of total [3H] acetate pre-labeled chicken reticulocyte histones (4 mg/mL) were added, resulting in a concentration of 41 μM. This mixture was incubated at 37°C for 1 h. The reaction was stopped by addition of 50 μL of 1 M HCl/0.4 M acetylacetate and 1 mL ethylacetate. After centrifugation at 10000 x g for 5 min an aliquot of 600 μL of the upper phase was counted for radioactivity in 3 mL liquid scintillation cocktail.For inhibition assays, partially purified HDAC1 (70–80 fold enriched relative to crude samples) from mouse A20 cells was used as enzyme source. HDAC1 activity was purified by a combination of anion exchange chromatography (Mono Q), affinity chromatography  and TSK size exclusion chromatography . HDAC1 protein was identified by immunoblotting using anti mouse HDAC1 antibodies . HDAC activity was determined as described by Sendra ueous One kit per the manufacturer’s protocol. All doses were tested in 4–8 replicates. IC50 values were calculated using the in-house software DIVISA.Lung cancer cells  werFor cell cycle analysis, cells were washed with PBS, fixed in 70% ethanol for 2 h, washed with PBS and resuspended in staining solution  and incubated at 37C for 15 min. Histograms were acquired on a BD FACSCalibur flow cytometer at UT Southwestern’s core facility. Apoptosis was measured using a FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen) according to manufacturer’s protocol after treating cells with compounds as indicated in Figure legends.For the direct comparison of HDAC1 vs. HDAC6 inhibition, HDAC activity was analyzed using the HDAC Assay Kit from Millipore (17–356). Briefly, recombinant HDAC1 or recombinant HDAC6 . was incubated with the fluorometric HDAC substrate according to the manufacturer’s protocol in the presence of DMSO vehicle or increase compound doses. In a secondary activator reaction, the fluorophore is only cleaved from the deacetylated substrate, allowing for quantification. Fluorescence was quantified on a FLUOstar-Optima or a FLUOstar Omega plate reader (BMG Biosciences). To ensure the compounds do not interfere with the detection system control experiments with standard deacetylated substrate were performed in the presence of high doses of all compounds.2+ ion were set and two conserved water molecules located inside the binding pocket were considered during the docking. Post-docking filtering has been carried out using the Python script distance_to_smarts . Only docking poses with distances between both oxygen atoms of the hydroxamic acid of the ligand and the Zn2+ ion inside the binding pocket of the protein below 2.7 Å have been selected. Rescoring of the docking poses was done applying the MM-GBSA approach. The MMF94 force field and a GBSA solvation model within the MOE software package were used. Ligands were minimized inside the binding pocket until a gradient of 0.05 kcal/mol was reached. Protein heavy atoms were tethered using a force constant of 100 kcal/mol Å.The crystal structure of human HDAC1  (PDB ID:9b) treatment (A). We confirmed that the signal was not coming from compound auto-fluorescence by co-inducing with estradiol which causes the nuclear translocation of the GFP chimeric protein sulfonylbenzenecarbohydro xamic acid , a zinc binding enzyme inhibition group (CO(NHOH) functionality) and a spacer (piperazine-sulfonylbenzene moiety) that links the cap to the binding system sulfonylbenzenecarbohydroxamic acid and its analogues might be selective HDAC6 inhibitors. Confirmatory studies showed that these compounds are indeed selective HDAC6 inhibitors compared to HDAC1 by a factor 6–40. In addition, in vivo toxicities associated with some of these compounds due to their molecular promiscuity may well prevent them from ever reaching clinical efficacy. The current hypothesis is that the development of selective inhibitors targeting only one member or a subclass of the HDAC family will lead to improved overall efficacy and fewer side effects. In view of this, the compounds described herein, particularly 9b, can serve as lead structures for the development of selective HDAC6 inhibitors with improved and more targeted activity. HDAC6 selective inhibitors have been described including Tubacin, Tubastatin A, ACY-1215 and several others [Several HDACi are in preclinical development and clinical trials for the treatment of a wide range of diseases, including cancer. The l others . These il others .9b and its analogs. The in vitro specificity of R306465 suggested a preference for HDAC1 vs. HDAC8 and cellular studies determined that both histone and tubulin acetylation were increased by the compound though the effects on histone acetylation were stronger and detected at lower doses compared to tubulin acetylation [9b-d configuration switches the selectivity of the compounds towards HDAC6. Clinical studies with R306465 have been reported in abstract form and toxicities were moderate with some indication of target involvement and efficacy in regimens below the maximal tolerated doses [9b related compounds have clinical promise and should be further developed.Among known HDAC inhibitors, R306465 developed by Johnson & Johnson  shares stylation . Consisted doses . This, ted doses , 33 indiS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig9c and 9d which exhibited significant growth inhibition were evaluated against the 60 cell panel at five concentration levels. This information can be at https://dtp.cancer.gov/.Compounds (PDF)Click here for additional data file.


The new catalyst contains an NHC ligand symmetrically substituted with chromanyl moieties. The complex was tested in model CM and RCM reactions. It showed very high activity in CM reactions with electron-deficient α,β-unsaturated compounds even at 0 °C. It was also examined in more demanding systems such as conjugated dienes and polyenes. The catalyst is stable, storable and easy to purify.The synthesis of a new type of Hoveyda–Grubbs 2 Z-selective catalyst , Ho, Hond gealyst 2,  16] and andnd gest 2, 3,  17] the thend ge4–6 , which was prepared by the reaction between 2,3,5-trimethylphenol and 3-methylbut-2-enol =CHCOX, which is kinetically favourable. As a result, a stronger electron-donating ligand should stabilise the electron-deficient enoic carbene + calcd for C14H19NNaO3: 272.1257, found: 272.1252. Literature mp 125–126 °C; IR, 1H NMR and 13C NMR data are compatible with the data in + calcd for C14H22NO: 220.1696, found: 220.1691. Literature mp 51.5–53.5 °C; IR, 1H NMR and 13C NMR data are compatible with the data in + calcd for C30H45N2O2: 465.3476, found: 465.3465.To a solution of 2,2,5,7,8-pentamethylchromanyl-6-amine  in trimethyl orthoformate (5 mL), ammonium chloride  was added in a similar manner as described in + calcd for C31H43N2O2: 475.3319, found: 475.3308. Some signals in 13C NMR spectrum are doubled (see text “Synthesis of the carbene precursor”).To a solution of ethylenediamine ribed in . The rea14  was dried under vacuum at 80 °C for 1 h. The flask was then cooled to rt and dry toluene (3 mL) was added under argon atmosphere. To the resulting suspension potassium tert-amylate  was added and the reaction mixture was stirred for 20 min at rt. Then a solution of Hoveyda–Grubbs 1st generation catalyst  in toluene (3 mL) was added and the reaction was carried out at 65 °C for 2 h. The reaction mixture was purified by column chromatography (hexane/ethyl acetate 10:1) to give a green solid  Mp 162–164 °C; IR (KBr) ν: 2973, 2925, 2854, 1475, 1457, 1274 cm−1; 1H NMR  δ 16.74 , 7.47, 6.94, 6.84 and 6.78 , 4.89 , 4.16 , 2.69 , 2.42, 2.29 and 2.20 (3s (2 br s and 1s), 18H, 3s H-5a, 7a, 8b), 1.88 , 1.44 , 1.28  ppm; 13C NMR  δ 297.5 (CH=Ru), 211.7 (CNHC), 152.1 (IVCAr), 145.4 (IVCAr), 135.0 (IVCAr weak), 133.5 (IVCAr weak), 132.2 (IVCAr weak), 129.1 (IIICHAr), 129.0 (IVCweak), 123.3 (IVCAr weak), 122.9 (IIICHAr), 122.4 (IIICHAr), 117.1 (IVCAr weak), 112.8 (IIICHAr), 74.7 (CHIPr), 73.3 (Cchroman-2), 53.1 and 51.8 (N-(CH2)2-N), 33.1 (Cchroman-3), 27.9 (Cchroman-2a and 2b), 25.7 (CH3 IPr), 21.2 (Cchroman-4), 18.3 (CH3 chroman weak), 15.2 (CH3 chroman weak), 12.0 (CH3 chroman) ppm; EIMS m/z: 796 (10), 794 (10), 610 (14), 572 (7), 473 (41), 472 (100), 416 (31), 243 (7), 181 (8), 108 (12), 69 (14), 44 (14%); HRMS (EI): [M]+ calcd for C41H54Cl2N2O3102Ru: 794.2549, found: 794.2571. Some signals in 13C NMR spectrum are weak .In a Schlenk flask imidazolinium salt File 11H and 13C NMR spectra of new compounds.Experimental procedures of the testing of the new catalyst and copies of


AbstractGnaphosa khovdensissp. n. (♂) and G. esyuninisp. n. (♂♀) are described from Khovd Aimag of Mongolia. Descriptions, figures, diagnosis and map of the records are provided. The new species are assigned to a monophyletic group together with the recently described G. ustyuzhanini Fomichev et al., 2013.Two new species:  PageBreakGnaphosa Latreille, 1804 is the third largest genus of ground spiders with 141 species , Institute of Zoology, Chinese Academy of Sciences, Beijing (IZCAS) and in the Museum of Natural History, Budapest (MNHB).Photographs were taken in dishes of different sizes with paraffin at the bottom. Specimens were photographed using an Olympus Camedia E-520 camera attached to an Olympus SZX16 stereomicroscope and the SEM JEOL JSM-5200 scanning microscope at the Zoological Museum, University of Turku. Digital images were prepared using “CombineZP” image stacking software. Illustrations of epigynes were made after maceration in 20% potassium hydroxide aqueous solution and exposition for a few minutes in an alcohol/water solution of Chlorazol Black. Lengths of leg segments were measured on the dorsal side. While describing spination of legs we use the following abbreviations: Taxon classificationAnimaliaAraneaeGnaphosidaehttp://zoobank.org/03D9E1C9-6024-41AA-9049-4CB538E6C52CKhovd Aimag: holotype ♂ (ISEA), Arshantyn-Nuruu Mountain Range, 46°16'46"N, 91°16'53"E, 1560 m, mountain stony steppe, under stone, 14.05.2012 (A.A. Fomichev).MONGOLIA, The specific name derived from Khovd Aimag, adjective.Gnaphosa esyunini sp. n., Gnaphosa jucunda Thorell, 1875 and Gnaphosa ustyuzhanini by having a strong spine in the embolic base and a filamentous embolus with a serrated prolateral edge. From Gnaphosa ustyuzhanini  and paratypes 2♀ (NHMB) with label “Nr. 666. Chovd aimak: Chovd (Kobdo), ca 5 km SW von der Stadt, 1500 m, 10.VII.1966. – Von kahlen, felsigen Bergen umgegebener Tal mit sandigem Schotter, geeinzelt unter Steinen, vom Boden und zwischen den Pflansenwurzeln, sowie von den Pflanzen”; 1 ♀ (IZCAS), collecting number: mk408, 18.5 km of Monhhairhan Town, Monhhairhan Sum, Khovd Aimag, Mongolia, 47.22783°N, 91.89552°E, 1945 m, 22.07.2011 (Meng Kaibayier).MONGOLIA, Different maps provide different spelling of Monhhairhan, one alternative spelling is Monkh Khayrkhan.The species is named after our colleague Sergei L. Esyunin from Perm University (Russia), noun.Gnaphosa esyunini sp. n. substantially differ from other species by the very wide fovea and the shape and angle of the lateral pockets.Males of the new species differ from all congeners by the large embolic spine directed prolaterally. Females of Male. Total length 14.4. Carapace: 7.3 long, 5.6 wide. Coloration: carapace and legs brown. Labium, maxillae and sternum dark brown. Chelicerae dark brown, almost black. Abdomen grayish-brown. Spinnerets light brown. Spination: I: Fe d1-1-0, p0-0-1; Ti v0-1-1; Mt v0-2-0. II: Fe d1-1-0, p0-1-1; Ti v0-0-1; Mt v0-2-0. III: Fe d1-1-0, p0-1-1, r0-1-1; Pt p1, r1; Ti d1-0-0, p3-1-1, r2-1-1, v2-2-2; Mt d0-2-0, p2-1-0, r1-1-0, v0-3-2. IV: Fe d1-1-0, p0-1-1, r0-1-1; Pt r1; Ti p2-3-3, r3-2-3, v4-1-4; Mt d0-3-0, p3-4-1, r3-1-0, v3-3-3. Leg article length. I: 5.7+3.3+5.25+4.4+2.65. II: 5.3+3.0+4.75+4.4+2.75. III: 4.85+2.5+3.85+5.05+2.65. IV: 6.0+3.0+5.2+7.25+3.05.Palp as in PageBreakFemale. Total length: 15.5. Carapace: 8.3 long, 6.15 wide. Coloration as in male. Spination: I: Fe d1-1-0, p0-0-1; Ti v0-1-1; Mt v0-2-0. II: Fe d1-1-0, p0-1-1; Ti p0-0-1, v0-1-2; Mt p1-0-0, v0-2-0. III: Fe d1-1-0, p0-1-1, r0-1-1; Pt p1, r1; Ti d2-0-0, p3-1-1, r2-1-1, v2-2-2, Mt d0-2-0, p2-1-0, r2-1-0, v2-0-2. IV: Fe d1-1-0, p0-1-1, r0-1-1; Pt r1; Ti p3-1-1, r2-1-2, v2-3-2; Mt d0-2-0, p2-2-0, r2-1-1, v2-1-2. Leg length. I: 5.9+3.6+4.9+3.95+2.8. II: 5.55+3.35+4.55+4.0+2.8. III: 5.05+2.75+3.8+4.7+2.8. IV: 6.15+3.3+5.3+7.1+3.2.PageBreakEpigyne as in Known only from two localities in Khovd Aimag .Taxon classificationAnimaliaAraneaeGnaphosidaeFomichev, Marusik & Omelko, 2013Gnaphosa ustyuzhaniniPageBreakKhovd Aimag: 1♂ (NHMB) with label “Nr.649, Z.Kaszab, Chovd aimak: Mongol Altaj Gebirge, Tal des Flusses Uenč gol, cca 64 km N von Somon Uenč, 2100 m, 8.VII.1966. – Im Flusstal unter Steinen geeinzelt, resp. geschossen.”MONGOLIA: Gnaphosa khovdensis sp. n. is located. The newly examined specimen has a more prominent embolic spine than in the holotype and paratype males.The type series of this species was collected at the same place where the type locality of Gnaphosa can be considered as a well studied genus and contains almost 150 species it was never subdivided into monophyletic groups on a global scale. Asian species have been assigned into 10 informal species groups (Although s groups . This inPageBreakapophysis (not longer than tibia); raised base of embolus; base of embolus with spine in anterior part; filamentous and rounded embolus with serrated prolateral edge; and simple median apophysis with claw-like tip. Members of other Asian species groups may have any mentioned character but never all together. For example some species in the PageBreaklugubris-group have a triangle or claw-like spine on the base of embolus, but never an anterior spine and they have a modified median apophysis with an enlarged base. Members of the muscorum-group have a filamentous curved embolus and short tibial apophysis, but with the base of the embolus low, never having a spine but rather a kind of spur or serrated keel. Species of the rufula-group have a spine or claw-like embolic tooth or nearly a spine, but have a long tibial apophysis and straight embolus, and/or modified median apophysis.It seems that the three Mongolian species treated here, all from Khovd Aimag, form a monophyletic group, which shares the same characters such as the short tibial


The structures of these metabolites were determined by extensive analysis of spectroscopic data. Gibberoketosterol (5) exhibited cytotoxicity against P-388 (mouse lymphocytic leukemia) cell line with an ED50 of 6.9 μM.Four new cembrane-type diterpenes; numerosol A–D ( Sinularia have proven to be a rich source of diterpenes, sesquiterpenes, steroids, steroidal glycosides, sphingosine derivatives, glycolipids, and spermidine derivatives D25 −28.2 . HRESIMS, 13C-NMR, and DEPT spectroscopic data established the molecular formula of 1 as C20H34O4. The IR spectrum of 1 indicated the presence of the hydroxy functionality (νmax 3413 cm−1). The 1H-NMR spectrum of 1 also showed signals for three olefinic protons at δ 5.94 , 5.63 , and 5.20  ppm; one oxymethine proton at δ 3.56 ; and an olefinic methyl group at δ 1.69 . The spectral data of 1 indicated some similarities to those of -4,11-dihydroxy-1,12-oxidocembra-2,7-diene +, consistent with the molecular formula of C22H34O4, requiring six degrees of unsaturation. The IR spectrum of 2 revealed the presence of hydroxy (νmax 3440 cm−1) and carbonyl (νmax 1730 cm−1) moieties. The 13C-NMR spectrum of 2 , δ 5.35 , and δ 5.24 , were attributable to three trisubstituted double bonds. Moreover, an acetoxy group was determined based on NMR signals at δC 171.7 (qC), 21.3 (CH3), and δH 2.10 . The 1H-NMR spectrum also displayed two hydroxy-containing methine signals . Thus, the bicyclic structure of 2 was revealed. From the 1H–1H COSY spectrum , between H-7 and H-9a , and between H-14 and H3-17 (C 15.2 (C-18) and δC 16.4 (C-19) further supported the E configuration at C-3/C-4, C-7/C-8 +) established the molecular formula C20H32O2, requiring five degrees of unsaturation. The IR spectrum of 3 revealed the presence of a hydroxyl (νmax 3404 cm−1) moiety. Analysis of the 1H–1H COSY and HMBC correlations , δC 16.7 (C-19), and δC 15.9 (C-20) also supported the E configuration at C-3/C-4, C-7/C-8, and C-11/C-12 D25 = −28 ; IR (neat) νmax 3413, 2925, 2857, 1456, 1378, 1166, 1074, 952, 756, 665 cm−1; 1H and 13C-NMR data, see m/z 361 [M + Na]+; HRESIMS m/z 361.2357  (Numerosol A (61.2355) .2): Colorless oil; [α]D25 = −60 ; IR (neat) νmax 3440, 2919, 2851, 1730, 1575, 1371, 1240, 1017 cm−1; 1H and 13C-NMR data, see m/z 385 [M + Na]+; HRESIMS m/z 385.2353  (Numerosol B (85.2355) .3): Colorless oil; [α]D25 = −100 ; IR (neat) νmax 3404, 2922, 2853, 1564, 1417, 1255, 1016, 771 cm−1; 1H and 13C-NMR data, see m/z 327 [M + Na]+; HRESIMS m/z 327.2299  (Numerosol C (27.2300) .4): Colorless oil; [α]D25 = +8 ; IR (neat) νmax 3420, 2972, 2931, 1564, 1419, 1381, 998, 765 cm−1; 1H and 13C-NMR data, see m/z 327 [M + Na]+; HRESIMS m/z 327.2299  (Numerosol D (27.2300) .Cytotoxicity was determined on P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549  tumor cells using a modification of the MTT colorimetric method according to a previously described procedure ,14. The 50 (50% inhibitory concentration), or compound concentration required to reduce virus induced CPE by 50% after 7 days as compared with the untreated control. To monitor the cell growth upon treatment with natural products, an MTT-colorimetric assay was employed [To determine the effects of natural products upon HCMV cytopathic effect (CPE), confluent human embryonic lung (HEL) cells grown in 24-well plates were incubated for 1 h in the presence or absence of various concentrations of tested natural products with three replications. Ganciclovir was used as a positive control. Then, cells were infected with HCMV at an input of 1000 pfu (plaque forming units) per well of a 24-well dish. Antiviral activity was expressed as ICemployed .5) showed moderate cytotoxicity and selectivity against the growth of Hepa59T/VGH cancer cells was evaluated [S. munerosa collected at San-Hsian-Tai  has led to the isolation of four new cembrane-type diterpenoids, Numerosol A–D (1–4) and the known steroid gibberoketosterol (5). Compound 5 displayed moderate cytotoxicity and selectivity against P-388, with ED50 of 6.9 μM.In previous studies, gibberoketosterol (valuated . This in


The aim of our study is to assess the effect of amyloid deposition on myocardial function.28 patients with transthyretin mutation and a group of 14 controls underwent echocardiography to quantify left ventricular (LV) dimensions and function, and global (G) longitudinal (L), radial (R) and circumferential (C) strain (S). 99mTc-3, 3-diphosphono-1, 2-propanodicarboxylic-acid-scintigraphy (99mTc-DPD) was used to quantify cardiac amyloidosis (CA). 99mTc-DPD revealed accumulation in 14 of 28 patients (CA-group) and no accumulation (no CA-group) in 14 patients. Cardiac accumulation was mild-moderate in 5 (Mild-Moderate CA-group) and severe in 9 patients (Severe CA-group). Severe CA-group showed higher values of LV septal thickness (LVST), posterior wall thickness and E/E’ ratio than the no CA-group and the control group (adj. p<0.05). Ejection fraction was similar among groups (p=0.65). GLS was lower (p=<0.001) in severe CA-group (-12.2 ± 4.5) respect to no CA-group (-19.3 ± 3.0) and to the control group (-20.9 ± 2.5). On the contrary, GCS and GRS were lower (p=<0.05) in mild-moderate CA-group  respect to the severe CA-group (-18.9 ± 5.1 and 23.9 ± 6.3 respectively), no CA-group (-19.2 ± 4.1 and 28.4 ± 10.2 respectively) and the control group (-23.9 ± 4.4 and 29.9 ± 8.7 respectively). A correlation was found between the scintigraphic heart retention (HR) index and LVST  and E/E’ . An inverse tendency was observed between HR and GLS .99mTc-DPD HR is well correlated with LVST, diastolic and longitudinal systolic dysfunction.


Dicranostigma leptopodum (Maxim.) Fedde (Papaveraceae) by repeated silica gel column chromatography. Their chemical structures were identified on the basic of physicochemical and spectroscopic data. Among them, 10-O-methylhernovine (1), nantenine (2), corytuberine (3), lagesianine A (4), and dihydrocryptopine (9) were first isolated from this plant. With a series of cytotoxic tests, compounds 2, 3, and 7 displayed cytotoxicity against SMMC-7721 with IC50 values of 70.08 ± 4.63, 73.22 ± 2.35, and 27.77 ± 2.29 μM, respectively.Nine alkaloids with three different structural skeletons were isolated from  Dicranostigma is a genus in the poppy family Papaveraceae, which is widely distributed in highland areas, especially in Western China. The plants of Dicranostigma have been used in folk medicine for treatment of tonsillitis, hepatitis, and inflammation in China for a long time +. 1H-NMR  δ ppm: 7.04 , 6.63 , 6.63 , 3.65 , 3.40 , 3.33 , 4.31 , 6.93 , 6.88 , 3.18 , 3.05 , 3.86 , 3.87 , 5.97 , 6.30 , 2.62 . 13C-NMR  δ ppm: 107.4 (C-1), 151.3 (C-2), 146.1 (C-3), 109.0 (C-4), 130.9 (C-4a), 63.9 (C-5), 55.5 (C-6), 54.1 (C-8), 123.8 (C-8a), 147.0 (C-9), 148.5 (C-10), 125.7 (C-11), 112.3 (C-12), 124.3 (C-12a), 55.4 (C-13), 42.0 (C-14), 126.9 (C-14a), 60.8 (2-OCH3), 55.8 (3-OCH3), 101.5 , 42.6 (N-CH3). The spectrum matched the previous report [s report . 1–9 was determined by the CCK-8 assay [μg/mL streptomycin  at 37°C, 5% CO2, and 90% humidity. Cancer cells were seeded in the growth medium (100 μL) into 96-well microtiter plate (5 × 103 cells per each well). After 4–6 h preincubation in the incubator (Forma Series ΙΙ Water Jacket) to allow cellular attachment, various concentrations of test solution were added and cells were incubated for 36 h. At the end of the incubation, CCK-8 reagent  was added into each well followed by further incubation for 2 h. The optical density (OD) was measured at 450 nm using a multiscan microplate reader  [50) value was calculated by the line equation of the dose-dependent curve of each compound. The equation to calculate the inhibition rate wasThe cytotoxicity of compounds-8 assay . H1299 (


Standard spectroscopic techniques were used to determine the structure of new steroid 1. The absolute stereochemistry of steroid 2 was confirmed by X-ray diffraction analysis. Steroid 3 exhibited potent activity against MOLT-4 tumor cells.Three pregnane-type steroids, including a new metabolite, 3β-methoxy-5,20-pregnadiene ( Scleronephthya genus have been well-recognized as marine organisms containing large quantities of steroids that exhibit varying degrees of bioactivities, such as cytotoxicity and anti-inflammatory activity +; HRESIMS: m/z 337.25027 .3β-Methoxy-5,20-pregnadiene (2): colorless prisms; mp 143−145 °C;  −60 ; IR (neat) νmax 1727 cm−1; 1H NMR  δH 5.76 , 5.38 , 4.98 , 4.97 , 4.60 , 2.32 , 2.03 , 2.01 , 1.98 , 1.88 , 1.86 , 1.79 , 1.73 , 1.70 , 1.60 , 1.58 , 1.57 , 1.54 , 1.46 , 1.42 , 1.19 , 1.16 , 1.09 , 1.03 , 1.01 , 0.98 , 0.61 ; 13C NMR  δC 170.5 , 139.8 (CH-20), 139.7 (C-5), 122.5 (CH-6), 114.5 (CH2-21), 73.9 (CH-3), 55.8 (CH-14), 55.3 (CH-17), 50.3 (CH-9), 43.4 (C-13), 38.1 (CH2-4), 37.3 (CH2-12), 37.0 (CH2-1), 36.7 (C-10), 32.0 (CH-8), 32.0 (CH2-7), 27.8 (CH2-2), 27.2 (CH2-16), 24.9 (CH2-15), 21.4 , 20.6 (CH2-11), 19.3 (CH3-19), 12.7 (CH3-18); ESIMS: m/z 365 [M + Na]+; HRESIMS: m/z 365.2452 .3β-Acetoxy-5,20-pregnadiene (3): white powder; mp 116−118 °C;  +45 ; IR (neat) νmax 1682 cm−1; ESIMS: m/z 321 [M + Na]+. The 1H and 13C NMR data of 3 were in full agreement with those of reported previously [5α-Pregna-1,20-dien-3-one , with a = 7.3639(4) Å, b = 11.5782(6) Å, c = 22.7667(11) Å, V = 1941.10(17) Å3, Z = 4, Dcalcd = 1.172 g/cm3 and λ  = 1.54178 Å. Intensity data were measured on a Bruker APEX-II CCD diffractometer equipped with a micro-focus Cu radiation source and Montel mirror up to θmax of 66.7° at 100 K. All 12,991 reflections were collected. The structure was resolved by direct methods and refined by a full-matrix least-squares procedure. The refined structure model converged to a final R1 = 0.0279, wR2 = 0.0717 for 3358 observed reflections (I > 2σ(I)) and 229 variable parameters. The absolute configuration was determined by Flack’s method, with Flack’s parameter determined to be 0.09(6) [Suitable colorless prisms of RPMI-1640 medium, fetal calf serum (FCS), tryptan blue, penicillin G and streptomycin were obtained from Gibco BRL . 3--2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and all other chemicals were purchased from Sigma-Aldrich .2. Cells were seeded at 4 × 104 per well in 96-well culture plates before treatment with different concentrations of the tested compounds. The compounds were dissolved in DMSO (less than 0.02%) and made immediately to 1.25, 2.5, 5, 10 and 20 μg/μL prior to the experiments. After treatment for 72 h, the cytotoxicities of the tested compounds were determined using a MTT cell proliferation assay . The MTT is reduced by the mitochondrial dehydrogenases of viable cells to a purple formazan product. The MTT-formazan product was dissolved in DMSO. Light absorbance values (OD = OD570 − OD620) were recorded at wavelengths of 570 and 620 nm using an ELISA reader  to calculate the concentration that caused 50% inhibition (IC50), i.e., the cell concentration at which the light absorbance value of the experimental group was half that of the control group. These results were expressed as a percentage of the control ± SD established from n = 4 wells per one experiment from three separate experiments.MOLT-4 (human acute lymphoblastic leukemia), HL-60 (human acute promyelocytic leukemia) and K-562 (human chronic myelogenous leukemia) cells were obtained from the American Type Culture Collection . Rat PBMCs were generated as described previously . Cells w6 cells were grown in 35-mm-diameter plates and were labeled with annexin V-FITC (10 μg/mL) and PI (20 μg/mL) prior to harvesting. After labeling, all plates were washed with a binding buffer and harvested. Cells were resuspended in the binding buffer at a concentration of 2 × 105 cells/mL before analysis by a flow cytometer FACS-Calibur  and CellQuest software. Approximately 10,000 cells were counted for each determination.The externalization of phosphatidylserine (PS) and the membrane integrity were quantified using an annexin V-FITC staining kit . In brief, 106 cells/mL before analysis by flow cytometry.These assays were performed as described previously. MMP disruption was detected with JC-1 cationic dye (5 μg/mL) . In briet-test, and p values lower than 0.05 were considered to be statistically significant. .The results were expressed as mean ± standard deviation (SD). Comparison in each experiment was performed using an unpaired Student’s Scleronephthya flexilis, three pregnane-type steroids, including a new metabolite, 3β-methoxy-5,20-pregnadiene (1), along with two known compounds, 3β-acetoxy-5,20-pregnadiene (2) and 5α-pregna-1,20-dien-3-one (3), were isolated. The structure of new steroid 1 was elucidated on the basis of spectroscopic methods. The absolute configuration of steroid 2 was further confirmed by X-ray diffraction analysis for the first time. Our results also suggest that the use of steroid 3 induced MMP disruption, as well as apoptosis in Molt-4 cells. The current work clearly supports the potential application of 3 for leukemia therapy. The soft coral Scleronephthya flexilis has been transplanted to culturing tanks located in the National Museum of Marine Biology and Aquarium, Taiwan, for extraction of additional natural products to establish a stable supply of bioactive material.In the first study on the chemical constituents of soft coral


S)-4-[4-cyano-2-(methylsulfonyl)phenyl]-3,6-dimethyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carbonitrile) was shown to be efficacious in a rodent animal model related to ALI. BAY 85-8501 is currently being tested in clinical studies for the treatment of pulmonary diseases.Human neutrophil elastase (HNE) is a key protease for matrix degradation. High HNE activity is observed in inflammatory diseases. Accordingly, HNE is a potential target for the treatment of pulmonary diseases such as chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchiectasis (BE), and pulmonary hypertension (PH). HNE inhibitors should reestablish the protease–anti-protease balance. By means of medicinal chemistry a novel dihydropyrimidinone lead-structure class was identified. Further chemical optimization yielded orally active compounds with favorable pharmacokinetics such as the chemical probe BAY-678. While maintaining outstanding target selectivity, picomolar potency was achieved by locking the bioactive conformation of these inhibitors with a strategically positioned methyl sulfone substituent. An induced-fit binding mode allowed tight interactions with the S2 and S1 pockets of HNE. BAY 85-8501 ((4 Human neutrophil elastase  is a member of the chymotrypsin-like family of serine proteases and is stored in the azurophil granules in the neutrophil cytoplasm. This highly active protease is able to break down mechanically important structures of the body’s own cellular matrix , as well as proteins foreign to the body . Furthermore, the enzyme cleaves a variety of endogenous and exogenous proteins, tuning their biological activity such as activation of other bioactive proteases , liberation of growth factors, shedding of cell-surface-bound receptors, and degradation of endogenous proteinase inhibitors  or exogenous virulence factors. ho ho13] ho1)–16] or  or 1)1)-6-methyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (35)Allyl  and phosphorus pentoxide  were stirred at 50 °C overnight. The mixture was diluted with tert-butyl methyl ether . 4-Formyl-3-(methylsulfonyl)benzonitrile , 1-[3-(trifluoromethyl)phenyl]urea  and allyl acetoacetate  were added. The reaction mixture was heated at reflux overnight. MTBE (350 mL) was removed by distillation. The residue was heated at reflux for a further 4 h. The organic solvents were removed. The residue was suspended in Et2O and filtered. The solid was washed with H2O (350 mL) and Et2O (50 mL). Yield: 34.74 g (64 %); 1H NMR : δ=2.15 , 3.45 , 4.45 , 4.95 , 5.05 , 5.65 , 6.40 , 7.20 , 7.70 , 7.80 , 7.85 , 8.10 , 8.25 , 8.35  ppm; MS (ESI+) m/z: 520.2 [M+H]+.S)-4-[4-cyano-2-(methylsulfonyl)phenyl]-6-methyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (36)Allyl (4: Compound 35 (2.33 g) was separated into enantiomers by preparative chiral-phase HPLC . Yield: 0.8 g ; LC–MS : tR=2.11 min; chiral analytical HPLC : tR=1.45 min; [α]D20=+41.6 (c=0.485 in MeOH); 1H NMR : δ=2.10 , 3.45 , 4.45 , 4.95 , 5.05 , 5.65 , 6.40 , 7.20 , 7.70 , 7.80 , 7.85 , 8.10 , 8.25 , 8.35  ppm; MS (ESI+) m/z: 520.1 [M+H]+; MS (ESI−) m/z: 518.2 [M−H]−.S)-4-[4-Cyano-2-(methylsulfonyl)phenyl]-6-methyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (37) and morpholine  were dissolved in anhydrous THF (25 mL) at room temperature. Tetrakis(triphenylphosphine)palladium(0)  was added. The mixture was stirred at room temperature for 90 min, then concentrated. The residue was dissolved in EtOAc (500 mL) and washed with saturated aqueous NH4Cl solution (50 mL), H2O (50 mL) and brine (50 mL). The organic layer was dried over Na2SO4 and concentrated. The residue was purified by preparative HPLC . Yield: 696 mg (94 %); 1H NMR : δ=2.14 , 3.45 , 6.35 , 7.14 , 7.72 , 7.80 , 7.86 , 8.11 , 8.27 , 8.36 , 12.64  ppm; MS (ESI+) m/z: 480.1 [M+H]+; MS (ESI−) m/z: 478.1 [M−H]−.S)-4-[4-Cyano-2-(methylsulfonyl)phenyl]-6-methyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carboxamide (38) and HATU  were dissolved in anhydrous DMF (35 mL) at 0 °C. After 20 min, NH4Cl  and N,N-diisopropylethylamine  were added. The mixture was stirred at room temperature for 4 h, and was then concentrated. The residue was purified by preparative HPLC . Yield: 612 mg (88 %); 1H NMR : δ=1.80 , 3.40 , 6.35 , 7.20 , 7.25 , 7.45 , 7.65–7.80 , 8.10 , 8.30 , 8.35  ppm; MS (ESI+) m/z: 479.1 [M+H]+; MS (ESI−) m/z: 477 [M−H]−.Method A: S)-4-[4-Cyano-2-(methylsulfonyl)phenyl]-6-methyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (28) was dissolved in anhydrous THF (35 mL). (Methoxycarbonylsulfamoyl)triethylammonium hydroxide  was added, and the mixture was stirred at room temperature for 90 min. The solvent was removed in vacuo. The residue was purified by preparative HPLC . Yield: 470 mg (87 %).Method B: S)-4-[4-Cyano-2-(methylsulfonyl)phenyl]-6-methyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (28) and trimethylamine  were dissolved in anhydrous THF (50 mL). Trifluoroacetic anhydride  was added dropwise, ensuring a temperature <35 °C. The reaction mixture was stirred at room temperature for 15 min. A saturated aqueous solution of NaHCO3 (250 mL) was added dropwise. The mixture was extracted twice with EtOAc. The combined organic layers were washed with brine and dried over MgSO4. Silica gel (30 g) was added, and the mixture was concentrated. The residue was purified by column chromatography on silica gel (CH2Cl2/EtOAc 2:1). Yield: 6.46 g (65 %); mp: 258–259 °C; [α]D20=−222.0 (c=0.480 in DMF); 1H NMR : δ=1.80 , 3.40 , 6.45 , 7.70–7.85 , 7.95 , 8.30–8.40  ppm; MS (ESI+) m/z: 461.1 [M+H]+; MS (ESI−) m/z: 459.2 [M−H]−.Method A: S)-4-[4-Cyano-2-(methylsulfonyl)phenyl]-3,6-dimethyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carbonitrile  was dissolved in THF (2 mL), and NaH suspension in mineral oil  was added. After stirring at room temperature for 20 min, CH3I  was added. The reaction mixture was stirred at room temperature for 120 min. The mixture was purified directly by preparative HPLC . Yield: 18 mg (23 %).Method B: S)-4-[4-Cyano-2-(methylsulfonyl)phenyl]-3,6-dimethyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carbonitrile  was dissolved in absolute THF (10 mL) and cooled to −78 °C. A solution of LiHMDS  was added dropwise. After stirring for 20 min, CH3I  was added. The mixture was allowed to warm to room temperature over 60 h. The mixture was purified directly by preparative HPLC . Yield: 454 mg (96 %); 1H NMR : δ=1.80 , 2.65 , 3.40 , 6.45 , 7.65–8.40 , 8.45  ppm; MS (ESI+) m/z: 475.0 (100) [M+H]+; MS (ESI−) m/z: 473.2 [M−H]−.19, 20, 24, and 28 have been deposited at the RCSB Protein Data Bank (PDB) with the respective access codes 5A09, 5A0A, 5A0B, and 5A0C. All in vivo procedures conformed to European Community directives and national legislation  for the use of animals for scientific purposes and were approved by the competent regional authority. All other experimental data including chemical procedures and analytics are provided in the Supporting Information.The crystallographic data for the four protein co-crystallization structures with